CHIR99021对小鼠胚胎干细胞心肌细胞分化的双重作用

摘要

Objective To investigate the roles of CHIR99021 in differentiation of mouse embryonic stem cells ( mESCs) into cardiomyocytes. Methods Hanging drop culture was used to induce mESCs differentiating into car-diomyocytes through forming EBs. ESCs were induced differentiating into cardiomyocytes via administering GSK3βinhibitor CHIR99021 at different time points. The percentage of beating EBs was calculated by inverted phase con-trast microscope and the mRNA expression of Nkx2. 5 and α-MHC were detected by RT-PCR at different time points. Cardiac-specific protein TnT( cTnT) was detected by Western blot and immunofluorescence staining. The un-treated group,the CHIR99021-treated group during days 2 to 5 and during days 6 to 10 were referred to as control group, 2 to 5 d group and 6 to 10 d group respectively. Results Spontaneously beating EBs were positively stained with cTnT via hanging drop culture,and 2 to 5 d group produced higher lever of cTnT versus control, while 6 to 10 d group had the opposite results. 2 to 5 d group produced higher percentage of beating EBs and higher gene expres-sion of Nkx2. 5 andα-MHC compared with those in the control group,whereas 6 to 10 d group had the opposite re-sults. Conclusion Our results indicate that the function of GSK3βinhibitor CHIR99021 during cardiac differentia-tion of mouse embryonic stem cells is biphasic. The activation of CHIR99021 during the days 2 to 5 produces a higher level of myocardial differentiation than the inhibition of CHIR99021 during days 6 to 10 .%目的：研究CHIR99021在小鼠胚胎干细胞( mESCs)分化为心肌细胞的作用。方法运用经典的悬滴培养法促进 mESCs 形成拟胚体( EBs)。在不同分化时间段加入糖原合成激酶-3β(GSK-3β)抑制剂CHIR99021,诱导mESCs向心肌细胞分化,相差显微镜在不同时间点观察不同组EBs搏动百分率。 RT-PCR法检测心肌特异性转录因子Nkx2．5、α-肌球蛋白重链(α-MHC)基因表达水平的变化。 Western blot法和免疫荧光染色法检测心肌特异性蛋白cTnT的表达。实验时将不加入CHIR99021命名为对照组,分化第2~5天和第6~10天加入CHIR99021分别命名为2~5 d组和6~10 d组。结果通过悬滴培养法能够形成 EBs,且形成的EBs能够出现自发性搏动并有cTnT表达。在诱导分化的过程中,第2~5天加入CHIR99021的2~5 d组的搏动 EBs百分率比对照组更高,Nkx2．5、α-MHC基因表达的水平也比对照组高；6~10 d组的结果跟2~5 d组相反。各组中都可通过Western blot和免疫荧光染色法检测到cTnT的表达,但2~5 d组的cTnT的表达量高于对照组,6~10 d组的cTnT的表达量低于对照组。结论 GSK-3β抑制剂 CHIR99021对mESCs心肌细胞分化具有双重作用,即分化前期(2~5 d)促进分化,可获得更高的分化率,分化后期(6~10 d)结果相反。