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Akwai buƙatar ingantaccen tsarin in vitro wanda zai iya sake haifar da yanayin ilimin halittar zuciya daidai don gwajin magunguna. Rashin isasshen tsarin al'adar ƙwayoyin zuciya na ɗan adam ya haifar da fassarori marasa daidaito game da tasirin magungunan zuciya. A nan, mun ƙirƙiri samfurin al'adar ƙwayoyin zuciya (CTCM) wanda ke motsa sassan zuciya ta hanyar lantarki kuma yana yin motsi na jiki a lokacin matakan systolic da diastolic na zagayowar zuciya. Bayan kwanaki 12 na al'ada, wannan hanyar ta inganta rayuwar sassan zuciya kaɗan, amma bai kiyaye cikakken amincin tsarin su ba. Saboda haka, bayan ƙaramin gwajin ƙwayoyin halitta, mun gano cewa ƙara 100 nM triiodothyronine (T3) da 1 μM dexamethasone (Dex) zuwa ga hanyar sadarwarmu ta kiyaye tsarin ƙananan sassan na tsawon kwanaki 12. Tare da maganin T3/Dex, tsarin CTCM ya kiyaye bayanan rubutu, dorewa, aikin metabolism, da kuma daidaiton tsarin a daidai matakin da sabbin ƙwayoyin zuciya na tsawon kwanaki 12. Bugu da ƙari, miƙewar kyallen zuciya da yawa a cikin al'ada yana haifar da siginar zuciya mai ƙarfi, yana ba da shaida cewa ikon CTCM na kwaikwayon yanayin hypertrophic da bugun zuciya ke haifarwa. A ƙarshe, CTCM na iya yin kwaikwayon ilimin halittar jiki da cututtukan zuciya a cikin al'ada na tsawon lokaci, wanda ke ba da damar yin gwajin magunguna masu inganci.
Kafin a fara binciken asibiti, ana buƙatar ingantattun tsarin in vitro waɗanda za su iya sake haifar da yanayin ilimin halittar zuciyar ɗan adam daidai. Irin waɗannan tsarin ya kamata su yi kwaikwayon canjin yanayin motsa jiki, bugun zuciya, da halayen electrophysiological. Ana amfani da samfuran dabbobi a matsayin dandamali na tantance ilimin halittar zuciya tare da ƙarancin aminci wajen nuna tasirin magunguna a cikin zuciyar ɗan adam1,2. A ƙarshe, Tsarin Gwaji na Al'adar Zuciya Mai Kyau (CTCM) samfurin da ke da matuƙar tasiri kuma takamaiman don hanyoyin magancewa da magunguna daban-daban, yana sake kwaikwayon ilimin halittar jiki da cututtukan zuciya na ɗan adam3 daidai. Rashin irin wannan tsarin yana iyakance gano sabbin magunguna don gazawar zuciya4,5 kuma ya haifar da gubar zuciya a matsayin babban dalilin fita daga kasuwa6.
A cikin shekaru goma da suka gabata, an janye magunguna takwas marasa zuciya daga amfani da su a asibiti saboda suna haifar da tsawaita tazara ta QT wanda ke haifar da arrhythmias na ventricular da mutuwar kwatsam7. Don haka, akwai buƙatar ƙarin dabarun tantancewa kafin asibiti don tantance ingancin zuciya da guba. Amfani da kwanan nan na cardiomyocytes na ƙwayoyin zuciya (hiPS-CM) da ɗan adam ya haifar a cikin gwajin magunguna da guba yana ba da mafita ga wannan matsala. Duk da haka, yanayin rashin girma na hips-CMs da rashin sarkakiyar ƙwayoyin zuciya da yawa sune manyan ƙuntatawa na wannan hanyar. Nazarin da aka yi kwanan nan ya nuna cewa ana iya shawo kan wannan iyakancewa ta hanyar amfani da farkon hiPS-CM don samar da hydrogels nama na zuciya jim kaɗan bayan fara matsewa kwatsam da kuma ƙara yawan kuzarin lantarki a hankali akan lokaci. Duk da haka, waɗannan ƙananan ƙwayoyin hips-CM ba su da halayen electrophysiological da contractile na myocardium na manya. Bugu da ƙari, ƙwayar zuciyar ɗan adam tana da tsari mai rikitarwa, wanda ya ƙunshi cakuda daban-daban na nau'ikan ƙwayoyin halitta, gami da ƙwayoyin endothelial, neurons, da stromal fibroblasts, waɗanda aka haɗa su ta hanyar takamaiman saitin furotin na extracellular matrix. Wannan bambancin da ke tsakanin yawan ƙwayoyin halittar da ba sa cikin zuciya11,12,13 a cikin zuciyar dabbobi masu shayarwa ta manya babban cikas ne ga yin kwaikwayon ƙwayar zuciya ta amfani da nau'ikan ƙwayoyin halitta daban-daban. Waɗannan manyan ƙuntatawa suna jaddada mahimmancin haɓaka hanyoyin haɓaka ƙwayoyin zuciya marasa lalacewa a ƙarƙashin yanayin ilimin halittar jiki da na cututtuka.
Sassan zuciya masu siriri (300 µm) da aka ƙera sun tabbatar da cewa samfuri ne mai kyau na myocardium na ɗan adam. Wannan hanyar tana ba da damar samun cikakken tsarin ƙwayoyin halitta na 3D mai kama da nama na zuciyar ɗan adam. Duk da haka, har zuwa 2019, amfani da sassan zuciya masu al'ada an iyakance shi ta hanyar ɗan gajeren lokacin rayuwa na al'ada (awa 24). Wannan ya faru ne saboda dalilai da dama, ciki har da rashin shimfiɗa jiki da na'ura, haɗin iska da ruwa, da kuma amfani da kafofin watsa labarai masu sauƙi waɗanda ba sa tallafawa buƙatun nama na zuciya. A cikin 2019, ƙungiyoyin bincike da yawa sun nuna cewa haɗa abubuwan injiniya a cikin tsarin al'adun nama na zuciya na iya tsawaita rayuwar al'ada, inganta bayyanar zuciya, da kuma kwaikwayon cututtukan zuciya. Nazari biyu masu kyau 17 da 18 sun nuna cewa ɗorawa na injiniya na uniaxial yana da tasiri mai kyau akan yanayin zuciya yayin al'ada. Duk da haka, waɗannan nazarin ba su yi amfani da ɗaukar nauyin jiki-na'ura mai ƙarfi na zagayen zuciya ba, tunda sassan zuciya suna da ƙarfin tensile na isometric 17 ko ɗaukar nauyin auxotonic linear 18. Waɗannan hanyoyin shimfiɗa nama sun haifar da danne ƙwayoyin halittar zuciya da yawa ko kuma yawan ƙwayoyin halittar da ke da alaƙa da amsawar miƙewa mara kyau. Abin lura, Pitoulis et al. 19 sun haɓaka wani wanka mai ƙarfi na al'adar yanke zuciya don sake gina zagayowar zuciya ta amfani da martanin mai canza ƙarfi da kuma abubuwan da ke motsa jiki. Kodayake wannan tsarin yana ba da damar yin ƙirar zagayowar zuciya ta in vitro daidai, rikitarwa da ƙarancin ƙarfin da ake samu na hanyar suna iyakance amfani da wannan tsarin. Kwanan nan dakin gwaje-gwajenmu ya ƙirƙiro tsarin al'adu mai sauƙi ta amfani da motsa jiki na lantarki da kuma ingantaccen matsakaici don kiyaye wanzuwar sassan naman alade da nama na zuciyar ɗan adam har zuwa kwanaki 620,21.
A cikin rubutun da aka yi a yanzu, mun bayyana samfurin al'adar nama na zuciya (CTCM) ta amfani da sassan zuciyar alade wanda ya haɗa da alamun nishaɗi don sake maimaita ilimin halittar zuciya mai girma uku da kuma yaduwar cututtuka a lokacin zagayowar zuciya. Wannan CTCM na iya ƙara daidaiton hasashen magunguna kafin asibiti zuwa matakin da ba a taɓa cimmawa ba ta hanyar samar da tsarin zuciya mai inganci, mai matsakaicin aiki wanda ke kwaikwayon ilimin halittar jiki/fasaha na zuciyar dabbobi masu shayarwa don gwajin magunguna kafin asibiti.
Siginar injina ta hemodynamic suna taka muhimmiyar rawa wajen kiyaye aikin zuciya a cikin vitro 22,23,24. A cikin rubutun da aka buga a yanzu, mun ƙirƙiro CTCM (Hoto na 1a) wanda zai iya kwaikwayon yanayin zuciya na manya ta hanyar haifar da motsawar lantarki da na inji a mitoci na jiki (1.2 Hz, bugun 72 a minti ɗaya). Don guje wa tsawaita nama mai yawa yayin diastole, an yi amfani da na'urar bugawa ta 3D don ƙara girman nama da 25% (Hoto na 1b). An tsara lokacin tafiyar lantarki da tsarin C-PACE ya haifar don fara 100 ms kafin systole ta amfani da tsarin tattara bayanai don sake zagayowar zuciya gaba ɗaya. Tsarin al'adar nama yana amfani da mai kunna iska mai shirye-shirye (LB Engineering, Jamus) don faɗaɗa membrane mai sassauƙa na silicone ta hanyar zagaye don haifar da faɗaɗa sassan zuciya a cikin ɗakin sama. An haɗa tsarin zuwa layin iska na waje ta hanyar na'urar watsa matsin lamba, wanda ya ba da damar daidaita matsin lamba daidai (± 1 mmHg) da lokaci (± 1 ms) (Hoto na 1c).
a Haɗa sashin nama zuwa zoben tallafi na mm 7, wanda aka nuna da shuɗi, a cikin ɗakin al'ada na na'urar. An raba ɗakin al'ada daga ɗakin iska ta hanyar siririn membrane mai laushi na silicone. Sanya gasket tsakanin kowane ɗaki don hana zubewa. Murfin na'urar ya ƙunshi electrodes na graphite waɗanda ke ba da motsin lantarki. b Wakiltar tsarin babban na'urar nama, zoben jagora da zoben tallafi. An sanya sassan nama (launin ruwan kasa) akan babban na'urar tare da zoben jagora da aka sanya a cikin ramin a gefen waje na na'urar. Ta amfani da jagorar, a hankali sanya zoben tallafi da aka lulluɓe da manne acrylic nama akan sashin nama na zuciya. c Jadawali yana nuna lokacin motsawar lantarki azaman aikin matsin lamba na ɗakin iska wanda mai kunna iska mai iya shiryawa (PPD) ke sarrafawa. An yi amfani da na'urar tattara bayanai don daidaita motsawar lantarki ta amfani da na'urori masu auna matsin lamba. Lokacin da matsin lamba a cikin ɗakin al'ada ya kai iyakar da aka saita, ana aika siginar bugun jini zuwa C-PACE-EM don haifar da motsawar lantarki. d Hoton CTCM guda huɗu da aka sanya akan shiryayyen incubator. Ana haɗa na'urori huɗu zuwa PPD ɗaya ta hanyar da'irar iska, kuma ana saka na'urori masu auna matsin lamba a cikin bawul ɗin hemostatic don sa ido kan matsin lamba a cikin da'irar iska. Kowace na'ura tana ɗauke da sassan nama guda shida.
