CHIR99021对小鼠胚胎干细胞心肌细胞分化的双重作用

Biphasic effects of CHIR99021 on differentiation of mouse embryonic stem cells into cardiomyocytes

目的研究CHIR99021在小鼠胚胎干细胞（m ESCs）分化为心肌细胞的作用。方法运用经典的悬滴培养法促进m ESCs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m ESCs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Nkχ2.5、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c Tn T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c Tn T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,Nkχ2.5、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c Tn T的表达,但2~5d组的c Tn T的表达量高于对照组,6~10 d组的c Tn T的表达量低于对照组。结论GSK-3β抑制剂CHIR99021对m ESCs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。

Objective To investigate the roles of CHIR99021 in differentiation of mouse embryonic stem cells（ m ESCs） into cardiomyocytes. Methods Hanging drop culture was used to induce m ESCs differentiating into cardiomyocytes 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 contrast microscope and the mRNA expression of Nkχ2. 5 and α-MHC were detected by RT-PCR at different time points. Cardiac-specific protein Tn T（ c Tn T） was detected by Western blot and immunofluorescence staining. The untreated 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 c Tn T via hanging drop culture,and 2 to 5 d group produced higher lever of c Tn T 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 expression of Nkχ2. 5 and α-MHC compared with those in the control group,whereas 6 to 10 d group had the opposite results. Conclusion Our results indicate that the function of GSK3β inhibitor CHIR99021 during cardiac differentiation 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.