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Hakenewe sisitemu yizewe ya in vitro ishobora kongera neza imiterere y’umubiri w’umutima kugira ngo ikoreshwe mu gupima imiti. Kuba hari uburyo buke bwo gupima ingingo z’umutima bw’umuntu bwatumye habaho ibisobanuro bitari byo ku ngaruka z’imiti y’umutima. Aha, twakoze icyitegererezo cyo gupima ingingo z’umutima (CTCM) gikangura ibice by’umutima mu buryo bw’amashanyarazi kandi kikagenda gitera imbaraga mu gihe cy’ibyiciro bya systolic na diastolic by’umutima. Nyuma y’iminsi 12 yo gupima, ubu buryo bwarushijeho kunoza ubushobozi bw’ibice by’umutima, ariko ntibwakomeje neza imiterere yabyo. Kubwibyo, nyuma yo gusuzuma molekile nto, twasanze ko kongeramo 100 nM triiodothyronine (T3) na 1 μM dexamethasone (Dex) mu buryo bwacu byakomeje imiterere mito y’ibice mu minsi 12. Hamwe no kuvura T3/Dex, sisitemu ya CTCM yakomeje imiterere ya transcription, ubushobozi bwo kubaho, imikorere ya metabolism, n’ubushobozi bw’imiterere ku rwego rumwe n’ubw’ibice by’umutima bishya mu minsi 12. Byongeye kandi, kunagura cyane ingingo z'umutima mu muco bitera ibimenyetso by'umutima bikabije, bigatanga ibimenyetso byerekana ubushobozi bwa CTCM bwo kwigana indwara ziterwa no kunanuka k'umutima. Muri make, CTCM ishobora kwerekana imiterere y'umubiri n'indwara z'umutima mu muco mu gihe kirekire, bigatuma habaho gusuzuma imiti neza.
Mbere y'ubushakashatsi mu by'ubuvuzi, hakenewe sisitemu zizewe za "in vitro" zishobora kongera neza imiterere y'umubiri w'umutima w'umuntu. Izi sisitemu zigomba kwigana uburyo umutima uhindagurika, umuvuduko w'umutima, n'imiterere y'amashanyarazi. Inyamaswa zikunze gukoreshwa nk'urubuga rwo gusuzuma imiterere y'umutima, zikizewe cyane mu kugaragaza ingaruka z'imiti mu mutima w'umuntu1,2. Amaherezo, "Ideal Cardiac Tissue Experimental Model (CTCM)" ni icyitegererezo cyumvikana cyane kandi cyihariye ku buryo butandukanye bwo kuvura no kuvura, kikongera neza imiterere y'umubiri n'indwara z'umutima w'umuntu3. Kutagira ubwo buryo bigabanya kuvumburwa kw'uburyo bushya bwo kuvura indwara z'umutima4,5 kandi byatumye habaho uburozi bw'umutima n'imiti nk'impamvu ikomeye yo kuva ku isoko6.
Mu myaka icumi ishize, imiti umunani idakoresha umutima yakuwe mu ikoreshwa ry’ubuvuzi kuko itera igihe kirekire cya QT bigatera arrhythmias y’imitsi y’umutima n’urupfu rutunguranye7. Bityo, hakenewe ingamba zizewe zo gusuzuma mbere yo kuvura kugira ngo hamenyekane akamaro k’umutima n’uburozi. Gukoresha vuba aha imiti ikomoka ku ngirabuzima fatizo z’umutima (hiPS-CM) iterwa n’abantu mu gusuzuma no gupima uburozi bitanga igisubizo cy’igice cy’iki kibazo. Ariko, kuba hiPS-CM zitarakura ndetse no kutagira imiterere myinshi y’uturemangingo tw’umutima ni imbogamizi zikomeye z’ubu buryo. Ubushakashatsi buherutse bwerekanye ko iyi mbogamizi ishobora gukemurwa igice hakoreshejwe hiPS-CM ya mbere mu gukora hydrogels z’uturemangingo tw’umutima nyuma gato y’uko hatangiye kugabanuka kw’imitsi mu buryo bwikora kandi buhoro buhoro hakongerwa imbaraga z’amashanyarazi uko igihe kigenda gihita. Ariko, izi ngingo nto za hiPS-CM zidafite imiterere ikuze y’amashanyarazi n’iy’ingirabuzima fatizo z’umutima w’umuntu mukuru. Byongeye kandi, ingingo z'umutima w'umuntu zifite imiterere igoye cyane, igizwe n'uruvange rw'ubwoko butandukanye bw'uturemangingo, harimo uturemangingo tw'inyuma, uturemangingo tw'ubwonko, na fibroblasts za stromal, zihujwe n'amatsinda yihariye ya poroteyine zo mu gice cy'inyuma cy'uturemangingo. Ubu bwoko butandukanye bw'uturemangingo tutari utw'umutima11,12,13 mu mutima w'inyamabere zikuze ni imbogamizi ikomeye mu kwerekana uturemangingo tw'umutima hakoreshejwe ubwoko bw'uturemangingo. Izi mbogamizi zikomeye zishimangira akamaro ko guteza imbere uburyo bwo guteza imbere ingingo z'umutima zitarangirika mu gihe cy'imiterere y'umubiri n'indwara.
Ibice by'umutima byoroheje (300 µm) by'umutima w'umuntu byagaragaye ko ari icyitegererezo cyiza cy'umutima w'umuntu udafite indwara. Ubu buryo butanga uburyo bwo kugera ku buryo bwuzuye bwa 3D multicellular system isa n'ingingo z'umutima w'umuntu. Ariko, kugeza mu 2019, ikoreshwa ry'ibice by'umutima byubatswe ryagabanijwe n'igihe gito (amasaha 24) cyo kubaho kw'imiti. Ibi biterwa n'ibintu byinshi birimo kutagira uburyo bworoshye bwo gukora, aho umwuka n'amazi bihurira, no gukoresha ibikoresho byoroshye bidashyigikira ibyo ingingo z'umutima zikeneye. Mu 2019, amatsinda menshi y'ubushakashatsi yagaragaje ko gushyira ibintu by'imikorere mu miti mu miti y'imiti y'umutima bishobora kongera ubuzima bw'imiti, kunoza imikorere y'umutima, no kwigana indwara y'umutima. Ubushakashatsi bubiri bwiza bwa 17 na 18 bwerekana ko umutwaro umwe w'imikorere ufite ingaruka nziza ku miterere y'umutima mu gihe cyo gukora. Ariko, ubu bushakashatsi ntibwakoresheje umutwaro uhoraho w'imiti y'umutima, kubera ko ibice by'umutima byari byuzuyemo imbaraga zo gukurura 17 cyangwa umutwaro wa auxotonic winjira 18. Ubu buryo bwo kunagura ingingo bwatumye habaho kugabanya imiterere y’uturemangingo twinshi tw’umutima cyangwa kwiyongera cyane kw’uturemangingo dufitanye isano n’imikorere idasanzwe yo kunagura. Igishimishije ni uko Pitoulis n’abandi 19 bakoze uburyo bwo koza umutima bukoresha uburyo bwo kongera kubaka imiterere y’umutima hakoreshejwe uburyo bwo gusubiza imbaraga no guhangayika. Nubwo ubu buryo bwemerera gukora icyitegererezo cy’imikorere y’umutima mu buryo buboneye, uburyo bugoye n’uburyo buke bwo gukoresha ubu buryo bigabanya ikoreshwa ry’ubu buryo. Laboratwari yacu iherutse gukora uburyo bwo koroshya imiterere y’uturemangingo hakoreshejwe uburyo bw’amashanyarazi n’uburyo bworoshye bwo kubungabunga ubuzima bw’ibice by’ingurube n’imibiri y’umutima w’umuntu mu gihe cy’iminsi 6.
Mu nyandiko iriho ubu, turasobanura icyitegererezo cyo guteza imbere imiterere y'imitsi y'umutima (CTCM) dukoresheje ibice by'umutima w'ingurube birimo ibimenyetso by'ubuhumyi kugira ngo twongere imiterere y'imiti y'umutima mu buryo butatu n'uburyo umutima utera mu gihe cy'imitsi. Iyi CTCM ishobora kongera ukuri kw'ibivugwa mbere y'uko habaho imiti kugeza ku rwego rutari rwagerwaho mbere binyuze mu gutanga sisitemu y'umutima ihendutse kandi ikoreshwa hagati mu gihe cy'imitsi, ikaba isa n'imiterere y'imiti y'umutima w'inyamabere mu gupima imiti mbere yo kuyipima.
Ibimenyetso bya hemodynamic mechanical bifite uruhare runini mu kubungabunga imikorere y’umutima mu buryo bwa vitro 22,23,24. Mu nyandiko iriho ubu, twakoze CTCM (Ishusho ya 1a) ishobora kwigana imiterere y’umutima w’umuntu mukuru binyuze mu gutera imbaraga z’amashanyarazi n’iz’imikorere ku buryo bw’umubiri (1.2 Hz, beats 72 ku munota). Kugira ngo hirindwe kurengerwa cyane kw’uturemangingo mu gihe cyo gutera imbaraga, hakoreshejwe icyuma gicapa cya 3D kugira ngo cyongere ingano y’uturemangingo ho 25% (Ishusho ya 1b). Ingufu z’amashanyarazi ziterwa na sisitemu ya C-PACE zagenwe gutangira 100 ms mbere ya sistole hakoreshejwe sisitemu yo kubona amakuru kugira ngo yongere neza uruhererekane rw’umutima. Sisitemu yo gukusanya uturemangingo ikoresha actuator ya pneumatic ishobora gukoreshwa (LB Engineering, Germany) kugira ngo yongere uruhererekane rwa silicone kugira ngo itume ibice by’umutima mu cyumba cyo hejuru bikura. Sisitemu yahujwe n’umurongo w’umwuka wo hanze binyuze muri transducer y’umuvuduko, byatumye bishoboka guhindura neza umuvuduko (± 1 mmHg) n’igihe (± 1 ms) (Ishusho ya 1c).
a Shyira igice cy'uruti ku mpeta y'inkunga ya mm 7, igaragara mu ibara ry'ubururu, imbere mu cyumba cy'inkunga cy'igikoresho. Icyumba cy'inkunga gitandukanywa n'icyumba cy'umwuka n'agace gato ka silikoni gafite ubushobozi bworoshye bworoshye. Shyira gasket hagati ya buri cyumba kugira ngo wirinde ko amazi ava. Umupfundikizo w'igikoresho urimo electrodes za grafiti zitanga amashanyarazi. b Ishusho ya shusho y'igikoresho kinini cy'uruti, impeta y'inyobora n'impeta y'inkunga. Ibice by'uruti (umukara) bishyirwa ku gikoresho kinini cyane impeta y'inyobora ishyizwe mu mwobo uri ku nkengero z'icyuma. Ukoresheje ikigo, shyira witonze impeta y'inkunga itwikiriwe na kole ya acrylic hejuru y'igice cy'uruti rw'umutima. c Imbonerahamwe igaragaza igihe cyo gukangura amashanyarazi nk'igikorwa cy'umuvuduko w'icyumba cy'umwuka ugenzurwa na actuator ya pneumatic ishobora gukoreshwa (PPD). Igikoresho cyo kubona amakuru cyakoreshejwe mu guhuza gukangura amashanyarazi hakoreshejwe sensors z'umuvuduko. Iyo umuvuduko mu cyumba cy'inyongera ugeze ku mupaka washyizweho, ikimenyetso cy'impyiko cyoherezwa kuri C-PACE-EM kugira ngo gitere amashanyarazi. d Ishusho ya CTCM enye zishyizwe ku gikoresho cy'inyongera. Ibikoresho bine bihujwe na PPD imwe binyuze mu muyoboro w’umwuka, kandi ibikoresho by’umuvuduko bishyirwa muri valve ya hemostatic kugira ngo bigenzure umuvuduko uri mu muyoboro w’umwuka. Buri gikoresho kigizwe n’ibice bitandatu by’inyama.
