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Title:: Effects of Wnt3a on differentiation of mouse embryonic stem cells into cardiomyocytes

作者:: 陈明¹; 张光宇¹; 毕琳琳²; 赵芳¹; 王智泉¹; 干学东¹; 王扬淦¹; (1.武汉大学中南医院心内科，湖北武汉 430071；2.南方医科大学神经生物学教研室，广东广州 510515)

Author(s):: CHEN Ming¹; ZHANG Guang-yu¹; BI Lin-lin²; ZHAO Fang¹; WANG Zhi-quan¹; GAN Xue-dong¹; WANG Yang-gan¹; (1.Department of Cardiology, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei, China; 2.Department of Neurobiology, Southern Medical University, Guangzhou 510515, Guangdong, China)

关键词:: 胚胎干细胞; 心肌细胞; 分化; Wnt3a

Keywords:: embryonic stem cells; cardiomyocyte; differentiation; Wnt3a

分类号:: R329.1

DOI:: -

文献标识码:: A

摘要:: 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。

Abstract:: AIM:To investigate the roles of Wnt3a in differentiation of mouse embryonic stem cells (ESC) into cardiomyocytes. METHODS: Hanging drop culture was used to induce the differentiation of ESCs into cardiomyocytes through formation of embryoid bodies (EBs). Cardiac-specific protein TnT was detected by immunocytochemistry and Wnt3a and its inhibitor Dkk-1 were administered at different time points. The percentage of beating EBs was calculated at different time points. mRNA expression of Nkx2.5, GATA4 and β-MHC were detected by RT-PCR, and the protein expression of TnT was detected by Western blot. The untreated group, the Wnt3a-treated group during days 0 to 5 and the Wnt3a-treated group during days 5 to 10 were referred to as control group, D0-5 group and D5-10 group, respectively. The group treated with Dkk-1was referred to as Dkk-1 group. RESULTS: Spontaneously beating EBs were positively stained with TnT. Wnt3a treatment during EBs formation (days 0 to 5) produced higher percentage of beating EBs, higher gene expression of Nkx2.5, GATA4, β-MHC, ANP, and higher protein expression of TnT compared with those in control group, whereas Wnt3a treatment after EB formation (days 5 to 10) had the opposite results. Dkk-1 treatment during days 5 to 10 produced a higher percentage of beating EBs and higher protein expression of TnT compared with those in control group. Wnt3a treatment during days 0 to 5 plus Dkk-1 treatment during days 5 to 10 produced a higher percentage of beating EBs and higher protein expression of TnT compared with those treated with Wnt3a or Dkk-1 alone. CONCLUSION: Our results indicate that Wnt3a regulates the cardiac differentiation of ESCs in a time-dependent manner. The activation of Wnt3a during EB formation and the inhibition of Wnt3a after EB formation produce a higher degree of myocardial differentiation.

参考文献/References

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Wnt3a对小鼠胚胎干细胞心肌细胞分化的影响

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

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