Ta amfani da na'urar kunna iska guda ɗaya, mun sami damar sarrafa na'urori CTCM guda 4, kowannensu zai iya ɗaukar sassan nama 6 (Hoto na 1d). A cikin CTCM, matsin lamba na iska a cikin ɗakin iska ya koma matsin lamba mai daidaitawa a cikin ɗakin ruwa kuma yana haifar da faɗaɗa yanayin aikin zuciya (Hoto na 2a da Fim na Ƙarin 1). Kimanta miƙewar nama a 80 mm Hg. Art. ya nuna miƙewar sassan nama da 25% (Hoto na 2b). An nuna cewa wannan miƙewar kashi ya yi daidai da tsawon sarcomere na jiki na 2.2–2.3 µm don daidaitaccen sassa na zuciya17,19,25. An tantance motsin nama ta amfani da saitunan kyamara na musamman (Hoto na Ƙarin 1). Girma da saurin motsi na nama (Hoto na 2c, d) sun yi daidai da miƙewa a lokacin zagayowar zuciya da lokacin lokacin systole da diastole (Hoto na 2b). Miƙewa da saurin nama na zuciya yayin matsewa da shakatawa ya kasance iri ɗaya na tsawon kwanaki 12 a cikin al'ada (Hoto na 2f). Domin kimanta tasirin ƙarfafawar lantarki akan ƙarfin gwiwa yayin al'ada, mun ƙirƙiro wata hanya don tantance nakasar aiki ta amfani da tsarin launi (Ƙarin Hoto na 2a,b) kuma mun sami damar bambance tsakanin nakasar tare da ƙarfafawar lantarki da ba tare da shi ba. Sashe ɗaya na zuciya (Hoto na 2f). A yankin da aka yanke (R6-9), ƙarfin lantarki yayin ƙarfafawar lantarki ya fi 20% girma fiye da rashin ƙarfafawar lantarki, wanda ke nuna gudummawar ƙarfafawar lantarki ga aikin kwantar da hankali.
Alamun matsin lamba na ɗakin iska, matsin lamba na ɗakin ruwa, da kuma ma'aunin motsin nama sun tabbatar da cewa matsin lamba na ɗakin yana canza matsin lamba na ɗakin ruwa, yana haifar da motsi daidai da nama. b Alamun matsakaicin kashi na shimfiɗa (shuɗi) na sassan nama wanda ya yi daidai da kashi na shimfiɗa (lemu). c Motsin da aka auna na yanki na zuciya ya yi daidai da saurin motsi da aka auna. (d) Alamun wakilci na motsi na zagaye (layin shuɗi) da gudu (layin lemu mai dige-dige) a cikin yanki na zuciya. e Ƙididdige lokacin zagayowar (n = yanka 19 a kowace ƙungiya, daga aladu daban-daban), lokacin matsewa (n = yanka 19 a kowace ƙungiya), lokacin shakatawa (n = yanka 19 a kowace ƙungiya, daga aladu daban-daban), motsin nama (n = 25). yanka)/ƙungiya daga aladu daban-daban), saurin systolic mafi girma (n = 24(D0), yanka 25(D12)/ƙungiya daga aladu daban-daban) da ƙimar hutawa mafi girma (n = 24(D0), yanka 25(D12)/ƙungiya daga aladu daban-daban). Gwajin t-ɗalibi mai wutsiya biyu bai nuna wani bambanci mai mahimmanci a cikin kowane siga ba. f Binciken nau'in tsokar da aka yi wa lakabi da sassan nama tare da (ja) da kuma ba tare da (shuɗi) na lantarki ba, yankuna goma na sassan nama daga sashe ɗaya. Ƙananan bangarorin suna nuna ƙididdige bambancin kashi a cikin tsokar da aka yi a sassan nama tare da da kuma ba tare da tsokar da aka yi a sassa goma daga sassa daban-daban. (n = yanka 8/rukuni daga aladu daban-daban, ana yin gwajin t-test na ɗalibi mai wutsiya biyu; ****p < 0.0001, **p < 0.01, *p < 0.05). (n = yanka 8/rukuni daga aladu daban-daban, ana yin gwajin t-test na ɗalibi mai wutsiya biyu; ****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 <0,0,0,001, **p<0,01). (n = sassa 8/rukuni daga aladu daban-daban, gwajin t-test na ɗalibi mai wutsiya biyu; ****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： (n = 8 片/组，来自不同的猪，进行双尾学生t 检验： (n = 8 срезов/группу, от разных свиней, двусторонний критерий Стьюдента; **** p <0,0001, **p <0,01, *p <0,05). (n = sassa 8/rukuni, daga aladu daban-daban, gwajin t-test na ɗalibi mai wutsiya biyu; ****p<0.0001, **p<0.01, *p<0.05).Sandunan kuskure suna wakiltar matsakaicin ± karkacewar da aka saba.
A cikin tsarin al'adar yanke zuciya ta biomimetic da muka gabata [20, 21], mun kiyaye dorewar aiki, aiki, da kuma daidaiton tsarin yanka zuciya na tsawon kwanaki 6 ta hanyar amfani da motsa jiki na lantarki da kuma inganta yanayin matsakaici. Duk da haka, bayan kwana 10, waɗannan alkaluma sun ragu sosai. Za mu koma ga sassan da aka haɓaka a cikin tsarin al'adar biomimetic da muka gabata 20, 21 yanayin sarrafawa (Ctrl) kuma za mu yi amfani da matsakaicin da muka inganta a baya azaman yanayin MC da al'ada a ƙarƙashin motsa jiki na injiniya da lantarki a lokaci guda (CTCM). da ake kira . Da farko, mun gano cewa motsawar injiniya ba tare da motsa jiki na lantarki ba bai isa ya kula da dorewar nama na tsawon kwanaki 6 ba (Ƙarin Hoto na 3a,b). Abin sha'awa, tare da gabatar da motsa jiki na injiniya da lantarki ta amfani da STCM, wanzuwar sassan zuciya na kwanaki 12 ya kasance iri ɗaya kamar a cikin sassan zuciya na sabo a ƙarƙashin yanayin MS, amma ba a ƙarƙashin yanayin Ctrl ba, kamar yadda aka nuna ta hanyar nazarin MTT (Hoto na 1). 3a). Wannan yana nuna cewa motsawar injiniya da kwaikwayon zagayowar zuciya na iya kiyaye wanzuwar sassan nama na tsawon lokaci sau biyu kamar yadda aka ruwaito a cikin tsarin al'adar mu na baya. Duk da haka, kimanta ingancin tsarin sassan nama ta hanyar labeling na troponin na zuciya T da connexin 43 ya nuna cewa bayyanar connexin 43 ta fi girma sosai a cikin kyallen MC a rana ta 12 fiye da yadda ake sarrafawa a rana ɗaya. Duk da haka, ba a kiyaye cikakken tsarin bayyanar connexin 43 da samuwar Z-diski ba (Hoto na 3b). Muna amfani da tsarin hankali na wucin gadi (AI) don auna ingancin tsarin nama26, bututun koyo mai zurfi bisa hoto bisa ga troponin-T da connexin staining43 don auna ingancin tsarin da hasken zuciya ta atomatik dangane da ƙarfin wurin zama. Wannan hanyar tana amfani da Convolutional Neural Network (CNN) da tsarin ilmantarwa mai zurfi don tantance ingancin tsarin nama na zuciya ta hanyar atomatik da rashin son kai, kamar yadda aka bayyana a cikin bayanin. 26. Nama na MC ya nuna ingantaccen kamanceceniya da rana ta 0 idan aka kwatanta da sassan sarrafawa marasa tsauri. Bugu da ƙari, tabon trichrome na Masson ya nuna ƙarancin kashi na fibrosis a ƙarƙashin yanayin MS idan aka kwatanta da yanayin sarrafawa a rana ta 12 ta al'ada (Hoto na 3c). Duk da cewa CTCM ta ƙara ƙarfin sassan ƙwayoyin zuciya a rana ta 12 zuwa matakin da ya yi kama da na sabbin ƙwayoyin zuciya, bai inganta tsarin sassan zuciya sosai ba.
Jadawalin Bar yana nuna ƙididdige yuwuwar MTT na sabbin yanka na zuciya (D0) ko al'adar yanka na zuciya na tsawon kwanaki 12 ko dai a cikin al'adar tsaye (D12 Ctrl) ko a cikin CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) yanka/rukuni daga aladu daban-daban, hanya ɗaya da ake yin gwajin ANOVA; ####p < 0.0001 idan aka kwatanta da D0 da **p < 0.01 idan aka kwatanta da D12 Ctrl). Jadawalin Bar yana nuna ƙididdige yuwuwar MTT na sabbin yanka na zuciya (D0) ko al'adar yanka na zuciya na tsawon kwanaki 12 ko dai a cikin al'adar tsaye (D12 Ctrl) ko a cikin CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), yanka 12 (D12 MC) daga aladu daban-daban, hanya ɗaya da ake yin gwajin ANOVA; ####p < 0.0001 idan aka kwatanta da D0 da **p < 0.01 idan aka kwatanta da D12 Ctrl).Tarihin yana nuna ƙididdige yuwuwar sassan zuciya na MTT sabo (D0) ko al'adar sassan zuciya na tsawon kwanaki 12 a cikin al'adar tsaye (ikon D12) ko CTCM (D12 MC) (n = 18 (D0), 15 (ikon D12). ) ), sassan/rukuni 12 (D12 MC) daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya;####p <0,0001 pо сравнению с D0 и **p <0,01 pо сравнению с D12 Ctrl). ####p < 0.0001 idan aka kwatanta da D0 da **p < 0.01 idan aka kwatanta da 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(D0)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)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(D0) ，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 ， trlhistogram wanda ke nuna ƙimar ƙarfin MTT a cikin sassan zuciya masu sabo (D0) ko sassan zuciya waɗanda aka noma na tsawon kwanaki 12 a cikin al'adar tsaye (ikon D12) ko CTCM (D12 MC) (n = 18 (D0), 15 (ikon D12)), sassan/rukuni 12 (D12 MC) daga aladu daban-daban, gwajin ANOVA na hanya ɗaya;####p <0,0001 pо сравнению с D0, **p <0,01 pо сравнению с D12 Ctrl). ####p < 0.0001 idan aka kwatanta da D0, **p < 0.01 idan aka kwatanta da D12 Ctrl).b Troponin-T (kore), connexin 43 (ja) da DAPI (shuɗi) a cikin sassan zuciya da aka ware sabo (D0) ko sassan zuciya waɗanda aka haɓaka a ƙarƙashin yanayin da ba ya canzawa (Ctrl) ko yanayin CTCM (MC) na tsawon kwanaki 12) na hotunan immunofluorescence masu wakiltar (ma'aunin blank = 100 µm). Ƙididdige hankali na wucin gadi na daidaiton tsarin kyallen zuciya (n = 7 (D0), 7 (D12 Ctrl), yanka 5 (D12 MC) kowanne daga alade daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ####p < 0.0001 idan aka kwatanta da D0 da ****p < 0.0001 idan aka kwatanta da D12 Ctrl). Ƙididdige hankali na wucin gadi na daidaiton tsarin kyallen zuciya (n = 7 (D0), 7 (D12 Ctrl), yanka 5 (D12 MC) kowanne daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ####p < 0.0001 idan aka kwatanta da D0 da ****p < 0.0001 idan aka kwatanta da D12 Ctrl). Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D15), 7 (D12) срезов/групп от разных свиней, проводится однофакторный тест ANOVA; D12 Ctrl). Ƙididdige daidaiton tsarin kyallen zuciya ta hanyar amfani da hankali na wucin gadi (n = 7 (D0), 7 (D12 Ctrl), sassan/ƙungiyoyi 5 (D12 MC) daga aladu daban-daban, an yi gwajin ANOVA ta hanya ɗaya; ####p < 0.0001 idan aka kwatanta da D0 da ****p < 0.0001 idan aka kwatanta da 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) yanka / rukuni kowane nau'in alade daban-daban, gwajin ANOVA mai hanya daya;#0#0相比，****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) yanka / rukuni kowane nau'in aladu daban-daban, gwajin ANOVA guda daya; #0#0与D0相比，****p <0.0001 与D12 Ctrl 相比）。 Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), 7 (D0), 7 (D0), 7 (D0) срезов/группу каждой из разных свиней, односторонний тест ANOVA; ####p <0,0001 vs. D0 Для сравнения ****p <0,000 Ctrl). Hankali na wucin gadi don auna daidaiton tsarin kyallen zuciya (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) sassan/rukunin kowanne daga cikin aladu daban-daban, gwajin ANOVA na hanya ɗaya; ####p<0.0001 vs .D0 Don kwatantawa ****p < 0.0001 idan aka kwatanta da D12 Ctrl). c Hotunan da aka wakilta (hagu) da ƙididdigewa (dama) don yanka zuciya da aka yi wa fenti da tabon Masson's trichrome (Sikeli bare = 500 µm) (n = yanka 10/rukuni kowanne daga alade daban-daban, an yi gwajin ANOVA ta hanya ɗaya; ####p < 0.0001 idan aka kwatanta da D0 da ***p < 0.001 idan aka kwatanta da D12 Ctrl). c Hotunan da aka wakilta (hagu) da ƙididdigewa (dama) don yanka zuciya da aka yi wa fenti da tabon Masson's trichrome (Sikeli bare = 500 µm) (n = yanka 10/rukuni kowanne daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; #### p < 0.0001 idan aka kwatanta da D0 da ***p < 0.001 idan aka kwatanta da D12 Ctrl). c Репрезентативные изображения (слева) Массона (масштаб без покрытия = 500 мкм) (n = 10 срезов/группу от разных свиней, выполняется одностор 0,0001 pо сравнению с D0 и ***p <0,001 pо сравнению с D12 Ctrl). c Hotunan da aka wakilta (hagu) da kuma ƙididdigewa (dama) na sassan zuciya da aka yi wa fenti da tabon Masson's trichrome (sikelin da ba a shafa ba = 500 µm) (n = sassa 10/rukuni daga aladu daban-daban, an yi gwajin ANOVA ta hanya ɗaya; #### p < 0 .0001 idan aka kwatanta da D0 da ***p < 0.001 idan aka kwatanta da D12 Ctrl). c 用Masson 三色染料染色的心脏切片的代表性图像）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，***0 1 <0相比） C 用 masson 三 色 染料 的 心脏 切片 的 代表性 （左 左） 量化裸尺度 = 500 µm） （n = 10 个 切片 组 每 组 来自 不同 猪 ， 进行 单向 单向 Anova#p 0.0001 与D0 相比，***p <0.001 与D12 Ctrl 相比）。 c Репрезентативные изображения (слева) Массона (чистая шкала = 500 мкм) (n = 10 срезов/группа, каждый от другой свиньи, протестировано с помї однофакторного дисперсионного анализа ;### #p <0,0001 pо сравнению с D0, ***p <0,001 по сравнению с D12 Ctrl). c Hotunan da aka wakilta (hagu) da kuma adadi (dama) na sassan zuciya da aka yi wa fenti da tabon Masson's trichrome (blank = 500 µm) (n = sassa 10/rukuni, kowanne daga alade daban-daban, an gwada shi ta hanyar nazarin bambancin hanya ɗaya;### # p < 0.0001 idan aka kwatanta da D0, ***p < 0.001 idan aka kwatanta da D12 Ctrl).Sandunan kuskure suna wakiltar matsakaicin ± karkacewar da aka saba.
