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Akwai buƙatar ingantaccen tsarin in vitro wanda zai iya haifar da daidaitaccen yanayin yanayin zuciya don gwajin ƙwayoyi.Iyakantaccen samuwa na tsarin al'adun nama na zuciyar ɗan adam ya haifar da rashin daidaiton fassarar tasirin magungunan zuciya.Anan, mun ƙirƙiri samfurin al'adun nama na zuciya (CTCM) wanda ke motsa jiki ta hanyar lantarki kuma yana jujjuya shimfidar jiki a lokacin systolic da diastolic matakan zagaye na zuciya.Bayan kwanaki 12 na al'ada, wannan hanyar ta ɗan inganta yanayin sassan zuciya, amma ba ta cika cikakkiyar amincin tsarin su ba.Saboda haka, bayan ƙananan ƙwayoyin ƙwayoyin cuta, mun gano cewa ƙarin 100 nM triiodothyronine (T3) da 1 μM dexamethasone (Dex) zuwa matsakaicinmu ya kiyaye ƙananan sassan sassan har tsawon kwanaki 12.A haɗe tare da jiyya na T3/Dex, tsarin CTCM ya kiyaye bayanan bayanan rubutu, yuwuwar, aiki na rayuwa, da daidaiton tsari a daidai matakin da sabbin nama na zuciya na kwanaki 12.Bugu da kari, wuce gona da iri na nama na zuciya a cikin al'ada yana haifar da siginar hypertrophic na zuciya, yana ba da shaida ga ikon CTCM don yin koyi da yanayin hypertrophic da ke haifar da bugun zuciya.A ƙarshe, CTCM na iya tsara tsarin ilimin halittar jiki da ilimin halittar jiki na zuciya a cikin al'ada na dogon lokaci, yana ba da damar ingantaccen gwajin magani.
Kafin binciken asibiti, ana buƙatar ingantaccen tsarin in vitro wanda zai iya haifar da daidaitaccen yanayin yanayin yanayin zuciyar ɗan adam.Irin waɗannan tsarin ya kamata su kwaikwayi sauye-sauyen miƙewa na inji, bugun zuciya, da kaddarorin electrophysiological.Ana amfani da nau'ikan dabbobi a matsayin dandamali na nunawa don ilimin lissafin zuciya tare da iyakancewar dogaro wajen nuna tasirin kwayoyi a cikin zuciyar ɗan adam1,2.A ƙarshe, mafi kyawun al'adun gargajiya na al'adun zuciya (CTCM) samfurin ne wanda yake da matukar damuwa da kuma hanyoyin magunguna, daidai sake fasalin kwakwalwa da cuta na zuciyar mutum 3.Rashin irin wannan tsarin yana iyakance gano sabbin hanyoyin magance cututtukan zuciya4,5 kuma ya haifar da cututtukan zuciya na ƙwayar cuta a matsayin babban dalilin fita kasuwa6.
A cikin shekaru goma da suka gabata, an cire magungunan takwas marasa na jijiyoyin jini daga amfani da asibiti saboda suna haifar da tsawaita tazara ta QT wanda ke haifar da arrhythmias na ventricular da mutuwa kwatsam7.Don haka, ana ƙara buƙatar ingantattun dabarun tantancewa na asali don tantance ingancin bugun jini da guba.Amfani da kwanan nan na ɗan adam wanda ya haifar da ɗimbin ƙarfi mai ƙarfi-samar cardiomyocytes (hiPS-CM) a cikin gwajin ƙwayar cuta da gwajin guba yana ba da mafita ga wannan matsala.Duk da haka, yanayin rashin girma na hiPS-CMs da kuma rashin hadaddun ƙwayoyin ƙwayoyin cuta na zuciya sune manyan iyakoki na wannan hanya.Binciken da aka yi kwanan nan ya nuna cewa za a iya shawo kan wannan iyakance ta hanyar amfani da farkon hiPS-CM don samar da hydrogels na nama na zuciya jim kaɗan bayan farawar ƙanƙancewa ba tare da bata lokaci ba kuma sannu a hankali yana haɓaka ƙarfin lantarki a kan lokaci.Koyaya, waɗannan microtissues na hiPS-CM ba su da manyan abubuwan electrophysiological da kaddarorin kwangila na manya myocardium.Bugu da ƙari, ƙwayar zuciya na ɗan adam yana da tsari mai rikitarwa, wanda ya ƙunshi cakuda nau'in tantanin halitta daban-daban, ciki har da sel na endothelial, neurons, da fibroblasts na stromal, wanda ke haɗuwa da takamaiman nau'i na furotin matrix na extracellular.Wannan iri-iri na yawan mutanen da ba na cardiomyocyte ba11,12,13 a cikin zuciyar manya masu shayarwa babban shamaki ne ga yin ƙirar nama na zuciya ta amfani da nau'ikan tantanin halitta ɗaya.Wadannan manyan iyakoki suna jaddada mahimmancin hanyoyin haɓakawa don haɓaka ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta a ƙarƙashin yanayin physiological da pathological.
Sassan ɓangarorin bakin ciki (300 µm) na zuciyar ɗan adam sun tabbatar da zama samfuri mai ban sha'awa na myocardium na ɗan adam.Wannan hanyar tana ba da damar samun cikakken tsarin 3D mai kama da naman zuciyar ɗan adam.Koyaya, har zuwa 2019, an iyakance amfani da sassan zuciya na al'ada ta gajeriyar rayuwar al'ada (24h).Wannan shi ne saboda dalilai masu yawa ciki har da rashin shimfidawa na jiki-nau'i-nau'i, mai amfani da ruwa na iska, da kuma amfani da kafofin watsa labaru masu sauƙi waɗanda ba su goyan bayan buƙatun ƙwayar zuciya.A cikin 2019, ƙungiyoyin bincike da yawa sun nuna cewa haɗa abubuwan inji cikin tsarin al'adun nama na zuciya na iya tsawaita rayuwar al'ada, haɓaka maganganun zuciya, da kwaikwayi cututtukan zuciya.Biyu m karatu 17 da 18 nuna cewa uniaxial inji loading yana da kyau sakamako a kan cardiac phenotype a lokacin al'ada.Duk da haka, waɗannan karatun ba su yi amfani da nauyin nauyin nau'i-nau'i-nau'i-nau'i-nau'i-nau'i na motsa jiki na zuciya ba, tun da an ɗora sassan zuciya tare da ko dai isometric tensile sojojin 17 ko linzamin auxotonic loading 18.Wadannan hanyoyin mikewar nama sun haifar da danne kwayoyin halittar zuciya da yawa ko kuma wuce gona da iri na kwayoyin halittar da ke hade da martanin mikewa mara kyau.Musamman, Pitoulis et al.19 sun haɓaka wankan al'adun yanki mai ƙarfi na zuciya don sake gina zagayowar zuciya ta amfani da ra'ayin mai canza kuzari da motsa tashin hankali.Ko da yake wannan tsarin yana ba da damar ƙarin daidaiton ƙirar zagayowar bugun zuciya a cikin vitro, ƙayyadaddun ƙayyadaddun ƙayyadaddun kayan aiki da ƙarancin hanyar hanyar suna iyakance aikace-aikacen wannan tsarin.Gidan dakin gwaje-gwajenmu kwanan nan ya haɓaka tsarin al'ada mai sauƙi ta amfani da kuzarin lantarki da ingantaccen matsakaici don kiyaye iyawar sassan porcine da nama na zuciyar ɗan adam har zuwa kwanaki 620,21.
A cikin rubutun na yanzu, mun bayyana samfurin al'adun nama na zuciya (CTCM) ta amfani da sassan zuciya na porcine waɗanda ke haɗa abubuwan ban dariya don sake fasalin ilimin halittar zuciya mai girma uku da ɓacin rai yayin zagayowar zuciya.Wannan CTCM na iya ƙara daidaiton tsinkayar magungunan magani na farko zuwa matakin da ba a taɓa samu ba ta hanyar samar da tsarin farashi mai tsada, tsaka-tsakin tsarin zuciya wanda ke kwaikwayon ilimin lissafi / pathophysiology na zuciyar mammalian don gwajin magunguna na farko.
Siginonin injiniya na hemodynamic suna taka muhimmiyar rawa wajen kiyaye aikin cardiomyocyte a cikin vitro 22,23,24.A cikin rubutun hannu na yanzu, mun ƙirƙira CTCM (Hoto 1a) wanda zai iya kwaikwayi yanayin zuciya na manya ta hanyar haifar da kuzarin lantarki da injina a mitocin ilimin lissafi (1.2 Hz, 72 bugun minti daya).Don kauce wa wuce gona da iri a lokacin diastole, an yi amfani da na'urar bugu na 3D don ƙara girman nama da 25% (Fig. 1b).Tafiyar wutar lantarki da tsarin C-PACE ya haifar ya kasance lokacin farawa 100 ms kafin systole ta amfani da tsarin sayan bayanai don sake sake sake zagayowar zuciya.Tsarin al'adun nama yana amfani da na'ura mai ɗaukar hoto (LB Engineering, Jamus) don faɗaɗa cyclically membrane silicone mai sassauƙa don haifar da faɗaɗa yankan zuciya a cikin ɗaki na sama.An haɗa tsarin zuwa layin iska na waje ta hanyar mai jujjuya matsa lamba, wanda ya sa ya yiwu a daidaita daidaitattun matsa lamba (± 1 mmHg) da lokaci (± 1 ms) (Fig. 1c).
Haɗa sashin nama zuwa zoben tallafi na mm 7, wanda aka nuna da shuɗi, cikin ɗakin al'ada na na'urar.An raba ɗakin al'ada daga ɗakin iska ta hanyar siliki mai laushi na bakin ciki.Sanya gasket tsakanin kowane ɗaki don hana yadudduka.Murfin na'urar yana ƙunshe da na'urorin lantarki na graphite waɗanda ke ba da kuzarin lantarki.b Tsarin tsari na babban na'urar nama, zoben jagora da zoben tallafi.Ana sanya sassan nama (launin ruwan kasa) akan na'ura mai girman gaske tare da zoben jagora da aka sanya a cikin tsagi a gefen waje na na'urar.Yin amfani da jagorar, a hankali sanya zoben tallafi wanda aka lulluɓe da nama acrylic m akan sashin nama na zuciya.c Graph yana nuna lokacin ƙarfafawar wutar lantarki a matsayin aikin matsa lamba na ɗakin iska wanda aka sarrafa ta na'urar kunnawa na pneumatic (PPD).An yi amfani da na'urar sayan bayanai don aiki tare da kuzarin lantarki ta amfani da na'urori masu auna matsi.Lokacin da matsa lamba a cikin ɗakin al'ada ya isa wurin da aka saita, ana aika siginar bugun jini zuwa C-PACE-EM don kunna kuzarin lantarki.d Hoton CTCM guda huɗu da aka sanya a kan shiryayye na incubator.Ana haɗa na'urori huɗu zuwa PPD ɗaya ta hanyar da'irar pneumatic, kuma ana shigar da firikwensin matsa lamba a cikin bawul ɗin hemostatic don saka idanu da matsa lamba a cikin da'irar pneumatic.Kowace na'ura ta ƙunshi sassan nama guda shida.
