【摘要】： 目的:1、探索脂肪間充質(zhì)干細(xì)胞(ADMSCs)的分離、體外培養(yǎng),觀察ADMSCs長(zhǎng)期傳代培養(yǎng)的遺傳穩(wěn)定性。2、以5-氮胞苷(5-aza)作為誘導(dǎo)劑,觀察5-aza對(duì)ADMSCs向心肌樣細(xì)胞誘導(dǎo)分化的作用。3、觀察不同濃度的5-aza誘導(dǎo)ADMSCs向心肌細(xì)胞分化過(guò)程中的毒理遺傳學(xué)影響。 方法:1、采用Ⅰ型膠原酶消化法自成人脂肪組織中分離培養(yǎng)ADMSCs,并在體外持續(xù)傳代培養(yǎng)。對(duì)第3代ADMSCs進(jìn)行表面標(biāo)志分子鑒定,對(duì)第3代及第25代細(xì)胞分別流式細(xì)胞儀檢測(cè)細(xì)胞增殖指數(shù)、常規(guī)核型分析及電鏡下觀察超微結(jié)構(gòu),同時(shí)對(duì)第3代、25代及30代細(xì)胞進(jìn)行腫瘤特異抗原的檢測(cè)。2、取生長(zhǎng)狀態(tài)良好的第3代ADMSCs以10μmol/L5-aza誘導(dǎo)作用24h后,用含體積分?jǐn)?shù)為5%胎牛血清的完全培養(yǎng)基繼續(xù)培養(yǎng),鏡下觀察細(xì)胞形態(tài)學(xué)變化,采用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)方法檢測(cè)心肌早期轉(zhuǎn)錄因子(NKX2.5、GATA4)及心肌特異性肌鈣蛋白Ⅰ(cTnI)基因的表達(dá),同時(shí)免疫細(xì)胞化學(xué)染色鑒定cTnI。3、分別用不同誘導(dǎo)濃度的5-aza 5μmol/L(Ⅰ組)、10μmol/L(Ⅱ組)、20μmol/L(Ⅲ組)對(duì)ADMSCs進(jìn)行誘導(dǎo),同時(shí)設(shè)立對(duì)照組(Ⅳ組),光鏡下觀察各組細(xì)胞的生長(zhǎng)情況及形態(tài)學(xué)變化,免疫細(xì)胞化學(xué)方法檢測(cè)cTn-Ⅰ,RT-PCR方法檢測(cè)cTn-Ⅰ基因的表達(dá),流式細(xì)胞儀檢測(cè)細(xì)胞周期及凋亡,并對(duì)試驗(yàn)各組常規(guī)核型分析。 結(jié)果:1、成人脂肪組織中含有大量間充質(zhì)干細(xì)胞,并易于分離、培養(yǎng),流式鑒定CD13、CD59、CD44陽(yáng)性表達(dá),CD34及HLA-DR陰性表達(dá)。20代時(shí)細(xì)胞增殖速度明顯加快,可見(jiàn)到形態(tài)略不規(guī)則的細(xì)胞出現(xiàn)。25代時(shí)形態(tài)不規(guī)則細(xì)胞增多,核漿比增加,染色體出現(xiàn)斷裂、畸變,呈亞二倍體核型或超二倍體核型,流式細(xì)胞儀檢測(cè)結(jié)果顯示細(xì)胞DNA合成旺盛,電鏡下超微結(jié)構(gòu)出現(xiàn)改變。2、5-aza作用ADMSCs后7d細(xì)胞形態(tài)及排列開(kāi)始發(fā)生變化,RT-PCR產(chǎn)物條帶即見(jiàn)NKX2.5、GATA4基因的表達(dá)。4周后可見(jiàn)胞體明顯增大,球形細(xì)胞增多,體積大小不等,胞漿粗糙,極個(gè)別細(xì)胞出現(xiàn)細(xì)微搏動(dòng),免疫細(xì)胞化學(xué)結(jié)果顯示cTnⅠ陽(yáng)性,RT-PCR產(chǎn)物條帶見(jiàn)cTnⅠ基因表達(dá)。3、Ⅰ、Ⅱ、Ⅲ組ADMSCs誘導(dǎo)后呈現(xiàn)類(lèi)似心肌細(xì)胞的形態(tài)學(xué)特征。4周后Ⅰ、Ⅱ、Ⅲ組免疫組化結(jié)果見(jiàn)cTnⅠ陽(yáng)性表達(dá),RT-PCR條帶見(jiàn)cTnⅠ基因表達(dá)。各組染色體結(jié)構(gòu)及數(shù)目均未見(jiàn)異常。 結(jié)論:1、建立了一種自人體脂肪組織分離、培養(yǎng)ADMSCs經(jīng)濟(jì)簡(jiǎn)便的方法,ADMSCs的長(zhǎng)期體外培養(yǎng)具有遺傳不穩(wěn)定性,有向腫瘤細(xì)胞突變的傾向。2、5-aza可以誘導(dǎo)ADMSCs向自發(fā)搏動(dòng)的心肌樣細(xì)胞分化。3、5-aza作為誘導(dǎo)劑對(duì)脂肪間充質(zhì)干細(xì)胞遺傳性無(wú)不良影響。
[Abstract]:Objective: to explore the isolation of adipose mesenchymal stem cells (ADMSCs) from adipose mesenchymal stem cells, culture in vitro, observe the genetic stability of ADMSCs, and use 5-azacytidine (5-aza) as inducer. To observe the effect of 5-aza on the differentiation of ADMSCs into cardiomyocyte-like cells, and to observe the toxicgenetic effects of different concentrations of 5-aza on the differentiation of ADMSCs into cardiomyocytes. Methods ADMSCs, was isolated from adult adipose tissue by type I collagenase digestion and cultured continuously in vitro. The surface marker molecules of the third generation ADMSCs were identified, the proliferation index of the third generation and the 25 generation cells were detected by flow cytometry, and the ultrastructure was observed by conventional karyotype analysis and electron microscopy. At the same time, the tumor specific antigens were detected in the cells of passage 25 and 30 of the third generation. The third generation of ADMSCs, which grew well, was induced by 10 渭 mol/L5-aza for 24 hours, and then cultured in a complete medium containing 5% fetal bovine serum by volume fraction. Morphological changes were observed under microscope. The expression of myocardial early transcription factor (NKX2.5,GATA4) and myocardial specific troponin 鈪，

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