miRNA-1和miRNA-133过表达对诱导性多能干细胞心肌分化的影响

Effect of miRNA-1 and miRNA-133 overexpression on myocardial differentiation of inducible pluripotent stem cells

目的探讨miRNA-1和miRNA-133过表达对诱导性多能干细胞(iPSC)心肌分化的影响。方法取2周龄美国国家癌症研究所(ICR)孕鼠的胚胎,0.5 g/L胰酶消化接种,传至3代后采用丝裂霉素进行处理,37℃孵育培养小鼠诱导性多能干细胞(miPSC),将生长于胚胎成纤维细胞上的miPSC消化传代;更换培养基,加入对照miRNA (U6)(10 nmol/L,记为对照组),miRNA-1 (10 nmol/L,记为miRNA-1组)、miRNA-133 (10 nmol/L,记为miRNA-133组)、miRNA-1和miRNA-133(分别为10 nmol/L,记为miRNA-1+133组)转染24 h后PBS洗涤,加入miPSC分化培养基,观察每日变化并计算细胞搏动率以表示分化效率。RT-PCR检测miRNA-1、miRNA-133及心肌肌钙蛋白T(cTnT)的表达。结果未分化的miPSC中较少表达cTnT,与分化0 d时比较,分化5 d后miRNA-1组及miRNA-133组内cTnT的表达水平明显升高(P <0.05),分化10 d时升高更明显(P <0.01)。分化10 d时,miRNA-1+133组cTnT表达水平较miRNA-1组及miRNA-133组更高(P <0.01)。与对照组比较,miRNA-1组及miRNA-133组细胞搏动率无变化(P> 0.05),但miRNA-1+133组搏动率显著提升(P <0.01)。结论单独应用miRNA-1或miRNA-133对心肌的分化无促进作用,共表达miRNA-1、miRNA-133可显著提高miPSC向心肌细胞定向分化的效率。

Objective To investigate the effect of miRNA-1 and miRNA-133 overexpression on myocardial differentiation of inducible pluripotent stem cells （iPSC）. Methods Embryos from two-week-old American Institute of Cancer Research （ICR） pregnant mice were collected, 0.5 g/L of trypsin was used for digestion of vaccination, mitomycin was used after spreading to three generations, mouse induced pluripotent stem cells （miPSC） was incubated and cultivated at 37℃, miPSC growed on embryonic fibroblasts was digested and passaged. Medium was replaced, miRNA （U6） （10 nmol/L, marked as control group）, miRNA-1 （10 nmol/L, marked as miRNA-1 group）, miRNA-133 （10 nmol/L, marked as miRNA-133 group）, miRNA-1 and miRNA-133 （10 nmol/L respectively, marked as miRNA-1＋133 group） were added and transfected for 24 hours, and were washed by PBS, then miPSC differentiation medium was added, and its differentiation was daily observed, cell pulse rate was calculated. RT-PCR was used to detect the expression of miRNA-1, miRNA-133 and cardiac troponin T （cTnT）. Results The cTnT was barely expressed in undifferentiated miPSC, compared with 0 day of differentiation, the expreesion level of cTnT of miRNA-1 group and miRNA-133 group was increased after 5 days of differentiation （P 〈 0.05）, the increase was more obvious after 10 days of differentiation （P 〈 0.01）. After 10 days of differentiation, the expression level of cTnT of miRNA-1＋133 group was obviously higher than those of miRNA-1 group and miRNA-133 group （P 〈 0.01）. Compared with control group, the cell pulse rate of miRNA-1 group and miRNA-133 group were not increased （P 〉 0.05）, while the cell pulse rate of miRNA-1＋133 group was significantly increased （P 〈 0.01）. Conclusion miRNA-1 or miRNA-133 alone does not promote myocardial differentiation, only after the co-expression of miRNA-1 and miRNA-133, it can significantly improve the directional differentiation efficiency of miPSC to cardiac cells.