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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。
Objective To investigate the effect of mechanical stretch on the efficiency of induced pluripotent stem cells to differentiate into cardiomyocytes. Methods After induced pluripotent stem cells to form embryoid bodies, the embryoid bodies were divided into four groups: control group (untreated), stretch group 1 (stretch for 5-6 days for 24 hours), stretch for group 2 Stretch for 5-6 days for 24 hours, stretch for 24 hours for 7-8 days), stretch group 3 (stretch for 5-6 days for 24 hours and stretch for 72 hours for 7-10 days) After 20% strain-induced mechanical strain was induced on mouse induced pluripotent stem cells, on the 15th day, the numbers of beating clones in each group were counted to select the highest induction efficiency group among the above four groups. Afterwards, immunofluorescence staining, Westernblot, RT-PCR and laser confocal microscopy were used to further identify the differences in the final differentiation efficiency and cell maturation between the control group and the initially screened stretch group. Results The number of beating clones in stretch group 2 increased (P <0.05) at 15 days after differentiation, and the stretch group 2 was initially screened to improve the differentiation efficiency. Statistical analysis of α-MHC immunofluorescent staining showed that stretch group 2 was 2.1-fold higher than that of the control group (P <0.05). The protein expression of 2Troponin I in the stretch group was 1.7 times that of the control group (P <0.05). The results of semi-quantitative PCR showed that the expressions of β-MHC, MLC- And the expression of Nkx2.5, an early transcription factor of cardiomyocytes, were increased 6.7 times, 4.4 times and 11.4 times respectively (all P <0.05). The α-actinin of single differentiated cardiomyocytes was observed by laser scanning confocal microscopy, Stretching group 2 is conducive to myocardial cell expansion and maturation. CONCLUSIONS: Mechanical stretch was used as a stimulus inducing factor. Tensile strain rate of 20%, stretch group 2 (adherent embryoid body 5-6 days for 24 hours stretch, 7-8 days for 24 hours Zhang) treatment can significantly promote the differentiation of induced pluripotent stem cells into cardiomyocytes, and provide the experimental basis for further research and clinical application.