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目的:观察Wnt3a信号对小鼠胚胎干细胞(embryonic stem cell,ESC)心肌细胞分化的影响。方法:用悬滴培养法促进ESCs形成拟胚体(embryoid bodies,EBs)。用免疫荧光染色法检测心肌特异性蛋白cTnT的表达。在不同分化时间加入Wnt3a及Wnt信号通路抑制剂Dkk-1观察对搏动EBs百分率的影响。用RT-PCR检测Nk同源异型盒5(Nkx2.5)、锌指转录因子-4(GATA-4)、β-肌球蛋白重链(β-MHC)及心房钠尿肽(ANP)基因表达水平的变化。用Western blot检测cTnT表达水平的变化。将不加入Wnt3a,分化第0~5天及5~10天加入Wnt3a的组分别命名为对照组,D0-5组及D5-10组。加入Dkk-1的组命名为Dkk-1组。结果:通过悬滴培养法形成的EBs能够出现自发性搏动并且有TnT表达。EBs形成过程中即分化第0~5天加入Wnt3a与对照组相比具有更高的搏动EBs百分率,Nkx2.5、GATA4、β-MHC和ANP基因表达的水平,以及cTnT蛋白表达水平,而EBs形成后即分化第5~10天加入Wnt3a的结果相反。分化第5~10天加入Dkk-1与对照组相比具有更高的搏动EBs百分率及cTnT蛋白表达水平,并且在分化第0~5天分别加入Wnt3a及Dkk-1与单独加入Wnt3a及Dkk-1相比,搏动EBs百分率及cTnT蛋白表达水平更高。结论:Wnt3a对ESCs向心肌细胞分化的调控呈时间依耐性,EBs形成过程中激活Wnt3a信号及EBs形成后阻断Wnt3a信号能够获得更多的心肌细胞。
Objective: To observe the effect of Wnt3a signaling on cardiomyocyte differentiation in mouse embryonic stem cells (ESC). Methods: ESCs were induced to form embryoid bodies (EBs) by hanging drop culture. The expression of myocardial specific protein cTnT was detected by immunofluorescence staining. Wnt3a and Wnt signaling pathway inhibitor Dkk-1 were added at different differentiation time to observe the influence on percentage of beating EBs. Nkx2.5, GATA-4, β-MHC and ANP genes were detected by RT-PCR. Changes in expression levels. Western blot was used to detect the expression of cTnT. The groups that did not join Wnt3a, the day 0 to 5, the day 5 to 5 and the group to which Wnt3a was added in 5 to 10 days were named as control group, D0-5 group and D5-10 group respectively. The group joining Dkk-1 is named Dkk-1 group. Results: EBs formed by hanging drop culture could spontaneously be pulsatile and had TnT expression. Compared with the control group, Wnt3a increased the percentage of beating EBs, the expression of Nkx2.5, GATA4, β-MHC and ANP, and the expression level of cTnT protein during EBs formation from 0 to 5 days after differentiation The result of the addition of Wnt3a on the 5th to 10th day after differentiation is opposite. Dkk-1 had a higher percentage of beating EBs and cTnT protein expression than control group on the 5th to 10th day of differentiation. Wnt3a and Dkk-1 were added separately on day 0 to day 5 of differentiation and Wnt3a and Dkk- 1, the percentage of beating EBs and cTnT protein expression were higher. CONCLUSION: Wnt3a regulates the differentiation of ESCs into cardiomyocytes in a time-dependent manner. Wnt3a signaling is activated during the formation of EBs and Wnt3a signaling blocks the formation of EBs, resulting in more cardiomyocytes.