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Me whai pūnaha pono i roto i te vitro ka taea te whakaputa tika i te taiao ā-tinana o te ngākau mō te whakamātautau tarukino. Nā te iti o te wātea o ngā pūnaha ahurea kiko ngākau tangata kua hua ake he whakamaoritanga hē o ngā pānga rongoā ngākau. I konei, kua whakawhanakehia e mātou he tauira ahurea kiko ngākau (CTCM) e whakaoho ana i ngā poro ngākau mā te hikohiko, ā, ka pāngia e te totoro ā-tinana i ngā wā systolic me te diastolic o te huringa ngākau. I muri i ngā rā 12 o te ahurea, i whakapai ake tēnei huarahi i te oranga o ngā wāhanga ngākau, engari kāore i tino tiakina tō rātou hanganga. Nō reira, i muri i te tirotiro i ngā ngota iti, i kitea e mātou ko te tāpiri o te 100 nM triiodothyronine (T3) me te 1 μM dexamethasone (Dex) ki tā mātou reo i pupuri i te hanganga moroiti o ngā wāhanga mō ngā rā 12. I te taha o te maimoatanga T3/Dex, i pupuri te pūnaha CTCM i ngā kōtaha whakawhiti, te oranga, te ngohe pākia, me te hanganga i te taumata kotahi ki te kiko ngākau hou mō ngā rā 12. Hei tāpiri, ko te totoro nui o te kiko ngākau i roto i te ahurea ka puta te tohu hypertrophic cardiac, e whakarato ana i te taunakitanga mō te kaha o te CTCM ki te whai i ngā āhuatanga hypertrophic i puta mai i te totoro o te ngākau. Hei whakamutunga, ka taea e te CTCM te whakatauira i te āhua o te tinana me te mate o te ngākau i roto i te ahurea mō ngā wā roa, kia taea ai te tirotiro pono i te rongoā.
I mua i te rangahau haumanu, e hiahiatia ana ngā pūnaha in vitro pono e taea ai te whakaputa tika i te taiao ā-tinana o te ngākau tangata. Me whai i ngā āhuatanga hikohiko kua whakarerekētia te totoro ā-pūnaha, te tere o te ngākau, me te hikohiko. He maha ngā wā ka whakamahia ngā tauira kararehe hei tūāpapa tirotiro mō te āhua o te ngākau me te iti o te pono ki te whakaata i ngā pānga o ngā rongoā ki te ngākau tangata1,2. Hei whakamutunga, ko te Tauira Whakamātautau Ahurea Tionga Ngākau Pai (CTCM) he tauira tino tairongo, ā, he motuhake hoki mō ngā wawaotanga rongoā me ngā rongoā, e whakaputa tika ana i te āhua o te ngākau me te mate o te ngākau tangata3. Ko te kore o tētahi pūnaha pēnei ka whakawhāiti i te kitenga o ngā maimoatanga hou mō te ngoikore o te ngākau4,5 ā, kua arahina ki te paitini o te ngākau rongoā hei take nui mō te wehenga atu i te mākete6.
I roto i te tekau tau kua pahure ake nei, e waru ngā rongoā kore-ngākau kua tangohia mai i te whakamahinga haumanu nā te mea ka roa ake te wā QT ka arahi ki ngā arrhythmias ventricular me te mate ohorere7. Nō reira, kei te piki haere te hiahia mō ngā rautaki tirotiro haumanu pono hei aromatawai i te whai huatanga o te ngākau me te paitini. Ko te whakamahinga hou o ngā cardiomyocytes i ahu mai i te pūtau take pluripotent tangata (hiPS-CM) i roto i te tirotiro rongoā me te whakamātautau paitini e whakarato ana i tētahi otinga wāhanga ki tēnei raruraru. Heoi, ko te āhua koretake o ngā hiPS-CM me te kore o te uaua maha-pūtau o te kiko ngākau he herenga nui o tēnei tikanga. Kua whakaatuhia e ngā rangahau hou ka taea te whakakore i tēnei herenga wāhanga mā te whakamahi i ngā hiPS-CM wawe ki te hanga hydrogels kiko ngākau i muri tata mai i te tīmatanga o ngā whakahekenga ohorere me te piki haere o te whakaoho hiko i roto i te wā. Heoi, kāore i roto i ēnei kiko moroiti hiPS-CM ngā āhuatanga electrophysiological me te contractile pakeke o te myocardium pakeke. Hei tāpiri, he hanganga uaua ake te kiko ngākau tangata, he ranunga rerekē o ngā momo pūtau rerekē, tae atu ki ngā pūtau endothelial, ngā neuron, me ngā fibroblasts stromal, e honoa ana e ngā huinga motuhake o ngā pūmua matrix extracellular. Ko tēnei rerekētanga o ngā taupori kore-cardiomyocyte11,12,13 i roto i te ngākau o ngā kararehe pakeke he arai nui ki te whakatauira i te kiko ngākau mā te whakamahi i ngā momo pūtau takitahi. E whakanui ana ēnei herenga nui i te hiranga o te whakawhanake i ngā tikanga mō te whakatipu i te kiko myocardial kāore i pakaru i raro i ngā āhuatanga ā-tinana me ngā āhuatanga mate.
Kua whakamātauhia ngā wāhanga angiangi (300 µm) o te ngākau tangata hei tauira whai hua mō te myocardium tangata kāore i pakaru. Mā tēnei tikanga ka taea te uru atu ki tētahi pūnaha maha-pūtau 3D katoa e rite ana ki te kiko ngākau tangata. Heoi, tae noa ki te tau 2019, i whakawhāitihia te whakamahinga o ngā wāhanga ngākau kua whakatipuria e te poto (24 hāora) o te oranga ahurea. Nā ētahi āhuatanga tēnei tae atu ki te korenga o te totoro ā-tinana-mīhini, te hononga hau-wai, me te whakamahinga o ngā pāpāho māmā kāore e tautoko i ngā hiahia o te kiko ngākau. I te tau 2019, i whakaatuhia e ētahi rōpū rangahau ko te whakauru i ngā āhuatanga mīhini ki roto i ngā pūnaha ahurea kiko ngākau ka taea te whakaroa i te oranga ahurea, te whakapai ake i te whakaaturanga o te ngākau, me te whai i te mate o te ngākau. E rua ngā rangahau huatau 17 me te 18 e whakaatu ana he pānga pai te utaina mīhini uniaxial ki te āhua o te ngākau i te wā e whakatipuria ana. Heoi, kāore ēnei rangahau i whakamahi i te utaina ā-tinana-mīhini-toru-ahu hihiri o te huringa ngākau, nā te mea i utaina ngā wāhanga ngākau ki ngā kaha kume isometric 17, ki te utaina auxotonic raina rānei 18. Nā ēnei tikanga totoro kiko i pehi te maha o ngā ira ngākau, i nui rawa rānei te whakaputanga o ngā ira e pā ana ki ngā urupare totoro rerekē. He mea nui, i whakawhanakehia e Pitoulis et al. 19 tētahi kaukau ahurea poro ngākau hihiri mō te hanga anō i te huringa ngākau mā te whakamahi i te urupare whakawhiti kaha me ngā taraiwa kume. Ahakoa ka taea e tēnei pūnaha te whakatauira huringa ngākau in vitro tika ake, ko te uaua me te iti o te putanga o te tikanga ka whakawhāiti i te tono o tēnei pūnaha. Nō nā tata nei i whakawhanakehia e tā mātou taiwhanga tētahi pūnaha ahurea māmā ake mā te whakamahi i te whakaoho hiko me tētahi reo kua arotauhia hei pupuri i te oranga o ngā wāhanga o te kiko ngākau poaka me te ngākau tangata tae atu ki te 6 rā20,21.
I roto i tēnei tuhinga, ka whakaahuahia e mātou tētahi tauira ahurea kiko ngākau (CTCM) mā te whakamahi i ngā wāhanga o te ngākau poaka e whakauru ana i ngā tohu humoral hei whakahoki anō i te āhua o te ngākau me te whānui o te pathophysiological i te huringa ngākau. Ka taea e tēnei CTCM te whakanui ake i te tika o te matapae rongoā i mua i te haumanu ki tētahi taumata kāore anō kia tutukihia i mua mā te whakarato i tētahi pūnaha ngākau whai hua, waenga-whakaputa, e whai ana i te āhua o te ngākau/pathophysiology o te ngākau kararehe mō te whakamātautau rongoā i mua i te haumanu.
He mahi nui tā ngā tohu ā-tinana mō te pupuri i te mahi a te cardiomyocyte i roto i te vitro 22,23,24. I roto i tēnei tuhinga, kua whakawhanakehia e mātou he CTCM (Pikitia 1a) ka taea te whai i te taiao ngākau pakeke mā te whakaoho i te whakaoho hiko me te whakaoho ā-tinana i ngā auau ā-tinana (1.2 Hz, 72 ngā pao i te meneti). Hei karo i te totoro nui o te kiko i te wā diastole, i whakamahia he taputapu tā 3D hei whakanui ake i te rahi o te kiko mā te 25% (Pikitia 1b). Ko te tere hiko i whakaohohia e te pūnaha C-PACE i whakaritea kia tīmata i te 100 ms i mua i te systole mā te whakamahi i tētahi pūnaha kohikohi raraunga hei whakaputa anō i te huringa ngākau. Ka whakamahia e te pūnaha ahurea kiko tētahi kaiwhakaoho hau ka taea te whakarite (LB Engineering, Tiamana) hei whakawhanui i tētahi kiriuhi silicone ngāwari hei whakawhanui i ngā poro ngākau i roto i te rūma o runga. I honoa te pūnaha ki tētahi raina hau o waho mā tētahi transducer pēhanga, i taea ai te whakarite tika i te pēhanga (± 1 mmHg) me te wā (± 1 ms) (Pikitia 1c).
a Tāpirihia te wāhanga kiko ki te mowhiti tautoko 7 mm, e whakaaturia ana i te kikorangi, i roto i te rūma ahurea o te taputapu. Kua wehea te rūma ahurea mai i te rūma hau e tētahi kiriuhi silicone angiangi ngāwari. Whakatakotoria he gasket i waenganui i ia rūma hei ārai i ngā turuturu. Kei roto i te taupoki o te taputapu ngā irahiko karāhe e whakarato ana i te whakaoho hiko. b Te whakaaturanga hoahoa o te taputapu kiko nui, te mowhiti ārahi me te mowhiti tautoko. Kua whakanohoia ngā wāhanga kiko (parauri) ki runga i te taputapu nui rawa atu me te mowhiti ārahi kua whakanohoia ki roto i te awaawa i te taha o waho o te taputapu. Mā te whakamahi i te aratohu, āta whakanohohia te mowhiti tautoko kua pania ki te piri kiriaku kiko ki runga i te wāhanga o te kiko ngākau. c Te kauwhata e whakaatu ana i te wā o te whakaoho hiko hei mahi a te pēhanga rūma hau e whakahaerehia ana e te kaiwhakaoho hau ka taea te whakarite (PPD). I whakamahia he taputapu kohikohi raraunga hei tukutahi i te whakaoho hiko mā te whakamahi i ngā pūoko pēhanga. Ina tae te pēhanga i roto i te rūma ahurea ki te paepae kua whakaritea, ka tukuna he tohu pāhekoheko ki te C-PACE-EM hei whakaoho i te whakaoho hiko. d Te ahua o ngā CTCM e whā kua whakanohoia ki runga i te whata incubator. E whā ngā taputapu e honoa ana ki te PPD kotahi mā te ara iahiko hau, ā, ka whakauruhia ngā pūoko pēhanga ki roto i te takirere hemostatic hei aroturuki i te pēhanga i roto i te ara iahiko hau. E ono ngā wāhanga kiko kei roto i ia taputapu.
Mā te whakamahi i tētahi pūwhakaoho hau kotahi, i taea e mātou te whakahaere i ngā taputapu CTCM e 4, ā, e 6 ngā wāhanga kiko e taea ana e ia taputapu te pupuri (Pikitia 1d). I roto i te CTCM, ka hurihia te pēhanga hau i roto i te rūma hau ki te pēhanga tukutahi i roto i te rūma wai, ā, ka puta te whānui ā-tinana o te poro ngākau (Pikitia 2a me te Kiriata Tāpiri 1). I te aromatawai i te totoro kiko i te 80 mm Hg. I whakaatuhia e te Toi te totoro o ngā wāhanga kiko mā te 25% (Pikitia 2b). Kua whakaatuhia tēnei totoro ōrau e rite ana ki te roa sarcomere ā-tinana o te 2.2–2.3 µm mō te kaha o te wāhanga ngākau17,19,25. I aromatawaihia te nekehanga kiko mā te whakamahi i ngā tautuhinga kāmera ritenga (Pikitia Tāpiri 1). Ko te kaha me te tere o te nekehanga kiko (Pikitia 2c, d) e rite ana ki te totoro i te huringa ngākau me te wā i te systole me te diastole (Pikitia 2b). I noho pūmau te totoro me te tere o te kiko ngākau i te wā o te whakaheke me te whakangā mō ngā rā 12 i roto i te ahurea (Pikitia 2f). Hei aromatawai i te pānga o te whakaoho hiko ki te kaha o te whakapūmau i te wā whakatipu, i whakawhanakehia e mātou he tikanga hei whakatau i te āhua ngoikore o te ngākau mā te whakamahi i tētahi tauira whakamarumaru (Pikitia Tāpiri 2a,b) ā, i taea e mātou te wehewehe i waenga i ngā āhua ngoikore me te kore whakaoho hiko. Ko te wāhanga kotahi o te ngākau (Pikitia 2f). I te rohe nekeneke o te tapahi (R6-9), ko te ngaohiko i te wā o te whakaoho hiko he 20% teitei ake i te kore o te whakaoho hiko, e tohu ana i te takoha o te whakaoho hiko ki te mahi whakapūmau.