Dukoresheje icyuma kimwe cya pneumatic actuator, twashoboye kugenzura ibikoresho 4 bya CTCM, buri kimwe muri byo gishobora gufata ibice 6 by'umubiri (Ishusho ya 1d). Muri CTCM, umuvuduko w'umwuka mu cyumba cy'umwuka uhinduka umuvuduko uhuza mu cyumba cy'amazi kandi bigatera kwaguka k'umubiri w'igice cy'umutima (Ishusho ya 2a n'Filime y'inyongera ya 1). Isuzuma ry'uburyo ingingo zirambaraye kuri 80 mm Hg. Art. ryagaragaje ko ibice by'umubiri byarambarayeho 25% (Ishusho ya 2b). Uku guhagarara kw'umubiri byagaragaye ko bihuye n'uburebure bwa sarcomere ya 2.2–2.3 µm kugira ngo igice cy'umutima gikomere neza17,19,25. Ingendo z'umubiri zapimwe hakoreshejwe kamera yihariye (Ishusho y'inyongera ya 1). Ubwinshi n'umuvuduko w'ingendo z'umubiri (Ishusho ya 2c, d) byahuye no guhagarara mu gihe cy'umutima n'igihe mu gihe cya systole na diastole (Ishusho ya 2b). Guhagarara n'umuvuduko w'umubiri w'umutima mu gihe cyo guhagarara no kuruhuka byakomeje kuba bihoraho mu minsi 12 mu gihe cyo gukurira (Ishusho ya 2f). Kugira ngo dusuzume ingaruka z'ubushyuhe bw'amashanyarazi ku guhagarara kw'ingufu mu gihe cyo gukuriramo, twakoze uburyo bwo kumenya ubushyuhe bukora dukoresheje uburyo bwo gusiga igicucu (Ishusho y'inyongera ya 2a, b) kandi twashoboye gutandukanya ubushyuhe burimo n'ubudafite ubushyuhe bw'amashanyarazi. Igice kimwe cy'umutima (Ishusho ya 2f). Mu gace kimukanwa k'agace (R6-9), ingufu z'amashanyarazi mu gihe cyo gukurura amashanyarazi zari hejuru ya 20% ugereranyije n'igihe nta bushyuhe bw'amashanyarazi bwabayeho, bigaragaza uruhare rw'ubushyuhe bw'amashanyarazi mu mikorere yo guhagarara kw'ingufu.
Ibimenyetso byerekana umuvuduko w'umwuka mu cyumba, umuvuduko w'amazi mu cyumba, n'ibipimo by'ingendo z'imitsi byemeza ko umuvuduko w'icyumba uhindura umuvuduko w'amazi mu cyumba, bigatera urujya n'uruza rw'igice cy'imitsi. b Ibimenyetso byerekana ijanisha ry'ubururu bw'ibice by'imitsi bihuye n'ijanisha ry'umuvuduko (icunga). c Ingendo yapimwe y'igice cy'umutima ihuye n'umuvuduko wapimwe w'ingendo. (d) Inzira zigaragaza ingendo z'uruziga (umurongo w'ubururu) n'umuvuduko (umurongo w'icunga ufite utudomo) mu gice cy'umutima. e Ingano y'igihe cy'uruziga (n = uduce 19 kuri buri tsinda, bivuye ku ngurube zitandukanye), igihe cyo gufunga (n = uduce 19 kuri buri tsinda, bivuye ku ngurube zitandukanye), ingendo z'imitsi (n = 25). uduce)/itsinda bivuye ku ngurube zitandukanye), umuvuduko w'umu ... Ikizamini cya T cy’Umunyeshuri ufite imirizo ibiri nta tandukaniro rigaragara cyagaragaje mu gipimo icyo ari cyo cyose. f Isesengura ry’imiterere y’ibice by’ingingo bifite (umutuku) kandi bidafite (ubururu) by’amashanyarazi, ibice icumi by’uturere by’ingingo z’ingingo biva mu gice kimwe. Udupapuro two hasi tugaragaza itandukaniro ry’ijanisha mu miterere y’ingingo mu bice by’ingingo bifite n’ibidafite amashanyarazi mu bice icumi biva mu bice bitandukanye. (n = ibice 8/itsinda bivuye mu ngurube zitandukanye, ikizamini cya T-Term cy'abanyeshuri gifite imirizo ibiri kirakorwa; ****p < 0.0001, **p < 0.01, *p < 0.05). (n = ibice 8/itsinda bivuye mu ngurube zitandukanye, ikizamini cya T-Term cy'abanyeshuri gifite imirizo ibiri kirakorwa; ****p < 0.0001, **p < 0.01, *p < 0.05). . (n = ibice 8/itsinda bivuye mu ngurube zitandukanye, ikizamini cya t cy'umunyeshuri gifite imirizo ibiri; ****p <0.0001, **p <0.01, *p <0.05). （N = 8 片 / 组，来自不同的猪，进行双尾学生 t 检验； **** p <0.0001 ， ** p <0.01 ， * p <0.05 ）。 （N = 8 片 / 组，来自不同的猪，进行双尾学生 t 检验； **** p <0.0001 ， ** p <0.01 ， * p <0.05 ）。 . (n = ibice 8/itsinda, bivuye mu ngurube zitandukanye, ikizamini cya t cy'umunyeshuri gifite imirizo ibiri; ****p <0.0001, **p <0.01, *p <0.05).Uturongo tw'amakosa tugaragaza impuzandengo ± ihinduka risanzwe.
Mu buryo bwacu bwa mbere bwo gukusanya ibice by'umutima bita static biomimetic [20, 21], twakomeje kubaho, imikorere, n'imiterere y'ibice by'umutima mu minsi 6 dukoresha uburyo bwo gukusanya amashanyarazi no kunoza imiterere y'ubugari. Ariko, nyuma y'iminsi 10, iyi mibare yagabanutse cyane. Tuzareba ibice byahinzwe muri sisitemu yacu ya mbere yo gukusanya static biomimetic conditions 20, 21 control conditions (Ctrl) kandi tuzakoresha uburyo bwacu bwakoreshejwe mbere nk'imiterere ya MC na culture munsi ya stimulation ya mechanical na electrical (CTCM). Ubwa mbere, twasanze uburyo bwo gukusanya amashanyarazi budafite stimulation y'amashanyarazi budahagije kugira ngo ingingo zikomeze kubaho mu minsi 6 (Ishusho y'inyongera ya 3a, b). Igishimishije ni uko, hamwe no gutangiza physio-mechanical na electrical stimulation hakoreshejwe STCM, ubushobozi bwo gukusanya ibice by'umutima by'iminsi 12 bwagumye ari bumwe no mu bice by'umutima mushya mu bihe bya MS, ariko ntabwo ari mu bihe bya Ctrl, nk'uko bigaragazwa n'isesengura rya MTT (Ishusho ya 1). 3a). Ibi bigaragaza ko uburyo bwo gukusanya no kwigana imikorere y'umutima bishobora gutuma ibice by'umubiri bikomeza kubaho kabiri nk'uko byavuzwe muri sisitemu yacu ya mbere yo gukusanya static. Ariko, isuzuma ry’imiterere y’ibice by’imitsi hakoreshejwe uburyo bwo gushyira mu majwi troponin T na connexin 43 mu maraso ryagaragaje ko imiterere ya connexin 43 yari hejuru cyane mu bice bya MC ku munsi wa 12 ugereranyije n’iy’igenzura ku munsi umwe. Ariko, imiterere ya connexin 43 n’imiterere ya Z-disc ntibyakomeje neza (Ishusho ya 3b). Dukoresha urwego rw’ubwenge bw’ubukorano (AI) kugira ngo dupime imiterere y’imitsi26, umuyoboro w’ubumenyi bwimbitse bushingiye ku ishusho ushingiye kuri troponin-T na connexin staining43 kugira ngo dupime imiterere y’imitsi n’urumuri rw’imitsi mu buryo bw’imbaraga z’aho umutima uherereye. Ubu buryo bukoresha Convolutional Neural Network (CNN) n’urwego rw’ubumenyi bwimbitse kugira ngo dupime imiterere y’imitsi y’umutima mu buryo bwikora kandi butabogamye, nk’uko byasobanuwe mu nyandiko. 26. Imitsi ya MC yagaragaje imiterere myiza n’iy’umunsi wa 0 ugereranije n’ibice by’ubuyobozi budahindagurika. Byongeye kandi, ibara rya Masson rya trichrome ryagaragaje ko igipimo gito cyane cya fibrosis mu ndwara za MS ugereranije n’indwara zo mu mutwe ku munsi wa 12 w’ivuriro (Ishusho ya 3c). Nubwo CTCM yongereye ubushobozi bwo kubaho kw’ibice by’umutima ku munsi wa 12 ku rugero rusa n’urw’ibice by’umutima bishya, ntabwo byarushijeho kunoza imiterere y’ibice by’umutima.
Imbonerahamwe y'imirongo igaragaza uburyo MTT ishobora kubaho mu bice by'umutima bishya (D0) cyangwa mu bice by'umutima mu minsi 12 haba mu gupima imiterere idahinduka (D12 Ctrl) cyangwa muri CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) ibice/itsinda bivuye mu ngurube zitandukanye, uburyo bumwe bwo gupima ANOVA bikorwa; ####p < 0.0001 ugereranije na D0 na **p < 0.01 ugereranije na D12 Ctrl). Imbonerahamwe y'imirongo igaragaza uburyo MTT ishobora kubaho mu bice by'umutima bishya (D0) cyangwa mu bice by'umutima mu minsi 12 haba mu gupima imiterere idahinduka (D12 Ctrl) cyangwa muri CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) ibice/itsinda bivuye mu ngurube zitandukanye, uburyo bumwe bwo gupima ANOVA bikorwa; ####p < 0.0001 ugereranije na D0 na **p < 0.01 ugereranije na D12 Ctrl).Histogram igaragaza uburyo ibice by'umutima bihagaze neza (D0) cyangwa ibice by'umutima bimara iminsi 12 mu miterere idahinduka (D12 control) cyangwa CTCM (D12 MC) (n = 18 (D0), 15 (D12 control). ) ), ibice 12 (D12 MC) / itsinda bivuye mu ngurube zitandukanye, hakorwa ikizamini cya ANOVA cy'inzira imwe;#### p <0,0001 по сравнению с D0 и ** p <0,01 по сравнению с D12 Ctrl). ####p < 0.0001 ugereranije na D0 na **p < 0.01 ugereranije na 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) 切片 / 组，进行单向 ANOVA 测试；与 D0 相比， #### p <0.0001 ，与 D12 Ctrl 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 ，与 D12 Ctrl 相比， ** p。)Histogram igaragaza igipimo cy'ubushobozi bwa MTT mu bice by'umutima mushya (D0) cyangwa ibice by'umutima byatewe mu gihe cy'iminsi 12 mu gupima imiterere idahinduka (D12 control) cyangwa CTCM (D12 MC) (n = 18 (D0), 15 (D12 control)), 12 (D12 MC) ibice/itsinda bivuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe;#### p <0,0001 по сравнению с D0, ** p <0,01 по сравнению с D12 Ctrl). ####p < 0.0001 ugereranije na D0, **p < 0.01 ugereranije na D12 Ctrl).b Troponin-T (icyatsi kibisi), connexin 43 (umutuku) na DAPI (ubururu) mu bice by'umutima bishya (D0) cyangwa ibice by'umutima byatewe mu bihe bidahinduka (Ctrl) cyangwa CTCM (MC) mu minsi 12) byerekana amashusho y'ubudahangarwa bw'umubiri (igipimo cy'ubusa = 100 µm). Gupima ubwenge bw'ubukorano bw'imiterere y'imitsi y'umutima (n = 7 (D0), 7 (D12 Ctrl), uduce 5 (D12 MC) buri gice giturutse ku ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ####p < 0.0001 ugereranije na D0 na ****p < 0.0001 ugereranije na D12 Ctrl). Gupima ubwenge bw'ubukorano bw'imiterere y'imitsi y'umutima (n = 7 (D0), 7 (D12 Ctrl), uduce 5 (D12 MC) buri kimwe kivuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ####p < 0.0001 ugereranije na D0 na ****p < 0.0001 ugereranije na D12 Ctrl). Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) срезов/групп от разных свиней, проводится однофакторный тест ANOVA; ####p < 0,0001 по сравнению с D0 и **** p <0,0001 по сравнению с D12 Ctrl). Gupima imiterere y’imitsi y’umutima hakoreshejwe ubwenge bw’ubukorano (n = 7 (D0), 7 (D12 Ctrl), ibice 5 (D12 MC) by’ingurube zitandukanye, ikizamini cya ANOVA cy’inzira imwe cyakozwe; ####p < 0.0001 ugereranije na D0 na ****p < 0.0001 ugereranije na 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) ibice / itsinda buri ngurube zitandukanye, ikizamini kimwe cya ANOVA; #### p <0.0001 与 D0 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) uduce / itsinda buri ngurube zitandukanye, ikizamini kimwe cya ANOVA; #### p <0.0001 与 D0 相比， **** p <0.0001 与 D12 Ctrl 相比）。 Искусственный интеллект для количественной оценки башной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) срезов / груу kaждой из разных свиней, одностороны сравнения **** p <0,0001 по сравнению с D12 Ctrl). Ubwenge bw'ubukorano kugira ngo harebwe imiterere y'imitsi y'umutima (n = 7 (D0), 7 (D12 Ctrl), ibice 5 (D12 MC) buri tsinda ry'ingurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe; ####p<0.0001 vs .D0 Kugereranya ****p < 0.0001 ugereranije na D12 Ctrl). c Amashusho agaragaza (ibumoso) n'ibipimo (iburyo) by'uduce tw'umutima twandujwe n'ikibabi cya Masson's trichrome (Scale bare = 500 µm) (n = uduce 10/itsinda rimwe na rimwe duturutse ku ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ####p < 0.0001 ugereranije na D0 na ***p < 0.001 ugereranije na D12 Ctrl). c Amashusho agaragaza (ibumoso) n'ibipimo (iburyo) by'uduce tw'umutima twandujwe n'ikibabi cya Masson's trichrome (Scale bare = 500 µm) (n = uduce 10/itsinda rimwe na rimwe duturutse ku ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; #### p < 0.0001 ugereranije na D0 na ***p < 0.001 ugereranije na D12 Ctrl). c Репрезент Канные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Маснаа (чин покрытия = 500 мкм) (n = 10 срезов / кну в ретсик с ANOVA #### p <0,0001 по сравнению с D0 и *** p <0,001 по сравнению с D12 Ctrl) c Amashusho agaragaza (ibumoso) n'ibipimo (iburyo) by'ibice by'umutima byandujwe n'ikibabi cya Masson cya trichrome (igipimo kitarapfukwa = 500 µm) (n = ibice 10/itsinda bivuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe cyakozwe; #### p < 0 .0001 ugereranije na D0 na ***p < 0.001 ugereranije na D12 Ctrl). c 用 Masson 三色染料染色的心脏切片的代表性图像（左）和量化（右）（裸尺度 = 500 µm ）（ n = 10 个切片 / 组，每组来自不同的猪，进行单向 ANOVA 测试； #### p <0.0001 与 D0 相比， *** p <0.001 与 D12 Ctrl 相比）。 C 用 masson 三 色 染料 的 心脏 切片 的 （左 左） 量化 = 500 µm） （n = 10 个 切片 组 每 组 来自 o 猪 ， 进行 单向 单向 Anova 测试； #### p <0.0001 与 D0 相比， *** p <0.001 12 D12 Ctrl 相比）。 c Репрезентныные изображения (слева) и количественный мат (справа) срезов сердца, окрашенных трихромным красителем Мас медиа (чя шкала = 500 мкм) (n = 10 срезов / группа, kaжки от сругов однофакторного ди ди барного мата; ### #p <0,0001 по сравнению с D0, *** p <0,001 по сравнению с D12 Ctrl). c Amashusho agaragaza (ibumoso) n'ingano (iburyo) y'ibice by'umutima byandujwe n'ikibabi cya Masson cya trichrome (ubusa = 500 µm) (n = ibice 10/itsinda, buri kimwe giturutse ku ngurube itandukanye, byageragejwe hakoreshejwe isesengura ry'uburyo bumwe bw'impinduka ;### # p < 0.0001 ugereranije na D0, ***p < 0.001 ugereranije na D12 Ctrl).Uturongo tw'amakosa tugaragaza impuzandengo ± ihinduka risanzwe.
