人骨髓间充质干细胞体外诱导分化及生物学特性探讨

发布时间：2018-01-18 01:03

本文关键词：人骨髓间充质干细胞体外诱导分化及生物学特性探讨　出处：《安徽医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:在体外将人骨髓间充质干细胞(hMSCs)进行纯化、扩增及向心肌样细胞定向诱导分化,对诱导后的细胞进行心肌样细胞相关特性分析并探讨体外诱导人骨髓间充质干细胞(hMSCs)向心肌样细胞分化过程中分化与凋亡的情况,检测分化过程中心肌细胞特征表达及膜电位变化的情况,为hMSCs临床移植治疗提供实验参考依据。方法: (1)采用体外纯化、扩增后的第4代hMSCs在体外经5μmol.l-1 5-杂氮胞苷诱导24小时后继续培养8周,相差显微镜下观察细胞形态变化,免疫组化方法鉴定心肌特异性蛋白心房钠尿肽(ANP)和连接蛋白(CX43)的表达,流式细胞仪检测诱导前后细胞表面抗原(CD31、CD34、CD45、CD90)的表达情况,全细胞膜片钳技术检测不同时期细胞膜电位的变化。(2)将纯化、扩增后的第4代hMSCs,分3组在体外分别经0μmol.l~(-1)、2.5μmol.l~(-1)、5.0μmol.l~(-1) 5-azacytidine(5-Aza)诱导24h后继续培养8周,相差显微镜下观察细胞形态变化,流式细胞仪检测各组细胞周期及凋亡指数,应用RT-PCR技术分析肌球蛋白重链(β-MHC)、肌钙蛋白T(CTNT)、凋亡相关基因(Bcl-2、Bax)的mRNA在分化过程中的动态表达。结果:hMSCs诱导前为纺锤形,诱导后第2天部分细胞即开始发生形变,呈球形或短棒状,1周后胞浆中颗粒增多,约20～30%细胞边缘呈毛刷样变化;hMSCs表面抗原CD31、CD34、CD45在诱导前后差异无统计学意义,CD90未诱导时表达呈弱阳性,诱导后明显增高(P0.05);ANP和CX43在诱导前无表达,诱导后第2周开始表达且表达随时间逐渐增强,但CX43在诱导后第5周表达量开始降低。hMSCs诱导后凋亡指数随诱导后培养时间增加,低浓度诱导组低于标准浓度诱导组,组间差异有统计学意义(P0.05),G_0/G_1期细胞比例诱导后较对照组显著减少(P0.05);β-MHC和CTNT基因分别在诱导后第1周和第4周时表达开始增强,在第6周时均达到高峰,第8周时表达开始衰减,Bcl-2、Bax基因表达呈时间依赖性变化,hMSCs经诱导后随心肌样细胞特征的表达膜静息电位、去极化幅值和去极化速率逐渐增高。结论:(1)骨髓间充质干细胞在体外经纯化、扩增和诱导后可表现心肌样细胞的生物学特性,可为hMSCs临床移植治疗提供可靠的细胞来源;(2)体外诱导过程中hMSCs可发生细胞凋亡,采用2.5μmol.l~(-1) 5-Aza低浓度诱导可减少细胞凋亡发生;(3)hMSCs移植后有致心律失常的潜在危险。
[Abstract]:Objective: in vitro human bone marrow mesenchymal stem cells (hMSCs) were purified, amplification and differentiation into cardiomyocyte like cells to cardiomyocytes oriented, characteristic analysis and explore the in vitro differentiation of human bone marrow mesenchymal stem cells induced cell differentiation and apoptosis (hMSCs) to differentiate into cardiomyocytes in the process the situation, to detect the expression changes of muscle cell differentiation characteristics of center and membrane potential, to provide experimental evidence for clinical transplantation in the treatment of hMSCs.
Methods: (1) by in vitro purification, amplification of the fourth generation of hMSCs in vitro by 5 mol.L-1 5- azacytidine induced 24 hours after cultured for 8 weeks, the morphological changes were observed under phase contrast microscope, immunohistochemical method and identification of cardiac specific protein atrial natriuretic peptide (ANP) and protein (CX43 connection) expression by flow cytometry before and after induction of cell surface antigens (CD31, CD34, CD45, CD90) expression, to detect the changes of the whole cell patch clamp technique in different periods of cell membrane potential. (2) the purification, amplification of the fourth generation of hMSCs, divided into 3 groups in vitro respectively by 0 mol.l~ (-1), 2.5 mol.l~ (-1), 5 mol.l~ (-1) 5-azacytidine (5-Aza) induced by 24h after cultured for 8 weeks, the morphological changes were observed under phase contrast microscope, detection of apoptosis rate index by flow cytometry and the application of RT-PCR technology on myosin heavy chain (beta -MHC), troponin T (CTNT),. The dynamic expression of mRNA of Bcl-2 (Bax) in the process of differentiation.
Results: hMSCs before induction of spindle shape, after second days of induction of some cells began to deform, spherical or short rod, after 1 weeks in the cytoplasm of particles increased from about 20 to 30% edge cells were brush like changes; hMSCs surface antigen CD31, CD34, CD45, no statistical significance in the difference of CD90 before and after induction. When not induced expression was weakly positive after induction was significantly increased (P0.05); ANP and CX43 expression in pre induction, and the expression increased gradually with time beginning second weeks after induction of CX43, but in fifth weeks after induction the expression began to decrease.HMSCs after induction of apoptosis index increased with incubation time increased after induction, induced by low concentration group is lower than the standard concentration induced group, there was significant difference between the groups (P0.05), the proportion of cells in G_0/G_1 phase after induction was significantly reduced than the control group (P0.05); beta -MHC and CTNT gene were induced after first weeks and fourth weeks to start in sixth weeks after enhancement At the eighth week, the expression began to decay, and the expression of Bcl-2 and Bax changed in a time-dependent manner. After hMSCs induction, the expression of membrane potential, depolarization amplitude and depolarization rate increased with the induction of cardiomyocyte like cells.
Conclusion: (1) bone marrow mesenchymal stem cells purified by in vitro amplification and biological characteristics of expression after induction of myocardial cells, which can provide a reliable source of cells for clinical transplantation in the treatment of hMSCs; (2) hMSCs in vitro during apoptosis, using 2.5 mol.l~ (-1) 5-Aza of low concentration induction of apoptosis; (3) hMSCs after the transplantation of potentially dangerous arrhythmias.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2

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