Ko ngā tohu e whakaatu ana i te pēhanga o te rūma hau, te pēhanga o te rūma wai, me ngā inenga nekehanga kiko e whakaū ana ka huri te pēhanga o te rūma i te pēhanga o te rūma wai, ka puta he nekehanga rite o te poro kiko. b Ngā tohu e whakaatu ana i te ōrau totoro (kikorangi) o ngā wāhanga kiko e rite ana ki te ōrau totoro (karaka). c Ko te nekehanga ine o te poro ngākau e rite ana ki te tere o te nekehanga ine. (d) Ngā ara e whakaatu ana i te nekehanga huringa (rārangi kikorangi) me te tere (rārangi ira karaka) i roto i te poro o te ngākau. e Te ine i te wā huringa (n = 19 poro ia rōpū, mai i ngā poaka rerekē), te wā whakaheke (n = 19 poro ia rōpū), te wā whakangā (n = 19 poro ia rōpū, mai i ngā poaka rerekē), te nekehanga kiko (n = 25 ). poro)/rōpū mai i ngā poaka rerekē), te tere systolic tihi (n = 24(D0), 25(D12) poro/rōpū mai i ngā poaka rerekē) me te tere whakangā tihi (n=24(D0), 25(D12) poro/rōpū mai i ngā poaka rerekē). Kāore i whakaatuhia e te whakamātautau-t a te Tauira-rua-hiku tētahi rerekētanga nui i roto i tētahi tawhā. f Ngā tohu tātari taumahatanga māngai o ngā wāhanga kiko me te whakaoho hiko (whero) me te kore (kikorangi), tekau ngā rohe rohe o ngā wāhanga kiko mai i te wāhanga kotahi. E whakaatu ana ngā panera o raro i te ine i te ōrau rerekētanga o te taumahatanga i roto i ngā wāhanga kiko me te whakaoho hiko, me te kore hoki i roto i ngā rohe tekau mai i ngā wāhanga rerekē. (n = 8 poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia te whakamātautau-t Tauira Hiku-Rua; ****p < 0.0001, **p < 0.01, *p < 0.05). (n = 8 poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia te whakamātautau-t Tauira Hiku-Rua; ****p < 0.0001, **p < 0.01, *p < 0.05). (n = 8 срезов/группу от разных свиней, проводится двусторонний t-критерий Стьюдента; ****p<0,0001, **p<0,01, *p<0,05). (n = 8 wāhanga/rōpū mai i ngā poaka rerekē, whakamātautau-t a te Tauira hiku-rua; ****p<0.0001, **p<0.01, *p<0.05). （n = 8 片/组，来自不同的猪，进行双尾学生t 检验；****p < 0.0001，**p < 0.01，*p < 0.05）。 （n = 8 片/组，来自不同的猪，进行双尾学生t 检验；****p < 0.0001，**p < 0.01，*p < 0.05）。 (n = 8 срезов/группу, от разных свиней, двусторонний критерий Стьюдента; ****p <0,0001, **p <0,01, *p <0,05). (n = 8 wāhanga/rōpū, mai i ngā poaka rerekē, whakamātautau-t a te Tauira hiku-rua; ****p<0.0001, **p<0.01, *p<0.05).Ko ngā pae hapa e tohu ana i te toharite ± paerewa rerekētanga.
I roto i tā mātou pūnaha whakatipu ngākau biomimetic pūmau o mua [20, 21], i pupuri mātou i te oranga, te mahi, me te hanganga o ngā poro ngākau mō ngā rā e 6 mā te whakamahi i te whakaoho hiko me te arotau i te hanganga o te reo. Heoi, i muri i ngā rā 10, ka tino heke iho ēnei tatauranga. Ka tirohia e mātou ngā wāhanga i whakatipuria i roto i tā mātou pūnaha whakatipu biomimetic pūmau o mua 20, 21 ngā tikanga whakahaere (Ctrl) ā, ka whakamahia e mātou tā mātou reo i arotauhia i mua hei tikanga MC me te ahurea i raro i te whakaoho miihini me te hiko kotahi (CTCM). i karangatia . Tuatahi, i whakatauhia e mātou kāore i te rawaka te whakaoho miihini me te kore whakaoho hiko hei pupuri i te oranga kiko mō ngā rā e 6 (Pikitia Tāpiri 3a,b). He mea whakamīharo, me te whakaurunga o te whakaoho tinana-mīhini me te hiko mā te whakamahi i te STCM, i noho tonu te oranga o ngā wāhanga ngākau 12-rā ki ngā wāhanga ngākau hou i raro i ngā tikanga MS, engari kāore i raro i ngā tikanga Ctrl, e whakaaturia ana e te tātaritanga MTT (Pikitia 1). 3a). E kī ana tēnei ka taea e te whakaoho miihini me te whakatauira o te huringa ngākau te pupuri i ngā wāhanga kiko kia ora mō te rua o te roa i te mea i pūrongohia i roto i tā mātou pūnaha whakatipu pūmau o mua. Heoi, i whakaatuhia e te aromatawai i te hanganga o ngā wāhanga kiko mā te immunolabeling o te troponin T me te connexin 43 o te ngākau he nui ake te whakaaturanga o te connexin 43 i roto i ngā kiko MC i te rā 12 i ngā mana whakahaere i taua rā anō. Heoi, kāore i tino mau tonu te whakaaturanga connexin 43 me te hanganga kōpae-Z (Pikitia 3b). Ka whakamahia e mātou he anga matauranga horihori (AI) hei ine i te hanganga o te kiko26, he paipa ako hohonu e ahu mai ana i te ahua e ahu mai ana i te troponin-T me te connexin staining43 hei ine aunoa i te hanganga me te fluorescence o ngā poro ngākau e pā ana ki te kaha o te localization. Ka whakamahia e tēnei tikanga he Whatunga Neural Convolutional (CNN) me te anga ako hohonu hei ine pono i te hanganga o te kiko ngākau i roto i te huarahi aunoa me te kore whakaaro, e ai ki te whakaahuatanga i roto i te tohutoro. 26. I whakaatuhia e te kiko MC te rite hanganga pai ake ki te rā 0 ki te whakataurite ki ngā wāhanga whakahaere tūmau. Hei tāpiri, i whakaatuhia e te tae trichrome a Masson he ōrau iti iho o te fibrosis i raro i ngā āhuatanga MS ki te whakataurite ki ngā āhuatanga whakahaere i te rā 12 o te ahurea (Pikitia 3c). Ahakoa i whakapiki ake te CTCM i te kaha o ngā wāhanga kiko o te ngākau i te rā 12 ki te taumata e rite ana ki te kaha o te kiko o te ngākau hou, kāore i tino whakapai ake i te hanganga o ngā wāhanga ngākau.
E whakaatu ana te kauwhata pae i te ine i te oranga MTT o ngā poro ngākau hou (D0) me ngā poro ngākau rānei mō ngā rā 12 i roto i te ahurea tūmau (D12 Ctrl) i roto rānei i te CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) poro/rōpū mai i ngā poaka rerekē, ka mahia te whakamātautau ANOVA kotahi te ara; ####p < 0.0001 ki te whakarite ki te D0 me **p < 0.01 ki te whakarite ki te D12 Ctrl). E whakaatu ana te kauwhata pae i te ine i te oranga MTT o ngā poro ngākau hou (D0) me ngā poro ngākau rānei mō ngā rā 12 i roto i te ahurea tūmau (D12 Ctrl) i roto rānei i te CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) poro/rōpū mai i ngā poaka rerekē, ka mahia te whakamātautau ANOVA kotahi te ara; ####p < 0.0001 ki te whakarite ki te D0 me **p < 0.01 ki te whakarite ki te D12 Ctrl).E whakaatu ana te kauwhata i te ine i te oranga o ngā wāhanga ngākau hou MTT (D0) i te ahurea rānei o ngā wāhanga ngākau mō ngā rā 12 i roto i te ahurea tūmau (mana D12) CTCM rānei (D12 MC) (n = 18 (D0), 15 (mana D12). ) ), 12 (D12 MC) wāhanga/rōpū mai i ngā poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara;####p < 0,0001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 ki te whakataurite ki a D0 me **p < 0.01 ki te whakataurite ki a D12 Ctrl). a 条形图显示在静态培养(D12 Ctrl) 或CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl) 中新鲜心脏切)片或心脏切片培养12 天的MTT 活力的量化)，来自不同猪的12 (D12 MC) 切片/组，进行单曑行单后从相比，####p < 0.0001，与D12 Ctrl 相比，**p < 0.01). a 条形图显示在静态培养(D12 Ctrl) 或CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl) 中新鲜心脏切)片，来自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；与D0 相比，####p < 0.0001，与与用 Ctrl)he kauwhata e whakaatu ana i te inenga o te oranga o te MTT i roto i ngā wāhanga ngākau hou (D0) i ngā wāhanga ngākau rānei i whakatōkia mō ngā rā 12 i roto i te ahurea tūmau (mana D12) CTCM rānei (D12 MC) (n = 18 (D0), 15 (mana D12)), 12 (D12 MC) wāhanga/rōpū mai i ngā poaka rerekē, whakamātautau ANOVA kotahi-ara;####p < 0,0001 по сравнению с D0, **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 ki te whakataurite ki a D0, **p < 0.01 ki te whakataurite ki a D12 Ctrl).b Troponin-T (kakariki), connexin 43 (whero) me te DAPI (kikorangi) i roto i ngā wāhanga ngākau hou kua wehea (D0) i ngā wāhanga ngākau rānei i whakatipuria i raro i ngā āhuatanga tūmau (Ctrl) i ngā āhuatanga CTCM rānei (MC) mō ngā rā 12) o ngā whakaahua immunofluorescence māngai (tauine pātea = 100 µm). Te ine i te hanganga o te kiko o te ngākau mā te whakamahi i te matauranga horihori (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) poro/rōpū mai i ia poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara; ####p < 0.0001 ki te whakataurite ki a D0 me ****p < 0.0001 ki te whakataurite ki a D12 Ctrl). Te ine i te hanganga o te kiko o te ngākau mā te whakamahi i te matauranga horihori (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) poro/rōpū ia poro mai i ngā poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara; ####p < 0.0001 ki te whakataurite ki a D0 me ****p < 0.0001 ki te whakataurite ki a D12 Ctrl). Количественная оценка структурной целостности сердечной тпкани искусственным интеллектом (n = 7 (D0), 7 (D12 Ctrl2), MC52 Ctrl от разных свиней, проводится однофакторный тест ANOVA; Te ine i te hanganga o te kiko o te ngākau mā te matauranga horihori (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) ngā wāhanga/rōpū mai i ngā poaka rerekē, i whakahaerehia he whakamātautau ANOVA kotahi-ara; ####p < 0.0001 ki te whakataurite ki te D0 me te ****p < 0.0001 ki te whakataurite ki te D12 Ctrl).人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) poro/rōpū o ia poaka rereke, kotahi-ara whakamatautau ANOVA;####p01 ， ####p01 ， ####p01 ， ****001 ， ####p01 ， ####p01 ， 0.****p < 0.0001 与D12 Ctrl 相比）。人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) poro/rōpū ia o nga poaka rereke, kotahi-ara whakamatautau ANOVA;##00 < 10.****p < 10. < 0.0001 与D12 Ctrl 相比）。 Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), MC12), MC (D0), 12 (D12) срезов/группу каждой из разных свиней, односторонний тест ANOVA ####p <0,0001 vs. D0 Для сравнения ****p < 0,0001 по сравню). Te mātauranga horihori hei ine i te hanganga o te kiko ngākau (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) wāhanga/rōpūhia ia poaka rerekē, whakamātautau ANOVA kotahi-ara; ####p<0.0001 vs .D0 Hei whakatairite ****p < 0.0001 ki te whakataurite ki te D12 Ctrl). c Ngā whakaahua māngai (maui) me te ine (matau) mō ngā poro ngākau i pania ki te tae trichrome a Masson (Tauine tahanga = 500 µm) (n = 10 ngā poro/rōpū mai i ia poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara; ####p < 0.0001 ki te whakataurite ki a D0 me ***p < 0.001 ki te whakataurite ki a D12 Ctrl). c Ngā whakaahua māngai (maui) me te ine (matau) mō ngā poro ngākau i pania ki te tae trichrome a Masson (Tauine tahanga = 500 µm) (n = 10 ngā poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara; #### p < 0.0001 ki te whakataurite ki a D0 me ***p < 0.001 ki te whakataurite ki a D12 Ctrl). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красмалете без покрытия = 500 мкм) (n = 10 срезов/группу от разных свиней, выполняется односторонний тест ANOVA; #### p < 0,0001 0,001 по сравнению с D12 Mana). c Ngā whakaahua māngai (maui) me te ine (matau) o ngā wāhanga ngākau i pania ki te tae trichrome a Masson (tauine kore pania = 500 µm) (n = 10 ngā wāhanga/rōpū mai i ngā poaka rerekē, i whakahaerehia he whakamātautau ANOVA kotahi-ara; #### p < 0 .0001 ki te whakataurite ki a D0 me ***p < 0.001 ki te whakataurite ki a D12 Ctrl). c 用Masson 三色染料染色的心脏切片的代表性图像（左）和量化（右）（裸尺度（裸尺度（（裸尺度 （裸尺度= 1个切片/组，每组来自不同的猪，进行单向ANOVA 测试；#### p < 0.0001 与D0 相比，***p < 0.0012 。 C 用 masson 三 色 染料 的 心脏 切片 的 代表性 （左 左） 量化 （右） 裸尺度 裸尺度 裸尺度 裸尺度裸尺度 = 500 µm） （n = 10 个 切片 组 每 组 来自 不同 猪 ， 进行 单向 单君向0.##0.与D0 相比，***p < 0.001 与D12 Ctrl 相比）. c Репрезентативные изображения (слева) и количественный анализ (справа) срезов сердца, окрашенных трихромным красителка = 500 мкм) (n = 10 срезов/группа, каждый от другой свиньи, протестировано с помощью однофакторного дисперси <###; 0,0001 по сравнению с D0, ***p < 0,001 по сравнению с D12 Ctrl). c Ngā whakaahua māngai (maui) me te ine (matau) o ngā wāhanga ngākau i pania ki te tae trichrome a Masson (kore = 500 µm) (n = 10 ngā wāhanga/rōpū, ia wāhanga mai i tētahi poaka rerekē, i whakamātauria mā te tātaritanga rerekētanga kotahi-ara;### # p < 0.0001 ki te whakarite ki a D0, ***p < 0.001 ki te whakarite ki a D12 Ctrl).Ko ngā pae hapa e tohu ana i te toharite ± paerewa rerekētanga.
