人骨髓间充质干细胞体外心肌样细胞诱导分化中细胞周期与凋亡的分析  

Analysis of cell cycle and apoptosis during directional differentiation of human mesenchymal stem cells to cadiomyocytes in vitro

作　　者：马健[1] 唐海沁[1] 翟志敏[2] 朱薇波[2] 李结华[1] 

机构地区：[1]安徽医科大学第一附属医院老年心血管科,合肥市230022 [2]安徽省立医院血液内科

出　　处：《中华老年医学杂志》2007年第2期120-123,共4页Chinese Journal of Geriatrics

基　　金：安徽省自然科学基金（0743771）

摘　　要：目的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，为hMSCs临床移植治疗提供数据。方法采用体外纯化、扩增后的第4代hMSCs，分3组在体外分别经0μmol／L、2．5μmol／L、5．0μmol／L 5-杂氮胞苷（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（P＜0．05），G0／G1期细胞比例诱导后较对照组显著减少（P＜0．05）；pIMHC和CTNT在诱导后表达开始增强，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。结论hMSCs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，但诱导过程中hMSCs发生细胞凋亡，采用2．5μmol／L 5-Aza低浓度诱导可减少细胞凋亡发生。Objective To investigate differentiation and apoptosis during the process of human mesenchymal stem cells （hMSCs） differentiating into cardiomyocytes in vitro and provide experimental data for clinical transplant therapy of hMSCs. Methods hMSCs were purified and expanded as undifferentiated cells in culture until the 4th passage, then were divided into 3 groups and treated with 0, 2.5, 5.0 μmol/l 5-azacytidine respectively for 24 h and then cultured for 8 weeks. The morphological changes in hMSCs were observed by phase-contrast microscope. The mRNA expressions of β-myosin heavy chain （β-MHC） , cardiac troponin T （cTNT） and apoptosis-related genes （Bcl- 2 and Bax）were determined by reverse translation polymerase chain reaction （RT-PCR） technique and the apoptosis index was analyzed by flow cytometry. Results The apoptosis index was correlated with the cultural time after induction, the apoptosis rate of induced hMSCs in 2.5 bμmol/L group was remarkably lower than in 5.0 μmol/L group （P〈0.05）. The cell cycle analysis showed that the percentages of G0/G1 phase were reduced significantly after induction compared with the control group （all P 〈 0. 05）. The expression levels of β-MHC and cTNT mRNA were undetectable before induction, but began to increase after induction, reached the peak at 6 th week and decreased gradually after that, the expressions of Bcl-2 and Bax mRNA varied in a time dependent manner after 5-azacytidine treatment. Conclusions hMSCs express characteristics of cardiomyocytes in vitro, but apoptosis is observed in the process of differentiation. Compared with nomal 5-azacytidine concentration, the low 5-azacytidine concentration （2.5 μmol/L） could reduce the ratio of cell aDOOtosis.

关 键 词：间质干细胞 诱导 肌细胞 心脏 细胞凋亡 

分 类 号：R686[医药卫生—骨科学]