Twatekereje ko kongeramo molecule nto mu buryo bw’imikurire, ubusugire bw’umutima bushobora kunozwa kandi iterambere rya fibrosis rikagabanuka mu gihe cyo gupima CTCM. Bityo twasuzumye molecule nto dukoresheje culture zacu zo kugenzura static20,21 bitewe n’umubare muto w’ibintu bitera urujijo. Dexamethasone (Dex), triiodothyronine (T3), na SB431542 (SB) ni byo byatoranyijwe kuri iki gipimo. Izi molecule nto zakoreshejwe mbere mu gupima hiPSC-CM kugira ngo zitere imbaraga mu gupima cardiomyocytes binyuze mu kongera uburebure bwa sarcomere, T-tubules, n’umuvuduko wo kuyobora. Byongeye kandi, Dex (glucocorticoid) na SB byombi bizwiho gukumira ububyimbirwe29,30. Kubwibyo, twagerageje niba gushyiramo imwe cyangwa uruvange rw’izi molecule nto byakongera ubusugire bw’imiterere y’ibice by’umutima. Mu gusuzuma bwa mbere, ingano ya buri kintu yatoranijwe hashingiwe ku bipimo bikunze gukoreshwa mu miterere y’uturemangingo (1 μM Dex27, 100 nM T327, na 2.5 μM SB31). Nyuma y'iminsi 12 yo gukorerwa isuzuma, guhuza T3 na Dex byatumye habaho imiterere myiza y'umutima n'imitsi ndetse no kugabanya ihinduka ry'imitsi (Ishusho y'inyongera ya 4 na 5). Byongeye kandi, gukoresha inshuro ebyiri cyangwa ebyiri z'iyi ngano ya T3 na Dex byagize ingaruka mbi ugereranije n'ingano zisanzwe (Ishusho y'inyongera ya 6a, b).
Nyuma yo gusuzuma bwa mbere, twakoze igereranya ry’imiterere 4 y’imiti (Ishusho ya 4a): Ctrl: ibice by’umutima byahinzwe mu miti yacu idahinduka twavuze mbere dukoresheje uburyo bwacu bworoshye; 20.21 TD: T3 na Ctrl s Added Dex kuwa gatatu; MC: ibice by’umutima byahinzwe muri CTCM dukoresheje uburyo bwacu bworoshye; na MT: CTCM hamwe na T3 na Dex byongewe muri ubwo buryo. Nyuma y’iminsi 12 yo guhingwa, ubushobozi bwo kubaho bw’uturemangingo twa MS na MT bwagumye ari bumwe no mu turemangingo dushya twapimwe hakoreshejwe isuzuma rya MTT (Ishusho ya 4b). Igishimishije ni uko kongeramo T3 na Dex mu miti ya transwell (TD) ntacyo byatanze ku ihinduka rikomeye mu kubaho ugereranije n’imiterere ya Ctrl, bigaragaza uruhare runini rwo gukangura imikorere mu kubungabunga ubushobozi bw’uturemangingo tw’umutima.
Ishusho y'igerageza igaragaza imiterere ine y'ubuhinzi yakoreshejwe mu gusuzuma ingaruka zo gukangura imikorere y'imashini no kongeramo T3/Dex ku buryo bworoshye mu gihe cy'iminsi 12. b Imbonerahamwe igaragaza uburyo bwo gupima ubuzima bw'umuntu nyuma y'iminsi 12 yo guhingwa mu miterere yose uko ari ine yo guhingwa (Ctrl, TD, MC, na MT) ugereranije n'uduce tw'umutima dushya (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD na D12 MT), uduce 12 (D12 MC) twavuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ####p < 0.0001, ###p < 0.001 ugereranije na D0 na **p < 0.01 ugereranije na D12 Ctrl). b Imbonerahamwe igaragaza uburyo bwo gupima ubuzima bw'ingurube mu minsi 12 nyuma yo guhingwa mu miterere yose uko ari ine yo guhingwa (Ctrl, TD, MC, na MT) ugereranije n'uduce tw'umutima dushya (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD na D12 MT), uduce 12 (D12 MC) twavuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ####p < 0.0001, ###p < 0.001 ugereranije na D0 na **p < 0.01 ugereranije na D12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после митингирования вохех 4 условиях медиатированя (дар, TD, MC na MT) по сравнению со свежим срезами срдча (D0) D12 TD na D12 MT), 12 (D12 MC) срезов / группу от разных свиней, про манится односторонний те ANOVA; #### p <0,0001 ,; ### p <0,001 по сравнению с D0 и ** p <0,01 по сравнению с D12 Ctrl). b Imbonerahamwe igaragaza uko ubuzima buhagaze mu minsi 12 nyuma yo guhingwa mu miterere yose uko ari ine yo guhingwa (igenzura, TD, MC, na MT) ugereranije n'ibice by'umutima mushya (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, na D12 MT), ibice 12 (D12 MC)/itsinda bivuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe; ####p < 0.0001, ###p < 0.001 ugereranije na D0 na **p < 0.01 ugereranije na D12 Ctrl). b 条形图显示所有 4 种培养条件（ Ctrl 、 TD 、 MC 和 MT ）与新鲜心脏切片 (D0) (n = 18 (D0) 、 15 (D12 Ctrl 、 D12 TD 和 D12 MT) ，来自不同猪的 12 (D12 MC) 切片 / 组，进行单向 ANOVA 测试； #### p <0.0001 ， ### p <0.001 与 D0 相比，b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия маттивирования (كان, TD, MC na MT) по сравнению со свежим срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD na D12 MT), от ср ср. односторонний ту ANOVA # # b Histogram igaragaza imiterere yose 4 y’imikurire (igenzura, TD, MC na MT) ugereranije n’ibice by’umutima mushya (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD na D12 MT), bivuye mu ngurube zitandukanye 12 (D12 MC) ibice/itsinda, ikizamini cya ANOVA cy’inzira imwe; ####p<0.0001, ###p<0.001 vs. D0, **p<0.01 vs. igenzura D12). c Imbonerahamwe y'ibice igaragaza igipimo cy'isukari mu maraso nyuma y'iminsi 12 yo gupima mu miterere yose uko ari ine yo gupima (Ctrl, TD, MC, na MT) ugereranije n'uduce tw'umutima dushya (D0) (n = uduce 6/itsinda ry'ingurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ###p < 0.001, ugereranije na D0 na ***p < 0.001 ugereranije na D12 Ctrl). c Imbonerahamwe y'ibice igaragaza igipimo cy'isukari mu maraso nyuma y'iminsi 12 yo gupima mu miterere yose uko ari ine yo gupima (Ctrl, TD, MC, na MT) ugereranije n'uduce tw'umutima dushya (D0) (n = uduce 6/itsinda ry'ingurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ###p < 0.001, ugereranije na D0 na ***p < 0.001 ugereranije na D12 Ctrl). c Гистограмма показывает количественную оценку потока глюкозы через 12 дней после митингирования вохех 4 условиях привирирования (дар, TD, MC и MT) по сравнению со свежим срезами срдова (с) свиней, односторонний Выполняется те ANOVA ### p <0,001 по сравнению с D0 и *** p <0,001 по сравнению с D12 Ctrl); c Histogram igaragaza igipimo cy'isukari iva mu mubiri nyuma y'iminsi 12 yo gupima mu buryo bune bwo gupima (igenzura, TD, MC na MT) ugereranije n'ibice by'umutima mushya (D0) (n = ibice 6/itsinda bivuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe cyakozwe; ###p < 0.001 ugereranije na D0 na ***p < 0.001 ugereranije na D12 Ctrl). c 条形图显示所有 4 种培养条件（ Ctrl 、 TD 、 MC 和 MT ）与新鲜心脏切片 (D0) 相比，培养后 12 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 ，与 D0 相比， *** p <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 切片 相比 相比 ， 后 后 天 n = 6 片 / 组 ， 来自 猪 ， ， ， 0.00 0.00 0.00 0.00 < 0.001 与 D12 Ctrl 相比）。 c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после митивированя для всех 4 условий митирированя (amateka, TD, MC и MT) по сравнению со свеж с с ср ср с разных свиней, односторонний Были проведены Тыы ANOVA; ### p <0,001 по сравнению с D0, *** p (контроль). c Histogram igaragaza igipimo cy'isukari mu maraso nyuma y'iminsi 12 yo gupima indwara ku ndwara zose uko ari enye zo gupima indwara (igenzura, TD, MC, na MT) ugereranije n'ibice by'umutima mushya (D0) (n = ibice 6/itsinda, bivuye mu ngurube zitandukanye, ku ruhande rumwe. Hakozwe ibizamini bya ANOVA, ###p < 0.001 ugereranije na D0, ***p < 0.001 ugereranije na D12 (igenzura).d Isesengura ry'imitsi ry'uturemangingo dushya (ubururu), umunsi wa 12 MC (icyatsi kibisi), n'umunsi wa 12 MT (umutuku) ku ngingo icumi z'agace k'uturemangingo (n = uduce 4/itsinda, ikizamini cya ANOVA cy'inzira imwe; nta tandukaniro rigaragara hagati y'amatsinda). e Ishusho y'ikirunga igaragaza gene zigaragara mu bice by'umutima mushya (D0) ugereranije n'ibice by'umutima byatewe mu bihe bidahinduka (Ctrl) cyangwa mu bihe bya MT (MT) mu minsi 10-12. f Ikarita y'ubushyuhe ya gene za sarcomere ku bice by'umutima byatewe muri buri kibazo cy'uburumbuke. Uduce tw'amakosa tugaragaza impuzandengo ± ihinduka risanzwe.