Mun yi hasashen cewa ta hanyar ƙara ƙananan ƙwayoyin halitta a cikin yanayin al'ada, ana iya inganta amincin zuciya da jijiyoyin jini kuma haɓakar fibrosis za ta ragu yayin al'adar CTCM. Saboda haka, mun tantance ƙananan ƙwayoyin halitta ta amfani da al'adun sarrafa mu na tsaye20,21 saboda ƙarancin adadin abubuwan da ke rikitar da mu. An zaɓi Dexamethasone (Dex), triiodothyronine (T3), da SB431542 (SB) don wannan allon. An riga an yi amfani da waɗannan ƙananan ƙwayoyin halitta a cikin al'adun hiPSC-CM don haifar da balaga na cardiomyocytes ta hanyar ƙara tsawon sarcomere, T-tubules, da saurin gudanarwa. Bugu da ƙari, duka Dex (glucocorticoid) da SB an san suna rage kumburi29,30. Saboda haka, mun gwada ko haɗa ɗaya ko haɗuwa da waɗannan ƙananan ƙwayoyin halitta zai inganta daidaiton tsarin sassan zuciya. Don tantancewa ta farko, an zaɓi adadin kowane mahaɗin bisa ga yawan da aka saba amfani da shi a cikin samfuran al'adun tantanin halitta (1 μM Dex27, 100 nM T327, da 2.5 μM SB31). Bayan kwana 12 na al'ada, haɗin T3 da Dex ya haifar da ingantaccen tsarin zuciya da kuma ƙarancin sake fasalin fibrous (Karin Hoto na 4 da 5). Bugu da ƙari, amfani da ninki biyu ko ninki biyu na waɗannan yawan T3 da Dex ya haifar da illa mai illa idan aka kwatanta da yawan da aka saba (Karin Hoto na 6a,b).
Bayan gwajin farko, mun yi kwatancen yanayi guda 4 na al'ada (Hoto na 4a): Ctrl: sassan zuciya da aka haɓaka a cikin al'adar da muka bayyana a baya ta amfani da hanyar da aka inganta; 20.21 TD: T3 da Ctrl s An ƙara Dex a ranar Laraba; MC: sassan zuciya da aka haɓaka a cikin CTCM ta amfani da hanyar da aka inganta a baya; da kuma MT: CTCM tare da T3 da Dex da aka ƙara a cikin hanyar. Bayan kwanaki 12 na noma, yuwuwar kyallen MS da MT ya kasance iri ɗaya kamar yadda yake a cikin sabbin kyallen da aka tantance ta hanyar gwajin MTT (Hoto na 4b). Abin sha'awa, ƙara T3 da Dex zuwa al'adun transwell (TD) bai haifar da babban ci gaba a cikin rayuwa ba idan aka kwatanta da yanayin Ctrl, yana nuna muhimmiyar rawa ta motsa jiki na injiniya wajen kiyaye wanzuwar sassan zuciya.
Tsarin zane na gwaji wanda ke nuna yanayin al'adu guda huɗu da ake amfani da su don tantance tasirin motsa jiki na injiniya da ƙarin T3/Dex akan matsakaici na tsawon kwanaki 12. b jadawali na sanduna yana nuna ƙididdige ƙarfin rayuwa kwanaki 12 bayan al'ada a cikin dukkan yanayi 4 na al'ada (Ctrl, TD, MC, da MT) idan aka kwatanta da sabbin yanka na zuciya (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD da D12 MT), yanka/rukuni 12 (D12 MC) daga aladu daban-daban, an yi gwajin ANOVA ta hanya ɗaya; ####p < 0.0001, ###p < 0.001 idan aka kwatanta da D0 da **p < 0.01 idan aka kwatanta da D12 Ctrl). b jadawali na sanduna yana nuna ƙididdige rayuwa kwanaki 12 bayan al'ada a cikin dukkan yanayi 4 na al'ada (Ctrl, TD, MC, da MT) idan aka kwatanta da sabbin yanka na zuciya (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD da D12 MT), yanka/rukuni 12 (D12 MC) daga aladu daban-daban, an yi gwajin ANOVA ta hanya ɗaya; ####p < 0.0001, ###p < 0.001 idan aka kwatanta da D0 da **p < 0.01 idan aka kwatanta da D12 ctrl). b Гистограмма показывает количественую оценку культивирования (контроль, TD, MC и MT) по сравнению со сравнению со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl 12 Ctrl), D12 Ctrl MC) срезов/группу от разных свиней, проводится односторонний тест ANOVA; ####p <0,0001, ###p <0,001 pо сравнению с D0 и **p <0,01 pо сравнению с D12 Ctrl). b Jadawalin sandunan yana nuna ƙididdige rayuwa a cikin kwanaki 12 bayan al'ada a cikin dukkan yanayi 4 na al'ada (iko, TD, MC, da MT) idan aka kwatanta da sassan zuciya masu sabo (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, da D12 MT), sassan/rukuni 12 (D12 MC) daga aladu daban-daban, gwajin ANOVA na hanya ɗaya; ####p < 0.0001, ###p < 0.001 vs. D0 da **p < 0.01 idan aka kwatanta da 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 (15)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 且0.01 与D12控制）b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC и MT) по сравнению (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD da D12 MT), da разных свиней 12 (D12 MC) срезы/группа, односто / группа, односто <0,001 по сравнению с D0, **p <0,01 pо сравнению с контролем D12). b Histogram yana nuna duk yanayin al'ada guda 4 (ikon sarrafawa, TD, MC da MT) idan aka kwatanta da sassan zuciya masu sabo (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD da D12 MT), daga aladu daban-daban 12 (D12 MC) sassan/rukuni, gwajin ANOVA na hanya ɗaya; ####p<0.0001, ###p<0.001 vs. D0, **p<0.01 vs. iko D12). C jadawali na sanduna yana nuna ƙididdige yawan kwararar glucose kwanaki 12 bayan al'ada a cikin dukkan yanayi 4 na al'ada (Ctrl, TD, MC, da MT) idan aka kwatanta da sabbin yanka na zuciya (D0) (n = yanka 6/rukuni daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ###p < 0.001, idan aka kwatanta da D0 da ***p < 0.001 idan aka kwatanta da D12 Ctrl). C jadawali na sanduna yana nuna ƙididdige yawan kwararar glucose kwanaki 12 bayan al'ada a cikin dukkan yanayi 4 na al'ada (Ctrl, TD, MC, da MT) idan aka kwatanta da sabbin yanka na zuciya (D0) (n = yanka 6/rukuni daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ###p < 0.001, idan aka kwatanta da D0 da ***p < 0.001 idan aka kwatanta da D12 Ctrl). c Гистограмма показывает количественую оценку потока глюкозы через 12 культивирования (контроль, TD, MC и MT) по сравнению односторонний Выполняется тест ANOVA; c Histogram yana nuna ƙididdige yawan kwararar glucose kwanaki 12 bayan al'ada a ƙarƙashin dukkan yanayin al'ada guda 4 (ikon sarrafawa, TD, MC da MT) idan aka kwatanta da sassan zuciya masu sabo (D0) (n = sassa 6/rukuni daga aladu daban-daban, an yi gwajin ANOVA ta hanya ɗaya; ###p < 0.001 idan aka kwatanta da D0 da ***p < 0.001 idan aka kwatanta da 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) 玻件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0)玻寔。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 0.001 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 切片培养 后 12 天 的 通量 定量 （n = 6 片/组 ， 来自 后测试；###p <0.001，与D0 相比，***p <0.001 与D12 Ctrl 相比）。 c Гистограмма, показывающая казывающая количественную условий культивирования (контроль, TD, MC и MT) по сравнению свиней, односторонний Были проведены тесты ANOVA; D12 (kwantena). c Histogram yana nuna ƙididdige yawan kwararar glucose a kwanaki 12 bayan al'ada ga dukkan yanayin al'adu guda 4 (ikon sarrafawa, TD, MC, da MT) idan aka kwatanta da sassan zuciya masu sabo (D0) (n = sassa 6/rukuni, daga aladu daban-daban, gefe guda. An gudanar da gwaje-gwajen ANOVA, ###p < 0.001 idan aka kwatanta da D0, ***p < 0.001 idan aka kwatanta da D12 (ikon sarrafawa).d Tsarin nazarin nau'ikan kyallen takarda na sabo (shuɗi), rana ta 12 MC (kore), da rana ta 12 MT (ja) a maki goma na sassan kyallen takarda na yanki (n = yanka 4/rukuni, gwajin ANOVA na hanya ɗaya; babu wani bambanci mai mahimmanci tsakanin ƙungiyoyi). e Tsarin dutsen mai aman wuta yana nuna kwayoyin halitta daban-daban a sassan zuciya na sabo (D0) idan aka kwatanta da sassan zuciya da aka noma a ƙarƙashin yanayi na tsaye (Ctrl) ko ƙarƙashin yanayin MT (MT) na tsawon kwanaki 10-12. f Taswirar zafi na kwayoyin halittar sarcomere don sassan zuciya da aka noma a ƙarƙashin kowane yanayin al'ada. Sandunan kuskure suna wakiltar matsakaicin ± daidaitaccen karkacewa.