Yin amfani da mai kunnawa na pneumatic guda ɗaya, mun sami damar sarrafa na'urorin CTCM 4, kowannensu zai iya ɗaukar sassan nama na 6 (Fig. 1d).A cikin CTCM, matsa lamba na iska a cikin ɗakin iska yana jujjuya zuwa matsa lamba mai daidaitawa a cikin ɗakin ruwa kuma yana haifar da faɗaɗa ƙwayar zuciya na yanki na zuciya (Hoto 2a da Ƙarin Fim 1).Ƙididdigar shimfiɗa nama a 80 mm Hg.Art.ya nuna shimfidar sassan nama da kashi 25% (Fig. 2b).An nuna wannan kaso na miƙewa yayi daidai da tsayin sarcomere na physiological na 2.2-2.3 µm don ƙaddamar da sashin zuciya na al'ada17,19,25.An tantance motsin nama ta amfani da saitunan kamara na al'ada (Ƙarin Hoto 1).Girman girma da saurin motsi na nama (Fig. 2c, d) ya dace da shimfiɗa a lokacin zagayowar zuciya da lokaci a lokacin systole da diastole (Fig. 2b).Miƙewa da saurin nama na zuciya a lokacin ƙanƙancewa da annashuwa sun kasance akai-akai har tsawon kwanaki 12 a cikin al'ada (Fig. 2f).Don kimanta tasirin tasirin wutar lantarki akan kwangila a lokacin al'ada, mun samar da wata hanya don ƙayyade nakasar aiki ta amfani da algorithm shading (Ƙarin Fig. 2a,b) kuma mun iya bambanta tsakanin nakasa tare da kuma ba tare da wutar lantarki ba.Sashe ɗaya na zuciya (Fig. 2f).A cikin yanki mai motsi na yanke (R6-9), ƙarfin lantarki a lokacin motsa jiki na lantarki ya kasance 20% mafi girma fiye da rashin haɓakar wutar lantarki, wanda ke nuna gudunmawar wutar lantarki zuwa aikin kwangila.
Alamun wakilci na matsa lamba na ɗakin iska, matsa lamba na ɗakin ruwa, da ma'aunin motsi na nama sun tabbatar da cewa matsa lamba ɗakin yana canza matsa lamba na ruwa, yana haifar da madaidaicin motsi na yanki na nama.b Wakilan burbushin kashi na shimfiɗa (blue) na sassan nama wanda ya dace da kashi ɗari (orange).c Motsin da aka auna na yanki na zuciya ya yi daidai da auna saurin motsi.(d) Matsayin wakilci na motsi na cyclic (layin shuɗi) da sauri (layin dige-dige-orange) a cikin yanki na zuciya.e Ƙayyadaddun lokaci na sake zagayowar (n = 19 yanka a kowace ƙungiya, daga aladu daban-daban), lokacin ƙaddamarwa (n = 19 yanka a kowace ƙungiya), lokacin shakatawa (n = 19 yanka a kowace ƙungiya, daga aladu daban-daban), motsi nama (n = 25).yanka) / rukuni daga aladu daban-daban), saurin systolic kololuwa (n = 24 (D0), 25 (D12) yanka / rukuni daga aladu daban-daban) da ƙimar kwanciyar hankali (n = 24 (D0), 25 (D12) yanka / rukuni daga aladu daban-daban).T-gwajin ɗalibi mai wutsiya biyu ya nuna babu wani muhimmin bambanci a kowane siga.f Wakilan bincike na nau'in nau'in nama tare da (ja) kuma ba tare da (blue) kuzarin lantarki ba, yankuna goma na sassan nama daga sashe ɗaya.Ƙungiyoyin ƙasa suna nuna ƙididdige yawan bambancin kashi a cikin nau'i a cikin sassan nama tare da kuma ba tare da motsa jiki na lantarki a wurare goma daga sassa daban-daban. (n = 8 yanka / rukuni daga aladu daban-daban, Ana yin gwajin t-gwajin ɗalibi mai wutsiya biyu; ****p <0.0001, ** p <0.01, *p <0.05). (n = 8 yanka / rukuni daga aladu daban-daban, Ana yin gwajin t-gwajin 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 = sassan 8 / rukuni daga aladu daban-daban, t-gwajin dalibi 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 = sassan 8 / rukuni, daga aladu daban-daban, t-gwajin dalibi mai wutsiya biyu; ****p <0.0001, ** p <0.01, *p <0.05).Kuskuren sanduna suna wakiltar ma'anar ± daidaitaccen karkacewa.
A cikin tsarin al'adun yanki na yanki na biomimetic na baya [20, 21], mun kiyaye iyawa, aiki, da amincin tsarin yankan zuciya na tsawon kwanaki 6 ta amfani da kuzarin lantarki da haɓaka matsakaiciyar abun ciki.Koyaya, bayan kwanaki 10, waɗannan alkalumman sun ragu sosai.Za mu koma ga sassan da aka tsara a cikin tsarin al'adun mu na baya-bayan nan na biomimetic 20, 21 yanayin sarrafawa (Ctrl) kuma za mu yi amfani da matsakaicin ingantaccen yanayin mu a baya azaman yanayin MC da al'ada ƙarƙashin injina da kuzari na lokaci guda (CTCM).ake kira .Na farko, mun ƙaddara cewa haɓakar injiniya ba tare da motsa jiki na lantarki bai isa ba don kula da lafiyar nama don kwanakin 6 (Ƙarin Hoton 3a,b).Abin sha'awa, tare da gabatarwar physio-mechanical da lantarki motsa jiki ta yin amfani da STCM, yiwuwar sassan zuciya na kwanaki 12 sun kasance iri ɗaya kamar a cikin sassan zuciya na zuciya a ƙarƙashin yanayin MS, amma ba a ƙarƙashin yanayin Ctrl ba, kamar yadda aka nuna ta hanyar bincike na MTT (Fig. 1).3 a).Wannan yana nuna cewa haɓakar injina da kwaikwaya na zagayowar zuciya na iya kiyaye sassan nama su yi aiki sau biyu muddin aka ruwaito a cikin tsarin al'adun mu na baya.Duk da haka, kima na tsarin tsarin sassan nama ta hanyar rigakafi na troponin T da connexin 43 ya nuna cewa connexin 43 magana ya kasance mafi girma a cikin kyallen takarda na MC a ranar 12 fiye da sarrafawa a rana guda.Duk da haka, maganganun haɗin kai na 43 na haɗin kai da kuma samuwar Z-faifan ba a cika su ba (Fig. 3b).Muna amfani da tsarin hankali na wucin gadi (AI) don ƙididdige mutuncin tsarin nama26, bututun ilmantarwa mai zurfi na tushen hoto dangane da troponin-T da ɓangarorin haɗin gwiwa43 don ƙididdige daidaiton tsari ta atomatik da walƙiya na yankan zuciya dangane da ƙarfin wuri.Wannan hanyar tana amfani da hanyar sadarwa ta Convolutional Neural Network (CNN) da tsarin ilmantarwa mai zurfi don ƙididdige ingantaccen tsarin nama na zuciya ta atomatik da rashin son zuciya, kamar yadda aka bayyana a cikin tunani.26. Nama na MC ya nuna ingantaccen tsarin kamanni zuwa rana ta 0 idan aka kwatanta da sassan sarrafawa.Bugu da kari, Masson ta trichrome tabo ya bayyana wani gagarumin ƙananan kashi na fibrosis a ƙarƙashin yanayin MS idan aka kwatanta da yanayin sarrafawa a ranar 12 na al'ada (Fig. 3c).Yayin da CTCM ya ƙãra yuwuwar sassan nama na zuciya a rana ta 12 zuwa wani mataki mai kama da na sabon ƙwayar zuciya, bai inganta ingantaccen tsarin sassan zuciya ba.
