多种方法诱导干细胞向心肌细胞分化的实验研究

发布时间：2018-06-29 00:40

本文选题：骨髓间充质干细胞 + 心肌细胞　；参考：《苏州大学》2009年硕士论文

【摘要】： 目的:通过体外实验,观察化学诱导剂和模拟的生物微环境诱导骨髓间充质干细胞向心肌细胞分化。方法:取6~8w龄SD大鼠股骨骨髓,培养至第二代,经流式细胞仪检测骨髓间充质干细胞纯度97%以上,然后分成五组:A组,单纯DMEM细胞培养组(对照组);B组,5-氮杂胞苷诱导组;C组,心肌细胞裂解液组;D组,心肌细胞裂解液组+5-氮杂胞苷组;E组,细胞间接接触组。根据各自实验条件诱导至第2w和第4w时,收获细胞。应用免疫组化法检测诱导后骨髓间充质干细胞表达心肌特异性肌钙蛋白T(cTnT)、连接蛋白43(CX-43),血管内皮细胞表面抗原标志CD31及肌性标志α-横纹肌肌动蛋白(α-SCA)的表达情况。应用RT-PCR法检测各组早期心肌细胞特异性转录因子GATA4、NKX2.5、β-MHC及成熟心肌细胞特异性转录因子α-MHC基因的表达情况,并进行简单量的比较。结果:免疫组化检测:实验组诱导2w后,cTnT、CX-43表达阴性或低表达,诱导4w后呈阳性表达,实验组较对照组有统计学差异(P0.05),实验组之间无显著差异(P0.05);对照组及B组无CD31表达,C, D, E组表达呈弱阳性,组间无统计学差异(P0.05);α-SCA诱导前骨髓间充质细胞中既有低度表达,诱导后浓度明显增高,实验组间无差异(P0.05)。 RT-PCR表达情况:实验组诱导2w后条带不明显;诱导4w后均表达GATA4, NKX2.5,β-MHC,无α-MHC表达,从条带密度和亮度分析,实验组较对照组有显著差异(P0.05),实验组间无显著差异(P0.05)。结论: 1. 5-氮杂胞苷可以诱导骨髓间充质干细胞向心肌细胞分化。 2.骨髓间充质干细胞因其“环境依赖性分化”特性,具有分化为心肌细胞的潜能。心肌细胞裂解液和细胞间接接触模拟的心肌微环境也可以诱导骨髓间充质干细胞分化成心肌样细胞,且诱导的效果与诱导的时间及诱导剂浓度相关,分化的细胞介于成熟的心肌细胞和心肌祖细胞之间的心肌细胞前体。
[Abstract]:Aim: to observe the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes induced by chemical inducers and simulated microenvironment in vitro. Methods: bone marrow of SD rats aged 6 to 8 weeks was cultured to the second generation. The purity of bone marrow mesenchymal stem cells was detected by flow cytometry. The bone marrow mesenchymal stem cells were divided into five groups: group A, group B (control group) and group C (group C) induced by 5-azacytidine. Group D was treated with cardiomyocyte lysate, group E with 5-azacytidine, group with indirect cell contact. According to the experimental conditions, the cells were harvested at the 2nd and 4th week. The expression of myocardial specific troponin T (cTnT), connexin 43 (CX-43), CD31 and 伪 -rhabdomyactin (伪 -SCA) in bone marrow mesenchymal stem cells (BMSCs) were detected by immunohistochemistry. RT-PCR was used to detect the expression of NKX2.5, 尾 -MHC and 伪 -MHC genes in the early cardiac myocyte specific transcription factor GATA4, NKX2.5, and mature cardiomyocyte specific transcription factor 伪 -MHC, and a simple comparison was made. Results: the expression of cTnTnTnTnTnTnCX-43 in the experimental group was negative or low after 2 weeks of induction, and was positive after 4 weeks of induction. There was statistical difference between the experimental group and the control group (P0.05), there was no significant difference between the experimental group and the experimental group (P0.05); in the control group and group B, the expression of CD31 was weakly positive, but there was no statistical difference between the two groups (P0.05); there was a low expression in the bone marrow mesenchymal cells before 伪 -SCA induction. After induction, the concentration of GATA4, NKX2.5, 尾 -MHC and 伪 -MHC were significantly increased (P0.05). The expression of RT-PCR: the bands were not obvious after 2 weeks of induction, but GATA4, NKX2.5, 尾 -MHCand 伪 -MHC were all expressed at 4 weeks after induction. There was significant difference between the experimental group and the control group (P0.05), but there was no significant difference between the experimental group (P0.05). Conclusion: 1. 5-azacytidine could induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes. 2. Bone marrow mesenchymal stem cells (BMSCs) have the potential to differentiate into cardiomyocytes due to their environmental dependent differentiation. Myocardial cell lysate and cell contact with the simulated myocardial microenvironment could also induce bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells, and the induction effect was related to the time of induction and the concentration of inducer. Differentiated cells are precursors of cardiac myocytes between mature cardiomyocytes and cardiac progenitor cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【引证文献】