Gushingira ku mikorere ya glucose kuva kuri oxidation ya aside irike kugera kuri glycolysis ni ikimenyetso cy’uko cardiomyocyte ihinduka. Cardiomyocytes zitarakura cyane cyane zikoresha glucose mu gukora ATP kandi zifite mitochondria ya hypoplastic ifite cristae nke5,32. Isesengura ry’ikoreshwa rya glucose ryagaragaje ko mu bihe bya MC na MT, ikoreshwa rya glucose ryari rimeze nk’iryo mu bice bya 0 by’umubiri (Ishusho ya 4c). Ariko, ingero za Ctrl zagaragaje ukwiyongera gukomeye mu ikoreshwa rya glucose ugereranije n’ibice bishya. Ibi bigaragaza ko guhuza CTCM na T3 / Dex byongera ubushobozi bwo kubaho kw’ibice by’umutima kandi bigakomeza imiterere ya metabolic y’ibice by’umutima byakuwe mu minsi 12. Byongeye kandi, isesengura ry’imikoreshereze y’ibice ryerekanye ko urugero rw’imikoreshereze y’ibice by’umutima byagumyeho nk’urwo mu bice by’umutima bishya mu minsi 12 mu bihe bya MT na MS (Ishusho ya 4d).
Kugira ngo dusesengure ingaruka rusange za CTCM na T3/Dex ku miterere y’umubiri w’umutima ku isi, twakoze RNAseq ku bice by’umutima bivuye mu miterere ine itandukanye y’umubiri (Amakuru y’inyongera 1). Igishimishije ni uko ibice bya MT byagaragaje ko bifitanye isano rikomeye n’imiterere y’umutima mushya, aho ibice 16 gusa byagaragaye mu buryo butandukanye muri 13.642. Ariko, nk'uko twabigaragaje mbere, ibice bya Ctrl byagaragaje imiterere 1229 yagaragaye mu buryo butandukanye nyuma y'iminsi 10-12 mu muco (Ishusho ya 4e). Aya makuru yemejwe na qRT-PCR y’uturemangingo tw’umutima na fibroblast (Ishusho y’inyongera 7a-c). Igishimishije ni uko ibice bya Ctrl byagaragaje ko imiterere y’uturemangingo tw’umutima n’uturemangingo igabanuka ndetse no gukora gahunda z’uturemangingo tw’umubiri. Aya makuru agaragaza ko gutandukana kw’uturemangingo, ubusanzwe bibaho nyuma yo guhingwa igihe kirekire, bigabanuka burundu bitewe n’imiterere ya MT (Ishusho y’inyongera 8a,b). Inyigo yitonze ku ngirabuzimafatizo za sarcomere yagaragaje ko mu bihe bya MT gusa, ngirabuzimafatizo zikora kuri sarcomere (Ishusho ya 4f) n'umuyoboro wa ion (Ishusho y'inyongera ya 9) zibikwa, zikazirinda gukandamizwa munsi y'imiterere ya Ctrl, TD, na MC. Aya makuru agaragaza ko iyo habayeho guhuza uburyo bwo gukangura imiterere n'ubuhumekero (T3 / Dex), transcriptome y'igice cy'umutima ishobora kuguma isa n'igice cy'umutima gishya nyuma y'iminsi 12 mu guhingwa.
Ibi byavumbuwe mu buryo bwa transcription bishyigikiwe n'uko imiterere y'umutima mu bice by'umutima ikomeza neza mu gihe cy'iminsi 12, nk'uko bigaragazwa na connexin 43 ihoraho kandi iri ahantu runaka (Ishusho ya 5a). Byongeye kandi, fibrosis mu bice by'umutima mu gihe cy'indwara ya MT yagabanutse cyane ugereranije na Ctrl kandi bisa n'ibice by'umutima mushya (Ishusho ya 5b). Aya makuru agaragaza ko guhuza uburyo bwo gukangura umubiri no kuvura T3 / Dex bibungabunga neza imiterere y'umutima mu bice by'umutima mu mico.
Amashusho agaragaza ubwiyongere bw'umubiri bw'umuntu (immunofluorescence) ya troponin-T (icyatsi), connexin 43 (umutuku), na DAPI (ubururu) mu bice by'umutima bishya (D0) cyangwa bigahingwa mu gihe cy'iminsi 12 mu bice bine by'umutima (urugero rwa scale bar = 100 µm). Gupima ubwenge bw'ubukorano bw'imiterere y'imitsi y'umutima (n = 7 (D0 na D12 Ctrl), 5 (D12 TD, D12 MC na D12 MT) ibice/itsinda bivuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ####p < 0.0001 ugereranije na D0 na *p < 0.05, cyangwa ****p < 0.0001 ugereranije na D12 Ctrl). Gupima ubwenge bw'ubukorano bw'imiterere y'imitsi y'umutima (n = 7 (D0 na D12 Ctrl), 5 (D12 TD, D12 MC na D12 MT) ibice/itsinda bivuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; #### p < 0.0001 ugereranije na D0 na *p < 0.05, cyangwa ****p < 0.0001 ugereranije na D12 Ctrl). Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 7 (D0 и D12 Ctrl), 5 (D12 TD, D12 MC и D12 MT) срезов/группу от разных свиней, проведен однофакторный тест ANOVA; #### p < 0,0001 по сравнению с D0 и * p <0,05 или **** p <0,0001 по сравнению с D12 Ctrl). Gupima imiterere y'imitsi y'umutima hakoreshejwe ubwenge bw'ubukorano (n = 7 (D0 na D12 Ctrl), ibice 5 (D12 TD, D12 MC na D12 MT) bivuye mu ngurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe cyakozwe; #### p < 0.0001 ugereranije na D0 na *p < 0.05 cyangwa ****p < 0.0001 ugereranije na D12 Ctrl).对不同猪的心脏组织结构完整性（ n = 7 （D0 和 D12 Ctrl ）、 5 （D12 TD 、 D12 MC 和 D12 MT ）切片 / 组）进行人工智能量化，进行单向 ANOVA 测试； #### p <0.0001 与 D0 和 * p <0.05 相比，或 **** p <0.0001 与 D12 Ctrl 相比）。对 不同 猪 的 心脏 结构 完整性 （n = 7 （d0 和 d12 ctrl） （5 （d12 td 、 d12 mc 和 d12 mt） 组） 人工 智能量 化 进行 单向 单向 单向 测试 # ########## p <0.0001 与 D0 和Ctrl 相比）。Gupima imiterere y’imitsi y’umutima hakoreshejwe ubwenge bw’ubukorano mu ngurube zitandukanye (n = 7 (D0 na D12 Ctrl), 5 (D12 TD, D12 MC na D12 MT) ibice/itsinda) hakoreshejwe ikizamini cya ANOVA cy’inzira imwe;#### p <0,0001 по сравнению с D0 и * p <0,05 или **** p <0,0001 по сравнению с D12 Ctrl). #### p < 0.0001 ugereranije na D0 na *p < 0.05 cyangwa ****p < 0.0001 ugereranije na D12 Ctrl). b Amashusho agaragaza n'ibipimo by'uduce tw'umutima twandujwe n'ikibabi cya Masson's trichrome (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, na D12 MC), uduce 9 (D12 MT) tw'ingurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ####p < 0.0001 ugereranije na D0 na ***p < 0.001, cyangwa ****p < 0.0001 ugereranije na D12 Ctrl). b Amashusho agaragaza n'ibipimo by'uduce tw'umutima twandujwe n'ikibabi cya Masson's trichrome (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, na D12 MC), uduce 9 (D12 MT) tw'ingurube zitandukanye, ikizamini cya ANOVA cy'inzira imwe kirakorwa; ####p < 0.0001 ugereranije na D0 na ***p < 0.001, cyangwa ****p < 0.0001 ugereranije na D12 Ctrl). b Репрезентныные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Мас ина (Кнаяяейей = 500 мкм) (n = 10 (D0, D12 Ctrl, D12 TD и D12 MC) выполняется односторонний тест ANOVA #### p <0,0001 по сравнению с D0 и *** p <0,001 или **** p <0,0001 по; сравнению с D12 Ctrl). b Amashusho agaragaza n'ibipimo by'ibice by'umutima byandujwe n'ikibabi cya Masson cya trichrome (umurongo w'ubugari = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD na D12 MC), ibice 9 (D12 MT)/itsinda bivuye mu ngurube zitandukanye, byakozwe mu buryo bumwe ANOVA; ####p < 0.0001 vs. D0 na ***p < 0.001 cyangwa ****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 （D0 、 D12 Ctrl 、 D12 TD 和 D12 MC ），来自不同猪的 9 个（ D12 MT ）切片 / 组，进行单因素方差分析； #### p <0.0001 与 D0 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 和 = 500 µm） （n = 10 （d0 、 d12 ctrl 、 d12 td 和 d12 mc） 来自 不同 的 9 个 12 12 切片 切片 切片 切片 切片 切片 / 组，进行单因素方差分析； #### p <0.0001 与 D0 相比， *** p <0.001 ，或 **** p <0.0001 与 D12 Ctrl 相比）。 b Репрезент Канные изображения и количественная оценка срезов сердца, окрашенных трихромом Мас хаа (шнаяяейей = 500 мкм) (n = 10 (D0, D12 Ctrl, D12 TD и D12 MC) ANOVA #### p <0,0001 по сравнению с D0, *** p <0,001 или **** p <0,0001 по сравненюю с D12 Ctrl). b Amashusho agaragaza n'ibipimo by'ibice by'umutima byandujwe na Masson's trichrome (umurongo w'ubugari = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD na D12 MC), ibice 9 (D12 MT) bivuye mu ngurube/itsinda ritandukanye, uburyo bumwe bwa ANOVA; ####p < 0.0001 ugereranije na D0, ***p < 0.001 cyangwa ****p < 0.0001 ugereranije na D12 Ctrl).Uturongo tw'amakosa tugaragaza impuzandengo ± ihinduka risanzwe.
Amaherezo, ubushobozi bwa CTCM bwo kwigana ubwiyongere bw'umutima bwapimwe hifashishijwe kongera ubwiyongere bw'imitsi y'umutima. Muri CTCM, umuvuduko w'umwuka wo mu cyumba cy'umwuka wiyongereye kuva kuri 80 mmHg kugera kuri 80 mmHg. Art. (kwiyongerera ubuziranenge) kugeza kuri 140 mmHg Art. (Ishusho ya 6a). Ibi bihuye n'ubwiyongere bwa 32% mu kwiyongerera ubuziranenge (Ishusho ya 6b), byagaragajwe mbere nk'ijanisha rijyanye n'ubwiyongere bw'imitsi y'umutima kugira ngo igere ku burebure bwa sarcomere nk'ubugaragara kuri hypertrophy. Kwiyongerera ubuziranenge n'umuvuduko w'imitsi y'umutima mu gihe cyo kwiyongerera ubuziranenge no kuruhuka byakomeje kuba bihoraho mu minsi itandatu yo kwiyongerera ubuziranenge (Ishusho ya 6c). Imitsi y'umutima yo mu bihe bya MT yashyizwe mu kwiyongerera ubuziranenge (MT (Normal)) cyangwa mu bihe byo kurenza urugero (MT (OS)) mu minsi itandatu. Nyuma y'iminsi ine yo kwiyongerera ubuziranenge, biomarker ya hypertrophic NT-ProBNP yazamutse cyane mu buryo buciriritse mu bihe bya MT (OS) ugereranije na MT (normal) (Ishusho ya 7a). Byongeye kandi, nyuma y'iminsi itandatu yo gucukura, ingano y'uturemangingo muri MT (OS) (Ishusho ya 7b) yariyongereye cyane ugereranije n'ibice by'umutima wa MT (bisanzwe). Byongeye kandi, ihererekanya rya NFATC4 ryariyongereye cyane mu ngingo zarambutse cyane (Ishusho ya 7c). Ibi bisubizo bigaragaza iterambere ry'ivugururwa ry'indwara nyuma yo kwangirika cyane kandi bishyigikira igitekerezo cy'uko igikoresho cya CTCM gishobora gukoreshwa nk'urubuga rwo kwiga ibimenyetso by'umutima bitera kwangirika cyane.