I whakapae mātou mā te tāpiri i ngā ngota iti ki te reo ahurea, ka taea te whakapai ake i te kounga o ngā cardiomyocyte me te whakaiti i te whanaketanga o te fibrosis i te wā o te ahurea CTCM. Nō reira, i tirotirohia e mātou ngā ngota iti mā te whakamahi i ā mātou ahurea whakahaere pūmau20,21 nā te iti o ngā āhuatanga whakapoauau. I whiriwhiria te Dexamethasone (Dex), te triiodothyronine (T3), me te SB431542 (SB) mō tēnei tirotiro. Kua whakamahia ēnei ngota iti i roto i ngā ahurea hiPSC-CM i mua hei whakaoho i te pakeketanga o ngā cardiomyocyte mā te whakanui ake i te roa o ngā sarcomere, ngā T-tubules, me te tere o te kawe. Hei tāpiri, e mōhiotia ana ko te Dex (he glucocorticoid) me te SB ka pehi i te mumura29,30. Nō reira, i whakamatautauria e mātou mēnā ka whakapai ake te whakaurunga o tētahi, o tētahi huinga rānei o ēnei ngota iti i te kounga hanganga o ngā wāhanga ngākau. Mō te tirotiro tuatahi, i whiriwhiria te horopeta o ia matū i runga i ngā kukū e whakamahia whānuitia ana i roto i ngā tauira ahurea pūtau (1 μM Dex27, 100 nM T327, me te 2.5 μM SB31). I muri i ngā rā 12 o te whakatipuranga, ko te whakakotahitanga o te T3 me te Dex i hua ake ai te hanganga pai o te cardiomyocyte me te iti o te hanga hou o te muka (Ngā Pikitia Tāpiri 4 me te 5). Hei tāpiri, ko te whakamahinga o te rua, te rua rānei o ēnei kukū o te T3 me te Dex i hua ake ai ngā pānga kino ki te whakataurite ki ngā kukū noa (Pikitia Tāpiri 6a,b).
I muri i te tirotiro tuatahi, i whakahaerehia e mātou he whakataurite kanohi ki te kanohi o ngā āhuatanga ahurea e 4 (Pikitia 4a): Ctrl: ngā wāhanga ngākau i whakatipuria i roto i tā mātou ahurea tūmau kua whakaahuatia i mua mā te whakamahi i tā mātou rauemi kua arotauhia; 20.21 TD: T3 me Ctrl s I tāpirihia a Dex i te Wenerei; MC: ngā wāhanga ngākau i whakatipuria i roto i te CTCM mā te whakamahi i tā mātou rauemi kua arotauhia i mua; me MT: CTCM me T3 me Dex i tāpirihia ki te rauemi. I muri i ngā rā 12 o te whakatipu, i noho tonu te oranga o ngā kiko MS me MT ki ngā kiko hou i aromatawaitia e te whakamātautau MTT (Pikitia 4b). He mea whakamīharo, ko te tāpiri o T3 me Dex ki ngā ahurea transwell (TD) kāore i hua ake he whakapainga nui o te oranga ki te whakataurite ki ngā āhuatanga Ctrl, e tohu ana i te tūranga nui o te whakaohooho ā-ringa ki te pupuri i te oranga o ngā wāhanga ngākau.
he hoahoa whakamātautau e whakaatu ana i ngā āhuatanga ahurea e whā i whakamahia hei aromatawai i ngā pānga o te whakaohooho ā-ringa me te tāpiringa T3/Dex ki te reo mō ngā rā 12. b E whakaatu ana te kauwhata pae i te ine i te oranga 12 rā i muri i te whakatipu i ngā āhuatanga whakatipu katoa e 4 (Ctrl, TD, MC, me MT) ki te whakataurite ki ngā poro ngākau hou (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD me D12 MT), 12 (D12 MC) poro/rōpū mai i ngā poaka rerekē, ka mahia te whakamātautau ANOVA kotahi-ara; ####p < 0.0001, ###p < 0.001 ki te whakataurite ki a D0 me **p < 0.01 ki te whakataurite ki a D12 Ctrl). b E whakaatu ana te kauwhata pae i te ine i te oranga 12 rā i muri i te whakatipu i ngā āhuatanga whakatipu katoa e 4 (Ctrl, TD, MC, me MT) ki te whakataurite ki ngā poro ngākau hou (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD me D12 MT), 12 (D12 MC) poro/rōpū mai i ngā poaka rerekē, ka mahia te whakamātautau ANOVA kotahi-ara; ####p < 0.0001, ###p < 0.001 ki te whakataurite ki a D0 me **p < 0.01 ki te whakataurite ki a D12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после культивирования во всельник 4 (контроль, TD, MC me MT) по сравнению со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD me D12 MT), 12 (D12 MT), 12 (D12 MT), MC разных свиней, проводится односторонний тест ANOVA; ####p < 0,0001, ###p < 0,001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). b E whakaatu ana te kauwhata pae i te inenga o te oranga i ngā rā 12 i muri i te whakatipu i ngā āhuatanga whakatipu katoa e 4 (mana whakahaere, TD, MC, me te MT) ki te whakataurite ki ngā wāhanga ngākau hou (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, me te D12 MT), 12 (D12 MC) wāhanga/rōpū mai i ngā poaka rerekē, whakamātautau ANOVA kotahi-ara; ####p < 0.0001, ###p < 0.001 vs. D0 me **p < 0.01 mā te whakataurite ki te D12 Ctrl). b 条形图显示所有4 种培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) (n = 18 (D0) 、 TD0和D12 MT)，来自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；####p < 0.0001，###p < 0.001 ，p<0.001 ，p<0.001 ，p**0.与D12控制）.b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC и MT) по сравнению со свежими (серди со свежими (серди) = 8 (D) 15 (D12 Ctrl, D12 TD и D12 MT), от разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA ####p <0,0001, ###p <0,0001, ###p <0,0001, ###p <0,0001, ###p <0. <0,01 по сравнению с контролем D12). b He kauwhata e whakaatu ana i ngā āhuatanga ahurea e 4 (mana whakahaere, TD, MC me MT) i whakaritea ki ngā wāhanga ngākau hou (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD me D12 MT), mai i ngā poaka rerekē 12 (D12 MC) wāhanga/rōpū, whakamātautau ANOVA kotahi-ara; ####p<0.0001, ###p<0.001 vs. D0, **p<0.01 vs. mana whakahaere D12). E whakaatu ana te kauwhata pae i te inenga o te rerenga huka 12 rā i muri i te whakatipu i ngā āhuatanga whakatipu katoa e 4 (Ctrl, TD, MC, me MT) ki te whakataurite ki ngā poro ngākau hou (D0) (n = 6 poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia te whakamātautau ANOVA kotahi-ara; ###p < 0.001, ki te whakataurite ki te D0 me te ***p < 0.001 ki te whakataurite ki te D12 Ctrl). E whakaatu ana te kauwhata pae i te inenga o te rerenga huka 12 rā i muri i te whakatipu i ngā āhuatanga whakatipu katoa e 4 (Ctrl, TD, MC, me MT) ki te whakataurite ki ngā poro ngākau hou (D0) (n = 6 poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia te whakamātautau ANOVA kotahi-ara; ###p < 0.001, ki te whakataurite ki te D0 me te ***p < 0.001 ki te whakataurite ki te D12 Ctrl). c Гистограмма показывает количественную оценку потока глюкозы через 12 дней после культивирования во всехол 4 лусих (koнтроль, TD, MC me MT) na roto i te сравнению со свежими срезами сердца (D0) (n = 6 срезов/группу от разных свиней, родниностяство ANOVA; ###p < 0,001 по сравнению с D0 me ***p < 0,001 по сравнению с D12 Ctrl). E whakaatu ana te kauwhata c i te inenga o te rerenga huka 12 rā i muri i te whakatipu i raro i ngā tikanga whakatipu katoa e 4 (mana whakahaere, TD, MC me MT) ki te whakataurite ki ngā wāhanga ngākau hou (D0) (n = 6 ngā wāhanga/rōpū mai i ngā poaka rerekē, i whakahaerehia he whakamātautau ANOVA kotahi-ara; ###p < 0.001 ki te whakataurite ki te D0 me te ***p < 0.001 ki te whakataurite ki te D12 Ctrl). c 条形图显示所有4 种培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) 相比，厹天的葡萄糖通量定量（n = 6 片/组，来自不同猪，单向执行ANOVA 测试；###p < 0.001 ，盎01 ，***0D1与D12 Ctrl 相比）. C 条形图 显示 所有 4 种 条件 （（ctrl 、 td 、 mc 和 mt） 新鲜 心脏 切片 切片 切片培养 后 后 12 天 的 通量 定量 （n = 6 片/组 ， 来自 猪 ， ， ， ， ， ， ， ， ， 猪 猪单行行< 0.001，与D0 相比，***p < 0.001 与D12 Ctrl 相比）. c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после культивирования для 4 всех культивирования (контроль, TD, MC и MT) по сравнению со свежими срезами сердца (D0) (n = 6 срезов/группа, от разинных Были проведены тесты ANOVA ###p < 0,001 по сравнению с D0, ***p < 0,001 по сравнению с D12 (контроль). c He kauwhata e whakaatu ana i te inenga o te rerenga huka i ngā rā 12 i muri i te whakatipuranga mō ngā āhuatanga whakatipuranga e 4 (mana whakahaere, TD, MC, me MT) ki te whakataurite ki ngā wāhanga ngākau hou (D0) (n = 6 ngā wāhanga/rōpū, mai i ngā poaka rerekē, taha kotahi. I te wā i whakahaerehia ai ngā whakamātautau ANOVA, ###p < 0.001 ki te whakataurite ki a D0, ***p < 0.001 ki te whakataurite ki a D12 (mana whakahaere).d Ngā kauwhata tātari taumahatanga o ngā kiko hou (kikorangi), MC (kakariki) o te rā 12, me MT (whero) o te rā 12 i ngā pūwāhi wāhanga kiko rohe tekau (n = 4 poro/rōpū, whakamātautau ANOVA kotahi-ara; kāore he rerekētanga nui i waenga i ngā rōpū). e Kauwhata puia e whakaatu ana i ngā ira rerekē te whakaaturanga i roto i ngā wāhanga ngākau hou (D0) ki te whakataurite ki ngā wāhanga ngākau i whakatipuria i raro i ngā āhuatanga pūmau (Ctrl) i raro rānei i ngā āhuatanga MT (MT) mō ngā rā 10-12. f Mahere wera o ngā ira sarcomere mō ngā wāhanga ngākau i whakatipuria i raro i ia o ngā āhuatanga ahurea. Ko ngā pae hapa e tohu ana i te toharite ± paerewa rerekētanga.