jadawali na Bar yana nuna ƙididdige ƙimar MTT na sabbin yankan zuciya (D0) ko al'adun yankan zuciya na 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 guda 1, an yi gwajin pigs / NO #0 # 1. zuwa D0 da ** p <0.01 idan aka kwatanta da D12 Ctrl). jadawali na Bar yana nuna ƙididdige ƙimar MTT na sabbin yankan zuciya (D0) ko al'adun yankan zuciya na 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) Pigs guda 12 (D12 MC) pigs / rukuni daban, NO # 0 # 0. idan aka kwatanta da D0 da ** p <0.01 idan aka kwatanta da D12 Ctrl).histogram yana nuna ƙididdige ƙimar MTT sabbin sassan zuciya (D0) ko al'adun sassan zuciya na kwanaki 12 a cikin al'adun a tsaye (D12 control) ko CTCM (D12 MC) (n = 18 (D0), 15 (D12 iko) )), 12 (D12 MC) sassan / VA gwajin da aka yi daban-daban;####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) 中新鲜心心脏切片(D0)天的MTT 活力的量化)，来自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；与D0 ，#12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) 的12 (D0) MC) 切片/组，进行单向ANOVA 测试；与D0 相比，####p <0.0001，与D12 Ctrl 相比，**p。)histogram yana nuna ƙididdige ƙimar MTT a cikin sabbin sassan zuciya (D0) ko sassan zuciya waɗanda aka tsara don kwanaki 12 a cikin al'adar tsaye (ikon D12) ko CTCM (D12 MC) (n = 18 (D0), 15 (D12 iko)) , 12 (D12 MC) sassan / rukuni AVA daga aladu daban-daban, gwaji guda 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 (blue) a cikin sabbin sassan zuciya da aka keɓe (D0) ko sassan zuciya waɗanda aka tsara a ƙarƙashin yanayi na tsaye (Ctrl) ko yanayin CTCM (MC) na kwanaki 12) na hotuna na wakilcin immunofluorescence (sikelin blank = 100 µm). Ƙididdigar hankali na wucin gadi na tsarin tsarin nama na zuciya (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) yanki / rukuni kowanne daga alade daban-daban, ana yin gwajin ANOVA ta hanya daya; ####p <0.0001 idan aka kwatanta da D0 da ****p <0.0001 C idan aka kwatanta da D0 da ****p <0.0001 C. Ƙididdigar hankali na wucin gadi na tsarin tsarin nama na zuciya (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) yanka / rukuni kowanne daga aladu daban-daban, ana yin gwajin ANOVA ta hanya daya; ####p <0.0001 idan aka kwatanta da D0 da ****p <0.0001 C idan aka kwatanta da D0 da ****p <0.0001 C. Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D15), 2 (D15) п от разных свиней, проводится однофакторный тест ANOVA; ####p <0,0001 по сравнению с D0 и ****p <0,0001 пню. Ƙididdigar tsarin tsarin tsarin ƙwayar zuciya ta hanyar basirar wucin gadi (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) sassan / ƙungiyoyi daga aladu daban-daban, gwajin ANOVA guda ɗaya da aka yi; ####p <0.0001 vs. tare da D0 da **** p <1 idan aka kwatanta da D0.00C.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 ɗaya, ，#0#0p <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 mai hanya ɗaya, ，#0#0p 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) руппу каждой из разных свиней, односторонний тест ANOVA; ####p <0,0001 vs. D0 Для сравнения ****p <0,0001 пни срав . Hankali na wucin gadi don ƙididdige daidaiton tsarin tsarin ƙwayar zuciya (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) sassan / rukuni kowane nau'i na aladu daban-daban, gwajin ANOVA guda ɗaya; ####p <0.0001 vs .D0 Don kwatanta ****p <1 idan aka kwatanta da D.000C. c Hotunan wakilci (hagu) da ƙididdigewa (dama) don yankan zuciya da aka lalata tare da tabon Masson's trichrome (Scale bare = 500 µm) (n = 10 yanka / rukuni kowanne daga alade daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ####p <0.0001 idan aka kwatanta da D0 da 02p = C = 0.1 p = 0.1 p = 0.02 p = 0. c Hotunan wakilci (hagu) da ƙididdigewa (dama) don yankan zuciya da aka lalata tare da tabon Masson's trichrome (Scale bare = 500 µm) (n = 10 yanka / rukuni kowanne daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; #### p <0.0001 idan aka kwatanta da D0 da 02p = C idan aka kwatanta da D0 da 0. c Репрезентативные изображения ( слева ) сштаб без покрытия = 500 мкм) (n = 10 срезов/группу от разных свиней, выполняется односторытия одностороняется одностороняется одностороняется одностороняется одностороняется односторонний тест ANO#0; с D0 и ***p <0,001 pо сравнению с D12 Ctrl). c Hotunan wakilci (hagu) da ƙididdigewa (dama) na sassan zuciya da aka lalata tare da tabo trichrome Masson (ma'auni maras kyau = 500 µm) (n = sassan 10 / rukuni daga aladu daban-daban, gwajin ANOVA guda daya da aka yi; #### p <0 .0001 idan aka kwatanta da D0 da *** 2 <0. 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）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 < 0.001 丯 C 用 masson 三 色 染料 的 心脏 切片 的 代表性 （左 左） 量化 （右） 心脏500 µm） （n = 10 个 切片 组 每 组 来自 不同 猪 ， 进行 单向 单向 Anova 测试；#0 ***p <0.001 与D12 Ctrl 相比） c Репрезентативные изображения (слева) стая шкала = 500 мкм) (n= 10 срезов/группа анализа ;### #p <0,0001 по сравнению с D0, ***p <0,001 по сравнению с D12 Ctrl). c Hotunan wakilci (hagu) da ƙididdigewa (dama) na sassan zuciya da aka lalata tare da tabo trichrome Masson (blank = 500 µm) (n = 10 sassan / rukuni, kowanne daga alade daban-daban, an gwada ta hanyar bincike guda ɗaya na bambance-bambance; ### # p <0.0001 idan aka kwatanta da D0.0.0001).Kuskuren sanduna suna wakiltar ma'anar ± daidaitaccen karkacewa.
Mun yi hasashen cewa ta hanyar ƙara ƙananan ƙwayoyin cuta zuwa matsakaicin al'ada, za a iya inganta amincin cardiomyocyte kuma rage ci gaban fibrosis yayin al'adun CTCM.Don haka mun bincika ƙananan ƙwayoyin cuta ta amfani da al'adun sarrafa mu na tsaye20,21 saboda ƙarancin adadin abubuwan ruɗani.Dexamethasone (Dex), triiodothyronine (T3), da SB431542 (SB) an zaɓi don wannan allon.An yi amfani da waɗannan ƙananan ƙwayoyin cuta a baya a cikin al'adun hiPSC-CM don haifar da maturation na cardiomyocytes ta hanyar haɓaka tsayin sarcomere, T-tubules, da saurin tafiyarwa.Bugu da ƙari, duka Dex (glucocorticoid) da SB an san su don kashe kumburi29,30.Saboda haka, mun gwada ko haɗa ɗaya ko haɗin waɗannan ƙananan ƙwayoyin cuta zai inganta ingantaccen tsarin sassan zuciya.Don nunawa na farko, an zaɓi kashi na kowane fili bisa ƙididdiga da aka saba amfani da su a cikin ƙirar al'adun tantanin halitta (1 μM Dex27, 100 nM T327, da 2.5 μM SB31).Bayan kwanaki 12 na al'ada, haɗuwa da T3 da Dex sun haifar da ingantaccen tsarin tsarin cardiomyocyte da ƙaramin gyare-gyaren fibrous (Ƙarin Figures 4 da 5).Bugu da ƙari, yin amfani da sau biyu ko sau biyu waɗannan ƙididdiga na T3 da Dex sun haifar da sakamako mai lalacewa idan aka kwatanta da abubuwan da suka dace (Ƙarin Hoto 6a,b).
Bayan gwajin farko, mun yi kwatancen kai-da-kai na yanayin al'adu 4 (Hoto na 4a): Ctrl: sassan zuciya da aka tsara a cikin al'adun mu da aka bayyana a baya ta amfani da ingantaccen matsakaici;20.21 TD: T3 da Ctrl s Added Dex ranar Laraba;MC: sassan zuciya da aka haɓaka a cikin CTCM ta amfani da ingantaccen matsakaicin mu na baya;da MT: CTCM tare da T3 da Dex da aka ƙara zuwa matsakaici.Bayan kwanaki 12 na noma, yiwuwar MS da MT kyallen takarda sun kasance iri ɗaya kamar a cikin sabbin kyallen takarda da aka tantance ta hanyar MTT assay (Fig. 4b).Abin sha'awa shine, ƙari na T3 da Dex zuwa al'adun transwell (TD) ba su haifar da gagarumin ci gaba a cikin iyawa ba idan aka kwatanta da yanayin Ctrl, yana nuna muhimmiyar rawa na ƙarfafawar injiniya don kiyaye yiwuwar sassan zuciya.
zane-zane na gwaji wanda ke nuna yanayin al'adu guda hudu da aka yi amfani da su don kimanta tasirin ƙarfafawar injiniya da T3/Dex a kan matsakaici na kwanaki 12. b Bar jadawali yana nuna ƙididdige yiwuwar 12 days post al'ada a cikin duk yanayin al'adu 4 (Ctrl, TD, MC, da MT) idan aka kwatanta da sabbin zuciya yanka (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD da D12 MT), 12 (D12 TD da D12 MT), 12 (D12 MC) pigs guda 0. 01, ###p <0.001 idan aka kwatanta da D0 da **p <0.01 idan aka kwatanta da D12 Ctrl). b Bar jadawali yana nuna ƙididdige yiwuwar 12 days post al'ada a cikin duk yanayin al'adu 4 (Ctrl, TD, MC, da MT) idan aka kwatanta da sabbin zuciya yanka (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD da D12 MT), 12 (D12 TD da D12 MT), 12 (D12 MC) pigs guda 0. 01, ###p <0.001 idan aka kwatanta da D0 da **p <0.01 idan aka kwatanta da D12 ctrl). b Гистограмма показывает количественую оценку ирования (контроль, TD, MC и MT) по сравнению со сравнению со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD), MT 12 Ctrl, D12 (TD) уппу от разных свиней, проводится односторонний тест ANOVA; ####p <0,0001, ###p <0,001 по сравнению с D0 и **1 сравнению с D0 и ** ). b Hoton mashaya yana nuna ƙididdiga na iya aiki a cikin kwanaki 12 bayan al'ada a cikin duk yanayin al'adu na 4 (ikon, TD, MC, da MT) idan aka kwatanta da sassan zuciya na sabo (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, da D12 MT), 12 (D12 MT); 12 (D12 MC) daga sassa daban-daban na pig0. 01, ###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) 12 MT), 来自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA控制).b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC и MT) по сравнению (MT) 18 (D0), 15 (D12 Ctrl, D12 TD da D12 MT), от разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA; #, ю с D0, **p <0,01 pо сравнению с контролем D12). b Histogram yana nuna duk yanayin al'adu na 4 (iko, TD, MC da MT) idan aka kwatanta da sassan zuciya na sabo (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD da D12 MT), daga aladu daban-daban 12 (D12 MC) sassan / rukuni, gwajin ANOVA guda daya; # . 0 # 0. p <0.01 vs. sarrafawa D12). c Bar jadawali yana nuna ƙididdige ƙimar glucose na kwanaki 12 bayan al'ada a cikin duk yanayin al'adun 4 (Ctrl, TD, MC, da MT) idan aka kwatanta da sabbin zuciya yanka (D0) (n = 6 yanka / rukuni daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ###p <0.001, idan aka kwatanta da D0 da 0.0p <0.1). c Bar jadawali yana nuna ƙididdige ƙimar glucose na kwanaki 12 bayan al'ada a cikin duk yanayin al'adun 4 (Ctrl, TD, MC, da MT) idan aka kwatanta da sabbin zuciya yanka (D0) (n = 6 yanka / rukuni daga aladu daban-daban, ana yin gwajin ANOVA ta hanya ɗaya; ###p <0.001, idan aka kwatanta da D0 da 0.0p <0.1). c Гистограмма показывает количественую оценку потока глюкозы через 12. ирования (контроль, TD, MC и MT) по сравнению со сравнению няется тест ANOVA; ###p <0,001 по сравнению с D0 и ***p <0,001 по сравнению с D12 Ctrl). c Histogram yana nuna ƙididdige ƙimar glucose na kwanaki 12 bayan al'ada a ƙarƙashin duk yanayin al'adun 4 (control, TD, MC da MT) idan aka kwatanta da sabbin sassan zuciya (D0) (n = sassan 6 / rukuni daga aladu daban-daban, gwajin ANOVA ta hanya ɗaya da aka yi; ###p <0.001 idan aka kwatanta da D0 da *** p <0.021 C idan aka kwatanta da D0 da *** p <0.001). 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) 玡件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) 玡件各的1通量定量（n = 6 片/组，来自不同猪，单不同猪，单向执行ANOVA . C 条形图 显示 所有 4 种 条件 （（ctrl 、 td 、 mc 和 mt） 新鲜后 后 12 天 的 通量 定量 （n = 6相比，***p <0.001 与D12 Ctrl 相比）。 c Гистограмма, показывающая казывающая количественую оценку льтивирования (контроль, TD, MC и MT) по сравнению Были проведены тесты ANOVA; ###p <0,001 pо сравнению с D0, ***p <0,001 по сравнению с D12 (контроль). c Histogram yana nuna ƙididdigar ƙwayar glucose a cikin kwanaki 12 bayan al'ada don duk yanayin al'adun 4 (control, TD, MC, da MT) idan aka kwatanta da sassan zuciya na sabo (D0) (n = sassan 6 / rukuni, daga aladu daban-daban, an yi gwajin ANOVA na unilateral, ###p <0.001 idan aka kwatanta da D0,1 ***1).d Shirye-shiryen nazarin nau'i na sabo (blue), ranar 12 MC (kore), da rana 12 MT (ja) kyallen takarda a wuraren yanki na yanki goma (n = 4 yanka / rukuni, gwajin ANOVA guda ɗaya; babu wani bambanci mai mahimmanci tsakanin kungiyoyi).e Siffar dutsen mai aman wuta tana nuna nau'ikan halittu daban-daban a cikin sabbin sassan zuciya (D0) idan aka kwatanta da sassan zuciya waɗanda aka al'adarsu a ƙarƙashin yanayi na tsaye (Ctrl) ko ƙarƙashin yanayin MT (MT) na kwanaki 10-12.f Taswirar zafafan kwayoyin halittar sarcomere don sassan zuciya da aka tsara a ƙarƙashin kowane yanayin al'ada.Kuskuren sanduna suna wakiltar ma'anar ± daidaitaccen karkacewa.