Ibimenyetso byerekana umuvuduko w'umwuka mu cyumba, umuvuduko w'amazi mu cyumba, n'ibipimo by'imitsi byemeza ko umuvuduko w'icyumba uhindura umuvuduko w'amazi mu cyumba, bigatera urujya n'uruza rw'igice cy'umubiri. b Ijanisha ryerekana ijanisha ryo gutambuka n'umuvuduko wo gutambuka ku bice bisanzwe byagutse (icunga) n'ibinini byarambuye (ubururu). c Imbonerahamwe y'umurongo igaragaza igihe cyo kuzenguruka (n = uduce 19 kuri buri tsinda, bivuye ku ngurube zitandukanye), igihe cyo gufunga (n = uduce 18-19 kuri buri tsinda, bivuye ku ngurube zitandukanye), igihe cyo kuruhuka (n = uduce 19 kuri buri tsinda, bivuye ku ngurube zitandukanye) ), uburebure bw'imitsi (n = uduce 14/itsinda, bivuye ku ngurube zitandukanye), umuvuduko wo hejuru wa sistolike (n = uduce 14/itsinda, bivuye ku ngurube zitandukanye) n'umuvuduko wo hejuru wo kuruhuka (n = 14 (D0), 15 (D6) ) ibice/amatsinda) bivuye ku ngurube zitandukanye), ikizamini cya t cy'umunyeshuri ufite imirizo ibiri nta tandukaniro rigaragara ryagaragaje mu gipimo icyo ari cyo cyose, bigaragaza ko ibi bipimo byagumye bihoraho mu minsi 6 yo guhingwa hamwe n'amashanyarazi menshi. Uduce tw'amakosa tugaragaza impuzandengo ± ihinduka risanzwe.
Isuzuma rya Bar graph ryerekana ubwinshi bwa NT-ProBNP mu miterere y’imitsi ikomoka ku bice by’umutima byakuwe mu miterere ya MT normal stretch (Norm) cyangwa overstretching (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, na D4 MTOS) ibice/itsinda bivuye mu ngurube zitandukanye, ANOVA y’inzira ebyiri irakorwa; **p < 0.01 ugereranije no normal stretch). Isuzuma rya Bar graph ryerekana ubwinshi bwa NT-ProBNP mu miterere y’imitsi ikomoka ku bice by’umutima byakuwe mu miterere ya MT normal stretch (Norm) cyangwa overstretching (OS) (n ​​= 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, na D4 MTOS) ibice/itsinda bivuye mu ngurube zitandukanye, ANOVA y’inzira ebyiri irakorwa; **p < 0.01 ugereranije no normal stretch).Histogramu y'ingano y'ubwinshi bwa NT-ProBNP mu buryo bwo gutubura amatungo buturutse ku bice by'umutima byatewe mu buryo busanzwe bwa MT stretch (normal) cyangwa overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, na D4).MTOS) ibice /itsinda bivuye ku ngurube zitandukanye, isesengura ry'uburyo bubiri bw'impinduka rikorwa;** p <0 **p < 0.01 ugereranije no kwaguka gusanzwe). a 在 MT 正常拉伸 (Ubusanzwe) 或过度拉伸 (OS) 条件下培养的心脏切片培养基中 NT-ProBNP 浓度的条形图量化（ n = 4 (D2 MTNorm) 、 3 （D2 MTOS 、 D4 MTNorm 和 D4 MTOS ）来自不同猪的切片 / 组，进行双向方差分析； ** 与正常拉伸相比， p <0.01 ）。 Quantification yibitekerezo bya NT-ProBNP mubice byumutima byimico munsi ya MT isanzwe (Norm) cyangwa kurenza urugero (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm 和 D4 MTOS) bitandukanye na 猪的切片 / 组，可以双向方方发发动; ** ugereranije no kurambura bisanzwe, p <0.01).Histogram Isuzuma ry'ibipimo bya NT-ProBNP mu bice by'umutima byahinzwe mu buryo busanzwe bwo kwaguka kwa MT (bisanzwe) cyangwa kurenza urugero (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) na D4 MTOS) ibice / itsinda bivuye mu ngurube zitandukanye, isesengura ry'uburyo bubiri bw'impinduka;** p <0 **p < 0.01 ugereranije no kwaguka gusanzwe). b Amashusho agaragaza ibice by'umutima byandujwe na troponin-T na WGA (ibumoso) hamwe n'ingano y'uturemangingo (iburyo) (n = 330 (D6 MTOS), 369 (D6 MTNorm) uturemangingo/itsinda bivuye mu bice 10 bitandukanye bivuye mu ngurube zitandukanye, Ikizamini cya T-T cy'umunyeshuri ufite imirizo ibiri kirakorwa; ****p < 0.0001 ugereranije no kwaguka bisanzwe). b Amashusho agaragaza ibice by'umutima byandujwe na troponin-T na WGA (ibumoso) hamwe n'ingano y'uturemangingo (iburyo) (n = 330 (D6 MTOS), 369 (D6 MTNorm) uturemangingo/itsinda bivuye mu bice 10 bitandukanye bivuye mu ngurube zitandukanye, Ikizamini cya T-Umunyeshuri ufite imirizo ibiri kirakorwa; ****p < 0.0001 ugereranije no kwaguka bisanzwe). b Репрезентныные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного определения размера клеток (справа) (n = 330 (D6 MTOS), рот рот р свиней, два- проитится хвостовой t-критерий Стьюдента; **** p <0,0001 по сравнению с ки b Amashusho agaragaza ibice by'umutima byandujwe na troponin-T na AZP (ibumoso) n'ingano y'uturemangingo (iburyo) (n = 330 (D6 MTOS), 369 (D6 MTNorm) uturemangingo/itsinda ryaturutse mu bice 10 bitandukanye bivuye mu ngurube zitandukanye, hakozwe ikizamini cya T-cy'umunyeshuri gifite imirizo ibiri; ****p < 0.0001 ugereranije n'ubwoko busanzwe). 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图像（ n = 330 （D6 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 Amashusho agaragaza ibice by'umutima byandujwe na calcarein-T na WGA (ibumoso) n'ingano y'uturemangingo (iburyo) (n = 330 (D6 MTOS), 369 bivuye mu bice 10 bitandukanye (D6 MTNorm)) Isuzuma ry'uturemangingo/组，两方法有尾学生t；ugereranije no kunanura bisanzwe，****p < 0.0001). b Репрезент Канные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оценка размера клеток (справа) (n = 330 (D6 MTOS), 369 (D6 MTNorm) из 10 ски скичч. дву данонние критерий Стьюдента; **** p b Amashusho agaragaza ibice by'umutima byandujwe na troponin-T na AZP (ibumoso) n'ingano y'uturemangingo (iburyo) (n = 330 (D6 MTOS), 369 (D6 MTNorm) bivuye mu bice 10 bitandukanye bivuye mu ngurube zitandukanye) Uturemangingo/itsinda, ibipimo by'imirizo ibiri T y'umunyeshuri; ****p < 0.0001 ugereranije n'uburyo busanzwe bwo kugabanya). c Amashusho agaragaza ibice by'umutima bya MTOS byo ku munsi wa 0 n'uwa 6 byashyizwemo immunolabel kuri troponin-T na NFATC4 hamwe n'isuzuma ry'ihererekanya rya NFATC4 mu ntungamubiri za CM (n = 4 (D0), ibice 3 (D6 MTOS) by'ingurube zitandukanye, hakorwa ikizamini cya T-Umunyeshuri ufite imirizo ibiri; *p < 0.05). c Amashusho agaragaza ibice by'umutima bya MTOS byo ku munsi wa 0 n'uwa 6 byashyizwemo immunolabel kuri troponin-T na NFATC4 hamwe n'isuzuma ry'ihererekanya rya NFATC4 mu ntungamubiri za CM (n = 4 (D0), ibice 3 (D6 MTOS) / itsinda bivuye mu ngurube zitandukanye, Ikizamini cya T-Umunyeshuri gifite imirizo ibiri kirakorwa; *p < 0.05). c Репрезент Канные изображения для срезов сердца 0 и 6 дней MTOS, им матомеченых для тропонина-Т и NFATC4, и количественная оценка кинлоккации NFATC4 в ядра kaвернозных клеток (n = 4) разных свиней, виполняется дву Кононний t-критерий Стьюдента; * p <0,05); c Amashusho agaragaza ibice by'umutima mu minsi 0 na 6 ya MTOS, yashyizwemo immunolabel kuri troponin-T na NFATC4, hamwe n'isuzuma ry'impinduka za NFATC4 mu gice cy'uturemangingo tw'imitsi (n = 4 (D0), uduce 3 (D6 MTOS) twavuye mu ngurube zitandukanye) bakoze ikizamini cya T cy'Umunyeshuri gifite imirizo ibiri; *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) 、 3 (D6 MTOS) 切片 / 组, 进行双尾学生 t 检验； * p <0.05)。 c Amashusho yerekana calcanin-T na NFATC4 immunolabeling 第 0 天和第 6 天 Ibice byumutima bya MTOS, na NFATC4 kuva NFATC4 易位至 CM selile nucleus 的 ingano 化 (n = 4 (D0), 3 (D6 MTOS) 切礼 / 组, 时间双尾学生 et 电影； * p <0.05). c Репрезент Канные изображения срезов сердца MTOS на 0 и 6 день для им матомарировки тропонином-Т и NFATC4 и количественная оценка нуклокации NFATC4 в ядра CM от разных свиней (n = 4 (D0) хвостатый t-критерий Стьюдента; * p <0,05); c Amashusho agaragaza ibice by'umutima bya MTOS ku munsi wa 0 na 6 ku bijyanye no gushyira ibimenyetso bya troponin-T na NFATC4 mu maraso no gupima uburyo NFATC4 ihinduka mu gice cy'umubiri cya CM kiva mu ngurube zitandukanye (n = 4 (D0), ibice 3 (D6 MTOS)/itsinda, t-criterion y'abanyeshuri; *p < 0.05).Uturongo tw'ikosa tugaragaza impuzandengo ± ihinduka risanzwe.
Ubushakashatsi ku ndwara z'umutima n'imitsi busaba ingero z'uturemangingo zikora neza imiterere y'umutima. Muri ubu bushakashatsi, hakozwe igikoresho cya CTCM kandi cyaragaragajwe gishobora gukurura ibice by'umutima bito cyane. Sisitemu ya CTCM irimo gukangura amashanyarazi mu buryo bw'umubiri hamwe no kongera T3 na Dex fluid. Igihe ibice by'umutima by'ingurube byahuraga n'ibi bintu, ubushobozi bwabyo bwo kubaho, imiterere yabyo, imikorere ya metabolic, n'imikorere ya transcription byakomeje kuba bimwe nk'uko byari bimeze mu ngingo nshya z'umutima nyuma y'iminsi 12 yo gukangura. Byongeye kandi, kunagura cyane ingingo z'umutima bishobora gutera hypertrophy y'umutima iterwa no kwiyongera k'umutima. Muri rusange, ibi bisubizo bishyigikira uruhare runini rw'imiterere y'umubiri mu kubungabunga imiterere y'umutima isanzwe kandi bitanga urubuga rwo gusuzuma imiti.
Ibintu byinshi bigira uruhare mu gushyiraho ibidukikije byiza ku mikorere no kubaho kw'imitsi y'umutima. Ibintu bigaragara cyane muri ibi bifitanye isano na (1) imikoranire hagati y'uturemangingo, (2) gukangura amashanyarazi, (3) ibintu by'ubuhumekero, na (4) ibice by'imikorere y'umubiri. Imikoranire y'uturemangingo hagati y'uturemangingo isaba urusobe rw'ibice bitatu rw'uturemangingo twinshi dushyigikiwe na matrix yo hanze y'uturemangingo. Iyo mikoranire y'uturemangingo iragoye kongera kubaka muri vitro binyuze mu guhuza ubwoko bw'uturemangingo, ariko ishobora kugerwaho byoroshye hakoreshejwe imiterere y'ibice by'umutima.