Ko te whakawhirinakitanga pākia ki te whakawhiti mai i te waikura waikawa ngako ki te glycolysis he tohu o te wehenga o te cardiomyocyte. Ko ngā cardiomyocyte kāore anō kia pakari ka whakamahi i te hukahuka hei hanga ATP, ā, he iti noa iho ngā mitochondria he iti noa iho ngā cristae5,32. I whakaatuhia e ngā tātaritanga whakamahinga hukahuka i raro i ngā āhuatanga MC me MT, he rite te whakamahinga hukahuka ki tērā i roto i ngā kiko o te rā 0 (Pikitia 4c). Heoi, i whakaatuhia e ngā tauira Ctrl he pikinga nui o te whakamahinga hukahuka ki te whakaritea ki te kiko hou. E tohu ana tēnei ko te whakakotahitanga o te CTCM me te T3/Dex ka whakarei ake i te oranga o te kiko, ā, ka tiakina te momo pākia o ngā wāhanga ngākau kua whakatipuria i te 12-rā. Hei tāpiri, i whakaatuhia e te tātaritanga taumahatanga he rite tonu ngā taumata taumahatanga ki te kiko ngākau hou mō ngā rā 12 i raro i ngā āhuatanga MT me MS (Pikitia 4d).
Hei tātari i te pānga whānui o CTCM me T3/Dex ki te whenua whakawhiti ā-ao o te kiko poro ngākau, i whakahaerehia e mātou te RNAseq ki ngā poro ngākau mai i ngā āhuatanga ahurea rerekē e whā (Raraunga Tāpiri 1). He mea whakamīharo, i whakaatuhia e ngā wāhanga MT te rite nui o te whakawhiti ki te kiko ngākau hou, me te 16 noa iho o ngā ira rerekē te whakaputanga mai i roto i te 13,642. Heoi, e ai ki tā mātou i whakaatu i mua ake nei, i whakaatuhia e ngā poro Ctrl te 1229 ira rerekē te whakaputanga i muri i te 10–12 rā i roto i te ahurea (Pikitia 4e). I whakaūhia ēnei raraunga e te qRT-PCR o ngā ira ngākau me ngā ira fibroblast (Pikitia Tāpiri 7a-c). He mea whakamīharo, i whakaatuhia e ngā wāhanga Ctrl te whakahekenga o ngā ira ngākau me te huringa pūtau me te whakahohenga o ngā kaupapa ira mumura. E kī ana ēnei raraunga ko te dedifferentiation, e puta ana i muri i te whakatipuranga roa, ka tino ngoikore i raro i ngā āhuatanga MT (Pikitia Tāpiri 8a,b). I whakaatuhia e te rangahau āta o ngā ira sarcomere i raro i ngā āhuatanga MT anake ka tiakina ngā ira e whakamārama ana i te sarcomere (Pikitia 4f) me te hongere katote (Pikitia Tāpiri 9), e tiaki ana i a rātou mai i te pehi i raro i ngā āhuatanga Ctrl, TD, me MC. E whakaatu ana ēnei raraunga mā te whakakotahitanga o te whakaohooho ā-pūkaha me te whakaohooho humoral (T3/Dex), ka taea e te transcriptome poro ngākau te noho rite tonu ki ngā poro ngākau hou i muri i ngā rā 12 i roto i te ahurea.
E tautokona ana ēnei kitenga whakawhiti e te meka ko te hanganga o ngā cardiomyocytes i roto i ngā wāhanga ngākau ka tiakina pai i raro i ngā āhuatanga MT mō ngā rā 12, e whakaaturia ana e te connexin 43 kāore i pakaru, ā-rohe hoki (Pikitia 5a). Hei tāpiri, ko te fibrosis i roto i ngā wāhanga ngākau i raro i ngā āhuatanga MT i tino heke iho i te whakataurite ki te Ctrl, ā, he rite ki ngā wāhanga ngākau hou (Pikitia 5b). E whakaatu ana ēnei raraunga ko te whakakotahitanga o te whakaohooho ā-pūkaha me te maimoatanga T3/Dex ka tiaki pai i te hanganga ngākau i roto i ngā wāhanga ngākau i roto i te ahurea.
he whakaahua immunofluorescence māngai o te troponin-T (kakariki), te connexin 43 (whero), me te DAPI (kikorangi) i roto i ngā wāhanga ngākau hou kua wehea (D0) kua whakatōkia rānei mō ngā rā 12 i roto i ngā āhuatanga whakatō wāhanga ngākau e whā (pae tauine = 100 µm). ). Te ine i te hanganga o te kiko o te ngākau mā te whakamahi i te matauranga horihori (n = 7 (D0 me te D12 Ctrl), 5 (D12 TD, D12 MC me te D12 MT) poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara; ####p < 0.0001 ki te whakataurite ki te D0 me te *p < 0.05, ****p < 0.0001 rānei ki te whakataurite ki te D12 Ctrl). Te ine i te hanganga o te kiko o te ngākau mā te whakamahi i te matauranga horihori (n = 7 (D0 me te D12 Ctrl), 5 (D12 TD, D12 MC me te D12 MT) poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara; #### p < 0.0001 ki te whakataurite ki te D0 me te *p < 0.05, ****p < 0.0001 rānei ki te whakataurite ki te D12 Ctrl). Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 7 (D0 и TD12), D15 D12 MT) срезов/группу от разных свиней, проведен однофакторный тест ANOVA #### p < 0,0001 по сравнению с D0 ,**** и и с D0 ,**** и0 и по < 0,**** и0 и по < 0,**** и по < 0,**** и по сравнению ki D12 Ctrl). Te ine i te hanganga o te kiko o te ngākau mā te whakamahi i te mātauranga horihori (n = 7 (D0 me te D12 Ctrl), 5 (D12 TD, D12 MC me te D12 MT) wāhanga/rōpū mai i ngā poaka rerekē, i whakahaerehia he whakamātautau ANOVA kotahi-ara; #### p < 0.0001 ki te whakataurite ki te D0 me te *p < 0.05, ****p < 0.0001 rānei ki te whakataurite ki te D12 Ctrl).对不同猪的心脏组织结构完整性（n = 7（D0 和D12 Ctrl）、5（D12 TD、D12 MC 和D12 MT）切片/组）进行人工智能量化，进行单向ANOVA 测试；#### p < 0.0001 与D0 和*p < 0.05 ，書所 0.05 ，書时与D12 Ctrl 相比）.对 不同 猪 的 心脏 结构 完整性 （n = 7 （d0 和 d12 ctrl） （5 （d12 td 、 d12 mc 和 d1智能量 化 进行 单向 单向 单向 测试 ； ########## p < 0.0001 与D0 和*p < 0.05 相月0 ， 我l 0.05 相比 ， 戸l 0.相比）.Te ine i te hanganga o te kiko ngākau mā te whakamahi i te mātauranga horihori i roto i ngā poaka rerekē (n = 7 (D0 me te D12 Ctrl), 5 (D12 TD, D12 MC me te D12 MT) wāhanga/rōpū) me te whakamātautau ANOVA kotahi-ara;#### p <0,0001 по сравнению с D0 и *p <0,05 ranei ****p <0,0001 по сравнению с D12 Ctrl). #### p < 0.0001 ki te whakataurite ki a D0 me *p < 0.05 rānei ****p < 0.0001 ki te whakataurite ki a D12 Ctrl). b Ngā whakaahua māngai me te ine ine mō ngā poro ngākau i pania ki te tae trichrome a Masson (Pae tauine = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, me te D12 MC), 9 (D12 MT) poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara; ####p < 0.0001 ki te whakataurite ki te D0 me te ***p < 0.001, ****p rānei < 0.0001 ki te whakataurite ki te D12 Ctrl). b Ngā whakaahua māngai me te ine ine mō ngā poro ngākau i pania ki te tae trichrome a Masson (Pae tauine = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, me te D12 MC), 9 (D12 MT) poro/rōpū mai i ngā poaka rerekē, ka whakahaerehia he whakamātautau ANOVA kotahi-ara; ####p < 0.0001 ki te whakataurite ki te D0 me te ***p < 0.001, ****p rānei < 0.0001 ki te whakataurite ki te D12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Массона 0 = массона мкм) (n = 10 (D0, D12 Ctrl, D12 TD и D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполняется односторонни , ##0 ANOVA; сравнению с D0 me ***p <0,001 ranei ****p < 0,0001 по сравнению с D12 Ctrl). b Ngā whakaahua māngai me te ine i ngā wāhanga ngākau i pania ki te tae trichrome a Masson (pae tauine = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD me D12 MC), 9 (D12 MT) ngā wāhanga/rōpū mai i ngā poaka rerekē, i whakahaerehia he ANOVA kotahi-ara; ####p < 0.0001 vs. D0 me ***p < 0.001 rānei ****p < 0.0001 vs. D12 Ctrl). b 用Masson 三色染料染色的心脏切片的代表性图像和量化（比例尺= 500 µm）！1D = 10（D10） TD 和D12 MC），来自不同猪的9 个（D12 MT）切片/组，进行单因素方差分析；####p < 0.0001 0.001，或****p < 0.0001 与D12 Ctrl 相比）. b 用 masson 三 色 染料 的 心脏 切片 的 代表性 和 量化 （比例 尺 尺 尺 = 500 µm） = 500 µm） = 500 µm） ctrl 、 d12 td 和 d12 mc） 来自 不同 的 9 个 d12 mt 切片 切片 切片 切片 切片 切片 切片 切片 切片切片 切片 切片 切片 切片 切片 切片 切片 切片/组，进行单因素方差分析；####p < 0.0001 与D0 相1比，我0**p <0，**** 0.0001 与D12 Ctrl 相比）. b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом Массона (масштабная = лим50) (D0, D12 Ctrl, D12 TD и D12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA ####p < 0,0001 Dе свиней / группы, один- способ ANOVA ####p < 0,0001 Dе л0, *** лю по, *** лю сра; ****p < 0,0001 по сравнению ki D12 Ctrl). b Ngā whakaahua māngai me te ine i ngā wāhanga ngākau i pania ki te Masson's trichrome (pae tauine = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD me D12 MC), 9 (D12 MT) ngā wāhanga mai i ngā poaka/rōpū rerekē, kotahi te tikanga ANOVA; ####p < 0.0001 ki te whakataurite ki a D0, ***p < 0.001, ****p rānei < 0.0001 ki te whakataurite ki a D12 Ctrl).Ko ngā pae hapa e tohu ana i te toharite ± paerewa rerekētanga.
Hei whakamutunga, i aromatawaihia te kaha o te CTCM ki te whai i te hypertrophy o te ngakau mā te whakanui ake i te totoro o te kiko ngakau. I roto i te CTCM, i piki ake te pēhanga o te rūma hau teitei mai i te 80 mmHg ki te 80 mmHg. Toi. (toto noa) tae atu ki te 140 mmHg Toi. (Pikitia 6a). E rite ana tēnei ki te pikinga 32% o te totoro (Pikitia 6b), i whakaaturia i mua ko te ōrau totoro e hiahiatia ana kia tutuki ai te roa o te sarcomere i ngā wāhanga ngakau e rite ana ki tērā i kitea i roto i te hypertrophy. I noho tonu te totoro me te tere o te kiko ngakau i te wā e whakaheke ana me te whakangā i roto i ngā rā e ono o te ahurea (Pikitia 6c). Ko te kiko ngakau mai i ngā āhuatanga MT i tukuna ki te totoro noa (MT (Noa)) ngā āhuatanga totoro rawa rānei (MT (OS)) mō ngā rā e ono. I muri i ngā rā e whā i roto i te ahurea, kua tino piki ake te tohu koiora hypertrophic NT-ProBNP i roto i te reo i raro i ngā āhuatanga MT (OS) ki ngā āhuatanga MT (noa) (Pikitia 7a). Hei tāpiri, i muri i ngā rā e ono o te whakatipu, kua tino piki ake te rahi o te pūtau i roto i te MT (OS) (Pikitia 7b) ki ngā wāhanga o te ngakau MT (noa). Hei tāpiritanga, i tino piki ake te nekehanga karihi NFATC4 i roto i ngā kiko kua totoro rawa (Pikitia 7c). E whakaatu ana ēnei hua i te whanaketanga haere tonu o te hanga hou pathological i muri i te hyperdistension, ā, e tautoko ana i te ariā ka taea te whakamahi i te taputapu CTCM hei tūāpapa hei ako i te tohu hypertrophy cardiac i puta mai i te totoro.