Dogarowar ƙwayar cuta akan sauyawa daga fatty acid oxidation zuwa glycolysis alama ce ta rarrabuwar cardiomyocyte.Cardiomyocytes marasa girma da farko suna amfani da glucose don samar da ATP kuma suna da hypoplastic mitochondria tare da 'yan cristae5,32.Nazarin amfani da glucose ya nuna cewa a ƙarƙashin yanayin MC da MT, amfani da glucose yayi kama da wanda ke cikin kyallen takarda na rana (Hoto 4c).Koyaya, samfuran Ctrl sun nuna babban haɓakar amfani da glucose idan aka kwatanta da nama mai sabo.Wannan yana nuna cewa haɗin CTCM da T3/Dex yana haɓaka ƙarfin nama kuma yana adana nau'in phenotype na sassan zuciya na kwanaki 12.Bugu da ƙari, nazarin ƙwayar cuta ya nuna cewa matakan damuwa sun kasance iri ɗaya kamar a cikin sabon ƙwayar zuciya don kwanaki 12 a ƙarƙashin yanayin MT da MS (Fig. 4d).
Don nazarin tasirin CTCM gabaɗaya da T3/Dex akan shimfidar wuri mai faɗi na duniya na yanki na zuciya, mun yi RNAseq akan yankan zuciya daga duk yanayin al'adu daban-daban guda huɗu (Ƙarin Bayanai 1).Abin sha'awa shine, sassan MT sun nuna kamanceceniya mai girma zuwa sabbin nama na zuciya, tare da 16 kaɗai aka bayyana daga cikin kwayoyin halitta 13,642.Duk da haka, kamar yadda muka nuna a baya, Ctrl yanka ya nuna 1229 daban-daban da aka bayyana kwayoyin halitta bayan 10-12 kwanakin a cikin al'ada (Fig. 4e).An tabbatar da waɗannan bayanan ta hanyar qRT-PCR na zuciya da ƙwayoyin fibroblast (Ƙarin Hoton 7a-c).Abin sha'awa, sassan Ctrl sun nuna raguwar tsarin kwayoyin halittar zuciya da na kwayar halitta da kunna shirye-shiryen kwayar cutar kumburi.Waɗannan bayanan sun nuna cewa rarrabuwa, wanda yakan faru bayan al'ada na dogon lokaci, an rage shi gaba ɗaya a ƙarƙashin yanayin MT (Ƙarin Hoton 8a,b).Nazarin hankali game da kwayoyin halittar sarcomere ya nuna cewa kawai a ƙarƙashin yanayin MT sune kwayoyin da ke ɓoye sarcomere (Fig. 4f) da tashar ion (Ƙarin Hoton 9) sun kiyaye su, suna kare su daga ƙuntatawa a ƙarƙashin yanayin Ctrl, TD, da MC.Waɗannan bayanai sun nuna cewa tare da haɗin injina da motsa jiki (T3/Dex), rubutun yanki na zuciya na iya kasancewa kama da sabbin yankan zuciya bayan kwanaki 12 a cikin al'ada.
Wadannan binciken kwafi suna goyan bayan gaskiyar cewa tsarin tsarin tsarin cardiomyocytes a cikin sassan zuciya yana da kyau a kiyaye shi a ƙarƙashin yanayin MT na kwanaki 12, kamar yadda aka nuna ta hanyar haɗin kai da kuma na gida 43 (Fig. 5a).Bugu da ƙari, fibrosis a cikin sassan zuciya a ƙarƙashin yanayin MT ya ragu sosai idan aka kwatanta da Ctrl kuma kama da sassan zuciya na sabo (Fig. 5b).Wadannan bayanan sun nuna cewa haɗuwa da motsa jiki na inji da kuma maganin T3 / Dex yadda ya kamata ya kiyaye tsarin zuciya a cikin sassan zuciya a cikin al'ada.
Hotunan wakilcin immunofluorescence na troponin-T (kore), connexin 43 (ja), da DAPI (blue) a cikin sassan zuciya da aka ware (D0) ko al'ada don kwanaki 12 a cikin duk yanayin al'adun sashe na zuciya huɗu (masanin sikelin = 100 µm).). Ƙididdigar hankali na wucin gadi na tsarin tsarin tsarin nama na 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 guda ɗaya; ####p <0.0001 idan aka kwatanta da D0 da 0.0p <0.0p, ko 0.0p <0p. rl). Ƙididdigar hankali na wucin gadi na tsarin tsarin 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 guda ɗaya; #### p <0.0001 idan aka kwatanta da D0 da 0.0p <0.0p <0. rl). Количественная Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта интеллекта (n = 2), 1 (D1) и D12 MT) срезов/группу от разных свиней, проведен однофакторный тест ANOVA; #### p <0,0001 по сравнению 01 по сравнению с D12 Ctrl). Daidaifiyar amincin tsarin zuciya ta amfani da bayanan wucin gadi (N0) Ctrl), 5 (D0).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 、D12 MC 和D12 MT智能量化，进行单向ANOVA 测试；#### p <0.0001 与D0 和*p <0.05 相比，或****p < 0.0001 与对 不同猪 的 心脏结构 完整性 （n = 7 （d0 和 d12 ctrl）量 化 进行 单向 单向 单向 测试 ； ######## p < 0.0001 ）Ƙididdigar tsarin tsarin tsarin ƙwayar zuciya ta amfani da basirar wucin gadi a cikin aladu daban-daban (n = 7 (D0 da D12 Ctrl), 5 (D12 TD, D12 MC da D12 MT) sassan / rukuni) tare da gwajin ANOVA guda 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 wakilci da ƙididdigewa don yankan zuciya da aka lalata tare da tabon trichrome Masson (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, da D12 MC), 9 (D12 MT) yanka / rukuni daga aladu daban-daban, an yi gwajin ANOVA guda ɗaya # 0 # 0 zuwa <1 # 0. .001, ko ****p <0.0001 idan aka kwatanta da D12 Ctrl). b Hotunan wakilci da ƙididdigewa don yankan zuciya da aka lalata tare da tabon trichrome Masson (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, da D12 MC), 9 (D12 MT) yanka / rukuni daga aladu daban-daban, an yi gwajin ANOVA guda ɗaya # 0 # 0 zuwa <1 # 0. .001, ko ****p <0.0001 idan aka kwatanta da D12 Ctrl). b Репрезентативные изображения количественная ка = 500 мкм) (n = 10 (D0, D12 Ctrl, D12 TD da D12 MC), 9 (D12 MT) 0001 по сравнению с D0 и ***p <0,001 ko ****p <0,0001 da сравнению с D12 Ctrl). b Hotunan wakilci da ƙididdige sassan sassan zuciya da aka lalata tare da tabo trichrome Masson (ma'auni = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD da D12 MC), 9 (D12 MT) sassan / rukuni daga aladu daban-daban, an yi ANOVA ta hanya daya da kuma <0.0.0. 1 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 = 100 TD10 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 丛行单因素方差分析；#*##p < 0.0001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/组，进行单因素方差分析；####p < 0.0001 0.0001或****p <0.0001 与D12 Ctrl 相比）。 b Репрезентативные изображения и количественная ) (n = 10 (D0, D12 Ctrl, D12 TD da D12 MC), 9 (D12 MT) срезов от разных свиней / групппы, один- способ , 0 пос 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 p <10. 0,001 ko ****p <0,0001 ko сравнению с D12 Ctrl). b Hotunan wakilci da ƙididdige sassan zuciya da aka lalata tare da trichrome na Masson (masanin sikelin = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD da D12 MC), sassan 9 (D12 MT) daga aladu / rukuni daban-daban, hanyar ANOVA guda ɗaya, <1##0p. ****p <0.0001 idan aka kwatanta da D12 Ctrl).Kuskuren sanduna suna wakiltar ma'anar ± daidaitaccen karkacewa.
A ƙarshe, an ƙididdige ikon CTCM don kwaikwayi hawan jini na zuciya ta hanyar haɓaka nama na zuciya.A cikin CTCM, matsakaicin ɗakin ɗakin iska ya ƙaru daga 80 mmHg zuwa 80 mmHg.Art.(daidaitaccen shimfidawa) har zuwa 140 mmHg Art.(Hoto na 6 a).Wannan ya dace da haɓakar 32% na shimfiɗawa (Fig. 6b), wanda aka nuna a baya a matsayin daidaitaccen adadin da ake buƙata don sassan zuciya don cimma tsayin sarcomere mai kama da wanda aka gani a hypertrophy.Miƙewa da saurin nama na zuciya a lokacin ƙanƙancewa da annashuwa sun kasance masu tsayi a cikin kwanaki shida na al'ada (Fig. 6c).Nama na zuciya daga yanayin MT an ƙaddamar da shi zuwa madaidaiciyar al'ada (MT (Na al'ada)) ko yanayin jujjuyawa (MT (OS)) na kwanaki shida.Tuni bayan kwanaki hudu a cikin al'ada, hypertrophic biomarker NT-ProBNP ya kasance mai girma a cikin matsakaici a karkashin yanayin MT (OS) idan aka kwatanta da yanayin MT (na al'ada) (Fig. 7a).Bugu da ƙari, bayan kwanaki shida na al'ada, girman tantanin halitta a cikin MT (OS) (Fig. 7b) ya karu sosai idan aka kwatanta da sassan MT zuciya (na al'ada).Bugu da ƙari, fassarar nukiliya ta NFATC4 ya karu sosai a cikin kyallen takarda (Fig. 7c).Wadannan sakamakon suna nuna ci gaba na ci gaba da gyaran gyare-gyaren cututtuka bayan hawan jini da kuma goyan bayan ra'ayi cewa za a iya amfani da na'urar CTCM a matsayin dandamali don nazarin siginar hypertrophy na zuciya mai shimfiɗa.