Dogaro kan metabolism akan sauyawa daga oxidation na fatty acid zuwa glycolysis alama ce ta rashin bambance-bambancen zuciya. Kwayoyin halittar zuciya marasa girma galibi suna amfani da glucose don samar da ATP kuma suna da hypoplastic mitochondria tare da ƙarancin cristae5,32. Nazarin amfani da glucose ya nuna cewa a ƙarƙashin yanayin MC da MT, amfani da glucose yayi kama da na kyallen rana ta 0 (Hoto na 4c). Duk da haka, samfuran Ctrl sun nuna ƙaruwa mai yawa a amfani da glucose idan aka kwatanta da sabbin kyallen. Wannan yana nuna cewa haɗin CTCM da T3/Dex yana haɓaka wanzuwar kyallen kuma yana kiyaye yanayin metabolism na sassan zuciya na kwanaki 12 da aka haɓaka. Bugu da ƙari, nazarin tsoka ya nuna cewa matakan tsoka sun kasance iri ɗaya kamar na kyallen zuciya na sabo na tsawon kwanaki 12 a ƙarƙashin yanayin MT da MS (Hoto na 4d).
Domin yin nazarin tasirin CTCM da T3/Dex gabaɗaya akan yanayin rubutun zuciya na duniya, mun yi RNAseq akan yanka zuciya daga dukkan yanayi huɗu na al'ada (Bayani na Ƙarin 1). Abin sha'awa, sassan MT sun nuna babban kamanceceniya da sabbin kyallen zuciya, tare da 16 kawai aka bayyana daban-daban daga cikin kwayoyin halitta 13,642. Duk da haka, kamar yadda muka nuna a baya, yanka Ctrl sun nuna 1229 kwayoyin halitta da aka bayyana daban-daban bayan kwanaki 10-12 a cikin al'ada (Hoto na 4e). An tabbatar da waɗannan bayanai ta hanyar qRT-PCR na kwayoyin halitta na zuciya da fibroblast (Hoto na Ƙarin 7a-c). Abin sha'awa, sassan Ctrl sun nuna raguwar kwayoyin halitta na zagayowar zuciya da ƙwayoyin halitta da kunna shirye-shiryen kwayoyin halitta masu kumburi. Waɗannan bayanan sun nuna cewa rashin bambance-bambance, wanda yawanci yakan faru bayan dogon lokaci na al'ada, an rage shi gaba ɗaya a ƙarƙashin yanayin MT (Hoto na Ƙarin 8a,b). Binciken da aka yi a kan kwayoyin halittar sarcomere ya nuna cewa a ƙarƙashin yanayin MT ne kawai kwayoyin halittar da ke ɗauke da sarcomere (Hoto na 4f) da kuma tashar ion (Hoto na Ƙarin 9) ke adanawa, suna kare su daga dannewa a ƙarƙashin yanayin Ctrl, TD, da MC. Waɗannan bayanai sun nuna cewa tare da haɗin motsa jiki na injiniya da na humoral (T3/Dex), rubutun zuciya na iya zama iri ɗaya da sabbin sassan zuciya bayan kwana 12 a cikin al'ada.
Waɗannan binciken da aka yi a rubuce-rubucen an tabbatar da su ta hanyar gaskiyar cewa ingancin tsarin ƙwayoyin zuciya a cikin sassan zuciya ya fi kyau a kiyaye shi a ƙarƙashin yanayin MT na tsawon kwanaki 12, kamar yadda aka nuna ta hanyar connexin 43 mara lahani kuma na gida (Hoto na 5a). Bugu da ƙari, fibrosis a cikin sassan zuciya a ƙarƙashin yanayin MT ya ragu sosai idan aka kwatanta da Ctrl kuma yayi kama da sassan zuciya sabo (Hoto na 5b). Waɗannan bayanai sun nuna cewa haɗin motsa jiki na injiniya da maganin T3/Dex yana kiyaye tsarin zuciya yadda ya kamata a sassan zuciya a cikin al'ada.
Hotunan immunofluorescence masu wakiltar troponin-T (kore), connexin 43 (ja), da DAPI (shuɗi) a cikin sabbin sassan zuciya da aka ware (D0) ko kuma an dasa su na tsawon kwanaki 12 a cikin dukkan yanayin yanayin aikin sassan zuciya guda huɗu (ma'aunin sikelin = 100 µm). Ƙididdige girman hankali na wucin gadi na tsarin ƙwayar zuciya (n = 7 (D0 da D12 Ctrl), 5 (D12 TD, D12 MC da D12 MT) yanka/rukuni daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ####p < 0.0001 idan aka kwatanta da D0 da *p < 0.05, ko ****p < 0.0001 idan aka kwatanta da D12 Ctrl). Ƙididdige girman hankali na wucin gadi na tsarin ƙwayar zuciya (n = 7 (D0 da D12 Ctrl), 5 (D12 TD, D12 MC da D12 MT) yanka/rukuni daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; #### p < 0.0001 idan aka kwatanta da D0 da *p < 0.05, ko ****p < 0.0001 idan aka kwatanta da D12 Ctrl). Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 2), 1 (D) D12 MC и D12 MT) срезов/группу от разных свиней, проведен однофакторный тест ANOVA; ko ****p <0,0001 da сравнению с D12 Ctrl). Ƙididdige daidaiton tsarin kyallen zuciya ta amfani da basirar wucin gadi (n = 7 (D0 da D12 Ctrl), sassan/rukuni 5 (D12 TD, D12 MC da D12 MT) daga aladu daban-daban, an yi gwajin ANOVA ta hanya ɗaya; #### p < 0.0001 idan aka kwatanta da D0 da *p < 0.05 ko ****p < 0.0001 idan aka kwatanta da 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 世****D0 和*p <0.05与D12 Ctrl 相比）对 不同猪 的 心脏结构 完整性 （n = 7 （d0 和 d12 ctrl）智能量 化 进行 单向 单向 单向 测试 ； ######### p <0.0001 与D0 和*p < 0.05 相比1 Ctrl 相比）Ƙididdige ingancin tsarin kyallen zuciya ta amfani da basirar wucin gadi a cikin aladu daban-daban (n = 7 (D0 da D12 Ctrl), sassan/rukuni 5 (D12 TD, D12 MC da D12 MT) tare da gwajin ANOVA na hanya ɗaya;#### p <0,0001 pо сравнению с D0 и *p <0,05 ko ****p <0,0001 pо сравнению с D12 Ctrl). #### p < 0.0001 idan aka kwatanta da D0 da *p < 0.05 ko ****p < 0.0001 idan aka kwatanta da D12 Ctrl). b Hotunan da aka wakilta da kuma ƙididdigewa don yanka zuciya da aka yi wa fenti da tabon Masson's trichrome (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, da D12 MC), yanka/rukuni 9 (D12 MT) daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ####p < 0.0001 idan aka kwatanta da D0 da ***p < 0.001, ko ****p < 0.0001 idan aka kwatanta da D12 Ctrl). b Hotunan da aka wakilta da kuma ƙididdigewa don yanka zuciya da aka yi wa fenti da tabon Masson's trichrome (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, da D12 MC), yanka/rukuni 9 (D12 MT) daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ####p < 0.0001 idan aka kwatanta da D0 da ***p < 0.001, ko ****p < 0.0001 idan aka kwatanta da D12 Ctrl). b Репрезентативные изображения и количественная линейка = 500 мкм) (n = 10 (D0, D12 Ctrl, D12 TD da D12 MC), 9 (D12 MT) ANOVA ; Ctrl D12). b Hotunan da aka wakilta da kuma auna sassan zuciya da aka yi wa fenti da tabon Masson's trichrome (ma'aunin sikelin = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD da D12 MC), sassan/rukuni 9 (D12 MT) daga aladu daban-daban, an yi su ta hanya ɗaya ta ANOVA; ####p < 0.0001 vs. D0 da ***p < 0.001 ko ****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）（n = 10！ 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 <10 .相比, *** p < 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） 1 ctrl d12 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/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 Репрезентативные изображения и количественная 500 мкм) (n = 10 (D0, D12 Ctrl, D12 TD da D12 MC), 9 (D12 MT) срезов от разных свиней / группы, один-# способ ANOVA <0; сравнению с D0, ***p <0,001 ko ****p <0,0001 ko сравнению с D12 Ctrl). b Hotunan da aka wakilta da kuma ƙididdige sassan zuciya da aka yi wa fenti da ma'aunin Masson's trichrome (ma'aunin sikelin = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD da D12 MC), sassan 9 (D12 MT) daga aladu/rukuni daban-daban, hanyar ANOVA ɗaya; ####p < 0.0001 idan aka kwatanta da D0, ***p < 0.001 ko ****p < 0.0001 idan aka kwatanta da D12 Ctrl).Sandunan kuskure suna wakiltar matsakaicin ± karkacewar da aka saba.
A ƙarshe, an tantance ikon CTCM na kwaikwayon hawan jini na zuciya ta hanyar ƙara yawan miƙewar kyallen zuciya. A cikin CTCM, matsin lamba na ɗakin iska mafi girma ya karu daga 80 mmHg zuwa 80 mmHg. Art. (miƙewa na al'ada) har zuwa 140 mmHg Art. (Hoto na 6a). Wannan ya yi daidai da ƙaruwar miƙewa da kashi 32% (Hoto na 6b), wanda a baya aka nuna a matsayin miƙewar kashi da ya dace da sassan zuciya don cimma tsawon sarcomere mai kama da wanda aka gani a cikin hawan jini. Miƙewa da saurin kyallen zuciya yayin matsewa da shakatawa sun kasance iri ɗaya a cikin kwanaki shida na al'ada (Hoto na 6c). An sanya yanayin kyallen zuciya daga MT a cikin miƙewa na al'ada (MT (Normal)) ko yanayin miƙewa (MT (OS)) na tsawon kwanaki shida. Bayan kwanaki huɗu a cikin al'ada, alamar cutar hypertrophic NT-ProBNP ta ƙaru sosai a cikin matsakaici a ƙarƙashin yanayin MT (OS) idan aka kwatanta da yanayin MT (na al'ada) (Hoto na 7a). Bugu da ƙari, bayan kwana shida na haɓaka, girman ƙwayar halitta a cikin MT (OS) (Hoto na 7b) ya ƙaru sosai idan aka kwatanta da sassan zuciya ta MT (na al'ada). Bugu da ƙari, an ƙara yawan canjin ƙwayoyin halitta na NFATC4 a cikin kyallen da suka miƙe (Hoto na 7c). Waɗannan sakamakon suna nuna ci gaba da ci gaba na sake fasalin cututtukan fata bayan hauhawar jini kuma suna goyon bayan ra'ayin cewa ana iya amfani da na'urar CTCM a matsayin dandamali don nazarin siginar hauhawar jini ta zuciya da ke haifar da shimfiɗawa.
