机械牵张对诱导多能干细胞向心肌细胞分化的研究

目的研究机械牵张对诱导多能干细胞向心肌细胞分化效率的影响。方法诱导多能干细胞形成拟胚体后将拟胚体分为四组:对照组(未行牵张处理),牵张组1(5-6天行24小时牵张),牵张组2(5-6天行24小时牵张,7-8天再行24小时牵张),牵张组3(5-6天行24小时牵张,7-10天再行72小时牵张),通过对小鼠诱导多能干细胞施加20%形变率的机械牵张力后,于第15天,分别计数每组跳动克隆数目从而初步在上述四组中选出诱导效率最高组,此后用荧光免疫染色、Westernblot、RT-PCR和激光共聚焦法,进一步鉴定对照组和初步筛选出的牵张组最终分化效率和细胞成熟度差异。结果机械牵张刺激下,分化15天时,牵张组2的跳动克隆数上升(P<0.05),初步筛选出牵张组2可以提高分化效率;统计α-MHC免疫荧光染色面积发现牵张组2是对照组的2.1倍(P<0.05);牵张组2TroponinI的蛋白表达量为对照组的1.7倍(P<0.05);半定量PCR结果发现,心肌细胞标志基因β-MHC,MLC-2v及心肌细胞早期转录因子Nkx2.5的表达量分别提高了6.7倍、4.4倍和11.4倍(P值均<0.05);激光扫描共聚焦显微镜对分化来的单个心肌细胞进行α-actinin观察发现,牵张组2有利于心肌细胞的伸展和成熟。结论初步验证机械牵张力作为一种刺激诱导因素,20%拉伸形变率,牵张组2(贴壁的拟胚体5-6天行24小时牵张,7-8天再行24小时牵张)的处理方法可以显著促进诱导多能干细胞向心肌细胞的分化效率,为以后的深入研究和临床应用提供了实验基础。

Effects of Mechanical stress on differentiation of induced pluripotent stem stem cells into cardiomyocytes

Objective To investigate the effects of mechanical stress on differentiation of induced pluripotent stem cells into cardiomyo- cytes in vitro. Methods Embryoid bodies （EBs） formed from Induced pluripotent stem cells were divided into 4 groups： control, mechanical stress groupl（mechanical stretched at 5-6d）,mechanical stress group2（mechanieal stretched at 5-6d, 7-8d ）, mechanical stress group3（mechanical stretched at 5-6d, 7-10d）. After subjected to 20% elongation by using mechanical stress system, at day 15, contracting EBs was counted and the highest me- chanical stress group was selected for further research, Immunofluresent staining of a -MHC in EBs, Western blot, RT-PCR and immunofluorescence by confocal laser scanning microscope were used to further identify the differences between the highest induced efficiency mechanical stress group and control group. Results At day l 5, the contract EBs in mechanical stress group2was increased （P〈0.05）, 2.1-fold increase in a-MHC positive staining（P〈0.05）, 1.7-fold increase in cardiac Troponin I expression level（P〈0.05）, 6.7-fold increase in the IS-MHC expression（P〈0.05）, 4.4-fold increase in the MLC-2v expression（P〈0.05）, 11.4-fold increase in the Nkx2.5expression（P〈0.05）.The morphology of cardiomyocytes showed more spread and mature in mechanical stress group2 evidenced by the observation obtaining from the confocal laser scanning microscope. Conclusions Mechanical stress system of the 20% elongation and mechanical stress group2 （mechanical stretched at 5-6d, 7-8d ） treatment protocol can effectively promote the differentiation of mouse induced pluripotent stem cells into cardiomyocytes effectively. Our study may lay the foundation for further re-