Ko ngā tohu tohu o te pēhanga rūma hau, te pēhanga rūma wai, me ngā inenga nekehanga kiko e whakaū ana ka huri te pēhanga rūma i te pēhanga rūma wai, ka puta he nekehanga rite o te poro kiko. b Ngā ānau ōrau totoro me te tere totoro tohu mō ngā wāhanga kiko kua totoro noa (karaka) me ngā wāhanga kiko kua totoro rawa (kikorangi). c Kauwhata pae e whakaatu ana i te wā huringa (n = 19 poro mō ia rōpū, mai i ngā poaka rerekē), te wā whakaheke (n = 18-19 poro mō ia rōpū, mai i ngā poaka rerekē), te wā whakangā (n = 19 poro mō ia rōpū, mai i ngā poaka rerekē) ), te kaha o te nekehanga kiko (n = 14 poro/rōpū, mai i ngā poaka rerekē), te tere systolic tihi (n = 14 poro/rōpū, mai i ngā poaka rerekē) me te tere whakangā tihi (n = 14 (D0), 15 (D6) ) ngā wāhanga/rōpū) mai i ngā poaka rerekē), kāore i whakaatuhia e te whakamātautau-t a te Tauira hiku-rua tētahi rerekētanga nui i roto i tētahi tawhā, e tohu ana i noho pūmau ēnei tawhā i roto i ngā rā e 6 o te ahurea me te ngaohiko nui. Ko ngā pae hapa e tohu ana i te toharite ± paerewa rerekētanga.
he tātaitanga kauwhata pae o te kukū NT-ProBNP i roto i ngā pāpāho ahurea mai i ngā poro ngākau i whakatipuria i raro i ngā tikanga totoro noa MT (Norm) me te totoro rawa (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, me te D4 MTOS) poro/rōpū mai i ngā poaka rerekē, ka mahia te ANOVA ara-rua; **p < 0.01 ki te whakataurite ki te totoro noa). He kauwhata pae e ine ana i te kukū NT-ProBNP i roto i ngā pāpāho ahurea mai i ngā poro ngākau i whakatipuria i raro i ngā tikanga totoro noa MT (Norm) me te totoro rawa (OS) (n ​​= 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, me te D4 MTOS) poro/rōpū mai i ngā poaka rerekē, ka mahia he ANOVA ara-rua; **p < 0.01 ki te whakataurite ki te totoro noa).He histogram tauanga o te kukū NT-ProBNP i roto i te reo ahurea mai i ngā poro ngākau i whakatipuria i raro i ngā āhuatanga o te totoro MT noa (noa) te totoro nui rānei (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, me D4).MTOS) poro /rōpū mai i ngā poaka rerekē, ka mahia te tātaritanga rerekētanga tauwehe-rua;**p < 0,01 по сравнению с нормальным растяжением). **p < 0.01 ki te whakarite ki te totoro noa). a 在MT 正常拉伸(Norm) 或过度拉伸(OS) 条件下培养的心脏切片培养基中NT-ProBNP 浓度的曾度4 (D2 MTNorm)、3（D2 MTOS、D4 MTNorm 和D4 MTOS）来自不同猪的切片/组，进行双向方差分析；p中中中文中中文0.01） a Te inenga o te kukū NT-ProBNP i roto i nga poro o te ngakau i mahia i raro i nga tikanga totoro noa MT (Norm) me te toronga nui (OS) tikanga (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm和D4 MTOS) mai i nga momo rereke rereke. **whakaritea ki te totoro noa, p <0.01).histogram Te ine i ngā kukū NT-ProBNP i roto i ngā poro ngākau i whakatōkia i raro i ngā āhuatanga o te totoro MT noa (noa) te totoro nui rānei (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) me te D4 MTOS) poro/rōpū mai i ngā poaka rerekē, tātaritanga rerekētanga rua-ara;**p < 0,01 по сравнению с нормальным растяжением). **p < 0.01 ki te whakarite ki te totoro noa). b Ngā whakaahua māngai mō ngā poro ngākau i pania ki te troponin-T me te WGA (maui) me te ine rahi pūtau (matau) (n = 330 (D6 MTOS), 369 (D6 MTNorm) pūtau/rōpū mai i ngā poro rerekē e 10 mai i ngā poaka rerekē, ka mahia te whakamātautau-t Tauira Hiku-Rua; ****p < 0.0001 ki te whakataurite ki te totoro noa). b Ngā whakaahua māngai mō ngā poro ngākau i pania ki te troponin-T me te WGA (maui) me te ine rahi pūtau (matau) (n = 330 (D6 MTOS), 369 (D6 MTNorm) pūtau/rōpū mai i ngā poro rerekē e 10 mai i ngā poaka rerekē, i mahia te whakamātautau-t Tauira-rua; ****p < 0.0001 ki te whakarite ki te totoro noa). b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного опеникрадер (справа) (n = 330 (D6 MTOS), 369 (D6 MTNorm) клеток/группу из 10 разных срезов от разных свиней, два- проводится х t-стой хтвится ****p < 0,0001 по сравнению с нормальным растяжением). b Ngā whakaahua māngai o ngā wāhanga ngākau i pania ki te troponin-T me te AZP (maui) me te ine rahi pūtau (matau) (n = 330 (D6 MTOS), 369 (D6 MTNorm) pūtau/rōpū mai i ngā wāhanga rerekē e 10 mai i ngā poaka rerekē, i whakahaerehia te whakamātautau-t a te Tauira hiku-rua; ****p < 0.0001 ki te whakataurite ki te momo noa). b 用肌钙蛋白-T 和WGA（左）和细胞大小量化（右）染色的心脏切片的代表性（小量化（右）染色的心脏切片的代表性（图图MTOS），来自不同猪的10 个不同切片的369（D6 MTNorm）细胞/组，两进行有尾学生t检验；与正常拉伸相比，****p < 0.0001）。 b Ngā whakaahua māngai o ngā poro ngākau i pania ki te calcarein-T me te WGA (maui) me te rahi o te pūtau (matau) (n = 330 (D6 MTOS), 369 mai i ngā poro rerekē e 10 (D6 MTNorm)) Ngā Pūtau/组，两方法有尾学生t whakamātautau；whakatairite ki te totoro noa，****p < 0.0001). b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оцленка (расленка) 330 (D6 MTOS), 369 (D6 MTNorm) из 10 различных срезов от разных свиней Клетки/группа, двусторонние критерий Ста;юд0 критерий С;юд0 сравнению с нормальным растяжением). b Ngā whakaahua māngai o ngā wāhanga ngākau i pania ki te troponin-T me te AZP (maui) me te ine i te rahi o te pūtau (matau) (n = 330 (D6 MTOS), 369 (D6 MTNorm) mai i ngā wāhanga rerekē e 10 mai i ngā poaka rerekē) Ngā pūtau/rōpū, paearu hiku-rua a te ākonga t; ****p < 0.0001 ki te whakataurite ki te momo noa). c Ngā whakaahua māngai mō ngā poro ngākau MTOS o te rā 0 me te rā 6 kua tohua mō te troponin-T me te NFATC4 me te ine i te nekehanga o te NFATC4 ki ngā karihi o ngā CM (n = 4 (D0), 3 (D6 MTOS) poro/rōpū mai i ngā poaka rerekē, ka mahia te whakamātautau-t Tauira Hiku-rua; *p < 0.05). c Ngā whakaahua māngai mō ngā poro ngākau MTOS o te rā 0 me te rā 6 kua tohua mō te troponin-T me te NFATC4 me te ine i te nekehanga o te NFATC4 ki ngā karihi o ngā CM (n = 4 (D0), 3 (D6 MTOS) poro/rōpū mai i ngā poaka rerekē, ka mahia te whakamātautau-t Tauira Hiku-rua; *p < 0.05). c Репрезентативные изображения для срезов сердца 0 и 6 дней MTOS, иммуномеченых для тропонина-Т и NFATC4, и колицяч транслокации NFATC4 в ядра кавернозных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней , выполнядяетскриятся Стьюдента; *p < 0,05). c Ngā whakaahua māngai mō ngā wāhanga ngākau i te 0 me te 6 rā MTOS, i tohua mō te troponin-T me te NFATC4, me te ine i te nekehanga NFATC4 i roto i te karihi o ngā pūtau cavernous (n = 4 (D0), 3 (D6 MTOS) poro/rōpū mai i ngā poaka rerekē) i mahia i te whakamātautau-t a te Tauira hiku-rua; *p < 0.05). c 用于肌钙蛋白-T 和NFATC4 免疫标记的第0 天和第6 天MTOS心脏切片的代表性图像，以及来自不同猪的NFATC4 易位至CM 细胞核的量化（n = 4 (D0)片)进行双尾学生t 检验；*p < 0.05). c Nga whakaahua o te calcanin-T me te NFATC4 immunolabeling 第0天和第6天MTOS poro ngakau, me te NFATC4 mai i nga momo NFATC4 易位至CM pūtau nucleus的 quantity化 (n = 4 (D0), 3 (D6 MTOS) /爇时间双尾学生et 电影；*p < 0.05). c Репрезентативные изображения срезов сердца MTOS ki te 0 me te 6 o nga ra ki te иммуномаркировки тропонином-Т и NFATC4 и колицнч транслокации NFATC4 в ядра CM от разных свиней (n = 4 (D0), 3 (D6 MTOS) срез/группа, два- хвостатый t-критерий Сть0, *0). c Ngā whakaahua māngai o ngā poro ngākau MTOS i te rā 0 me te 6 mō te tohu ārai mate troponin-T me te NFATC4 me te ine i te nekehanga NFATC4 i roto i te karihi o CM mai i ngā poaka rerekē (n = 4 (D0), 3 (D6 MTOS) poro/rōpū, tauira paearu-t hiku-rua; *p < 0.05).Ko ngā pae hapa e tohu ana i te toharite ± paerewa rerekētanga.
Ko te rangahau mō te ngākau whakamāori e hiahia ana ki ngā tauira pūtau e whakaputa tika ana i te taiao ngākau. I roto i tēnei rangahau, i whakawhanakehia, i whakaahuatia hoki he taputapu CTCM ka taea te whakaoho i ngā wāhanga tino kikokore o te ngākau. Kei roto i te pūnaha CTCM te whakaoho hikohiko ā-tinana me te whakarei ake i te wai T3 me te Dex. I te wā i tukuna ai ngā wāhanga ngākau poaka ki ēnei āhuatanga, i noho tonu tō rātou oranga, te hanganga, te mahi pākia, me te whakaaturanga whakawhiti ki te kiko ngākau hou i muri i ngā rā 12 o te ahurea. Hei tāpiri, ko te totoro nui o te kiko ngākau ka taea te whakaputa i te hypertrophy o te ngākau i puta mai i te hyperextension. I te nuinga, e tautoko ana ēnei hua i te tūranga nui o ngā āhuatanga ahurea ā-tinana ki te pupuri i te āhua noa o te ngākau, ā, e whakarato ana i tētahi tūāpapa mō te tirotiro rongoā.
He maha ngā āhuatanga e whai wāhi ana ki te waihanga i tētahi taiao tino pai mō te mahi me te oranga o ngā cardiomyocytes. Ko ngā mea tino kitea o ēnei āhuatanga e pā ana ki (1) ngā taunekeneke i waenga i ngā pūtau, (2) te whakaoho hikohiko, (3) ngā āhuatanga humoral, me (4) ngā matū matūkai. Ko ngā taunekeneke pūtau-ki-pūtau ā-tinana e hiahia ana ki ngā whatunga matatini toru-ahu o ngā momo pūtau maha e tautokona ana e tētahi matrix extracellular. He uaua ki te hanga anō i ēnei taunekeneke pūtau matatini i roto i te vitro mā te whakatipu tahi i ngā momo pūtau takitahi, engari ka taea te whakatutuki ngāwari mā te whakamahi i te āhua organotypic o ngā wāhanga ngākau.
He mea nui te totoro ā-ringa me te whakaoho hiko o ngā cardiomyocytes hei pupuri i te āhua o te ngākau33,34,35. Ahakoa kua whānuitia te whakamahinga o te whakaoho ā-ringa mō te whakatikatika me te pakeketanga o te hiPSC-CM, he maha ngā rangahau huatau kua ngana ki te whakaoho ā-ringa i ngā poro ngākau i roto i te ahurea mā te whakamahi i te uta uniaxial. E whakaatu ana ēnei rangahau he pānga pai te uta ā-ringa uniaxial 2D ki te āhua o te ngākau i te wā o te ahurea. I roto i ēnei rangahau, i utaina ngā wāhanga o te ngākau ki ngā kaha kume isometric17, ki te uta auxotonic linear18, i hangaia anō rānei te huringa ngākau mā te whakamahi i te urupare transducer kaha me ngā taraiwa kume. Heoi, ka whakamahia e ēnei tikanga te totoro kiko uniaxial me te kore e arotau i te taiao, ka hua ake te pehi i te maha o ngā ira ngākau, te whakaputa nui rānei o ngā ira e pā ana ki ngā urupare totoro rerekē. Ko te CTCM kua whakaahuahia i konei e whakarato ana i te whakaoho hiko 3D e whai ana i te huringa ngākau tūturu e pā ana ki te wā huringa me te totoro ā-tinana (25% totoro, 40% systole, 60% diastole, me te 72 ngā pao ia meneti). Ahakoa kāore e ranea ana tēnei whakaohooho ā-tinana toru-ahu anake hei pupuri i te pumau o te kiko, me whakakotahi te whakaohooho humoral me te whakaohooho ā-tinana mā te whakamahi i te T3/Dex hei pupuri i te ora, te mahi, me te pumau o te kiko.