Alamun wakilci na matsa lamba na ɗakin iska, matsa lamba na ɗakin ruwa, da ma'aunin motsi na nama sun tabbatar da cewa matsa lamba ɗakin yana canza matsa lamba na ruwa, yana haifar da madaidaicin motsi na yanki na nama.b Yawan miƙewa na wakilci da madaidaicin ƙididdigewa ga sassan nama na yau da kullun (orange) da maɗauri (blue).c Bar jadawali yana nuna lokacin sake zagayowar (n = 19 yanka a kowace rukuni, daga aladu daban-daban), lokacin ƙanƙancewa (n = 18-19 yanka a kowace ƙungiya, daga aladu daban-daban), lokacin shakatawa (n = 19 yanka a kowace ƙungiya, daga aladu daban-daban)), girman motsin nama (n = 14 yanka / rukuni, daga aladu daban-daban), peak systolic veak da peak systolic veak = systolic veak = systolic veak da peak systolic. ƙimar (n = 14 (D0), 15 (D6) ) sassan / ƙungiyoyi) daga aladu daban-daban), T-gwajin ɗalibi mai wutsiya biyu bai nuna wani bambanci mai mahimmanci a cikin kowane ma'auni ba, yana nuna cewa waɗannan sigogi sun kasance masu tsayi a cikin kwanakin 6 na al'ada tare da overvoltage.Kuskuren sanduna suna wakiltar ma'anar ± daidaitaccen karkacewa.
a Bar jadawali ƙididdiga na NT-ProBNP maida hankali a cikin kafofin watsa labarai na al'adu daga zuciya yanka al'adu karkashin MT al'ada stretch (Norm) ko overstretching (OS) yanayi (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, da D4 MTOS) yanka / rukuni daga daban-daban aladu, Biyu-hanyar ANOVA aka yi idan aka kwatanta da al'ada;). a Bar jadawali ƙididdiga na NT-ProBNP maida hankali a cikin kafofin watsa labarai na al'adu daga zuciya yankan al'adu a karkashin MT al'ada stretch (Norm) ko overstretching (OS) yanayi (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, da D4 MTOS) yanka / rukuni daga daban-daban aladu, Biyu-hanyar ANOVA aka yi idan aka kwatanta da al'ada0;).Ƙididdigar ƙididdiga na NT-ProBNP maida hankali a cikin al'ada matsakaici daga zuciya yankan al'adu a karkashin yanayi na al'ada MT stretch (na 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 abubuwa biyu;**p <0,01 pо сравнению с нормальным растяжением). ** p <0.01 idan aka kwatanta da shimfiɗar al'ada). 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 rm), 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；**与手析；**与手析；* . Ƙididdigar ƙaddamarwar NT-ProBNP a cikin yankan zuciya da aka tsara a ƙarƙashin yanayin MT na al'ada (Norm) ko overstretch (OS) yanayi (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm和D4 MTOS) daga daban-daban ed tare da shimfidawa na yau da kullun, p <0.01).histogram Quantification na NT-ProBNP maida hankali a cikin zuciya yanka al'adu karkashin yanayi na al'ada MT stretch (na al'ada) ko overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) da D4 MTOS) yanka / rukuni daga daban-daban aladu, biyu-hanyar bincike na bambance-bambancen;**p <0,01 pо сравнению с нормальным растяжением). ** p <0.01 idan aka kwatanta da shimfiɗar al'ada). b Hotunan wakilai don yankan zuciya da aka lalata tare da troponin-T da WGA (hagu) da ƙididdige girman tantanin halitta (dama) (n = 330 (D6 MTOS), 369 (D6 MTNorm) sel / rukuni daga 10 daban-daban yanka daga aladu daban-daban, T-gwajin Student Biyu mai wutsiya an yi; 0**** p <10. b Hotunan wakilci don yankan zuciya da aka lalata tare da troponin-T da WGA (hagu) da ƙididdige girman tantanin halitta (dama) (n = 330 (D6 MTOS), 369 (D6 MTNorm) sel / rukuni daga 10 daban-daban yanka daga aladu daban-daban, T-gwajin Student Biyu mai wutsiya an yi; 0****p <10). b Репрезентативные изображения срезов сердца к (справа) (n = 330 (D6 MTOS), 369 (D6 MTNorm) юдента; ****p <0,0001 по сравнению с нормальным растяжением). b Hotunan wakilci na sassan zuciya da aka lalata tare da troponin-T da AZP (hagu) da ƙididdigar girman tantanin halitta (dama) (n = 330 (D6 MTOS), 369 (D6 MTNorm) sel / rukuni daga sassan 10 daban-daban daga aladu daban-daban, nau'i-nau'i biyu-biyu-T-gwajin 0 0). 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 ） 3）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 ） b Hotunan wakilai na yankan zuciya da aka lalata da calcarein-T da WGA (hagu) da girman tantanin halitta (dama) (n = 330 (D6 MTOS), 369 daga nau'i daban-daban 10 (D6 MTNorm)) Kwayoyin / 组, 两方法有尾学生 法有尾学生 法有尾学生 法有尾学生 法有尾学生 an gwada shi tare da al'ada 0. b Репрезентативные изображения срезов сердца. а) (n = 330 (D6 MTOS), 369 (D6 MTNorm) из 10 различных срезов от разных свиней Клетки/группа, двусторонтьте , двусторонть кри0; 01 по сравнению с нормальным растяжением). b Hotunan wakilci na sassan zuciya da aka lalata tare da troponin-T da AZP (hagu) da ƙididdige girman tantanin halitta (dama) (n = 330 (D6 MTOS), 369 (D6 MTNorm) daga sassa daban-daban 10 daga aladu daban-daban) Kwayoyin / rukuni, nau'i mai nau'i biyu na Student's t;****p <0.0001 idan aka kwatanta da iri na al'ada). c Hotunan wakilci na rana 0 da rana 6 MTOS zuciya yankakken immunolabeled ga troponin-T da NFATC4 da ƙididdigewa na fassarar NFATC4 zuwa tsakiya na CMs (n = 4 (D0), 3 (D6 MTOS) yanka / rukuni daga daban-daban aladu, Biyu-tailed Student yi t. - 5. c Hotunan wakilci na rana 0 da ranar 6 MTOS zuciya yankakken immunolabeled ga troponin-T da NFATC4 da ƙididdigewa na fassarar NFATC4 zuwa tsakiya na CMs (n = 4 (D0), 3 (D6 MTOS) yanka / rukuni daga daban-daban aladu, Biyu-tailed Stu ; - 0. c Репрезентативные изображения для срезов сердца 0 и 6 дней MTOS NFATC4 в ядра кавернозных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней тьюдента; *p <0,05). c Hotunan wakilci don sassan zuciya a 0 da 6 days MTOS, immunolabeled for troponin-T da NFATC4, da kuma ƙididdiga na NFATC4 canzawa a cikin tsakiya na cavernous Kwayoyin (n = 4 (D0), 3 (D6 MTOS) yanka / rukuni daga daban-daban aladu) yi biyu-tailed ttutest.*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易位至CM 细胞核的量化（n = 4 (D0)、3 (D6 MTOS) 切片/组, 进行双尾学生t 检验；*0) 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学生et 电影；*p <0.05). c Репрезентативные изображения срезов сердца MTOS на 0 6 день для ранслокации NFATC4 в ядра CM от разных свиней (n = 4 (D0), 3 (D6 MTOS) срез/группа, два- хвостатый t-критеридет; 0, 5. c Hotunan wakilci na yankan zuciya na MTOS a ranar 0 da 6 don troponin-T da NFATC4 immunolabeling da ƙididdigewa na NFATC4 fassarar a cikin tsakiya na CM daga daban-daban aladu (n = 4 (D0), 3 (D6 MTOS) yanka / rukuni, biyu-teiled t -criterion; 0 Student.Kuskuren sanduna suna wakiltar ma'anar ± daidaitaccen karkata.
Binciken bugun zuciya na fassara yana buƙatar ƙirar salon salula waɗanda ke haifar da yanayin yanayin zuciya daidai.A cikin wannan binciken, an ƙirƙiri na'urar CTCM da siffa wacce za ta iya motsa sassan zuciya.Tsarin CTCM ya haɗa da haɓakar injin lantarki da aka daidaita ta jiki da T3 da haɓakar ruwa na Dex.Lokacin da sassan zuciya na porcine suka fallasa ga waɗannan abubuwan, yuwuwar su, amincin tsarin su, ayyukan rayuwa, da bayanin rubutu ya kasance iri ɗaya da namarar zuciya bayan kwanaki 12 na al'ada.Bugu da kari, wuce gona da iri na nama na zuciya na iya haifar da hauhawar jini wanda ke haifar da hauhawar jini.Gabaɗaya, waɗannan sakamakon suna goyan bayan muhimmiyar rawar yanayin yanayin al'adar ilimin lissafi don kiyaye yanayin yanayin zuciya na yau da kullun da kuma samar da dandamali don tantance magunguna.
Abubuwa da yawa suna taimakawa wajen ƙirƙirar yanayi mafi kyau don aiki da rayuwa na cardiomyocytes.Mafi bayyanannen waɗannan abubuwan suna da alaƙa da (1) hulɗar tsakanin salula, (2) kuzarin lantarki, (3) abubuwan ban dariya, da (4) abubuwan da ke haifar da rayuwa.Ma'amalar tantanin halitta zuwa tantanin halitta na buƙatar hadaddun hanyoyin sadarwa mai girma uku na nau'ikan tantanin halitta masu goyan bayan matrix na waje.Irin wannan hadaddun hulɗar salon salula yana da wuya a sake ginawa a cikin vitro ta hanyar al'adu na nau'ikan tantanin halitta guda ɗaya, amma ana iya samun sauƙi ta amfani da yanayin organotypic na sassan zuciya.
Ƙwararren injiniya da ƙarfin lantarki na cardiomyocytes suna da mahimmanci don kiyaye phenotype na zuciya33,34,35.Duk da yake an yi amfani da haɓakar injiniya sosai don daidaitawa na hiPSC-CM da maturation, kyawawan karatu da yawa kwanan nan sun yi ƙoƙarin haɓaka injinan yankan zuciya a cikin al'ada ta amfani da lodin uniaxial.Wadannan nazarin sun nuna cewa 2D uniaxial inji loading yana da tasiri mai kyau a kan phenotype na zuciya a lokacin al'ada.A cikin waɗannan karatun, an ɗora sassan zuciya tare da ƙarfin tensile isometric17, linzamin auxotonic loading18, ko kuma an sake sake zagayowar bugun zuciya ta hanyar amfani da martani mai juzu'i da motsin tashin hankali.Duk da haka, waɗannan hanyoyin suna amfani da shimfiɗar nama na uniaxial ba tare da inganta muhalli ba, wanda ke haifar da danne yawancin kwayoyin cututtukan zuciya ko wuce gona da iri na kwayoyin da ke da alaƙa da amsawar mikewa mara kyau.CTCM da aka bayyana anan yana ba da abin motsa jiki na lantarki na 3D wanda ke kwaikwayi yanayin zagayowar zuciya dangane da lokacin zagayowar da kuma shimfidar jiki (miƙewar 25%, 40% systole, 60% diastole, da bugun 72 a minti daya).Ko da yake wannan motsi na inji mai girma uku kawai bai isa ba don kula da mutuncin nama, haɗuwa da motsa jiki da motsa jiki ta hanyar amfani da T3 / Dex ana buƙata don kiyaye lafiyar nama, aiki, da mutunci.