Alamun matsin lamba na ɗakin iska, matsin lamba na ɗakin ruwa, da ma'aunin motsin nama sun tabbatar da cewa matsin lamba na ɗakin yana canza matsin lamba na ɗakin ruwa, yana haifar da motsi mai dacewa na yanki na nama. b Kashi mai wakiltar miƙewa da lanƙwasa na miƙewa na sassan nama da aka shimfiɗa (orange) da waɗanda aka shimfiɗa (shuɗi). c Jadawalin sanda yana nuna lokacin zagayowar (n = yanka 19 a kowace ƙungiya, daga aladu daban-daban), lokacin matsewa (n = yanka 18-19 a kowace ƙungiya, daga aladu daban-daban), lokacin shakatawa (n = yanka 19 a kowace ƙungiya, daga aladu daban-daban) ), girman motsin nama (n = yanka 14/ƙungiya, daga aladu daban-daban), saurin systolic mafi girma (n = yanka 14/ƙungiya, daga aladu daban-daban) da ƙimar shakatawa mafi girma (n = 14 (D0), sassan/ƙungiyoyi 15 (D6)) daga aladu daban-daban), gwajin t-test na ɗalibi mai wutsiya biyu bai nuna wani bambanci mai mahimmanci a cikin kowane siga ba, yana nuna cewa waɗannan sigogi sun kasance daidai a cikin kwanaki 6 na al'ada tare da ƙarfin lantarki mai yawa. Sandunan kuskure suna wakiltar matsakaicin ± karkacewar daidaito.
A tantance girman jadawali na yawan NT-ProBNP a cikin kafofin watsa labarai na al'ada daga yanka na zuciya da aka noma a ƙarƙashin yanayin MT na al'ada (Na al'ada) ko yanayin shimfiɗawa (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, da D4 MTOS) yanka/rukuni daga aladu daban-daban, Ana yin ANOVA ta hanyoyi biyu; **p < 0.01 idan aka kwatanta da shimfiɗa na al'ada). A tantance girman jadawali na yawan NT-ProBNP a cikin kafofin watsa labarai na al'ada daga yanka na zuciya da aka noma a ƙarƙashin yanayin MT na al'ada (Na al'ada) ko yanayin shimfiɗawa (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, da D4 MTOS) yanka/rukuni daga aladu daban-daban, Ana yin ANOVA ta hanyoyi biyu; **p < 0.01 idan aka kwatanta da shimfiɗa na al'ada).Histogram na ƙididdiga na yawan NT-ProBNP a cikin matsakaicin al'ada daga yanka na zuciya da aka noma a ƙarƙashin yanayin shimfiɗa MT na al'ada (al'ada) ko overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, da D4).MTOS) yanka /rukuni daga aladu daban-daban, ana yin nazarin bambance-bambancen abubuwa biyu;**p <0,01 pо сравнению с нормальным растяжением). **p < 0.01 idan aka kwatanta da mikewa na yau da kullun). 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 (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；**与正常拉伸相比，p <0.01）。 Ƙididdigar ƙididdiga na NT-ProBNP a cikin yankan zuciya da aka al'ada a ƙarƙashin MT al'ada stretch (Norm) ko overstretch (OS) yanayi (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm和D4 MTOS) daga daban-daban猪的切片/组，可以双向方方发发动;histogram Kimanta yawan NT-ProBNP a cikin yanka na zuciya da aka noma a ƙarƙashin yanayin shimfiɗa MT na al'ada (al'ada) ko kuma shimfiɗawa (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) da D4 MTOS) yanka/rukuni daga aladu daban-daban, nazarin bambance-bambancen hanyoyi biyu;**p <0,01 pо сравнению с нормальным растяжением). **p < 0.01 idan aka kwatanta da mikewa na yau da kullun). b Hotunan da aka wakilta don yanka zuciya da aka yi wa fenti da troponin-T da WGA (hagu) da kuma ƙididdige girman tantanin halitta (dama) (n = 330 (D6 MTOS), ƙwayoyin 369 (D6 MTNorm)/rukuni daga yanka 10 daban-daban daga aladu daban-daban, an yi gwajin t-dalibi mai wutsiya biyu; ****p < 0.0001 idan aka kwatanta da miƙewa na yau da kullun). b Hotunan da aka wakilta don yanka zuciya da aka yi wa fenti da troponin-T da WGA (hagu) da kuma ƙididdige girman tantanin halitta (dama) (n = 330 (D6 MTOS), ƙwayoyin 369 (D6 MTNorm)/rukuni daga yanka 10 daban-daban daga aladu daban-daban, an yi gwajin t-ɗalibi mai wutsiya biyu; ****p < 0.0001 idan aka kwatanta da miƙewa na yau da kullun). b Репрезентативные изображения срезов сердца клеток (справа) (n = 330 (D6 MTOS), 369 (D6 MTNorm) t-критерий Стьюдента; ****p <0,0001 по сравнению с нормальным растяжением). b An yi wa hotunan sassan zuciya fenti da troponin-T da AZP (hagu) da kuma ƙididdige girman tantanin halitta (dama) (n = 330 (D6 MTOS), ƙwayoyin halitta 369 (D6 MTNorm)/rukuni daga sassa 10 daban-daban daga aladu daban-daban, gwajin t-test na ɗalibi mai wutsiya biyu; ****p < 0.0001 idan aka kwatanta da nau'in da aka saba). 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3） 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 Hotunan da aka nuna na yanka zuciya da aka yi wa fenti da calcarein-T da WGA (hagu) da girman tantanin halitta (dama) (n = 330 (D6 MTOS), 369 daga yanka 10 daban-daban (D6 MTNorm)) Gwajin ƙwayoyin halitta/gwajin miƙewa na al'ada； idan aka kwatanta da miƙewa na al'ada；****p < 0.0001). b Репрезентативные изображения срезов сердца (sправа) (n = 330 (D6 MTOS), 369 (D6 MTNorm) Стьюдента; ****p <0,0001 по сравнению с нормальным растяжением). b Hotunan da aka nuna na sassan zuciya da aka yi wa fenti da troponin-T da AZP (hagu) da kuma ƙididdige girman tantanin halitta (dama) (n = 330 (D6 MTOS), 369 (D6 MTNorm) daga sassa 10 daban-daban daga aladu daban-daban) Kwayoyin halitta/rukuni, ma'aunin wutsiya biyu na ɗalibi t; ****p < 0.0001 idan aka kwatanta da nau'in da ya dace). c Hotunan da aka wakilta don rana ta 0 da rana ta 6 Yanka zuciya ta MTOS da aka yiwa lakabi da rigakafi don troponin-T da NFATC4 da kuma auna canjin NFATC4 zuwa tsakiya na CMs (n = 4 (D0), yanka 3 (D6 MTOS)/rukuni daga aladu daban-daban, An yi gwajin t-dalibi mai wutsiya biyu; *p < 0.05). c Hotunan da aka wakilta don rana ta 0 da rana ta 6 Yanka zuciya ta MTOS da aka yiwa lakabi da rigakafi don troponin-T da NFATC4 da kuma auna canjin NFATC4 zuwa tsakiya na CMs (n = 4 (D0), yanka 3 (D6 MTOS)/rukuni daga aladu daban-daban, An yi gwajin t-dalibi mai wutsiya biyu; *p < 0.05). c Репрезентативные изображения для срезов сердца 0 и 6 дней MTOS оценка транслокации NFATC4 в ядра кавернозных клеток (n = 4 (D0), 3 (D6 MTOS) двусторонний t-критерий Стьюдента; c Hotunan da aka wakilta don sassan zuciya a kwanaki 0 ​​da 6 na MTOS, waɗanda aka yiwa lakabi da rigakafi don troponin-T da NFATC4, da kuma ƙididdige canjin NFATC4 a cikin ƙwayar sel na rami (n = 4 (D0), yanka 3 (D6 MTOS)/rukuni daga aladu daban-daban) an yi gwajin t-test na ɗalibi mai wutsiya biyu; *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)进行双尾学生t 检验；*p <0.05)). Hotunan wakilci na calcanin-T da NFATC4 immunolabeling 第0天和第6天MTOS yankan zuciya, da NFATC4 daga NFATC4 daban-daban 易位至CM cell nucleus的 quntity时间双尾学生et 电影；*p <0.05). c Репрезентативные изображения срезов сердца MTOS на 0 и 6 день для оценка транслокации NFATC4 в ядра CM от разныh свиней (n = 4 (D0), 3 (D6 MTOS) 0,05). c Hotunan da aka wakilta na yanka zuciyar MTOS a rana ta 0 da 6 don labeling na troponin-T da NFATC4 da kuma auna canjin NFATC4 a cikin tsakiya na CM daga aladu daban-daban (n = 4 (D0), yanka 3 (D6 MTOS)/rukuni, ƙa'idodin ɗalibi masu wutsiya biyu; *p < 0.05).Sandunan kuskure suna wakiltar matsakaicin ± karkacewar da aka saba.
Binciken zuciya da jijiyoyin jini na fassara yana buƙatar samfuran ƙwayoyin halitta waɗanda ke sake haifar da yanayin zuciya daidai. A cikin wannan binciken, an ƙirƙiri na'urar CTCM kuma an siffanta ta wanda zai iya motsa sassan zuciya masu siriri sosai. Tsarin CTCM ya haɗa da motsa jiki na lantarki mai daidaitawa da kuma wadatar da ruwa na T3 da Dex. Lokacin da sassan zuciya na alade suka fuskanci waɗannan abubuwan, yuwuwarsu, amincin tsarinsu, aikin metabolism, da bayyanar rubutun sun kasance iri ɗaya kamar yadda yake a cikin sabbin kyallen zuciya bayan kwana 12 na al'ada. Bugu da ƙari, shimfiɗa kyallen zuciya da yawa na iya haifar da hauhawar zuciya sakamakon ƙaruwar zuciya. Gabaɗaya, waɗannan sakamakon suna tallafawa muhimmiyar rawar da yanayin al'adun jiki ke takawa wajen kiyaye yanayin zuciya na yau da kullun kuma suna samar da dandamali don tantance magunguna.
Abubuwa da yawa suna taimakawa wajen ƙirƙirar yanayi mafi kyau don aiki da rayuwar ƙwayoyin zuciya. Mafi bayyananne daga cikin waɗannan abubuwan suna da alaƙa da (1) hulɗar tsakanin ƙwayoyin halitta, (2) motsa jiki na lantarki, (3) abubuwan humoral, da (4) abubuwan da ke cikin metabolism. Hulɗar ƙwayoyin halitta zuwa ƙwayoyin halitta tana buƙatar hanyoyin sadarwa masu girma uku masu rikitarwa na nau'ikan ƙwayoyin halitta da yawa waɗanda ke da goyon bayan matrix na extracellular. Irin waɗannan hulɗar ƙwayoyin halitta masu rikitarwa suna da wahalar sake ginawa a cikin vitro ta hanyar haɗin gwiwa na nau'ikan ƙwayoyin halitta, amma ana iya cimma su cikin sauƙi ta amfani da yanayin organotypic na sassan zuciya.