He mea nui te mahi a ngā āhuatanga humoral ki te whakarerekē i te āhua o te ngākau pakeke. I whakamāramahia tēnei i roto i ngā rangahau HiPS-CM i tāpirihia ai te T3 me te Dex ki ngā pāpāho ahurea hei whakatere i te pakeketanga o te pūtau. Tērā pea ka pā te T3 ki te kawe i ngā waikawa amino, ngā huka me te konupūmā puta noa i ngā kiriuhi pūtau36. Hei tāpiri, ka whakatairanga te T3 i te whakaaturanga MHC-α me te whakahekenga o te MHC-β, ka whakatairanga i te hanganga o ngā myofibrils tere tere i roto i ngā cardiomyocytes pakeke ki te whakataurite ki ngā myofibrils puhoi tere i roto i te CM fetal. Ko te korenga o te T3 i roto i ngā tūroro hypothyroid ka hua ake te ngaronga o ngā whitiki myofibrillar me te whakahekenga o te tere o te whanaketanga o te oro37. Ka mahi te Dex ki ngā kaiwhakawhiwhi glucocorticoid, ā, kua whakaaturia ka whakanui ake i te kaha o te uaua o te uaua i roto i ngā ngākau kua wehea te toto; 38 e whakaarohia ana ko tēnei whakapainga e pā ana ki te pānga ki te urunga putunga-konupūmā (SOCE) 39,40. Hei tāpiri, ka herea e te Dex ki ōna kaiwhakawhiwhi, ka puta he urupare whānui ki roto i te pūtau e pehi ana i te mahi ārai mate me te mumura30.
E whakaatu ana ā mātou hua i whakapai ake te whakaohooho ā-tinana (MS) i te mahi whānui o te ahurea i whakaritea ki a Ctrl, engari kāore i taea te pupuri i te oranga, te hanganga pai, me te whakaaturanga o te ngākau i roto i ngā rā 12 i roto i te ahurea. I whakaritea ki a Ctrl, ko te tāpiri o te T3 me te Dex ki ngā ahurea CTCM (MT) i whakapai ake i te oranga, ā, i pupuri i ngā kōtaha whakawhiti rite, te hanganga pai, me te mahi pākia me te kiko ngākau hou mō ngā rā 12. Hei tāpiri, mā te whakahaere i te nui o te totoro kiko, i hangaia he tauira hypertrophy ngākau i puta mai i te hyperextension mā te whakamahi i te STCM, e whakaatu ana i te maha o ngā mahi a te pūnaha STCM. Me mōhio ahakoa ko te whakahoutanga o te ngākau me te fibrosis he maha ngā wā ka uru ngā okana kāore anō kia pakaru, ā, ka taea e ngā pūtau e rere ana te whakarato i ngā cytokines tika me te phagocytosis me ētahi atu āhuatanga whakahou, ka taea tonu e ngā wāhanga o te ngākau te whai i te tukanga fibrotic hei whakautu ki te ahotea me te whara. ki roto i ngā myofibroblasts. Kua arotakehia tēnei i mua i roto i tēnei tauira poro ngākau. Me mōhio ka taea te whakarerekē i ngā tawhā CTCM mā te huri i te kaha me te auau o te pēhanga/hiko hei whai i ngā āhuatanga maha pēnei i te tachycardia, te bradycardia, me te tautoko rere toto ā-tinana (ngākau kore utaina ā-tinana). Nā tēnei ka waiho te pūnaha hei pūnaha waenga-rahi mō te whakamātautau tarukino. Mā te kaha o te CTCM ki te whakatauira i te hypertrophy o te ngakau i puta mai i te kaha rawa o te mahi ka taea te whakamātautau i tēnei pūnaha mō te rongoā whaiaro. Hei whakamutunga, e whakaatu ana tēnei rangahau he mea nui te totoro ā-ringa me te whakaohooho humoral mō te pupuri i te ahurea o ngā wāhanga kiko ngakau.
Ahakoa e kī ana ngā raraunga e whakaaturia ana i konei he tūāpapa tino whai hua te CTCM mō te whakatauira i te myocardium kāore i pakaru, he iti noa iho ngā herenga o tēnei tikanga ahurea. Ko te herenga matua o te ahurea CTCM ko tana tuku i ngā ahotea miihini hihiri tonu ki runga i ngā poro, e aukati ana i te kaha ki te aroturuki kaha i ngā whakahekenga poro ngākau i ia huringa. Hei tāpiri, nā te iti o ngā wāhanga ngākau (7 mm), he iti noa iho te kaha ki te aromatawai i te mahi systolic i waho o ngā pūnaha ahurea mā te whakamahi i ngā pūoko kaha tuku iho. I roto i te tuhinga o nāianei, kua wikitoriahia e mātou tēnei herenga mā te aromatawai i te ngaohiko whatu hei tohu mō te mahi whakahekenga. Heoi, me mahi anō tēnei herenga, ā, tera pea ka whakatikahia i te heke mai mā te whakauru i ngā tikanga mō te aroturuki whatu i te mahi o ngā poro ngākau i roto i te ahurea, pērā i te mahere whatu mā te whakamahi i te konupūmā me ngā tae tairongo-ngaohiko. Ko tētahi atu herenga o te CTCM ko te kore e whakahaere te tauira mahi i te ahotea ā-tinana (preload me afterload). I roto i te CTCM, i whakaohohia te pehanga ki ngā taha rerekē hei whakaputa i te 25% o te totoro ā-tinana i te diastole (totoro katoa) me te systole (te roa o te whakahekenga i te wā whakaoho hiko) i roto i ngā kiko tino nui. Me whakakore tēnei herenga i ngā hoahoa CTCM a meake nei mā te pēhanga tika ki runga i te kiko o te ngākau mai i ngā taha e rua, me te whakamahi i ngā whanaungatanga pēhanga-rahinga tuturu e puta ana i roto i ngā rūma o te ngākau.
Ko te whakarerekētanga i puta mai i te totoro nui rawa i kōrerotia i roto i tēnei tuhinga he iti noa iho ki te whai i ngā tohu totoro nui rawa atu. Nō reira, ka taea e tēnei tauira te āwhina i te ako i ngā tohu totoro nui rawa atu i puta mai i te totoro me te kore e hiahiatia he āhuatanga humoral, he āhuatanga neural rānei (kāore i roto i tēnei pūnaha). Me whai atu rangahau hei whakanui ake i te maha o te CTCM, hei tauira, ko te whakatipu tahi me ngā pūtau ārai mate, ngā āhuatanga humoral plasma e rere ana, me te innervation ina whakatipu tahi me ngā pūtau neuronal ka whakapai ake i ngā āheinga o te whakatauira mate me te CTCM.
Tekau mā toru ngā poaka i whakamahia i roto i tēnei rangahau. I mahia ngā tikanga kararehe katoa i runga anō i ngā aratohu whare wānanga, ā, i whakaaetia e te Komiti Tiaki me te Whakamahi Kararehe Whare Wānanga o Louisville. I herea te āwhata o te aorta, ā, i ringihia te hinu ki te ngākau ki te 1 rita o te cardioplegia sterile (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U/mL heparin, pH tae atu ki te 7.4); I tiakina ngā ngākau i roto i te wai whakapūmau mō te manawa me te ngākau makariri tae noa ki te wā i kawea ai ki te taiwhanga i runga i te hukapapa, he iti iho i te 10 meneti te roa i te nuinga o te wā. I tiakina ngā ngākau i roto i te wai whakapūmau mō te manawa me te ngākau makariri tae noa ki te wā i kawea ai ki te taiwhanga i runga i te hukapapa, he iti iho i te 10 meneti te roa i te nuinga o te wā. сердца хранили в ледяном кардиоплегическом растворе до транспортировки в лабораторию на льду, что обычно занимает <. I rongoatia ngā ngākau ki roto i te wai whakapūmau mō te ngākau me te manawa tae noa ki te wā i kawea ai ki te taiwhanga i runga i te hukapapa, e iti iho ana i te 10 meneti te roa.将心脏保存在冰冷的心脏停搏液中，直到冰上运送到实验室，通常<10分钟。将心脏保存在冰冷的心脏停搏液中，直到冰上运送到实验室，通常<10分钟。 Держите сердца в ледяной кардиоплегии до транспортировки в лабораторию на льду, обычно <10 min. Puritia te ngākau ki runga i te hukapapa kia tae rā anō ki te wā e kawea ai ki te taiwhanga i runga i te hukapapa, i te nuinga o te wā <10 meneti.
I whakawhanakehia te taputapu CTCM i roto i te pūmanawa hoahoa āwhina-rorohiko (CAD) a SolidWorks. He mea hanga ngā rūma ahurea, ngā wehewehe me ngā rūma hau ki te kirihou kiriaku mārama CNC. Ko te mowhiti tautoko 7mm te whānui he mea hanga ki te polyethylene kiato teitei (HDPE) i waenganui, ā, he awaawa mowhiti-o hei whakauru i te mowhiti-o silicone hei hiri i ngā pāpāho i raro. He kiriuhi silica angiangi e wehe ana i te rūma ahurea mai i te pereti wehewehe. Ka tapahia te kiriuhi silicone ki te laser mai i te rau silicone matotoru 0.02″, ā, he pakeke 35A. Ko ngā hiri silicone o raro me o runga ka tapahia ki te laser mai i te rau silicone matotoru 1/16″, ā, he pakeke 50A. Ka whakamahia ngā tīwiri maitai kore waikura 316L me ngā nati parirau hei whakamau i te poraka me te hanga hiri ā-hau.
Kua hangaia he papa hiko tānga motuhake (PCB) kia whakauruhia ki te pūnaha C-PACE-EM. Ko ngā tūhono hononga mīhini Swiss i runga i te PCB ka honoa ki ngā irahiko karāhe mā te whakamahi i ngā waea parahi whakakikoruatia ki te hiriwa me ngā tīwiri parahi 0-60 kua whiria ki roto i ngā irahiko. Ka whakanohoia te papa hiko tānga ki roto i te taupoki o te pūreretā 3D.
Ko te taputapu CTCM e whakahaeretia ana e te kaiwhakaoho hau ka taea te whakarite (PPD) e hanga ana i te pēhanga tohanga toto e whakahaeretia ana, he rite ki te huringa ngakau. I te pikinga ake o te pēhanga i roto i te rūma hau, ka whānui ake te kiriuhi silicone ngāwari ki runga, ka panaia te reo ki raro i te wāhi kiko. Kātahi ka totoro te horahanga o te kiko e tēnei pananga wai, e whai ana i te whānui ā-tinana o te ngakau i te wā diastole. I te tihi o te whakangā, ka whakamahia te whakaoho hiko mā roto i ngā irahiko karāhe, i whakaiti i te pēhanga i roto i te rūma hau, ā, ka hua te whakawhāiti i ngā wāhanga kiko. Kei roto i te paipa he takirere hemostatic me te pūoko pēhanga hei kite i te pēhanga i roto i te pūnaha hau. Ko te pēhanga e rongohia ana e te pūoko pēhanga ka whakamahia ki te kaikohi raraunga e hono ana ki te pona. Mā tēnei ka taea te aroturuki tonu i te pēhanga i roto i te rūma hau. I te taenga ki te pēhanga rūma mōrahi (paerewa 80 mmHg, 140 mmHg OS), ka whakahaua te taputapu kohikohi raraunga kia tukuna he tohu ki te pūnaha C-PACE-EM hei whakaputa i te tohu ngaohiko biphasic mō te 2 ms, kua whakatakotoria ki te 4 V.