Abubuwan ban dariya suna taka muhimmiyar rawa wajen daidaita yanayin halittar zuciyar manya.An bayyana wannan a cikin nazarin HiPS-CM wanda aka ƙara T3 da Dex zuwa kafofin watsa labarai na al'ada don haɓaka balaga tantanin halitta.T3 na iya rinjayar jigilar amino acid, sugars da calcium a cikin membranes cell36.Bugu da ƙari, T3 yana inganta maganganun MHC-α da kuma MHC-β downregulation, yana inganta samar da sauri myofibrils a cikin balagagge cardiomyocytes idan aka kwatanta da jinkirin myofibrils a cikin CM tayi.Rashin ƙarancin T3 a cikin marasa lafiya na hypothyroid yana haifar da asarar ƙungiyoyin myofibrillar da rage yawan ci gaban sautin37.Dex yana aiki akan masu karɓa na glucocorticoid kuma an nuna shi don ƙara ƙarfin zuciya na zuciya a cikin keɓaɓɓen zukata;38 ana tsammanin wannan haɓakar yana da alaƙa da tasiri akan shigar da ajiyar kuɗin calcium (SOCE) 39,40.Bugu da ƙari, Dex yana ɗaure ga masu karɓar sa, yana haifar da amsa mai zurfi na ciki wanda ke hana aikin rigakafi da kumburi30.
Sakamakonmu yana nuna cewa haɓakar injina ta jiki (MS) ya inganta aikin al'ada gabaɗaya idan aka kwatanta da Ctrl, amma ya kasa kiyaye iyawa, daidaiton tsari, da maganganun zuciya sama da kwanaki 12 a cikin al'ada.Idan aka kwatanta da Ctrl, ƙari na T3 da Dex zuwa al'adun CTCM (MT) sun inganta iyawa da kiyaye bayanan bayanan rubutu iri ɗaya, daidaiton tsari, da ayyukan rayuwa tare da sabbin nama na zuciya na kwanaki 12.Bugu da ƙari, ta hanyar sarrafa nauyin shimfiɗar nama, an ƙirƙiri samfurin hypertrophy na zuciya da aka haifar da hyperextension ta hanyar amfani da STCM, yana nuna bambancin tsarin STCM.Ya kamata a lura cewa ko da yake gyare-gyaren zuciya da fibrosis yawanci sun haɗa da gabobin da ba su da kyau wanda kwayoyin halitta zasu iya samar da cytokines masu dacewa da kuma phagocytosis da sauran abubuwan gyaran gyare-gyare, sassan zuciya na iya har yanzu kwaikwayon tsarin fibrotic don mayar da martani ga damuwa da rauni.cikin myofibroblasts.An kimanta wannan a baya a cikin wannan ƙirar yanki na zuciya.Ya kamata a lura cewa ana iya daidaita sigogin CTCM ta hanyar canza matsa lamba / girman wutar lantarki da mitar don daidaita yanayi da yawa kamar tachycardia, bradycardia, da goyan bayan bugun jini na inji (zuciya da ba ta da kayan inji).Wannan ya sa tsarin ya zama matsakaicin kayan aiki don gwajin ƙwayoyi.Ƙarfin CTCM don yin ƙirƙira haɓakar hawan jini na zuciya wanda ke haifar da wuce gona da iri yana buɗe hanya don gwada wannan tsarin don keɓancewar jiyya.A ƙarshe, binciken da aka yi a yanzu ya nuna cewa shimfiɗar injiniya da motsa jiki na ban dariya suna da mahimmanci don kiyaye al'adun sassan nama na zuciya.
Kodayake bayanan da aka gabatar a nan sun nuna cewa CTCM dandamali ne mai ban sha'awa don yin ƙirar myocardium mara kyau, wannan hanyar al'ada tana da wasu iyakoki.Babban ƙayyadaddun al'adun CTCM shine cewa yana sanya ci gaba da damuwa na inji akan yankan, wanda ke hana ikon sa ido kan ƙanƙancewar yanki na zuciya yayin kowane zagayowar.Bugu da ƙari, saboda ƙananan sassan sassan zuciya (7 mm), ikon kimanta aikin systolic a waje da tsarin al'ada ta amfani da na'urori masu auna karfin gargajiya yana iyakance.A cikin rubutun hannu na yanzu, mun shawo kan wannan iyaka ta wani yanki ta hanyar kimanta ƙarfin lantarki a matsayin mai nunin aikin kwangila.Koyaya, wannan ƙayyadaddun yana buƙatar ƙarin aiki kuma ana iya magance shi nan gaba ta hanyar gabatar da hanyoyin don saka idanu na gani na aikin yankan zuciya a cikin al'ada, kamar taswirar gani ta amfani da alli da dyes masu ƙarfi.Wani ƙayyadaddun CTCM shine cewa ƙirar aiki baya sarrafa damuwa ta jiki (saɓani da kaya).A cikin CTCM, an jawo matsa lamba a wasu wurare dabam dabam don sake haifar da 25% shimfidar jiki a cikin diastole (cikakken shimfiɗa) da systole (tsawon ƙanƙancewa yayin ƙarfafawar lantarki) a cikin kyallen takarda masu girma sosai.Ya kamata a cire wannan iyakance a cikin ƙira na CTCM na gaba ta hanyar isassun matsa lamba akan nama na zuciya daga ɓangarorin biyu da kuma amfani da madaidaicin matsi-ƙarar alaƙar da ke faruwa a cikin ɗakunan zuciya.
Gyaran da aka haifar da wuce gona da iri da aka ruwaito a cikin wannan rubutun yana iyakance ga kwaikwayi siginar hypertrophic hyperstretch.Don haka, wannan samfurin zai iya taimakawa wajen nazarin siginar hypertrophic mai shimfiɗawa ba tare da buƙatar abubuwan ban dariya ko jijiyoyi (wanda ba ya wanzu a cikin wannan tsarin).Ana buƙatar ƙarin karatu don ƙara yawan CTCM, alal misali, haɗakarwa tare da ƙwayoyin rigakafi, rarraba abubuwan jin daɗi na plasma, da innervation lokacin da aka haɗa tare da ƙwayoyin neuronal zasu inganta yiwuwar yin samfurin cututtuka tare da CTCM.
An yi amfani da aladu goma sha uku a cikin wannan binciken.Dukkan hanyoyin dabba an yi su daidai da jagororin hukumomi kuma Jami'ar Louisville Cibiyar Kula da Dabbobi da Kwamitin Amfani ta amince da su.An danne baka aortic kuma an lullube zuciyar da 1 L na bakararre na zuciya (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 cututtukan zuciya mai sanyi mai sanyi har sai an kai su dakin gwaje-gwaje akan kankara wanda yawanci <10 min. An adana zukata a cikin maganin cututtukan zuciya mai sanyi mai sanyi har sai an kai su dakin gwaje-gwaje akan kankara wanda yawanci <10 min. сердца хранили в ледяном кардиоплегическом растворе до транспортиртировки в лабораторию на льду, что обымы. An adana zuciyoyin a cikin maganin cututtukan zuciya mai sanyi mai sanyi har sai an kai su dakin gwaje-gwaje akan kankara, wanda yawanci yana ɗaukar <10 min.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. Ci gaba da zukata kan kankara cardioplegia har sai an kai su dakin gwaje-gwaje akan kankara, yawanci <10 min.
An ƙirƙira na'urar CTCM a cikin software na SolidWorks mai taimakon kwamfuta (CAD).Ƙungiyoyin al'adu, masu rarrabawa da ɗakunan iska an yi su ne da filastik CNC mai tsabta.Zoben baya na diamita na 7mm an yi shi da babban yawa polyethylene (HDPE) a tsakiya kuma yana da tsagi na o-ring don ɗaukar zoben silicone o-ring da ake amfani da shi don rufe kafofin watsa labarai a ƙasa.Silica membrane na bakin ciki yana raba ɗakin al'ada daga farantin rabuwa.Membran silicone an yanke Laser daga takardar silicone mai kauri 0.02 inci kuma yana da taurin 35A.Gas ɗin silicone na ƙasa da saman an yanke Laser daga takardar silicone mai kauri 1/16 ″ kuma suna da taurin 50A.316L bakin karfe sukurori da reshe kwayoyi ana amfani da su ɗaure toshe da kuma haifar da wani airtight hatimi.
An kera kwamitocin da'ira da aka keɓe (PCB) don haɗawa da tsarin C-PACE-EM.Ana haɗa kwas ɗin haɗin na'ura na injin Swiss akan PCB zuwa na'urorin lantarki na graphite ta wayoyin jan ƙarfe da aka ɗora da azurfa da sukurori 0-60 na tagulla da aka cusa cikin na'urorin lantarki.Ana sanya allon da'ira da aka buga a cikin murfin firinta na 3D.
Na'urar CTCM tana sarrafa na'urar mai ɗaukar hoto mai ɗaukar hoto (PPD) wanda ke haifar da matsa lamba mai sarrafawa mai kama da zagayowar zuciya.Yayin da matsin lamba a cikin ɗakin iska ya karu, murfin silicone mai sassauƙa yana faɗaɗa sama, yana tilasta matsakaici a ƙarƙashin wurin nama.Sa'an nan kuma za a shimfiɗa yankin nama ta hanyar wannan fitar da ruwa, yana yin kwaikwayon faɗaɗawar zuciya a lokacin diastole.A kololuwar shakatawa, an yi amfani da kuzarin lantarki ta hanyar lantarki na graphite, wanda ya rage matsin lamba a cikin ɗakin iska kuma ya haifar da raguwar sassan nama.A cikin bututu akwai bawul ɗin hemostatic tare da firikwensin matsa lamba don gano matsa lamba a cikin tsarin iska.Ana amfani da matsi da firikwensin matsa lamba akan mai tattara bayanai da aka haɗa da kwamfutar tafi-da-gidanka.Wannan yana ba da damar ci gaba da lura da matsa lamba a cikin ɗakin gas.Lokacin da aka kai matsakaicin matsa lamba na ɗakin (misali 80 mmHg, 140 mmHg OS), an ba da umarnin na'urar samun bayanan don aika sigina zuwa tsarin C-PACE-EM don samar da siginar wutar lantarki na biphasic don 2 ms, saita zuwa 4 V.