Miƙewa ta injina da kuma motsa jiki ta lantarki na ƙwayoyin zuciya suna da matuƙar muhimmanci don kiyaye yanayin zuciya33,34,35. Duk da cewa an yi amfani da motsa jiki ta injina sosai don daidaita yanayin hipSC-CM da balaga, wasu bincike masu kyau sun yi ƙoƙarin motsa jiki ta injina na yanka zuciya a cikin al'ada ta amfani da nauyin uniaxial. Waɗannan nazarin sun nuna cewa ɗaukar nauyin injiniya na 2D uniaxial yana da tasiri mai kyau akan yanayin zuciya yayin al'ada. A cikin waɗannan nazarin, an ɗora sassan zuciya da ƙarfin tensile na isometric17, ɗaukar nauyin auxotonic na layi18, ko kuma an sake ƙirƙirar zagayowar zuciya ta amfani da martanin mai canza ƙarfi da kuma ƙarfin tashin hankali. Duk da haka, waɗannan hanyoyin suna amfani da shimfiɗar nama ta uniaxial ba tare da inganta muhalli ba, wanda ke haifar da danne ƙwayoyin zuciya da yawa ko kuma yawan bayyanar kwayoyin halitta da ke da alaƙa da martanin shimfiɗa marasa kyau. CTCM da aka bayyana a nan yana ba da motsin lantarki na 3D wanda ke kwaikwayon zagayowar zuciya ta halitta dangane da lokacin zagayowar da shimfiɗar jiki (25% shimfiɗa, 40% systole, 60% diastole, da bugun 72 a minti ɗaya). Ko da yake wannan motsi na inji mai girma uku kawai bai isa ba don kiyaye amincin nama, haɗin motsa jiki na humoral da na inji yana buƙatar amfani da T3/Dex don kiyaye dorewar nama, aiki, da mutunci yadda ya kamata.
Abubuwan ban dariya suna taka muhimmiyar rawa wajen daidaita yanayin zuciyar manya. An nuna wannan a cikin nazarin HiPS-CM inda aka ƙara T3 da Dex a cikin hanyoyin al'adu don hanzarta balaga tantanin halitta. T3 na iya yin tasiri ga jigilar amino acid, sukari da calcium a cikin membranes na tantanin halitta36. Bugu da ƙari, T3 yana haɓaka bayyanar MHC-α da raguwar MHC-β, yana haɓaka samuwar myofibrils masu sauri a cikin manyan ƙwayoyin zuciya idan aka kwatanta da myofibrils masu jinkirin juyawa a cikin tayin CM. Rashin T3 a cikin marasa lafiya da ke da hypothyroid yana haifar da asarar madaurin myofibrillar da raguwar saurin haɓakar sautin37. Dex yana aiki akan masu karɓar glucocorticoid kuma an nuna yana ƙara ƙanƙantar zuciya a cikin zukata masu perfused; 38 ana tsammanin wannan ci gaban yana da alaƙa da tasirin shigarwar calcium-driven deposit-driven (SOCE) 39,40. Bugu da ƙari, Dex yana ɗaure ga masu karɓar sa, yana haifar da martani mai faɗi a cikin ƙwayoyin halitta wanda ke hana aikin garkuwar jiki da kumburi30.
Sakamakonmu ya nuna cewa motsa jiki na injiniyanci (MS) ya inganta aikin al'ada gabaɗaya idan aka kwatanta da Ctrl, amma ya kasa kiyaye rayuwa, daidaiton tsari, da bayyanar zuciya tsawon kwanaki 12 a cikin al'ada. Idan aka kwatanta da Ctrl, ƙara T3 da Dex zuwa al'adun CTCM (MT) sun inganta rayuwa kuma sun ci gaba da riƙe irin waɗannan bayanan rubutu, daidaiton tsari, da aikin metabolism tare da sabbin kyallen zuciya na tsawon kwanaki 12. Bugu da ƙari, ta hanyar sarrafa matakin shimfiɗa nama, an ƙirƙiri samfurin hypertrophy na zuciya wanda hyperextension ya haifar ta amfani da STCM, yana nuna sauƙin tsarin STCM. Ya kamata a lura cewa kodayake gyaran zuciya da fibrosis yawanci suna shafar gabobin da ke kewaye waɗanda ƙwayoyin da ke zagayawa za su iya samar da cytokines masu dacewa da kuma phagocytosis da sauran abubuwan gyara, sassan zuciya har yanzu suna iya kwaikwayon tsarin fibrotic don mayar da martani ga damuwa da rauni. zuwa cikin myofibroblasts. An riga an kimanta wannan a cikin wannan samfurin yanke zuciya. Ya kamata a lura cewa ana iya daidaita sigogin CTCM ta hanyar canza matsin lamba/girma da mita don kwaikwayon yanayi da yawa kamar tachycardia, bradycardia, da tallafin zagayawa na inji (zuciya mara saukarwa ta inji). Wannan ya sa tsarin ya zama matsakaiciyar hanyar gwajin magunguna. Ikon CTCM na yin kwaikwayon hauhawar zuciya da ke haifar da yawan motsa jiki yana buɗe hanya don gwada wannan tsarin don maganin da aka keɓance. A ƙarshe, wannan binciken ya nuna cewa miƙewa ta injina da motsa jiki na humoral suna da mahimmanci don kiyaye al'adun sassan ƙwayoyin zuciya.
Duk da cewa bayanan da aka gabatar a nan sun nuna cewa CTCM dandamali ne mai matuƙar kyau don yin kwaikwayon myocardium, wannan hanyar al'ada tana da wasu iyakoki. Babban iyakancewar al'adar CTCM shine yana sanya damuwa ta injiniya mai ci gaba a kan yanka, wanda ke hana ikon sa ido kan matsewar yanka na zuciya a kowane zagaye. Bugu da ƙari, saboda ƙaramin girman sassan zuciya (7 mm), ikon kimanta aikin systolic a wajen tsarin al'ada ta amfani da na'urori masu auna ƙarfi na gargajiya yana da iyaka. A cikin rubutun da ake amfani da shi a yanzu, mun shawo kan wannan iyakancewa ta wani ɓangare ta hanyar kimanta ƙarfin lantarki na gani a matsayin alamar aikin matsewa. Duk da haka, wannan iyakancewa zai buƙaci ƙarin aiki kuma ana iya magance shi a nan gaba ta hanyar gabatar da hanyoyin sa ido kan aikin yanka na zuciya a cikin al'ada, kamar taswirar gani ta amfani da launuka masu ɗauke da sinadarin calcium da ƙarfin lantarki. Wani iyakancewar CTCM shine cewa samfurin aiki baya sarrafa damuwa ta jiki (kayan aiki kafin kaya da bayan kaya). A cikin CTCM, an haifar da matsin lamba a akasin haka don haifar da matsewar jiki na 25% a cikin diastole (cikakken miƙewa) da systole (tsawon matsewa yayin motsawar lantarki) a cikin manyan kyallen takarda. Ya kamata a cire wannan iyakancewa a cikin zane-zanen CTCM na gaba ta hanyar isasshen matsin lamba akan kyallen zuciya daga ɓangarorin biyu da kuma ta hanyar amfani da ainihin alaƙar matsin lamba da ke faruwa a cikin ɗakunan zuciya.
Gyaran da aka yi ta hanyar tsawaitawa da aka ruwaito a cikin wannan rubutun ya takaita ne kawai ga kwaikwayon siginar tsawaitawa ta hypertrophic. Don haka, wannan samfurin zai iya taimakawa wajen nazarin siginar tsawaitawa da ke haifar da tsawaitawa ba tare da buƙatar abubuwan humoral ko jijiyoyi ba (waɗanda ba sa wanzuwa a cikin wannan tsarin). Ana buƙatar ƙarin bincike don ƙara yawan CTCM, misali, haɓaka tare da ƙwayoyin rigakafi, abubuwan humoral da ke yawo a cikin plasma, da kuma shiga cikin jiki lokacin da ake haɓaka tare da ƙwayoyin jijiyoyi zai inganta yuwuwar yin samfurin cuta tare da CTCM.
An yi amfani da aladu goma sha uku a cikin wannan binciken. An yi dukkan hanyoyin da aka bi na dabbobi bisa ga ka'idojin hukuma kuma Kwamitin Kula da Dabbobi na Jami'ar Louisville ya amince da su. An daure baka na aortic kuma an zuba wa zuciya lita 1 na zuciya mai tsafta (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U/mL heparin, pH har zuwa 7.4); An adana zukata a cikin maganin zuciya da sanyi har sai an kai su dakin gwaje-gwaje a kan kankara wanda yawanci bai wuce minti 10 ba. An adana zukata a cikin maganin zuciya da sanyi har sai an kai su dakin gwaje-gwaje a kan kankara wanda yawanci bai wuce minti 10 ba. сердца хранили в ледяном кардиоплегическом растворе до транспортиртировки в лабораторию на льду, что обымы. An adana zukata a cikin maganin zuciya da sanyi har sai an kai su dakin gwaje-gwaje a kan kankara, wanda yawanci yakan ɗauki ƙasa da minti 10.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 мин. A ajiye zukata a kan kankara a kan zuciya har sai an kai su dakin gwaje-gwaje a kan kankara, yawanci ƙasa da minti 10.
An ƙirƙiro na'urar CTCM ta hanyar amfani da manhajar SolidWorks mai taimakon kwamfuta (CAD). An yi ɗakunan al'adu, masu rabawa da ɗakunan iska da filastik mai tsabta na CNC. Zoben baya mai diamita 7mm an yi shi ne da polyethylene mai yawa (HDPE) a tsakiya kuma yana da ramin zobe don ɗaukar zoben silicone da ake amfani da shi don rufe kafofin da ke ƙasa. Wani siririn membrane na silica yana raba ɗakin al'adu da farantin rabuwa. An yanke membrane na silicone daga laser daga takardar silicone mai kauri 0.02" kuma yana da tauri na 35A. Gasket ɗin silicone na ƙasa da na sama an yanke su ne da laser daga takardar silicone mai kauri 1/16" kuma suna da tauri na 50A. Ana amfani da sukurori na bakin ƙarfe 316L da goro na fikafikai don ɗaure toshe da ƙirƙirar hatimin da ba ya shiga iska.
An tsara wani allon da'ira na musamman (PCB) don a haɗa shi da tsarin C-PACE-EM. An haɗa soket ɗin haɗin injin swiss akan PCB zuwa electrodes na graphite ta hanyar wayoyi masu launin azurfa da sukurori na tagulla 0-60 da aka sumu a cikin electrodes. An sanya allon da'ira na bugawa a cikin murfin firintar 3D.
Ana sarrafa na'urar CTCM ta hanyar na'urar kunna iska mai iya aiki da iska (PPD) wanda ke haifar da matsin lamba na zagayawa wanda aka sarrafa kamar zagayowar zuciya. Yayin da matsin lamba a cikin ɗakin iska ke ƙaruwa, membrane mai laushi na silicone yana faɗaɗa sama, yana tilasta matsakaiciyar ƙarƙashin wurin nama. Za a shimfiɗa yankin nama ta hanyar wannan fitar da ruwa, yana kwaikwayon faɗaɗawar jiki na zuciya yayin diastole. A lokacin kololuwar annashuwa, an yi amfani da ƙarfin lantarki ta hanyar lantarki na lantarki ta hanyar lantarki na graphite, wanda ya rage matsin lamba a cikin ɗakin iska kuma ya haifar da matsewar sassan nama. A cikin bututun akwai bawul ɗin hemostatic tare da firikwensin matsa lamba don gano matsin lamba a cikin tsarin iska. Ana amfani da matsin lambar da firikwensin matsin lamba ke ji akan mai tattara bayanai da aka haɗa da kwamfutar tafi-da-gidanka. Wannan yana ba da damar ci gaba da sa ido kan matsin lamba a cikin ɗakin iskar gas. Lokacin da aka kai matsakaicin matsin lamba na ɗakin (daidaitaccen 80 mmHg, 140 mmHg OS), an umarci na'urar tattara bayanai ta aika sigina zuwa tsarin C-PACE-EM don samar da siginar ƙarfin lantarki na biphasic na 2 ms, wanda aka saita zuwa 4 V.