I tangohia ngā wāhanga o te ngākau, ā, i mahia ngā tikanga whakatipu i roto i ngā puna e 6 penei: Whakawhitihia ngā ngākau kua hauhaketia mai i te ipu whakawhiti ki tētahi tirei kei roto ko te cardioplegia makariri (4° C.). I wehea te ventricle maui ki tētahi mata ma, ā, i tapahia kia 1-2 cm3 te whānui. I tāpirihia ēnei poraka kiko ki ngā tautoko kiko me te piripiri kiko, ā, i whakanohoia ki roto i tētahi kaukau kiko microtome wiri kei roto ko te otinga Tyrode, ā, i whakakīia tonutia ki te hāora (3 g/L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g) . ), 6 mM KCl (0.447 g), 10 mM D-glucose (1.86 g), 10 mM HEPES (2.38 g), 1 mM MgCl2 (1 ml 1 M otinga), 1.8 mM CaCl2 (1.8 ml 1 M otinga), tae atu ki te 1 L ddH2O). I whakatakotoria te microtome wiri ki te tapahi i ngā poro matotoru 300 µm i te auau o te 80 Hz, he kaha wiri whakapae o te 2 mm, me te tere whakamua o te 0.03 mm/s. I karapotia te kaukau kiko ki te hukapapa hei pupuri i te matao o te otinga, ā, i puritia te pāmahana ki te 4°C. Whakawhitihia ngā wāhanga kiko mai i te kaukau microtome ki tētahi kaukau whakatō kei roto ko te otinga Tyrode kua whakakīia tonutia ki te hāora i runga i te hukapapa kia whiwhi rā anō i ngā wāhanga e ranea ana mō tētahi pereti ahurea. Mō ngā ahurea transwell, i tāpirihia ngā wāhanga kiko ki ngā tautoko polyurethane parakore 6 mm te whānui, ā, i whakanohoia ki roto i te 6 ml o te reo kua arotauhia (199 reo, 1x tāpiringa ITS, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-kawa me te 2X paturopi-antihoko-antihoko). I whakamahia te whakaoho hiko (10 V, auau 1.2 Hz) ki ngā wāhanga kiko mā te C-Pace. Mō ngā āhuatanga TD, i tāpirihia te T3 hou me te Dex ki te 100 nM me te 1 μM i ia whakawhiti rauemi. Ka whakakīia te rauemi ki te hāora i mua i te whakakapinga e 3 ngā wā i te rā. I whakatōngia ngā wāhanga kiko ki roto i te incubator i te 37°C me te 5% CO2.
Mō ngā ahurea CTCM, i whakatakotoria ngā wāhanga kiko ki runga i tētahi pūreretā 3D i hangaia i roto i tētahi rihi Petri kei roto ko te otinga Tyrode kua whakarerekētia. Kua hangaia te taputapu hei whakanui ake i te rahi o te poro o te ngākau mā te 25% o te horahanga o te mowhiti tautoko. Ka mahia tēnei kia kore ai ngā wāhanga o te ngākau e totoro i muri i te whakawhiti mai i te otinga Tyrode ki te reo me te wā diastole. Mā te whakamahi i te kāpia histoacrylic, i whakapirihia ngā wāhanga 300 µm te matotoru ki runga i tētahi mowhiti tautoko 7 mm te whānui. I muri i te tāpiri i ngā wāhanga kiko ki te mowhiti tautoko, tapahia ngā wāhanga kiko taikaha ka whakahokia ngā wāhanga kiko kua piri ki roto i te kaukau o te otinga Tyrode i runga i te hukapapa (4°C) kia rite rā anō ngā wāhanga mō tētahi taputapu. Kaua te wā tukatuka katoa mō ngā taputapu katoa e neke atu i te 2 hāora. I muri i te tāpiri i ngā wāhanga kiko e 6 ki ō rātou mowhiti tautoko, ka huihuia te taputapu CTCM. Kua whakakīia te rūma ahurea CTCM ki te 21 ml o te reo kua whakakīia ki te hāora. Whakawhitihia ngā wāhanga kiko ki te rūma ahurea ka tangohia marietia ngā mirumiru hau ki tētahi pipette. Kātahi ka arahina te wāhanga kiko ki roto i te kōhao, ā, ka pēhia mārire ki tōna wāhi. Hei whakamutunga, whakatakotoria te taupoki hiko ki runga i te taputapu, ka whakawhitia te taputapu ki te incubator. Kātahi ka honoa te CTCM ki te ngongo hau me te pūnaha C-PACE-EM. Ka tuwhera te actuator hau, ā, ka whakatuwherahia te CTCM e te takirere hau. I whirihorahia te pūnaha C-PACE-EM ki te tuku 4 V i te 1.2 Hz i te wā e tere ana te whakawhiti rua-wāhanga mō te 2 ms. E rua ngā wā i te rā i whakawhitihia ai te reo, ā, kotahi te wā i te rā i whakawhitihia ai ngā hiko hei karo i te kohikohinga o te karāpeti ki runga i ngā hiko. Mena e tika ana, ka taea te tango i ngā wāhanga kiko mai i ō rātou puna ahurea hei pana i ngā mirumiru hau kua taka ki raro i a rātou. Mō ngā āhuatanga maimoatanga MT, i tāpirihia te T3/Dex hou me ia whakawhiti reo me te 100 nM T3 me te 1 μM Dex. I whakatipuria ngā taputapu CTCM ki roto i te incubator i te 37°C me te 5% CO2.
Hei tiki i ngā ara totoro o ngā poro ngākau, i whakawhanakehia he pūnaha kāmera motuhake. I whakamahia he kāmera SLR (Canon Rebel T7i, Canon, Tokyo, Hapani) me te arotahi makaro Navitar Zoom 7000 18-108mm (Navitar, San Francisco, CA). I ​​mahia te whakaaturanga i te pāmahana rūma i muri i te whakakapinga o te rauemi ki te rauemi hou. Ka whakanohoia te kāmera ki te koki 51°, ā, ka tuhia te ataata i te 30 anga ia hekona. Tuatahi, i whakamahia he pūmanawa pūtake tuwhera (MUSCLEMOTION43) me Image-J hei ine i te nekehanga o ngā poro ngākau. I hangaia te kopare mā te whakamahi i te MATLAB (MathWorks, Natick, MA, USA) hei tautuhi i ngā rohe e hiahiatia ana mō te patu i ngā poro ngākau kia kore ai e haruru. Ka whakamahia ngā kopare kua wehea ā-ringa ki ngā whakaahua katoa i roto i te raupapa anga, kātahi ka tukuna ki te mono-mai MUSCLEMOTION. Ka whakamahia e Muscle Motion te kaha toharite o ngā pika i roto i ia anga hei ine i tōna nekehanga e pā ana ki te anga tohutoro. I tuhia ngā raraunga, i tātarihia, ā, i whakamahia hei ine i te wā huringa me te aromatawai i te totoro kiko i te huringa ngākau. I tukatukahia te ataata i hopukina mā te whakamahi i tētahi tātari matihiko kore-wāhanga tuatahi. Hei ine i te totoro kiko (tihi-ki-tihi), i whakahaerehia he tātaritanga tihi-ki-tihi hei wehewehe i waenga i ngā tihi me ngā awaawa i roto i te tohu i hopukina. Hei tāpiri, ka whakahaerehia te tango-aukati mā te whakamahi i tētahi pūrau ono o te raupapa hei whakakore i te nekenekehanga tohu. I whakawhanakehia te waehere hōtaka i roto i te MATLAB hei whakatau i te nekehanga kiko ā-ao, te wā huringa, te wā whakangā, me te wā whakaheke (Waehere Hōtaka Tāpiri 44).
Mō te tātari taumahatanga, mā te whakamahi i ngā ataata i hangaia mō te aromatawai totoro ā-mīhini, i tuatahihia e mātou ngā whakaahua e rua e tohu ana i ngā tihi nekehanga (ngā pūwāhi nekehanga teitei (runga) me raro (raro)) e ai ki te pūmanawa MUSCLEMOTION. Kātahi ka wehewehea e mātou ngā rohe kiko, ā, ka whakamahia he momo tauira whakamarumaru ki te kiko kua wehewehea (Pikitia Tāpiri 2a). Kātahi ka wehewehea te kiko kua wehewehea kia tekau ngā mata o raro, ā, ka tatauhia te taumahatanga i runga i ia mata mā te whakamahi i te whārite e whai ake nei: Taumahatanga = (Sup-Sdown)/Sdown, ko Sup me Sdown ngā tawhiti o te āhua mai i ngā atarangi o runga me raro o te papanga, ia (Pikitia Tāpiri .2b).
I whakapūmauhia ngā wāhanga ngākau ki te 4% paraformaldehyde mō ngā hāora 48. I whakamaroketia ngā kiko i whakapūmauhia ki te 10% me te 20% sucrose mō te hāora 1, kātahi ki te 30% sucrose mō te pō. Kātahi ka whakatōkia ngā wāhanga ki roto i te matū pāmahana tapahi tino pai (pūhui OCT) ā, ka whakatio haeretia ki roto i te kaukau isopentane/hukapapa maroke. Penapenahia ngā poraka whakapūmau OCT ki te -80 °C tae noa ki te wehenga. I whakaritea ngā kiriata hei wāhanga me te matotoru o te 8 μm.
Hei tango i te OCT mai i ngā wāhanga ngākau, whakamahanahia ngā kiriata i runga i te poraka whakamahana i te 95 °C mō te 5 meneti. Tāpirihia te 1 ml PBS ki ia kiriata ka whakatōkia mō te 30 meneti i te pāmahana rūma, kātahi ka uru ki roto i ngā wāhanga mā te whakatakoto i te 0.1% Triton-X ki roto i te PBS mō te 15 meneti i te pāmahana rūma. Hei ārai i te here o ngā paturopi kore-motuhake ki te tauira, tāpirihia te 1 ml o te otinga BSA 3% ki ngā kiriata ka whakatōkia mō te 1 hāora i te pāmahana rūma. Kātahi ka tangohia te BSA, ā, ka horoia ngā kiriata ki te PBS. Tohua ia tauira ki te pene. I tāpirihia ngā paturopi tuatahi (i whakaranua ki te 1:200 i roto i te 1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) me te troponin-T (Thermo Scientific; #MA5-12960) mō ngā meneti 90, kātahi ka tāpirihia ngā paturopi tuarua (i whakaranua ki te 1:200 i roto i te 1% BSA) ki te kiore Alexa Fluor 488 (Thermo Scientific; #A16079), ki te rāpeti Alexa Fluor 594 (Thermo Scientific; #T6391) mō ētahi atu 90 meneti. I horoia kia 3 ngā wā ki te PBS. Hei wehewehe i te tae o te ūnga mai i te papamuri, i whakamahia e mātou te paturopi tuarua anake hei mana whakahaere. Hei whakamutunga, i tāpirihia te tae karihi DAPI, ā, i whakanohoia ngā kiriata ki roto i te vectashield (Vector Laboratories) ā, i hiritia ki te peita whao. (whakanuia -x) me te karu hiko Keyence me te whakanui 40x.
I whakamahia te WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) i te 5 μg/ml i roto i te PBS mō te tae WGA, ā, i pania ki ngā wāhanga pumau mō te 30 meneti i te pāmahana rūma. Kātahi ka horoia ngā kiriata ki te PBS, ā, ka tāpirihia te pango Sudan ki ia kiriata, ā, ka whakatōkia mō te 30 meneti. Kātahi ka horoia ngā kiriata ki te PBS, ā, ka tāpirihia he rauemi whakauru vectashield. I tirohia ngā kiriata ki runga i te karu hiko Keyence i te whakanui 40x.
I tangohia te OCT mai i ngā tauira e ai ki te whakaahuatanga i runga ake nei. Whai muri i te tango i te OCT, rumakina ngā kiriata ki roto i te wai whakapūmau a Bouin mō te pō. Kātahi ka horoia ngā kiriata ki te wai whakapūmau mō te 1 hāora, kātahi ka whakanohoia ki roto i te wai whakapūmau waikawa aloe Bibrich mō te 10 meneti. Kātahi ka horoia ngā kiriata ki te wai whakapūmau, ā, ka whakanohoia ki roto i te wai whakapūmau o te 5% phosphomolybdenum/5% waikawa phosphotungstic mō te 10 meneti. Me te kore e horoia, whakawhitia tika ngā kiriata ki roto i te wai whakapūmau kikorangi aniline mō te 15 meneti. Kātahi ka horoia ngā kiriata ki te wai whakapūmau, ā, ka whakanohoia ki roto i te wai whakapūmau waikawa acetic 1% mō te 2 meneti. I whakamaroketia ngā kiriata ki te 200 N etanoru, ā, i whakawhitia ki te xylene. I tirohia ngā kiriata kua pania mā te whakamahi i te karuārai Keyence me te arotahi 10x. I inehia te ōrau horahanga Fibrosis mā te whakamahi i te pūmanawa Keyence Analyzer.
Te Whakamātautau Oranga Pūtau MTT CyQUANT™ (Invitrogen, Carlsbad, CA), nama rarangi V13154, e ai ki te tikanga a te kaihanga me ētahi whakarerekētanga. Ina koa, i whakamahia he pokanga pokanga me te diameter o te 6 mm hei whakarite kia ōrite te rahi o te kiko i te wā tātaritanga MTT. I whakakikoruatia takitahitia ngā kiko ki roto i ngā poka o tētahi pereti 12-poka kei roto te papa MTT e ai ki te tikanga a te kaihanga. Ka whaowhia ngā wāhanga i te 37° C. mō ngā haora e 3, ā, ka whakapūmau te kiko ora i te papa MTT hei hanga i tētahi matū formazan papura. Whakakapia te otinga MTT ki te 1 ml DMSO ka whaowhia ki te 37 °C mō ngā meneti e 15 hei tango i te formazan papura mai i ngā wāhanga ngākau. I whakaranua ngā tauira 1:10 ki roto i te DMSO i roto i ngā pereti raro mārama 96-poka, ā, i inehia te kaha o te tae papura i te 570 nm mā te whakamahi i tētahi kaipānui pereti Cytation (BioTek). I whakatauritea ngā pānui ki te taumaha o ia poro o te ngākau.