Gukangura imitsi no gukangura umutima hakoreshejwe amashanyarazi ni ingenzi cyane mu kubungabunga imiterere y'umutima33,34,35. Nubwo gukangura imitsi byakoreshejwe cyane mu kuvura no gukura kwa hiPSC-CM, ubushakashatsi bwinshi bwiza buherutse kugerageza gukangura ibice by'umutima mu mitsi hakoreshejwe uburyo bwa uniaxial. Ubu bushakashatsi bwerekana ko imiterere ya 2D uniaxial ifite ingaruka nziza ku miterere y'umutima mu gihe cyo gukangura imitsi. Muri ubu bushakashatsi, ibice by'umutima byari byuzuyemo imbaraga zo gukurura umutima17, imiterere ya auxotonic ya linear18, cyangwa imiterere y'umutima yasubiwemo hakoreshejwe uburyo bwa force transducer hamwe n'imbaraga zo guhangayika. Ariko, ubu buryo bukoresha uburyo bwa uniaxial tissue stretch nta kurengera ibidukikije, bigatuma habaho kugabanya gene nyinshi z'umutima cyangwa kwiyongera cyane kwa genes bifitanye isano n'imikorere idasanzwe yo gukangura imitsi. CTCM ivugwa hano itanga uburyo bwa 3D electromechanical stimulation bugana imiterere y'umutima mu buryo busanzwe mu gihe cy'uruziga n'uburyo bwa physiological (25% stretch, 40% systole, 60% diastole, na 72 beats ku munota). Nubwo ubu buryo bwo gukangura imiterere y’umubiri mu buryo butatu gusa budahagije kugira ngo ingingo zigumane ubusugire, uruvange rw’uburyo bwo gukangura imiterere y’umubiri n’ubw’umubiri hakoreshejwe T3/Dex rurakenewe kugira ngo ingingo zigumane ubuzima, imikorere n’ubusugire bihagije.
Ibintu bisekeje bigira uruhare runini mu guhindura imiterere y'umutima w'umuntu mukuru. Ibi byagaragajwe mu bushakashatsi bwa HiPS-CM aho T3 na Dex byongewe mu buryo bwo gukurura ingirabuzimafatizo kugira ngo byihutishe gukura kw'uturemangingo. T3 ishobora kugira ingaruka ku gutwara aside amine, isukari na kalisiyumu mu turemangingo tw'uturemangingo36. Byongeye kandi, T3 iteza imbere ishyirwa mu bikorwa rya MHC-α no kugabanya MHC-β, bigateza imbere ikorwa rya myofibrils zihuta mu maraso y'umutima akuze ugereranije na myofibrils zihuta buhoro mu maraso y'umwana uri mu nda. Kubura T3 mu barwayi ba hypothyroid bitera kubura imigozi ya myofibrillar no kugabanuka k'umuvuduko w'ijwi37. Dex ikora kuri receptors za glucocorticoid kandi byagaragaye ko yongera ubushobozi bwo gufungana k'umutima mu mitima yakuwemo umwuka;38 iyi ntambwe ifatwa nk'ifitanye isano n'ingaruka ku kwinjira kwa kalisiyumu iterwa n'imizigo (SOCE) 39,40. Byongeye kandi, Dex ifatana na receptors zayo, igatera igisubizo kinini mu turemangingo kigabanya imikorere y'ubudahangarwa bw'umubiri no kubyimba30.
Ibisubizo byacu bigaragaza ko gukangura umubiri (MS) byazamuye imikorere y’imitsi muri rusange ugereranije na Ctrl, ariko ntibyashoboye gukomeza kubaho, ubusugire bw’imiterere, no kugaragaza umutima mu minsi 12 mu mitsi. Ugereranyije na Ctrl, kongeramo T3 na Dex muri CTCM (MT) byazamuye ubuzima kandi bikomeza imiterere isa ya transcription, ubusugire bw’imiterere, n’imikorere ya metabolism hamwe n’imitsi mishya y’umutima mu minsi 12. Byongeye kandi, binyuze mu kugenzura urwego rw’imitsi, hakozwe icyitegererezo cya hyperextension-induced cardiac hypertrophy hakoreshejwe STCM, bigaragaza uburyo STCM ikora neza. Ni ngombwa kumenya ko nubwo kuvugurura umutima na fibrosis akenshi biba birimo ingingo zidafite aho zihuriye n’uturemangingo dushobora gutanga cytokines zikwiye ndetse na phagocytosis n’ibindi bintu bihindura, ibice by’umutima bishobora kwigana inzira ya fibrotic mu gusubiza stress no gukomereka. Ibi byasuzumwe mbere muri iki cyitegererezo cy’imitsi. Ni ngombwa kumenya ko ibipimo bya CTCM bishobora guhindurwa hakoreshejwe guhindura umuvuduko/amashanyarazi n’inshuro kugira ngo byigane indwara nyinshi nka tachycardia, bradycardia, n’imbaraga z’imitsi zisimburana mu mitsi (umutima udafunguye). Ibi bituma iyi sisitemu iba uburyo bwo gupima imiti. Ubushobozi bwa CTCM bwo kugaragaza ubwiyongere bw'umutima buterwa no gukora cyane butanga inzira yo kugerageza iyi sisitemu kugira ngo habeho ubuvuzi bwihariye. Muri make, ubu bushakashatsi bwerekana ko kunanura imitsi no gukangura umutima ari ingenzi cyane mu kubungabunga imiterere y'ingingo z'umutima.
Nubwo amakuru yatanzwe hano agaragaza ko CTCM ari urubuga rwiza cyane rwo gukora ibishushanyo bya myocardium idafite ingaruka mbi, ubu buryo bwo gukora ibishushanyo bufite imbogamizi zimwe na zimwe. Imbogamizi nyamukuru ya CTCM ni uko ishyiraho imihangayiko ihoraho ya mekanike ku bice, ibi bikaba bibuza ubushobozi bwo gukurikirana neza imitsi y'umutima muri buri ruziga. Byongeye kandi, bitewe n'ingano nto y'ibice by'umutima (7 mm), ubushobozi bwo gusuzuma imikorere ya systolic hanze ya sisitemu yo gukora ibishushanyo hakoreshejwe sensors zisanzwe z'imbaraga ni buke. Mu nyandiko iriho ubu, twatsinze igice cy'iyi mbogamizi dusuzuma voltage y'amajwi nk'ikimenyetso cy'imikorere yo gukora ibishushanyo. Ariko, iyi mbogamizi izasaba akazi kenshi kandi ishobora gukemurwa mu gihe kizaza dushyiraho uburyo bwo kugenzura imikorere y'ibice by'umutima mu gukora ibishushanyo, nko gukora ibishushanyo mbonera bya calcium na voltage-sensitive. Indi mbogamizi ya CTCM ni uko moderi ikora idakoresha stress y'umubiri (preload na afterload). Muri CTCM, igitutu cyatewe mu byerekezo bitandukanye kugira ngo cyongere 25% by'imitsi yo gukora ibishushanyo mbonera muri diastole (full stretch) na systole (uburebure bwo gukora ibishushanyo mu gihe cyo gukangura amashanyarazi) mu bice binini cyane. Iyi mbogamizi igomba gukurwaho mu miterere ya CTCM y’ejo hazaza hakoreshejwe igitutu gihagije ku ngingo z’umutima ku mpande zombi no gukoresha isano nyayo y’igitutu n’ingano biba mu byumba by’umutima.
Ivugurura rikabije ryavuzwe muri iyi nyandiko rigarukira gusa ku kwigana ibimenyetso bya hyperstretch. Bityo, iyi moderi ishobora gufasha mu kwiga ibimenyetso bya hypertrophic bitera stretch hatabayeho ibintu bya humoral cyangwa neural (bitabaho muri iyi sisitemu). Hakenewe ubundi bushakashatsi kugira ngo hongerwe ubwinshi bwa CTCM, urugero, guhuza imiterere n'uturemangingo tw'umubiri, plasma humoral factors zizenguruka, no kwitabwaho mu gihe guhuza imiterere n'uturemangingo tw'imitsi bizamura amahirwe yo kwigana indwara na CTCM.
Muri ubu bushakashatsi hakoreshejwe ingurube 13. Uburyo bwose bwo kuvura inyamaswa bwakozwe hakurikijwe amabwiriza y’ikigo kandi byemejwe na Komite Ishinzwe Kwita ku Matungo ya Kaminuza ya Louisville. Umuyoboro w’amaraso wafashwe neza maze umutima ushyirwamo litiro 1 y’indwara z’umutima (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U/mL heparin, pH kugeza kuri 7.4); Imitima yabitswe mu gikoresho gikonje cyane cy’umutima kugeza igihe ijyanwa muri laboratwari ku rubura, ubusanzwe ikaba iri munsi y’iminota 10. Imitima yabitswe mu gikoresho gikonje cyane cy’umutima kugeza igihe ijyanwa muri laboratwari ku rubura, ubusanzwe ikaba iri munsi y’iminota 10. сердца хранили в ледяном кардиоп угическом растворе до зировки в лабораторию на льду, что обонно занимает <10 мин. imitima yabitswe mu gikoresho gikonje cyane cy’umutima kugeza igihe kijyanwa muri laboratwari ku rubura, ubusanzwe bifata iminota iri munsi ya 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 кардиоп огии до матировки в лабораторию на льду, обоно <10 мин. Komeza umutima kuri barafu kugeza igihe ujyanwa muri laboratwari kuri barafu, ubusanzwe munsi y'iminota 10.
Igikoresho cya CTCM cyakozwe muri porogaramu ya SolidWorks ikoresha mudasobwa (CAD). Ibyumba byo gukurura, ibice bigabanya n'ibyumba byo mu mwuka bikozwe muri pulasitiki ya acrylic ya CNC. Impeta yo gukingira ifite umurambararo wa mm 7 ikozwe muri polyethylene ifite ubucucike bwinshi (HDPE) hagati kandi ifite umuyoboro wa o-ring wo gushyiramo o-ring ya silicone ikoreshwa mu gufunga igice kiri munsi. Uduce duto twa silica dutandukanya icyumba cyo gukurura n'isahani yo gutandukanya. Uduce twa silica ducibwa na laser uhereye ku rupapuro rwa silica rufite ubugari bwa santimetero 0.02 kandi dufite ubukana bwa santimetero 35. Gasket za silica zo hepfo n'izo hejuru zicibwa na laser uhereye ku rupapuro rwa silica rufite ubugari bwa santimetero 1/16 kandi zifite ubukana bwa santimetero 50. Udupira twa silica twa litiro 316 n'amababa y'amababa bikoreshwa mu gufatanya blok no gukora agapfundikizo k'umwuka.
Urubaho rwagenewe ikoranabuhanga ryacapwe (PCB) rwagenewe guhuzwa na sisitemu ya C-PACE-EM. Uduce duhuza imashini zo mu Busuwisi kuri PCB duhujwe na electrodes za grafiti hakoreshejwe insinga z'umuringa zikozwe mu ifeza n'utugozi tw'umuringa 0-60 dushyizwe muri electrodes. Urubaho rwacapwe rushyirwa mu gifuniko cya printer ya 3D.
Igikoresho cya CTCM kigenzurwa na actuator ya pneumatic ishobora gukoreshwa (PPD) ikora umuvuduko w’amaraso ugenzurwa nk’uko umutima ugenda wiyongera. Uko umuvuduko uri mu cyumba cy’umwuka wiyongera, urukiramende rwa silicone rworoshye ruzamuka, rushyira umwuka munsi y’aho umubiri uherereye. Agace k’ingingo kazagenda karangizwa n’iyi mashini isohoka, bigana kwaguka k’umutima mu gihe cyo kwiyahura. Mu gihe cyo kuruhuka, hakoreshejwe amashanyarazi binyuze muri electrodes za grafiti, byagabanyije umuvuduko mu cyumba cy’umwuka bigatuma ibice by’ingingo bigabanuka. Imbere mu muyoboro hari valve ya hemostatic ifite sensor y’umuvuduko kugira ngo imenye umuvuduko uri mu mwuka. Umuvuduko wumvise na sensor y’umuvuduko ushyirwa ku gikoresho gikusanya amakuru gihujwe na mudasobwa igendanwa. Ibi bituma habaho gukurikirana umuvuduko uri mu cyumba cy’umwuka. Iyo umuvuduko ntarengwa w’icyumba wageraga (80 mmHg, 140 mmHg OS), igikoresho cyo gufata amakuru cyategetswe kohereza ikimenyetso kuri sisitemu ya C-PACE-EM kugira ngo hakorwe ikimenyetso cya voltage ya biphasic kuri 2 ms, gishyizwe kuri 4 V.
