成人脂肪间充质干细胞遗传稳定性及5-氮胞苷对成人脂肪间充质干细胞毒理遗传学影响的实验研究
发布时间：2018-10-09 16:13
【摘要】： 目的:1、探索脂肪间充质干细胞(ADMSCs)的分离、体外培养,观察ADMSCs长期传代培养的遗传稳定性。2、以5-氮胞苷(5-aza)作为诱导剂,观察5-aza对ADMSCs向心肌样细胞诱导分化的作用。3、观察不同浓度的5-aza诱导ADMSCs向心肌细胞分化过程中的毒理遗传学影响。 方法:1、采用Ⅰ型胶原酶消化法自成人脂肪组织中分离培养ADMSCs,并在体外持续传代培养。对第3代ADMSCs进行表面标志分子鉴定,对第3代及第25代细胞分别流式细胞仪检测细胞增殖指数、常规核型分析及电镜下观察超微结构,同时对第3代、25代及30代细胞进行肿瘤特异抗原的检测。2、取生长状态良好的第3代ADMSCs以10μmol/L5-aza诱导作用24h后,用含体积分数为5%胎牛血清的完全培养基继续培养,镜下观察细胞形态学变化,采用逆转录-聚合酶链反应(RT-PCR)方法检测心肌早期转录因子(NKX2.5、GATA4)及心肌特异性肌钙蛋白Ⅰ(cTnI)基因的表达,同时免疫细胞化学染色鉴定cTnI。3、分别用不同诱导浓度的5-aza 5μmol/L(Ⅰ组)、10μmol/L(Ⅱ组)、20μmol/L(Ⅲ组)对ADMSCs进行诱导,同时设立对照组(Ⅳ组),光镜下观察各组细胞的生长情况及形态学变化,免疫细胞化学方法检测cTn-Ⅰ,RT-PCR方法检测cTn-Ⅰ基因的表达,流式细胞仪检测细胞周期及凋亡,并对试验各组常规核型分析。 结果:1、成人脂肪组织中含有大量间充质干细胞,并易于分离、培养,流式鉴定CD13、CD59、CD44阳性表达,CD34及HLA-DR阴性表达。20代时细胞增殖速度明显加快,可见到形态略不规则的细胞出现。25代时形态不规则细胞增多,核浆比增加,染色体出现断裂、畸变,呈亚二倍体核型或超二倍体核型,流式细胞仪检测结果显示细胞DNA合成旺盛,电镜下超微结构出现改变。2、5-aza作用ADMSCs后7d细胞形态及排列开始发生变化,RT-PCR产物条带即见NKX2.5、GATA4基因的表达。4周后可见胞体明显增大,球形细胞增多,体积大小不等,胞浆粗糙,极个别细胞出现细微搏动,免疫细胞化学结果显示cTnⅠ阳性,RT-PCR产物条带见cTnⅠ基因表达。3、Ⅰ、Ⅱ、Ⅲ组ADMSCs诱导后呈现类似心肌细胞的形态学特征。4周后Ⅰ、Ⅱ、Ⅲ组免疫组化结果见cTnⅠ阳性表达,RT-PCR条带见cTnⅠ基因表达。各组染色体结构及数目均未见异常。 结论:1、建立了一种自人体脂肪组织分离、培养ADMSCs经济简便的方法,ADMSCs的长期体外培养具有遗传不稳定性,有向肿瘤细胞突变的倾向。2、5-aza可以诱导ADMSCs向自发搏动的心肌样细胞分化。3、5-aza作为诱导剂对脂肪间充质干细胞遗传性无不良影响。
[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，
本文编号：2259957
[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，
本文编号：2259957
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2259957.html
最近更新
教材专著