An samo sassan zuciya da kuma yanayin al'adu a cikin rijiyoyin 6 kamar haka: Canja wurin zukata da aka girbe daga jirgin ruwa zuwa tire mai sanyi (4 ° C.) cardioplegia.An keɓe ventricle na hagu tare da ruwa maras kyau kuma an yanke shi cikin guda 1-2 cm3.Wadannan tubalan nama an haɗa su zuwa goyon bayan nama tare da manne nama kuma an sanya su a cikin wanka na microtome na jiki mai girgiza wanda ke dauke da maganin Tyrode da ci gaba da oxygenated (3 g / L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g) . ), 6 mM KCl (0.1400 g.M ), 1-447 g. S (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 kauri na 300 µm a mitar 80 Hz, girman girgiza a kwance na 2 mm, da ƙimar gaba na 0.03 mm/s.An kewaye wankan nama da ƙanƙara don kiyaye ruwan sanyi kuma ana kiyaye zafin jiki a 4 ° C.Canja wurin sassan nama daga wanka na microtome zuwa wanka mai shiryawa mai ɗauke da ci gaba da maganin Tyrode oxygenated akan kankara har sai an sami isassun sassan farantin al'ada ɗaya.Don al'adun transwell, sassan nama an haɗa su zuwa bakararre 6 mm fadi da tallafin polyurethane kuma an sanya su a cikin 6 ml na ingantaccen matsakaici (matsakaicin 199, ƙarin 1x ITS, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-alkaline da 2X ƙwayoyin cuta-antifungal).An yi amfani da ƙarfin lantarki (10 V, mita 1.2 Hz) zuwa sassan nama ta hanyar C-Pace.Don yanayin TD, an ƙara sabo T3 da Dex a 100 nM da 1 μM a kowane canji na matsakaici.Matsakaicin yana cike da iskar oxygen kafin maye gurbin sau 3 a rana.An al'ada sassan nama a cikin incubator a 37 ° C da 5% CO2.
Don al'adun CTCM, an sanya sassan nama akan firinta na 3D na al'ada a cikin kwano na Petri mai ɗauke da ingantaccen maganin Tyrode.An tsara na'urar don ƙara girman yanki na zuciya da kashi 25% na yankin zoben tallafi.Ana yin haka ne don kada sassan zuciya su tashi bayan an canja su daga maganin Tyrode zuwa matsakaici da lokacin diastole.Yin amfani da manne na tarihi, an gyara sassan 300 µm kauri akan zoben tallafi na 7 mm a diamita.Bayan haɗa sassan nama zuwa zoben tallafi, yanke sassan nama da suka wuce kima kuma sanya sassan nama da aka haɗe a baya a cikin wanka na maganin Tyrode akan kankara (4 ° C) har sai an shirya isassun sassan don na'ura ɗaya.Jimlar lokacin aiki don duk na'urori bai kamata ya wuce sa'o'i 2 ba.Bayan an haɗa sassan nama guda 6 zuwa zoben tallafi, an haɗa na'urar CTCM.An riga an cika ɗakin al'adun CTCM da 21 ml pre-oxygenated matsakaici.Canja wurin sassan nama zuwa ɗakin al'ada kuma a hankali cire duk wani kumfa mai iska tare da pipette.Sa'an nan kuma ana jagorantar sashin nama zuwa cikin rami kuma a danna shi a hankali.A ƙarshe, sanya hular lantarki akan na'urar kuma canja wurin na'urar zuwa incubator.Sannan haɗa CTCM zuwa bututun iska da tsarin C-PACE-EM.Mai kunna pneumatic yana buɗewa kuma bawul ɗin iska yana buɗe CTCM.An saita tsarin C-PACE-EM don isar da 4V a 1.2 Hz yayin takin biphasic don 2 ms.Ana canza matsakaici sau biyu a rana kuma ana canza na'urorin lantarki sau ɗaya a rana don guje wa tarin graphite akan wayoyin.Idan ya cancanta, ana iya cire sassan nama daga rijiyoyin al'adunsu don fitar da duk wani kumfa mai yuwuwa ya faɗo a ƙarƙashinsu.Don yanayin jiyya na MT, an ƙara T3/Dex sabo tare da kowane canji na matsakaici tare da 100 nM T3 da 1 μM Dex.An haɓaka na'urorin CTCM a cikin incubator a 37°C da 5% CO2.
Don samun shimfidar wurare na yankan zuciya, an haɓaka tsarin kyamara na musamman.An yi amfani da kyamarar SLR (Canon Rebel T7i, Canon, Tokyo, Japan) tare da Navitar Zoom 7000 18-108mm macro lens (Navitar, San Francisco, CA).An yi hangen nesa a cikin zafin jiki bayan maye gurbin matsakaici tare da sabon matsakaici.Ana ajiye kyamarar a kusurwar 51° kuma ana yin rikodin bidiyo a firam 30 a sakan daya.Na farko, an yi amfani da software na buɗe tushen (MUSCLEMOTION43) tare da Image-J don ƙididdige motsin yankan zuciya.An ƙirƙiri abin rufe fuska ta amfani da MATLAB (MathWorks, Natick, MA, Amurka) don ayyana yankuna masu sha'awar bugun zuciya don guje wa hayaniya.Ana amfani da abin rufe fuska da hannu zuwa duk hotuna a cikin jerin firam sannan a wuce su zuwa filogin MUSCLEMOTION.Motsin Muscle yana amfani da matsakaicin ƙarfin pixels a kowane firam don ƙididdige motsinsa dangane da firam ɗin tunani.An yi rikodin bayanai, tacewa kuma an yi amfani da su don ƙididdige lokacin sake zagayowar da kuma tantance shimfiɗar nama yayin zagayowar zuciya.Bidiyon da aka yi rikodi an aiwatar da shi ta hanyar amfani da tacewa na dijital sifili-lokaci na farko.Don ƙididdige shimfiɗar nama (kolo-zuwa-ƙalo), an gudanar da bincike-bincike-kololuwa don bambanta tsakanin kololuwa da magudanar ruwa a cikin siginar da aka yi rikodi.Bugu da kari, ana aiwatar da detrending ta amfani da tsari na 6th polynomial don kawar da firar sigina.An ƙirƙiri lambar shirin a cikin MATLAB don ƙayyade motsin nama na duniya, lokacin zagayowar, lokacin hutu, da lokacin ƙanƙancewa (Ƙarin Tsarin Shirin 44).
Don nazarin damuwa, ta yin amfani da bidiyo iri ɗaya da aka ƙirƙira don ƙimanta shimfiɗar injina, mun fara gano hotuna biyu masu wakiltar kololuwar motsi (mafi girma (na sama) da mafi ƙasƙanci (ƙananan) maki na motsi) bisa ga software MUSCLEMOTION.Sa'an nan kuma muka rarraba yankunan nama kuma muka yi amfani da wani nau'i na shading algorithm zuwa nama da aka raba (Ƙarin Hoton 2a).Sa'an nan kuma an raba nau'in nau'in nau'in nau'in nau'in nau'in nau'i guda goma, kuma an ƙididdige damuwa akan kowane saman ta hanyar amfani da ma'auni mai zuwa: Strain = (Sup-Sdown) / Sdown, inda Sup da Sdown su ne nisa na siffar daga sama da inuwa na kasa na masana'anta, bi da bi (Ƙarin Fig. .2b).
An gyara sassan zuciya a cikin 4% paraformaldehyde na awanni 48.Kafaffen kyallen takarda an bushe su cikin 10% da 20% sucrose na awa 1, sannan a cikin 30% sucrose na dare.Sa'an nan an saka sassan a cikin mafi kyawun mahallin zafin jiki (OCT fili) kuma a hankali a daskare su a cikin wankan isopentane/bushewar kankara.Ajiye OCT tubalan a -80 °C har sai rabuwa.An shirya zane-zane a matsayin sassan da kauri na 8 μm.
Don cire OCT daga sassan zuciya, zazzage nunin faifai akan toshe mai dumama a 95 ° C na 5 min.Ƙara 1 ml PBS zuwa kowane nunin faifai kuma a sanya shi na mintuna 30 a zafin jiki, sa'an nan kuma kuɗa sassan ta hanyar saita 0.1% Triton-X a cikin PBS na minti 15 a zafin jiki.Don hana ƙwayoyin rigakafin da ba takamaiman ba daga ɗaure ga samfurin, ƙara 1 ml na 3% BSA bayani zuwa nunin faifai kuma a sanya shi na awa 1 a zafin jiki.An cire BSA kuma an wanke zane-zane tare da PBS.Alama kowane samfurin da fensir.Magungunan rigakafi na farko (diluted 1:200 a cikin 1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) da troponin-T (Thermo Scientific; #MA5-12960) an kara sama da mintuna 90, sa'an nan kuma a yi amfani da magungunan rigakafi na biyu na BSA a cikin 1% ko BSA. 488 (Thermo Scientific; #A16079), da zomo Alexa Fluor 594 (Thermo Scientific; #T6391) don ƙarin 90 mintuna An wanke sau 3 tare da PBS Don bambanta tabo daga bango, mun yi amfani da antibody na sakandare kawai a matsayin sarrafawa. nification) da microscope na Keyence tare da haɓaka 40x.
WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) a 5 μg/ml a cikin PBS an yi amfani da shi don lalata WGA kuma an yi amfani da shi zuwa sassan da aka gyara don minti 30 a dakin da zafin jiki.Sannan an wanke faifai tare da PBS kuma an saka baƙar fata Sudan a kowace faifai kuma a sanya su na tsawon mintuna 30.Sannan an wanke nunin faifai tare da PBS kuma an ƙara matsakaicin saka vectashield.An hango nunin faifai akan ma'aunin ma'aunin mahimmin mahimmin mahimmin mahimmin abu a girman girman 40x.
An cire OCT daga samfuran kamar yadda aka bayyana a sama.Bayan cire OCT, nutsar da nunin faifai a cikin maganin Bouin na dare.Bayan haka, an wanke zane-zane da ruwa mai narkewa na awa 1 sannan a sanya shi a cikin maganin fuchsin na Bibrich aloe acid na minti 10.Sa'an nan kuma an wanke zane-zane da ruwa mai tsabta kuma an sanya shi a cikin wani bayani na 5% phosphomolybdenum / 5% phosphotungstic acid na minti 10.Ba tare da kurkura ba, canja wurin nunin faifai kai tsaye zuwa cikin maganin blue aniline na minti 15.Sa'an nan kuma an wanke zane-zane tare da ruwa mai tsabta kuma an sanya shi a cikin 1% acetic acid bayani na minti 2.An bushe faifai a cikin 200 N ethanol kuma an canza shi zuwa xylene.An zana zane-zanen zane-zane ta amfani da microscope na Keyence tare da manufar 10x.An ƙididdige adadin yankin Fibrosis ta amfani da software na Keyence Analyzer.