Ibice by'umutima byabonetse kandi imiterere y'imitsi mu byobo 6 yakozwe ku buryo bukurikira: Kwimura imitima yakuwe mu gikoresho cyo kwimura ikajya mu gikapu kirimo ubukonje (4° C.) bw'umutima. Inkondo y'ibumoso yakuwemo icyuma gikozwe mu buryo butanduye maze icibwamo ibice bya cm3 1-2. Ibi bice by'imitsi byashyizwe ku byuma bishyigikira imitsi hakoreshejwe kole hanyuma bishyirwa mu bwogero bw'imitsi mito irimo umuti wa Tyrode kandi bikomeza gushyirwamo ogisijeni (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), 1.8 mM CaCl2 (1.8 ml 1 M), kugeza kuri L 1 ddH2O). Microtome ihindagurika yashyizweho kugira ngo ikate uduce twa µm 300 tw’ubugari ku muvuduko wa 80 Hz, amplitude ya 2 mm itambitse, n’umuvuduko wa 0.03 mm/s. Ubwogero bw’inyama bwari bukikijwe n’urubura kugira ngo umuti ugumane ubukonje kandi ubushyuhe buguma kuri 4°C. Hindura ibice by’inyama kuva mu bwogero bwa microtome ujya mu bwogero bwo gushyushya burimo umuti wa Tyrode uhora uhumekwa kuri barafu kugeza igihe habonetse ibice bihagije byo gukoresha isahani imwe yo gukuriraho. Ku bijyanye no gukuriraho, ibice by’inyama byashyizwe ku nkunga ya polyurethane ya mm 6 y’ubugari maze bishyirwa muri ml 6 z’ubugari bwabugenewe (199 medium, 1x ITS supplement, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-alkaline na 2X antibiyotike-antifungal). Gukangura amashanyarazi (10 V, umuvuduko wa 1.2 Hz) byashyizwe ku bice by’inyama binyuze muri C-Pace. Ku bijyanye n’indwara za TD, T3 nshya na Dex byongewe kuri 100 nM na 1 μM buri mpinduka isanzwe. Ingufu zishyirwamo ogisijeni mbere yo gusimburwa inshuro 3 ku munsi. Ibice by’uturemangingo byashyizwe mu cyuma gishyushya ubushyuhe bwa 37°C na 5% bya CO2.
Ku bijyanye no gupima CTCM, ibice by'imitsi byashyizwe ku gishushanyo cya 3D cyakozwe mu gikoresho cya Petri kirimo umuti wa Tyrode wahinduwe. Iki gikoresho cyagenewe kongera ingano y'agace k'umutima ho 25% by'ubuso bw'impeta yo gushyigikira. Ibi bikorwa kugira ngo ibice by'umutima bitarambuka nyuma yo kwimurwa kuva kuri umuti wa Tyrode ukajya kuri mashini no mu gihe cyo guterana. Hakoreshejwe kole ya histoacrylic, ibice bifite ubugari bwa µm 300 byashyizwe ku mpeta yo gushyigikira ifite umurambararo wa mm 7. Nyuma yo gushyira ibice by'imitsi ku mpeta yo gushyigikira, gabanya ibice by'imitsi birenga hanyuma usubize ibice by'imitsi mu bwogero bwa Tyrode ku rubura (4°C) kugeza igihe ibice bihagije biteguwe ku gikoresho kimwe. Igihe cyose cyo gutunganya ibikoresho byose ntikigomba kurenza amasaha 2. Nyuma y'uko ibice 6 by'imitsi bifatanye n'impeta zo gushyigikira, igikoresho cya CTCM cyarateranijwe. Icyumba cyo kwigisha cya CTCM cyuzuzwa mbere na mbere 21 ml ya ogisijeni. Himura ibice by'imitsi mu cyumba cyo kwigisha kandi ukureho witonze uduce tw'umwuka ukoresheje pipette. Igice cy'uturemangingo gishyirwa mu mwobo hanyuma kigashyirwa ahantu hacyo buhoro buhoro. Hanyuma, shyira umupfundikizo wa electrode ku gikoresho hanyuma wimurire igikoresho kuri count. Hanyuma huza CTCM n'umuyoboro w'umwuka na sisitemu ya C-PACE-EM. Pneumatic actuator irakinguka hanyuma valve y'umwuka igafungura CTCM. Sisitemu ya C-PACE-EM yashyizweho kugira ngo itange 4 V kuri 1.2 Hz mu gihe cyo gupima biphasic mu buryo bwa 2 ms. Imashini yahinduwe kabiri ku munsi kandi electrode zahinduwe rimwe ku munsi kugira ngo hirindwe graphite yirundanye kuri electrode. Iyo bibaye ngombwa, ibice by'uturemangingo bishobora gukurwa mu byobo byabyo byo gukurura kugira ngo hasohorwe utubumbe tw'umwuka dushobora kuba twaguye munsi yatwo. Ku bijyanye n'uburyo bwo kuvurwa na MT, T3/Dex yongewemo buri mpinduka ikoreshwa na 100 nM T3 na 1 μM Dex. Ibikoresho bya CTCM byashyizwe muri count kuri 37°C na 5% CO2.
Kugira ngo haboneke inzira zirambuye z'uduce tw'umutima, hakozwe sisitemu yihariye ya kamera. Kamera ya SLR (Canon Rebel T7i, Canon, Tokyo, Ubuyapani) yakoreshejwe hamwe na lensi ya Navitar Zoom 7000 18-108mm macro (Navitar, San Francisco, CA). Ishusho yakozwe ku bushyuhe bw'icyumba nyuma yo gusimbuza icyuma gishya. Kamera ishyirwa ku nguni ya 51° kandi videwo ifatwa kuri frame 30 ku isegonda. Ubwa mbere, porogaramu ifunguye (MUSCLEMOTION43) yakoreshejwe hamwe na Image-J kugira ngo igereranye imikorere y'uduce tw'umutima. Iyo mask yakozwe hakoreshejwe MATLAB (MathWorks, Natick, MA, USA) kugira ngo hamenyekane uturere dushimishije two gukubita uduce tw'umutima kugira ngo hirindwe urusaku. Udupfukamunwa dushyirwa ku mashusho yose mu buryo bwa frame hanyuma twoherezwa kuri plug-in ya MUSCLEMOTION. Muscle Motion ikoresha ubukana bw'impuzandengo bwa pixels muri buri frame kugira ngo igereranye imikorere yayo ugereranije n'ishusho. Amakuru yafashwe, ayungururwa kandi akoreshwa mu gupima igihe cy'uruziga no gusuzuma kurekura kw'imitsi mu gihe cy'uruziga rw'umutima. Videwo yafashwe yakozwe nyuma hakoreshejwe akayunguruzo k'ikoranabuhanga ka zero-phase. Kugira ngo harebwe kurekura kw'imitsi (kugera ku muvuduko), hakozwe isesengura ryo kurekura kw'imitsi kugira ngo hamenyekane aho imitsi igera n'aho igera mu kimenyetso cyafashwe. Byongeye kandi, gukuraho imiterere y'imitsi bikorwa hakoreshejwe polynomial ya 6th order kugira ngo hakurweho kurekura kw'imitsi. Kode ya porogaramu yakozwe muri MATLAB kugira ngo hamenyekane uko imitsi igenda ku isi, igihe cy'uruziga, igihe cyo kuruhuka, n'igihe cyo kugabanuka (Kode y'inyongera ya Porogaramu 44).
Mu gusesengura imitsi, dukoresheje videwo zimwe zakozwe kugira ngo dusuzume uburyo bwo kurekura, twabanje gukurikirana amashusho abiri agaragaza imiterere y'imitsi (ingingo zo hejuru (zo hejuru) n'izo hasi (zo hasi) z'imitsi) dukurikije porogaramu ya MUSCLEMOTION. Hanyuma twagabanyije uturere tw'imitsi dukoresha uburyo bwo gusiga igicucu ku ngirabuzimafatizo zagabanyijwemo ibice (Ishusho y'inyongera ya 2a). Hanyuma ngirabuzimafatizo zagabanyijwemo ibice icumi, maze uburemere kuri buri buso bubarwa hakoreshejwe ikigereranyo gikurikira: Imitsi = (Sup-Sdown) / Sdown, aho Sup na Sdown ari intera y'imiterere uhereye ku gicucu cyo hejuru n'icyo hasi cy'umwenda, uko bikurikirana (Ishusho y'inyongera ya .2b).
Ibice by'umutima byashyizwe muri 4% ya paraformaldehyde mu gihe cy'amasaha 48. Ibice byashyizweho byakuwemo amazi muri 10% na 20% bya sucrose mu gihe cy'isaha 1, hanyuma muri 30% bya sucrose mu ijoro ryose. Ibice byashyizwemo mu gice cy'ubushyuhe bwiza (OCT compound) hanyuma bigakonjeshwa buhoro buhoro mu bwogero bw'urubura bwa isopentane/ubwumutse. Bika ibice bya OCT embedding kuri -80 °C kugeza bitandukanijwe. Amaslayidi yateguwe nk'ibice bifite ubugari bwa 8 μm.
Kugira ngo ukureho OCT mu bice by'umutima, shyushya slides ku gikoresho gishyushya kuri 95 °C mu gihe cy'iminota 5. Shyiramo ml 1 ya PBS kuri buri slide hanyuma uyishyire mu bushyuhe bw'icyumba mu minota 30, hanyuma uyishyire mu bice ushyiremo 0.1% ya Triton-X muri PBS mu minota 15 ku bushyuhe bw'icyumba. Kugira ngo wirinde ko antibiyotike zitari zimwe zifatana n'icyitegererezo, ongeramo ml 1 ya 3% ya BSA ku slides hanyuma uyishyire mu bushyuhe bw'icyumba mu gihe cy'isaha 1. BSA yakuweho hanyuma slides zogezwa na PBS. Shyira akamenyetso kuri buri sampuli ukoresheje ikaramu. Antibodies z'ibanze (zavanzwe 1:200 muri 1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) na troponin-T (Thermo Scientific; #MA5-12960) byongeweho mu minota 90, hanyuma antibodies z'inyongera (zavanzwe 1:200 muri 1% BSA) ku mbeba Alexa Fluor 488 (Thermo Scientific; #A16079), ku rukwavu Alexa Fluor 594 (Thermo Scientific; #T6391) mu minota 90 y'inyongera. Yogejwe inshuro 3 na PBS Kugira ngo dutandukanye irangi ry'intego n'inyuma, twakoresheje antibodies z'inyongera gusa nk'uburyo bwo kugenzura. Amaherezo, hongeweho irangi rya nuclear rya DAPI maze slides zishyirwa muri vectashield (Vector Laboratories) kandi zifunze na hariri y'inzara. -x magnification) na mikroskopu ya Keyence ifite magnification ya 40x.
WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) kuri 5 μg/ml muri PBS yakoreshejwe mu gusiga irangi rya WGA hanyuma ishyirwa ku bice bihamye mu gihe cy'iminota 30 ku bushyuhe bw'icyumba. Hanyuma slides zogejwe na PBS hanyuma Sudani y'umukara yongerwa kuri buri slide hanyuma ishyirwa mu cyuma mu gihe cy'iminota 30. slides zogejwe na PBS hanyuma vectashield embedding medium yongerwaho. slides zashushanyijwe kuri mikorosikopi ya Keyence ku gipimo cya 40x.
OCT yakuwe mu bipimo nkuko byasobanuwe haruguru. Nyuma yo gukuraho OCT, shyiramo slides mu gikoresho cya Bouin ijoro ryose. Hanyuma slides zogejwe n'amazi yaciwe mu gihe cy'isaha 1 hanyuma zishyirwa mu gikoresho cya Bibrich aloe aside fuchsin mu gihe cy'iminota 10. Hanyuma slides zogejwe n'amazi yaciwe hanyuma zishyirwa mu gikoresho cya 5% phosphomolybdenum / 5% phosphotungstic acid mu gihe cy'iminota 10. Udasukuye, shyira slides mu gikoresho cy'ubururu cya aniline mu gihe cy'iminota 15. Hanyuma slides zogejwe n'amazi yaciwe hanyuma zishyirwa mu gikoresho cya 1% aside asetiki mu gihe cy'iminota 2. slides zogejwe muri 200 N ethanol hanyuma zishyirwa muri xylene. slides zaranduye zashushanyijwe hakoreshejwe mikorosikopi ya Keyence ifite intego inshuro 10. Ijanisha ry'ubuso bwa fibrosis ryapimwe hakoreshejwe porogaramu ya Keyence Analyzer.