An samo sassan zuciya kuma an yi yanayin da aka yi amfani da shi a cikin rijiyoyi 6 kamar haka: A mayar da zukatan da aka girbe daga cikin jirgin da aka canja zuwa tire mai dauke da sanyi (4° C.) cardioplegia. An ware ventricle na hagu da ruwan wuka mai tsafta sannan a yanka su guda 1-2 cm3. An haɗa waɗannan tubalan nama a kan tallafin nama tare da manne nama sannan a sanya su a cikin wani baho mai girgiza wanda ke ɗauke da maganin Tyrode kuma ana ci gaba da sanya iskar oxygen (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 bayani), 1.8 mM CaCl2 (1.8 ml 1 M bayani), har zuwa 1 L ddH2O). An saita microtome mai girgiza don yanke yanka mai kauri 300 µm a mita 80 Hz, girman girgiza a kwance na 2 mm, da kuma saurin ci gaba na 0.03 mm/s. An kewaye wankan nama da kankara don kiyaye maganin ya yi sanyi kuma an kiyaye zafin jiki a 4°C. Canja sassan nama daga baho na microtome zuwa baho mai ɗauke da maganin Tyrode mai iskar oxygen a kan kankara har sai an sami isassun sassan don faranti ɗaya na al'ada. Don al'adun transwell, an haɗa sassan nama zuwa tallafin polyurethane mai faɗi 6 mm kuma an sanya su a cikin 6 ml na matsakaici mai kyau (matsakaici 199, ƙarin ITS 1x, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-alkaline da 2X antibiotic-antifungal). An yi amfani da ƙarfin lantarki (10 V, mita 1.2 Hz) a sassan nama ta hanyar C-Pace. Ga yanayin TD, an ƙara sabo T3 da Dex a 100 nM da 1 μM a kowane canjin matsakaici. Ana cika maganin da iskar oxygen kafin a maye gurbinsa sau 3 a rana. An haƙa sassan nama a cikin incubator a 37°C da 5% CO2.
Ga al'adun CTCM, an sanya sassan nama a kan firintar 3D da aka yi musamman a cikin kwano na Petri wanda ke ɗauke da maganin Tyrode da aka gyara. An tsara na'urar don ƙara girman yanki na zuciya da kashi 25% na yankin zoben tallafi. Ana yin wannan ne don kada sassan zuciya su miƙe bayan an canja su daga maganin Tyrode zuwa matsakaici da kuma lokacin diastole. Ta amfani da manne na histoacrylic, an sanya sassan 300 µm kauri akan zoben tallafi mai diamita 7 mm. Bayan haɗa sassan nama zuwa zoben tallafi, yanke sassan nama da suka wuce gona da iri kuma a mayar da sassan nama da aka haɗa cikin wanka na maganin Tyrode akan kankara (4°C) har sai an shirya isassun sassa don na'ura ɗaya. Jimlar lokacin sarrafawa ga duk na'urori bai kamata ya wuce awanni 2 ba. Bayan an haɗa sassan nama 6 zuwa zoben tallafi, an haɗa na'urar CTCM. An cika ɗakin al'adun CTCM da matsakaici mai iskar oxygen 21 ml. Canja sassan nama zuwa ɗakin al'adu kuma a hankali cire duk wani kumfa mai iska tare da pipette. Sashen nama ana shirya shi zuwa cikin ramin sannan a matse shi a hankali. A ƙarshe, sanya murfin lantarki a kan na'urar sannan a mayar da na'urar zuwa incubator. Sannan a haɗa CTCM zuwa bututun iska da tsarin C-PACE-EM. Mai kunna iska yana buɗewa kuma bawul ɗin iska yana buɗe CTCM. An tsara tsarin C-PACE-EM don isar da 4 V a 1.2 Hz a lokacin biphasic pacing na tsawon ms 2. An canza matsakaici sau biyu a rana kuma an canza electrodes sau ɗaya a rana don guje wa taruwar graphite akan electrodes. Idan ya cancanta, ana iya cire sassan nama daga rijiyoyin al'adunsu don fitar da duk wani kumfa na iska da ka iya faɗuwa a ƙarƙashinsu. Don yanayin maganin MT, an ƙara T3/Dex sabo tare da kowane canjin matsakaici tare da 100 nM T3 da 1 μM Dex. An haɓaka na'urorin CTCM a cikin incubator a 37°C da 5% CO2.
Domin samun hanyoyin da aka shimfiɗa na yanka zuciya, an ƙirƙiri tsarin kyamara na musamman. An yi amfani da kyamarar SLR (Canon Rebel T7i, Canon, Tokyo, Japan) tare da ruwan tabarau na Navitar Zoom 7000 18-108mm (Navitar, San Francisco, CA). An yi amfani da na'urar hangen nesa a zafin ɗaki bayan an maye gurbin matsakaiciyar da sabon matsakaici. An sanya kyamarar a kusurwar 51° kuma ana ɗaukar bidiyo a firam 30 a kowace daƙiƙa. Da farko, an yi amfani da software na tushen buɗewa (MUSCLEMOTION43) tare da Image-J don auna motsin yanka zuciya. An ƙirƙiri abin rufe fuska ta amfani da MATLAB (MathWorks, Natick, MA, Amurka) don ayyana yankunan da ake sha'awa don bugun yanka zuciya don guje wa hayaniya. Ana amfani da abin rufe fuska da hannu a kan duk hotuna a cikin jerin firam sannan a wuce zuwa ga MUSCLEMOTION plugin. Motsin Muscle yana amfani da matsakaicin ƙarfin pixels a cikin kowane firam don auna motsinsa dangane da firam ɗin tunani. An yi rikodin bayanai, an tace su kuma an yi amfani da su don auna lokacin zagayowar da kuma tantance miƙewar nama a lokacin zagayowar zuciya. An yi amfani da matattarar dijital ta sifili ta farko. Don ƙididdige miƙewar nama (kololuwa-zuwa-kololuwa), an yi nazarin kololuwa-zuwa-kololuwa don bambance tsakanin kololuwa da magudanar ruwa a cikin siginar da aka yi rikodin. Bugu da ƙari, ana yin detrending ta amfani da polynomial na tsari na 6 don kawar da karkatar sigina. An haɓaka lambar shirin a cikin MATLAB don tantance motsin nama na duniya, lokacin zagayowar, lokacin shakatawa, da lokacin matsewa (Lambar Shirin Ƙarin 44).
Don nazarin nau'in, ta amfani da bidiyon da aka ƙirƙira don tantance miƙewa na injiniya, da farko mun gano hotuna biyu da ke wakiltar kololuwar motsi (mafi girma (na sama) da na ƙasa (ƙasa) na motsi) bisa ga software na MUSCLEMOTION. Sannan muka raba yankunan nama muka yi amfani da wani nau'in algorithm na inuwa ga nama da aka raba (Hoto na Ƙari na 2a). Daga nan aka raba nama da aka raba zuwa ƙananan wurare goma, kuma an ƙididdige matsin lamba akan kowane saman ta amfani da lissafin da ke ƙasa: Matsi = (Sup-Sdown)/Sdown, inda Sup da Sdown sune nisan siffar daga inuwar sama da ƙasa na masana'anta, bi da bi (Hoto na Ƙari .2b).
An gyara sassan zuciya a cikin kashi 4% na paraformaldehyde na tsawon awanni 48. An cire kyallen da aka gyara a cikin kashi 10% da kashi 20% na sucrose na tsawon awa 1, sannan a cikin kashi 30% na sucrose na dare ɗaya. Sannan an saka sassan a cikin mafi kyawun yanayin zafi (OCT compound) sannan a hankali a daskare a cikin wanka mai isopentane/busasshen kankara. Ajiye tubalan da aka saka OCT a -80 °C har sai an raba su. An shirya zamewar a matsayin sassan da kauri na 8 μm.
Don cire OCT daga sassan zuciya, a zafafa zamiya a kan toshewar dumama a digiri 95 na Celsius na tsawon minti 5. A ƙara PBS 1 ml a kowace zamiya sannan a saka a cikin zamiya na tsawon minti 30 a zafin ɗaki, sannan a shiga sassan ta hanyar sanya 0.1% Triton-X a cikin PBS na tsawon minti 15 a zafin ɗaki. Don hana ƙwayoyin rigakafi marasa takamaiman ɗaurewa ga samfurin, a ƙara 1 ml na maganin BSA 3% a cikin zamiya sannan a saka a cikin zamiya na tsawon awa 1 a zafin ɗaki. Sannan an cire BSA kuma an wanke zamiya da PBS. A yiwa kowanne samfurin alama da fensir. An ƙara manyan ƙwayoyin rigakafi (an narkar da su 1:200 a cikin 1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) da troponin-T (Thermo Scientific; #MA5-12960) a cikin mintuna 90, sannan aka ƙara ƙwayoyin rigakafi na biyu (an narkar da su 1:200 a cikin 1% BSA) a kan linzamin kwamfuta Alexa Fluor 488 (Thermo Scientific; #A16079), a kan zomo Alexa Fluor 594 (Thermo Scientific; #T6391) na ƙarin mintuna 90. An wanke sau 3 da PBS. Don bambance tabo mai manufa daga bango, mun yi amfani da ƙwayoyin rigakafi na biyu kawai a matsayin iko. A ƙarshe, an ƙara tabon nukiliya na DAPI kuma an sanya zamewar a cikin vectasheld (Vector Laboratories) kuma an rufe shi da goge ƙusa. -x girma) da kuma na'urar microscope ta Keyence tare da girman 40x.
An yi amfani da WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) a 5 μg/ml a cikin PBS don yin fenti na WGA kuma an shafa a kan sassan da aka gyara na tsawon minti 30 a zafin ɗaki. Sannan an wanke zane-zanen da PBS sannan aka ƙara Sudan black a kowane zane kuma aka saka shi a cikin injin na tsawon minti 30. Sannan an wanke zane-zanen da PBS kuma an ƙara kayan haɗin vectasheld. An nuna zane-zanen a kan na'urar hangen nesa ta Keyence a girman 40x.
An cire OCT daga samfuran kamar yadda aka bayyana a sama. Bayan cire OCT, a nutsar da zamiya a cikin ruwan Bouin cikin dare ɗaya. Sannan an wanke zamiya a cikin ruwan da aka tace na tsawon awa 1 sannan a sanya a cikin ruwan Bibrich aloe acid fuchsin na minti 10. Sannan an wanke zamiya a cikin ruwan da aka tace sannan a sanya a cikin ruwan phosphomolybdenum/5% phosphotungstic acid na minti 10. Ba tare da kurkura ba, a canza zamiya kai tsaye zuwa ruwan aniline blue na minti 15. Sannan an wanke zamiya a cikin ruwan da aka tace sannan a sanya a cikin ruwan acetic acid na 1% na minti 2. An busar da zamiya a cikin 200 N ethanol sannan a mayar da shi zuwa xylene. An yi amfani da na'urar hangen nesa ta Keyence microscope mai girman 10x. An auna kashi na yankin Fibrosis ta amfani da manhajar Keyence Analyzer.
Gwajin Rayuwar Kwayar CyQUANT™ MTT (Invitrogen, Carlsbad, CA), lambar kasida V13154, bisa ga ka'idar masana'anta tare da wasu gyare-gyare. Musamman ma, an yi amfani da bugun tiyata mai diamita na 6 mm don tabbatar da daidaiton girman kyallen yayin nazarin MTT. An saka kyallen a cikin ramukan farantin rijiyoyi 12 da ke ɗauke da sinadarin MTT bisa ga ka'idar masana'anta. An saka sassan a zafin jiki na 37°C na tsawon awanni 3 kuma kyallen mai rai yana haɓaka sinadarin MTT don samar da mahaɗin formazan mai launin shuɗi. Sauya maganin MTT da DMSO 1 ml kuma a saka a zafin jiki na 37°C na mintuna 15 don cire formazan mai launin shuɗi daga sassan zuciya. An narkar da samfuran 1:10 a cikin DMSO a cikin faranti masu haske 96 da ƙarfin launin shunayya wanda aka auna a 570 nm ta amfani da mai karanta farantin Cytation (BioTek). An daidaita karatun zuwa nauyin kowane yanki na zuciya.