CyQUANT™ MTT Kwayar cutar Kwayoyin Halitta (Invitrogen, Carlsbad, CA), lambar kasida V13154, bisa ga ka'idar masana'anta tare da wasu gyare-gyare.Musamman, an yi amfani da naushin tiyata tare da diamita na 6 mm don tabbatar da girman nau'in nama a yayin nazarin MTT.An jera nama daban-daban a cikin rijiyoyin faranti mai rijiya 12 mai ɗauke da MTT bisa ka'idar masana'anta.Ana shigar da sassan a 37 ° C. na tsawon sa'o'i 3 kuma nama mai rai yana metabolizes da MTT substrate don samar da fili na formazan purple.Sauya maganin MTT tare da 1 ml DMSO kuma a sanya shi a 37 ° C na mintina 15 don cire nau'in launin ruwan hoda daga sassan zuciya.An diluted samfurori 1:10 a cikin DMSO a cikin 96-da kyau bayyanannun faranti na ƙasa da launi mai launin shuɗi da 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 watsa labaru na yanki na zuciya tare da kafofin watsa labaru masu ƙunshe da 1 μCi / ml [5-3H] -glucose (Moravek Biochemicals, Brea, CA, USA) don amfani da glucose kamar yadda aka bayyana a baya.Bayan awa 4 na shiryawa, ƙara 100 µl na matsakaici zuwa buɗaɗɗen microcentrifuge bututu mai ɗauke da 100 µl na 0.2 N HCl.Sannan an sanya bututun a cikin bututun scintillation mai ɗauke da 500 μl na dH2O don ƙafe [3H] 2O na awanni 72 a 37°C.Sa'an nan cire microcentrifuge bututu daga scintillation tube da kuma ƙara 10 ml na scintillation ruwa.An yi kirga scintillation ta amfani da Tri-Carb 2900TR mai nazarin scintillation na ruwa (Kamfanin Packard Bioscience, Meriden, CT, Amurka).An ƙididdige yawan amfani da glucose a cikin la'akari da [5-3H] - takamaiman ayyuka na glucose, rashin cika ma'auni da bango, dilution na [5-3H] - zuwa glucose mara alama, da ingancin scintillation counter.An daidaita bayanan zuwa yawan sassan zuciya.
Bayan haɗin nama a cikin Trizol, an ware RNA daga sassan zuciya ta amfani da Qiagen miRNeasy Micro Kit #210874 bisa ga ka'idar masana'anta.An yi shirye-shiryen ɗakin karatu na RNAsec, jeri da nazarin bayanai kamar haka:
An yi amfani da 1 μg na RNA kowane samfurin azaman kayan farawa don shirya ɗakin karatu na RNA.An ƙirƙiro dakunan karatu na jeri ta amfani da NEBNext UltraTM RNA Library Preparation Kit don Illumina (NEB, Amurka) bin shawarwarin masana'anta, kuma an ƙara lambobin fihirisa zuwa jerin halayen kowane samfurin.A taƙaice, an tsarkake mRNA daga jimlar RNA ta amfani da beads na maganadisu da aka haɗe da poly-T oligonucleotides.Ana aiwatar da rarrabuwar kawuna ta amfani da cations masu yawa a babban zafin jiki a cikin NEBNext First Strand Synthesis Reaction Buffer (5X).An haɗa cDNA na farko ta amfani da bazuwar hexamer primers da M-MuLV reverse transcriptase (RNase H-).Sa'an nan kuma an haɗa cDNA na biyu ta amfani da DNA polymerase I da RNase H. Sauran overhangs an canza su zuwa ƙarewa ta hanyar exonuclease/polymerase aiki.Bayan karewa na 3′ ƙarshen DNA ɗin, adaftar NEBNext tare da tsarin madauki na gashi an haɗa shi don shirya shi don haɓakawa.Don zaɓin guntun cDNA na tsayin da aka fi so 150-200 bp.An tsarkake gutsuttsuran ɗakin karatu ta amfani da tsarin AMPure XP (Beckman Coulter, Beverly, Amurka).Bayan haka, 3 μl USER Enzyme (NEB, Amurka) tare da cDNA da aka zaɓa mai girma wanda aka haɗa tare da adaftan an yi amfani da shi na mintuna 15 a 37°C sannan kuma na mintuna 5 a 95°C kafin PCR.Daga nan aka yi PCR ta amfani da Phusion High-Fidelity DNA polymerase, PCR na duniya, da firamare na Index (X).A ƙarshe, an tsarkake samfuran PCR (tsarin AMPure XP) da ƙimar ɗakin karatu da aka tantance akan tsarin Agilent Bioanalyzer 2100.Sa'an nan an jera ɗakin karatu na cDNA ta amfani da mabiyi Novaseq.Fayilolin hoto na asali daga Illumina an canza su zuwa ɗanyen karantawa ta amfani da CASAVA Base Calling.Ana adana danyen bayanai a cikin fayilolin FASTQ(fq) waɗanda ke ƙunshe da jerin abubuwan karantawa da daidaitattun halayen tushe.Zaɓi HISAT2 don dacewa da matakan da aka tace ana karantawa zuwa ga genome na Sscrofa11.1.Gabaɗaya, HISAT2 tana goyan bayan genomes na kowane girman, gami da genomes mafi girma fiye da sansanonin biliyan 4, kuma ana saita ƙimar tsoho don yawancin sigogi.Ana iya daidaita karatun da aka karanta daga bayanan RNA Seq ta hanyar amfani da HISAT2, tsarin mafi sauri a halin yanzu, tare da daidaito iri ɗaya ko mafi inganci fiye da kowace hanya.
Yawancin rubuce-rubuce kai tsaye yana nuna matakin maganganun kwayoyin halitta.Ana ƙididdige matakan maganganun kwayoyin halitta ta hanyar ɗimbin rubuce-rubuce (ƙididdigar ƙididdiga) masu alaƙa da kwayoyin halitta ko exons.Adadin karantawa yayi daidai da matakan maganganun kwayoyin halitta, tsawon kwayoyin halitta, da zurfin jeri.An ƙididdige FPKM (guts ɗin kowane nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i guntu)) kuma an ƙaddara ƙimar P-darajar magana ta amfani da kunshin DSeq2.Sa'an nan kuma muka ƙididdige ƙimar gano ƙarya (FDR) ga kowane ƙimar P ta amfani da tsarin Benjamini-Hochberg9 dangane da ginanniyar aikin R-aiki "p.adjust".
RNA da aka ware daga sassan zuciya an canza su zuwa cDNA a matakin 200 ng/μl ta amfani da SuperScript IV Vilo Master mix daga Thermo (Thermo, cat. no. 11756050).An yi kididdigar RT-PCR ta amfani da Applied Biosystems Endura Plate Microamp 384-da kyau farantin amsa (Thermo, cat. no. 4483319) da microamp na gani na gani (Thermo, cat. no. 4311971).Cakudar amsa ta ƙunshi 5 µl Taqman Fast Advanced Master mix (Thermo, cat # 4444557), 0.5 µl Taqman Primer da 3.5 µl H2O gauraye kowace rijiyar.An gudanar da madaidaicin zagayowar qPCR kuma an auna ƙimar CT ta amfani da kayan aikin PCR da aka Aiwatar da su Quantstudio 5 (modul 384-riji; samfur # A28135).An siyi abubuwan farko na Taqman daga Thermo (GAPDH (Ss03375629_u1), PARP12 (Ss06908795_m1), PKDCC (Ss06903874_m1), CYGB (Ss06900188_m1), RGL1 (Ss03375629_u1) mH), GATA4 (Ss03383805_u1), GJA1 (Ss03374839_u1), COL1A2 (Ss03375009_u1)
An tantance sakin watsa labarai na NT-ProBNP ta amfani da NT-ProBNP kit (alade) (Cat. No. MBS2086979, MyBioSource) bisa ga ka'idar masana'anta.A taƙaice, 250 µl na kowane samfurin da ma'auni an ƙara shi cikin kwafi ga kowace rijiya.Nan da nan bayan ƙara samfurin, ƙara 50 µl na Assay Reagent A ga kowace rijiya.A hankali a girgiza farantin kuma a rufe da abin rufewa.Sannan an sanya allunan a zazzabi na 37 ° C na awa 1.Sannan azuba maganin sannan a wanke rijiyoyin sau 4 tare da 350 µl na maganin 1X, azuba ruwan wankan na tsawon mintuna 1-2 kowane lokaci.Sa'an nan kuma ƙara 100 µl na Assay Reagent B a kowace rijiya kuma a hatimi da farantin karfe.An girgiza kwamfutar hannu a hankali kuma an sanya shi a 37 ° C na minti 30.A nemi maganin kuma a wanke rijiyoyin sau 5 tare da 350 µl na 1X maganin wankin.Ƙara 90 µl na maganin substrate a kowace rijiyar kuma rufe farantin.Sanya farantin a zazzabi na 37 ° C na minti 10-20.Ƙara Magani 50 µl Tsaya zuwa kowace rijiya.Nan take aka auna farantin ta amfani da na'urar karanta farantin Cytation (BioTek) wanda aka saita a 450 nm.
An yi nazarin wutar lantarki don zaɓar girman rukuni wanda zai ba da> 80% iko don gano cikakken canji na 10% a cikin siga tare da ƙimar kuskuren Nau'in I na 5%. An yi nazarin wutar lantarki don zaɓar girman rukuni wanda zai ba da> 80% iko don gano cikakken canji na 10% a cikin siga tare da ƙimar kuskuren Nau'in I na 5%. Анализ мощности был выполнен для выбора размеров групп, которые обеспечат > 80% ения параметра с 5% частотой ошибок типа I. An yi nazarin wutar lantarki don zaɓar girman rukuni waɗanda za su ba da> 80% iko don gano cikakken canjin ma'auni na 10% tare da 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% менения параметров и 5% частоты ошибок типа I. An yi nazarin wutar lantarki don zaɓar girman rukuni wanda zai samar da> 80% iko don gano cikakken canjin siga 10% da nau'in kuskuren nau'in 5% na I.An zaɓi sassan nama ba da gangan ba kafin gwajin.Dukkanin nazarin sun kasance makafi kuma an yanke samfuran kawai bayan an bincika duk bayanan.An yi amfani da software na GraphPad Prism (San Diego, CA) don yin duk nazarin ƙididdiga. Ga duk kididdiga, p-darajar an yi la'akari da mahimmanci a ƙimar <0.05. Ga duk kididdiga, p-darajar an yi la'akari da mahimmanci a ƙimar <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Ga duk kididdiga, p-darajar an yi la'akari da 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 kididdiga, p-darajar an yi la'akari da mahimmanci a ƙimar <0.05.An yi t-gwajin ɗalibi mai wutsiya biyu akan bayanan tare da kwatancen 2 kawai.An yi amfani da ANOVA ta hanya ɗaya ko biyu don tantance mahimmanci tsakanin ƙungiyoyi da yawa.Lokacin yin gwaje-gwaje na baya-bayan nan, an yi amfani da gyaran Tukey don lissafin kwatancen da yawa.Bayanan RNAsec yana da abubuwan ƙididdiga na musamman lokacin ƙididdige FDR da p.daidaita kamar yadda aka bayyana a cikin sashin Hanyoyi.
Don ƙarin bayani game da ƙira na nazari, duba Ƙididdigar Rahoton Bincike mai alaƙa da wannan labarin.