I whakakapia te rauemi poro ngākau ki te rauemi kei roto te 1 μCi/ml [5-3H]-glucose (Moravek Biochemicals, Brea, CA, USA) mō te whakamātautau whakamahinga huka e ai ki te whakaahuatanga o mua. I muri i te 4 hāora o te whakatipu, tāpirihia te 100 µl o te reo ki tētahi ngongo microcentrifuge tuwhera kei roto te 100 µl o te 0.2 N HCl. Kātahi ka whakanohoia te ngongo ki roto i tētahi ngongo scintillation kei roto te 500 μl o te dH2O hei mimiti i te [3H]2O mō ngā hāora 72 i te 37°C. Kātahi ka tangohia te ngongo microcentrifuge mai i te ngongo scintillation ka tāpirihia te 10 ml o te wai scintillation. I whakahaerehia ngā tatau scintillation mā te whakamahi i tētahi tātari scintillation wai Tri-Carb 2900TR (Packard Bioscience Company, Meriden, CT, USA). Kātahi ka tatauhia te whakamahinga o te hukahuka me te whakaaro ki te ngohe motuhake o te hukahuka [5-3H], te taurite me te papamuri kāore i oti, te waimehatanga o te [5-3H]-ki te hukahuka kāore i tapaina, me te whai huatanga o te tatau kanapa. Ka whakatauritea ngā raraunga ki te papatipu o ngā wāhanga o te ngākau.
I muri i te whakakotahitanga o ngā kiko i roto i te Trizol, i wehea te RNA mai i ngā wāhanga ngākau mā te whakamahi i te Qiagen miRNeasy Micro Kit #210874 e ai ki te tikanga a te kaihanga. Ko te whakarite whare pukapuka RNAsec, te raupapatanga me te tātari raraunga i mahia penei:
I whakamahia te 1 μg o te RNA mō ia tauira hei rauemi tīmatanga mō te whakarite i te whare pukapuka RNA. I hangaia ngā whare pukapuka raupapatanga mā te whakamahi i te NEBNext UltraTM RNA Library Preparation Kit for Illumina (NEB, USA) i muri i ngā taunakitanga a te kaihanga, ā, i tāpirihia ngā waehere taupū ki ngā raupapatanga huanga mō ia tauira. I roto i te poto, i purea te mRNA mai i te RNA katoa mā te whakamahi i ngā piripiri aukume e piri ana ki ngā poly-T oligonucleotides. Ka mahia te wehewehenga mā te whakamahi i ngā kationa divalent i te pāmahana teitei i roto i te NEBNext First Strand Synthesis Reaction Buffer (5X). I hangaia te cDNA whenu tuatahi mā te whakamahi i ngā primers hexamer matapōkere me te M-MuLV reverse transcriptase (RNase H-). Kātahi ka hangaia te cDNA whenu tuarua mā te whakamahi i te DNA polymerase I me te RNase H. Ka hurihia ngā taupua e toe ana ki ngā pito pūngoi mā te mahi exonuclease/polymerase. I muri i te adenylation o te pito 3′ o te kongakonga DNA, ka piri he NEBNext Adapter me te hanganga koropiko makawe ki reira hei whakarite mō te whakahāngai. Mō te kōwhiringa o ngā kongakonga cDNA o te roa pai 150-200 bp. I purea ngā kongakonga whare pukapuka mā te whakamahi i te pūnaha AMPure XP (Beckman Coulter, Beverly, USA). Kātahi ka whakamahia te 3 μl USER Enzyme (NEB, USA) me te cDNA kua tīpakohia te rahi i herea ki te urutau mō te 15 meneti i te 37°C, ā, mō te 5 meneti i te 95°C i mua i te PCR. I mahia te PCR mā te whakamahi i te Phusion High-Fidelity DNA polymerase, ngā primer PCR whānui, me ngā primer Index (X). Hei whakamutunga, i purea ngā hua PCR (pūnaha AMPure XP) ā, i aromatawaihia te kounga o te whare pukapuka i runga i te pūnaha Agilent Bioanalyzer 2100. I raupapahia te whare pukapuka cDNA mā te whakamahi i te Novaseq sequencing. I hurihia ngā kōnae whakaahua mata mai i Illumina ki ngā pānui mata mā te whakamahi i te CASAVA Base Calling. Ka rongoatia ngā raraunga mata ki ngā kōnae hōputu FASTQ(fq) kei roto ngā raupapa pānui me ngā kounga turanga e rite ana. Tīpakohia te HISAT2 hei whakataurite i ngā pānui raupapatanga kua tātarihia ki te ira tohutoro Sscrofa11.1. I te nuinga o te wā, ka tautokohia e te HISAT2 ngā ira o ngā rahi katoa, tae atu ki ngā ira nui atu i te 4 piriona turanga, ā, ka whakatakotoria ngā uara taunoa mō te nuinga o ngā tawhā. Ka taea te whakarite pai i ngā pānui honohono mai i ngā raraunga RNA Seq mā te whakamahi i te HISAT2, te pūnaha tere rawa atu e wātea ana i tēnei wā, me te tika ōrite, pai ake rānei i tētahi atu tikanga.
Ko te nui o ngā tuhinga whakaputa e whakaata tika ana i te taumata o te whakaaturanga ira. Ka aromatawaihia ngā taumata whakaaturanga ira e te nui o ngā tuhinga whakaputa (te maha o ngā raupapatanga) e pā ana ki te ira, ki ngā exon rānei. He rite te maha o ngā pānui ki ngā taumata whakaaturanga ira, te roa o te ira, me te hohonu o te raupapatanga. I tatauhia te FPKM (ngā kongakonga mō ia mano takirua turanga o te tuhinga whakaputa kua raupapahia mō ia miriona takirua turanga) ā, i whakatauhia ngā uara-P o te whakaaturanga rerekētanga mā te whakamahi i te mōkihi DESeq2. Kātahi ka tatauhia e mātou te tere kitenga teka (FDR) mō ia uara P mā te whakamahi i te tikanga Benjamini-Hochberg9 i runga i te mahi-R "p.adjust" kua hangaia.
I hurihia te RNA i wehea mai i ngā wāhanga ngākau ki te cDNA i te kukū o te 200 ng/μl mā te whakamahi i te ranunga SuperScript IV Vilo Master mai i Thermo (Thermo, nama kāwai 11756050). I mahia te RT-PCR tauanga mā te whakamahi i te pereti tauhohenga mārama Applied Biosystems Endura Plate Microamp 384-puna (Thermo, nama kāwai 4483319) me te piripiri whatu microamp (Thermo, nama kāwai 4311971). Ko te ranunga tauhohenga he 5 µl ranunga Taqman Fast Advanced Master (Thermo, nama kāwai # 4444557), 0.5 µl Taqman Primer me te 3.5 µl H2O i konatunatua ki ia puna. I whakahaerehia ngā huringa qPCR paerewa, ā, i inehia ngā uara CT mā te whakamahi i te taputapu PCR wā-tūturu Applied Biosystems Quantstudio 5 (kōwae puna-384; nama hua A28135). I hokona ngā paraime Taqman mai i Thermo (GAPDH (Ss03375629_u1), PARP12 (Ss06908795_m1), PKDCC (Ss06903874_m1), CYGB (Ss06900188_m1), RGL1 (Ss06868890_m1), ACTN1 (Ss01009508_mH), GATA4 (Ss03383805_u1), GJA1 (Ss03374839_u1), COL1A2 (Ss03375009_u1), COL3A1 (Ss04323794_m1), ACTA2 (Ss04245588_m1). I whakatauritea ngā uara CT o ngā tauira katoa ki te ira tiaki whare GAPDH.
I aromatawaihia te tukunga pāpāho o te NT-ProBNP mā te whakamahi i te kete NT-ProBNP (poaka) (Tau Nama MBS2086979, MyBioSource) e ai ki te tikanga a te kaihanga. I roto i te poto, i tāpirihia he 250 µl o ia tauira me te paerewa ki ia puna. I muri tonu i te tāpiritanga o te tauira, tāpirihia he 50 µl o te Tauhohenga Whakamātautau A ki ia puna. Rūrūtia mārire te pereti ka hiritia ki te hiri. Kātahi ka whaowhia ngā papa ki te 37°C mō te 1 hāora. Kātahi ka mimitihia te otinga ka horoia ngā puna kia 4 ngā wā ki te 350 µl o te otinga horoi 1X, me te whaowhia te otinga horoi mō te 1-2 meneti i ia wā. Kātahi ka tāpirihia he 100 µl o te Tauhohenga Whakamātautau B mō ia puna ka hiritia ki te hiri pereti. I rūrūtia mārire te papa ka whaowhia ki te 37°C mō te 30 meneti. Mimia te otinga ka horoia ngā puna kia 5 ngā wā ki te 350 µl o te otinga horoi 1X. Tāpirihia te 90 µl o te otinga papa ki ia poka, ka hiritia te pereti. Whakatōkia te pereti ki te 37°C mō te 10-20 meneti. Tāpirihia te 50 µl o te Otinga Whakamutu ki ia poka. I inehia tonutia te pereti mā te whakamahi i te kaipānui pereti Cytation (BioTek) i whakatakotoria ki te 450 nm.
I whakahaerehia ngā tātaritanga mana hei whiriwhiri i ngā rahi o ngā rōpū ka whakarato i te mana >80% hei kite i te huringa tino 10% i roto i te tawhā me te reiti hapa Momo I 5%. I whakahaerehia ngā tātaritanga mana hei whiriwhiri i ngā rahi o ngā rōpū ka whakarato i te mana >80% hei kite i te huringa tino 10% i roto i te tawhā me te reiti hapa Momo I 5%. Анализ мощности был выполнен для выбора размеров групп, которые обеспечат >80% мощности для обнаружениблого 10% изменения параметра с 5% частотой ошибок типа I. I whakahaerehia te tātari mana hei tīpako i ngā rahi rōpū e whakarato ana i te mana >80% hei kite i te 10% huringa tawhā tino me te reiti hapa Momo I 5%.进行功效分析以选择将提供> 80％功效以检测参数中10％绝对变化和5％I功效以检测参数中10％绝对变化和5％I小田金。进行功效分析以选择将提供> 80％功效以检测参数中10％绝对变化和5％I功效以检测参数中10％绝对变化和5％I小田金。 Был проведен анализ мощности для выбора размера группы, который обеспечил бы > 80% мощности для обнаругожложениа изменения параметров и 5% частоты ошибок типа I. I whakahaerehia he tātaritanga mana hei tīpako i te rahi o te rōpū e whakarato ana i te mana >80% hei kite i te 10% huringa tawhā tino me te 5% te reiti hapa momo I.I tīpakohia matapōkeretia ngā wāhanga kiko i mua i te whakamātautau. I tirotirohia ngā āhuatanga katoa, ā, i wetewetehia ngā tauira i muri i te tātaritanga o ngā raraunga katoa. I whakamahia te pūmanawa GraphPad Prism (San Diego, CA) hei mahi i ngā tātaritanga tatauranga katoa. Mō ngā tatauranga katoa, i kiia he hiranga ngā uara-p i ngā uara <0.05. Mō ngā tatauranga katoa, i kiia he hiranga ngā uara-p i ngā uara <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Mō ngā tatauranga katoa, i kiia he hiranga ngā uara-p i ngā uara <0.05.对于所有统计数据，p 值在会<0.05 时被认为是显着的。对于所有统计数据，p 值在会<0.05 时被认为是显着的。 Для всей статистики p-значения считались значимыми при значениях <0,05. Mō ngā tatauranga katoa, i kiia he hiranga ngā uara-p i ngā uara <0.05.I whakahaerehia te whakamātautau-t a te Tauira-rua-hiku ki ngā raraunga me te rua noa iho o ngā whakataurite. I whakamahia te ANOVA kotahi-ara, rua-ara rānei hei whakatau i te hiranga i waenga i ngā rōpū maha. I te wā e whakahaerehia ana ngā whakamātautau muri-hoc, i whakamahia te whakatikatika a Tukey hei whakaaro mō ngā whakataurite maha. He whakaaro tatauranga motuhake kei ngā raraunga RNAsec ina tatauhia ana te FDR me te p.adjust e ai ki te whakaahuatanga i te wāhanga Tikanga.
Mō ētahi atu kōrero e pā ana ki te hoahoa rangahau, tirohia te whakarāpopototanga o te Pūrongo Rangahau Taiao e hono ana ki tēnei tuhinga.