Isuzuma ry’ubuzima bw’uturemangingo twa CyQUANT™ MTT (Invitrogen, Carlsbad, CA), nimero ya kataloge V13154, hakurikijwe amabwiriza y’uruganda hamwe n’impinduka zimwe na zimwe. By’umwihariko, hakoreshejwe uburyo bwo kubaga bufite umurambararo wa mm 6 kugira ngo harebwe ingano y’uturemangingo imwe mu gihe cyo gusesengura MTT. Uturemangingo twashyizwe mu mwobo w’isahani y’amabati 12 irimo MTT hakurikijwe amabwiriza y’uruganda. Utwo duce dushyirwa kuri 37°C. mu gihe cy’amasaha 3 maze utwo dusimba duhindura imiterere ya MTT kugira ngo tukore imvange y’umuhengeri ya formazan. Simbuza umuti wa MTT 1 ml DMSO hanyuma utwikirize kuri 37°C mu gihe cy’iminota 15 kugira ngo ukuremo formazan y’umuhengeri mu bice by’umutima. Ingero zavanzwe ku gipimo cya 1:10 muri DMSO mu masahani yo hasi afite ubuso busobanutse neza bwa 96-wells clear n’ubukana bw’ibara ry’umuhengeri bwapimwe kuri 570 nm hakoreshejwe uburyo bwo gusoma plate ya Cytation (BioTek). Ingano y’ibipimo yashyizwe ku buremere bwa buri gice cy’umutima.
Igikoresho cyo gukata umutima cyasimbuwe n'igikoresho kirimo 1 μCi/ml [5-3H]-glucose (Moravek Biochemicals, Brea, CA, USA) kugira ngo hakoreshwe isuzuma ry'ikoreshwa rya glucose nk'uko byasobanuwe mbere. Nyuma y'amasaha 4 yo gucukura, ongeramo 100 µl y'ubushyuhe buciriritse ku muyoboro wa microcentrifuge ufunguye urimo 100 µl ya 0.2 N HCl. Hanyuma umuyoboro washyizwe mu muyoboro wa scintillation urimo 500 μl ya dH2O kugira ngo uhinduke [3H]2O mu gihe cy'amasaha 72 kuri 37°C. Hanyuma ukuremo umuyoboro wa microcentrifuge mu muyoboro wa scintillation hanyuma wongeremo 10 ml y'amazi ya scintillation. Kubara scintillation byakozwe hakoreshejwe Tri-Carb 2900TR liquid scintillation analyzer (Packard Bioscience Company, Meriden, CT, USA). Hanyuma ikoreshwa rya glucose ryabazwe hashingiwe ku gikorwa cya [5-3H] - glucose yihariye, uburinganire budahagije n'inyuma, kugabanuka kwa [5-3H] - glucose idafite ikirango, no kugabanya ubushobozi bwa squinlation. Amakuru ashyirwa ku rugero rusanzwe ku bice by'umutima.
Nyuma yo guhuza ingirabuzimafatizo muri Trizol, RNA yakuwe mu bice by'umutima hakoreshejwe Qiagen miRNeasy Micro Kit #210874 hakurikijwe amabwiriza y'uwakoze. Gutegura isomero rya RNAsec, gukurikirana no gusesengura amakuru byakozwe ku buryo bukurikira:
1 μg ya RNA kuri buri ngero yakoreshejwe nk'ibikoresho byo gutangira gutegura isomero rya RNA. Amasomero yo gukurikirana yakozwe hakoreshejwe NEBHext UltraTM RNA Library Preparation Kit for Illumina (NEB, USA) hakurikijwe inama z'uwakoze, kandi kode z'ibipimo byongewe ku miterere y'ibiranga kuri buri ngero. Muri make, mRNA yasukuwe kuva kuri RNA yose hakoreshejwe uduce twa magnetic beads dufatanye na poly-T oligonucleotides. Gucikamo ibice bikorwa hakoreshejwe divalent cations ku bushyuhe bwinshi muri NEBHext First Strand Synthesis Reaction Buffer (5X). CDNA ya mbere yakozwe hakoreshejwe random hexamer primers na M-MuLV reverse transcriptase (RNase H-). CDNA ya kabiri yakozwe hakoreshejwe DNA polymerase I na RNase H. Ibindi birebire bihinduka impera zoroheje hakoreshejwe exonuclease/polymerase activity. Nyuma yo gukuraho impera ya 3′ y'agace ka ADN, ADAPTER YA NEBHext ifite imiterere ya hairpin loop irashyirwa kuri yo kugira ngo itegurirwe hybridization. Ku bijyanye no gutoranya ibice bya cDNA by'uburebure bwa 150-200 bp. ibice bya biblioteki byatunganyijwe hakoreshejwe sisitemu ya AMPure XP (Beckman Coulter, Beverly, USA). Hanyuma, hakoreshejwe 3 μl USER Enzyme (NEB, USA) ifite cDNA yatoranijwe ifite adaptateri mu minota 15 kuri 37°C hanyuma ikoreshwa iminota 5 kuri 95°C mbere ya PCR. PCR yakozwe hakoreshejwe polymerase ya Phusion High-Fidelity DNA, primers za PCR zisanzwe, na primers za Index (X). Amaherezo, ibikoresho bya PCR byaratunganyijwe (sisitemu ya AMPure XP) kandi ireme ry'ibiblioteki ryapimwe kuri sisitemu ya Agilent Bioanalyzer 2100. Ibitabo bya cDNA byakurikiranywe hakoreshejwe sequencer ya Novaseq. Dosiye z'amashusho yavuye muri Illumina zahinduwe mo isomwa ry'umwimerere hakoreshejwe CASAVA Base Calling. Amakuru yakoreshejwe abikwa muri dosiye za FASTQ (fq) zirimo isesengura ry'ibisomwa n'imiterere y'ibanze ijyanye nabyo. Hitamo HISAT2 kugira ngo uhuze uburyo bwo gusesengura buyunguruye na genome ya Sscrofa11.1. Muri rusange, HISAT2 ishyigikira genome z'ingano iyo ari yo yose, harimo na genome zirenga miliyari 4 z'ibanze, kandi agaciro gasanzwe kashyizweho kuri parameter nyinshi. Guhuza ibyanditswe bivuye kuri RNA Seq bishobora guhuzwa neza hakoreshejwe HISAT2, sisitemu yihuta iboneka ubu, ifite ubunyangamugayo bumwe cyangwa bwiza kurusha ubundi buryo ubwo aribwo bwose.
Ubwinshi bw'inyandiko zigaragaza neza urwego rw'igaragazwa ry'uturemangingo. Ingano y'igaragazwa ry'uturemangingo ipimwa hakurikijwe ubwinshi bw'inyandiko zigaragaza uturemangingo (umubare w'ikurikiranwa) zijyanye na genome cyangwa exons. Umubare w'ibisomwa ujyanye n'urwego rw'igaragazwa ry'uturemangingo, uburebure bwa gene, n'ubujyakuzimu bw'ikurikiranwa. FPKM (ibice kuri pauni igihumbi z'ishingiro ry'inyandiko zikurikirana kuri pauni miliyoni z'ishingiro) byarabazwe kandi agaciro ka P k'itandukaniro ry'imvugo kagenwe hakoreshejwe paki ya DESeq2. Hanyuma twabaze igipimo cyo kuvumbura ikinyoma (FDR) kuri buri gaciro ka P dukoresheje uburyo bwa Benjamini-Hochberg9 dushingiye ku buryo bwa R bwubatswemo "p.adjust".
RNA yakuwe mu bice by'umutima yahinduwe cDNA ku gipimo cya 200 ng/μl hakoreshejwe uruvange rwa SuperScript IV Vilo Master rwavuye muri Thermo (Thermo, cat. no. 11756050). RT-PCR y'ingano yakozwe hakoreshejwe plate ya reaction igaragara ya Applied Biosystems Endura Plate Microamp 384-well transparent (Thermo, cat. no. 4483319) na microamp optical adhesive (Thermo, cat. no. 4311971). Uruvange rw'irangizwa rwari rugizwe na 5 µl Taqman Fast Advanced Master mix (Thermo, cat # 4444557), 0.5 µl Taqman Primer na 3.5 µl H2O bivanze kuri buri ruzi. Imizunguko isanzwe ya qPCR yakozwe kandi agaciro ka CT kapimwe hakoreshejwe igikoresho cya PCR cya Applied Biosystems Quantstudio 5 real-time (384-well module; product # A28135). Imashini za Taqman primers zaguzwe muri 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). Agaciro ka CT k'ibipimo byose kari gasanzwe kuri GAPDH y'ubucukuzi bw'inzu.
Itangazo rya NT-ProBNP ryasuzumwe hakoreshejwe agakoresho ka NT-ProBNP (ingurube) (Cat. No. MBS2086979, MyBioSource) hakurikijwe amabwiriza y’uwakoze. Muri make, 250 µl za buri gipimo n’ibisanzwe byongeweho kabiri kuri buri cyobo. Ukimara kongeramo icyitegererezo, ongeramo 50 µl bya Assay Reagent A kuri buri cyobo. Kungura isahani witonze hanyuma ufunge ifu. Hanyuma ibinini byashyizwe kuri 37°C mu gihe cy'isaha 1. Hanyuma shyiramo umuti hanyuma woze amafu inshuro 4 ukoresheje 350 µl bya 1X woza, ushyiremo umuti woza mu gihe cy'iminota 1-2 buri gihe. Hanyuma ongeramo 100 µl bya Assay Reagent B kuri buri cyobo hanyuma ufunge ifu ya plate. Ikinini cyanyeganyejwe buhoro buhoro maze gishyirwa kuri 37°C mu gihe cy'iminota 30. Shyiramo umuti wogeje inshuro 5 ukoresheje µl 350 z'umuti wogeje inshuro 1. Shyiramo µl 90 z'umuti wogeje kuri buri mwobo hanyuma ufunge isahani. Shyira isahani kuri 37°C mu gihe cy'iminota 10-20. Shyiramo µl 50 z'umuti wogeje kuri buri mwobo. Isahani yapimwe ako kanya hakoreshejwe uburyo bwo gusoma plate bwa Cytation (BioTek) kuri 450 nm.
Isesengura ry’ingufu ryakozwe kugira ngo hatoranywe ingano z’amatsinda azatanga imbaraga zirenze 80% zo kubona impinduka ya 10% mu gipimo hamwe n’igipimo cy’ikosa rya 5% ry’ubwoko bwa I. Isesengura ry’ingufu ryakozwe kugira ngo hatoranywe ingano z’amatsinda azatanga imbaraga zirenze 80% zo kubona impinduka ya 10% mu gipimo hamwe n’igipimo cy’ikosa rya 5% ry’ubwoko bwa I. Анз мощности был выполнен для выбора размеров групп, кот кае обеспечат> 80% мощности для обнаружения 10% абсолтного изменения Параметра с 5% частотой чибобок ча I. Isesengura ry'ingufu ryakozwe kugira ngo hatoranywe ingano z'amatsinda azatanga imbaraga zirenze 80% zo kubona impinduka za parameter 10% hamwe n'igipimo cy'ikosa rya 5% ry'ubwoko bwa I.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 型错误率的组大小。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 型错误率的组大小。 Был проекты Isesengura ry'ingufu ryakozwe kugira ngo hatoranywe ingano y'itsinda ryatanga imbaraga zirenze 80% zo kubona impinduka za parameter 10% n'igipimo cy'ikosa rya type I cya 5%.Ibice by'uturemangingo byatoranijwe mu buryo butunguranye mbere y'igerageza. Isesengura ryose ryakozwe mu buryo butagaragara ku miterere y'umubiri kandi ingero zakozwe mu buryo burambuye nyuma y'uko amakuru yose asesenguwe. Porogaramu ya GraphPad Prism (San Diego, CA) yakoreshejwe mu gukora isesengura ryose ry'ibarurishamibare. Ku mibare yose, agaciro ka p kafashwe nk'akamaro ku gaciro ka <0.05. Ku mibare yose, agaciro ka p kafatwaga nk'akamaro ku gaciro <0.05. Для всей стати мати p-значения считались значимыми при значениях <0,05. Ku mibare yose, agaciro ka p kafatwaga nk'akamaro ku gaciro <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 стати мати p-значения считались значимыми при значениях <0,05. Ku mibare yose, agaciro ka p kafatwaga nk'akamaro ku gaciro <0.05.Ikizamini cya T-T cyakozwe n'umunyeshuri ufite imirizo ibiri cyakozwe ku makuru hakoreshwa igereranya 2 gusa. ANOVA y'inzira imwe cyangwa ebyiri yakoreshejwe kugira ngo hamenyekane akamaro hagati y'amatsinda menshi. Mu gihe cyo gukora ibizamini bya nyuma y'ibizamini, ikosora rya Tukey ryakoreshejwe kugira ngo harebwe igereranya ryinshi. Amakuru ya RNAsec afite ibarurishamibare ryihariye mu kubara FDR na p.adjust nk'uko byasobanuwe mu gice cya Methods.
Kugira ngo ubone amakuru arambuye ku gishushanyo mbonera cy'ubushakashatsi, reba incamake ya Raporo y'Ubushakashatsi bw'Ibintu ijyanye n'iyi nkuru.