An maye gurbin kafofin yanke zuciya da kafofin watsa labarai da ke ɗauke da 1 μCi/ml [5-3H]-glucose (Moravek Biochemicals, Brea, CA, Amurka) don gwajin amfani da glucose kamar yadda aka bayyana a baya. Bayan awanni 4 na haɗuwa, ƙara 100 µl na matsakaici zuwa bututun microcentrifuge mai buɗewa wanda ke ɗauke da 100 µl na 0.2 N HCl. Sannan an sanya bututun a cikin bututun scintillation wanda ke ɗauke da 500 μl na dH2O don ƙafe [3H]2O na tsawon awanni 72 a zafin jiki na 37°C. Sannan a cire bututun microcentrifuge daga bututun scintillation sannan a ƙara 10 ml na ruwan scintillation. An yi ƙididdigar scintillation ta amfani da na'urar nazarin scintillation na ruwa ta Tri-Carb 2900TR (Packard Bioscience Company, Meriden, CT, Amurka). An ƙididdige amfani da glucose ta hanyar la'akari da takamaiman aikin glucose [5-3H], rashin daidaituwa da asalin aiki, raguwar [5-3H] zuwa glucose mara lakabi, da kuma ingancin scintillation. Bayanan an daidaita su zuwa nauyin sassan zuciya.
Bayan an haɗa ƙwayoyin halitta a cikin Trizol, an ware RNA daga sassan zuciya ta amfani da Qiagen miRNeasy Micro Kit #210874 bisa ga ka'idar masana'anta. An gudanar da shirye-shiryen ɗakin karatu na RNAsec, jerin abubuwa da nazarin bayanai kamar haka:
An yi amfani da 1 μg na RNA a kowace samfuri a matsayin kayan farawa don shirya ɗakin karatu na RNA. An samar da ɗakunan karatu na jerin abubuwa ta amfani da Kayan Shirye-shiryen Laburare na NEBNext UltraTM RNA don Illumina (NEB, Amurka) bisa ga shawarwarin masana'anta, kuma an ƙara lambobin fihirisa zuwa jerin sifofin kowane samfuri. A takaice, an tsarkake mRNA daga jimlar RNA ta amfani da beads na maganadisu da aka haɗa da poly-T oligonucleotides. Ana gudanar da rarrabuwa ta amfani da cations na divalent a babban zafin jiki a cikin NEBNext First Strand Synthesis Reaction Buffer (5X). An haɗa cDNA na farko ta amfani da firam ɗin hexamer bazuwar da M-MuLV reverse transcriptase (RNase H-). Sannan an haɗa cDNA na biyu ta amfani da DNA polymerase I da RNase H. Sauran overhangs ana canza su zuwa ƙarshen ƙura ta hanyar aikin exonuclease/polymerase. Bayan adenylation na ƙarshen 3′ na ɓangaren DNA, an haɗa Adaftar NEBNext tare da tsarin madauki na gashi don shirya shi don haɗakarwa. Don zaɓar guntun cDNA masu tsayin da aka fi so, an tsarkake guntun ɗakin karatu ta amfani da tsarin AMPure XP (Beckman Coulter, Beverly, Amurka). Sannan, an yi amfani da μl USER Enzyme (NEB, Amurka) tare da cDNA da aka zaɓa da girman da aka haɗa da adaftar na tsawon mintuna 15 a 37°C sannan na mintuna 5 a 95°C kafin PCR. Daga nan aka yi amfani da PCR ta amfani da Phusion High-Fidelity DNA polymerase, na'urorin PCR na duniya, da na'urorin Index (X). A ƙarshe, an tsarkake samfuran PCR (tsarin AMPure XP) kuma an tantance ingancin ɗakin karatu akan tsarin Agilent Bioanalyzer 2100. Daga nan aka tsara ɗakin karatu na cDNA ta amfani da na'urar Novaseq sequencer. An canza fayilolin hoto na asali daga Illumina zuwa na asali ta amfani da CASAVA Base Calling. Ana adana bayanai na asali a cikin fayilolin tsarin FASTQ(fq) waɗanda ke ɗauke da jerin karatu da halayen tushe masu dacewa. Zaɓi HISAT2 don daidaita karatun jerin da aka tace zuwa ga tsarin halittar Sscrofa11.1. Gabaɗaya, HISAT2 yana tallafawa kwayoyin halitta na kowane girma, gami da kwayoyin halitta waɗanda suka fi girma sama da tushen biliyan 4, kuma an saita ƙimar tsoho don yawancin sigogi. Ana iya daidaita karatun haɗin bayanai daga bayanan RNA Seq yadda ya kamata ta amfani da HISAT2, tsarin da ya fi sauri a halin yanzu, tare da daidaito iri ɗaya ko mafi kyau fiye da kowace hanya.
Yawan rubuce-rubucen kai tsaye yana nuna matakin bayyanar kwayar halitta. Ana tantance matakan bayyanar kwayar halitta ta hanyar yawan rubutun (ƙidayar jerin kwayoyin halitta) da ke da alaƙa da kwayar halitta ko exons. Adadin karantawa yana daidai da matakan bayyanar kwayar halitta, tsawon kwayar halitta, da zurfin jerin kwayoyin halitta. An ƙididdige FPKM (guda-guda a kowace nau'i-nau'i dubu na rubutun da aka jera a kowace nau'i-nau'i miliyan) kuma an ƙayyade ƙimar P na bambancin bayyanar ta amfani da fakitin DESeq2. Sannan muka ƙididdige ƙimar gano ƙarya (FDR) don kowane ƙimar P ta amfani da hanyar Benjamini-Hochberg9 bisa ga aikin R-da aka gina a ciki "p.adjust".
An canza RNA da aka ware daga sassan zuciya zuwa cDNA a yawan 200 ng/μl ta amfani da cakuda SuperScript IV Vilo Master daga Thermo (Thermo, cat. no. 11756050). An yi amfani da RT-PCR mai ƙima ta amfani da farantin amsawa mai haske na Applied Biosystems Endura Plate Microamp 384-well (Thermo, cat. no. 4483319) da manne mai gani na microamp (Thermo, cat. no. 4311971). Haɗin amsawar ya ƙunshi cakuda Taqman Fast Advanced Master 5 µl (Thermo, cat # 4444557), Taqman Primer 0.5 µl da 3.5 µl H2O da aka gauraya a kowace rijiya. An gudanar da zagayawa na qPCR na yau da kullun kuma an auna ƙimar CT ta amfani da kayan aikin PCR na Applied Biosystems Quantstudio 5 na ainihin lokaci (module na rijiyar 384; samfurin # A28135). An sayi firam ɗin Taqman daga 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) An daidaita ƙimar CT na duk samfuran zuwa ga kwayar halittar kula da gida GAPDH.
An tantance fitowar NT-ProBNP ta hanyar amfani da kayan aikin NT-ProBNP (alade) (Cat. No. MBS2086979, MyBioSource) bisa ga ka'idar masana'anta. A takaice, an ƙara 250 µl na kowane samfurin da ma'auni a cikin kwafi a kowace rijiya. Bayan ƙara samfurin, ƙara 50 µl na Assay Reagent A ga kowace rijiya. A hankali girgiza farantin sannan a rufe da sealant. Sannan an sanya allunan a zafin jiki na 37°C na tsawon awa 1. Sannan a zuba a cikin sealant sannan a wanke rijiyoyin sau 4 da 350 µl na 1x na maganin wanke-wanke, a saka a cikin sealant na minti 1-2 kowane lokaci. Sannan a ƙara 100 µl na Assay Reagent B a kowace rijiya sannan a rufe da sealant na faranti. An girgiza allunan a hankali sannan a saka a cikin sealant a zafin jiki na 37°C na minti 30. A zuba ruwan maganin a cikin ruwan sannan a wanke rijiyoyin sau 5 da ruwan wankewa sau 350 µl sau 1. A zuba ruwan maganin substrate sau 90 µl a kowace rijiya sannan a rufe farantin. A zuba farantin a zafin digiri 37 na Celsius na tsawon minti 10-20. A zuba ruwan maganin Stop Solution guda 50 µl a kowace rijiya. Nan da nan aka auna farantin ta amfani da na'urar karanta faranti ta Cytation (BioTek) da ke 450 nm.
An gudanar da nazarin wutar lantarki don zaɓar girman rukuni wanda zai samar da wutar lantarki sama da kashi 80% don gano canji na kashi 10% a cikin sigogi tare da ƙimar kuskuren Nau'in I na 5%. An yi nazarin wutar lantarki don zaɓar girman rukuni wanda zai samar da wutar lantarki sama da kashi 80% don gano canji na kashi 10% a cikin sigogi tare da ƙimar kuskuren Nau'in I na 5%. Анализ мощности был выполнен для выбора размеров групп, которые обеспечат > 80% изменения параметра с 5% частотой ошибок типа I. An gudanar da nazarin wutar lantarki don zaɓar girman rukuni wanda zai samar da wutar lantarki sama da kashi 80% don gano canjin sigogi 10% na cikakken daidaito tare da ƙimar kuskuren Nau'in I na 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变化和5％I变化和5％I型百给。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变化和5％I变化和5％I型百给。 Был проведен анализ мощности для выбора размера группы, который обеспечил бы > 80% мощности для обнару абсолютного изменения параметров и 5% частоты ошибок типа I. An gudanar da nazarin wutar lantarki don zaɓar girman rukuni wanda zai samar da wutar lantarki sama da kashi 80% don gano canjin sigogi 10% na cikakken tsari da kuma ƙimar kuskuren nau'in I na 5%.An zaɓi sassan nama bazuwar kafin gwajin. Duk nazarin an yi su ne ba tare da an gano yanayin su ba kuma an fassara samfuran ne kawai bayan an yi nazarin dukkan bayanai. An yi amfani da manhajar GraphPad Prism (San Diego, CA) don yin duk nazarin kididdiga. Ga dukkan ƙididdiga, an ɗauki ƙimar p a matsayin mai mahimmanci a ƙimar <0.05. Ga duk ƙididdiga, an ɗauki ƙimar p a matsayin mahimmanci a ƙimar <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Ga duk ƙididdiga, an ɗauki ƙimar p a matsayin mahimmanci a ƙimar <0.05.对于所有统计数据，p 值在值<0.05 时被认为是显着的。对于所有统计数据，p 值在值<0.05 时被认为是显着的。 Для всей статистики p-значения считались значимыми при значениях <0,05. Ga duk ƙididdiga, an ɗauki ƙimar p a matsayin mahimmanci a ƙimar <0.05.An yi gwajin t-test na ɗalibi mai wutsiya biyu akan bayanan tare da kwatancen guda 2 kawai. An yi amfani da ANOVA mai hanya ɗaya ko biyu don tantance mahimmanci tsakanin ƙungiyoyi da yawa. Lokacin yin gwaje-gwajen bayan hoc, an yi amfani da gyaran Tukey don la'akari da kwatancen da yawa. Bayanan RNAsec suna da la'akari na musamman na ƙididdiga lokacin ƙididdige FDR da p.adjust kamar yadda aka bayyana a cikin sashin Hanyoyi.
Don ƙarin bayani game da ƙirar nazari, duba taƙaitaccen Rahoton Bincike na Yanayi wanda aka haɗa da wannan labarin.