CaSR在淫羊藿苷诱导的胚胎干细胞向心肌细胞分化中的作用  

Roles of calcium sensing receptor in icariin-induced differentiation of mouse embryonic stem cells to cardiomyocyte

作　　者：孙健[1] 白淑芝[2] 李爽[1] 许晓义[1] 袁辉[1] 魏韬[1] 徐长庆[2] 栾海蓉[1] 

机构地区：[1]牡丹江医学院,黑龙江牡丹江157011 [2]哈尔滨医科大学病理生理教研室,黑龙江哈尔滨150081

出　　处：《中国病理生理杂志》2016年第2期234-239,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81300163;No.81200160);黑龙江省教育厅科学技术研究项目(No.12531749;No.12531731);牡丹江医学院科学研究项目(No.IS201308)

摘　　要：目的:观察钙敏感受体(calcium sensing receptor,CaSR)对淫羊藿苷(ICA)诱导小鼠胚胎干细胞向心肌细胞分化的影响。方法:129小鼠ES-D3细胞经直接悬浮法形成拟胚体(EBs),应用ICA定向诱导,透射电镜观察分化细胞的超微结构;免疫荧光和Western blot分别检测细胞有无α-辅肌动蛋白(α-actinin)和肌钙蛋白I(cTnI)的表达;流式细胞术检测细胞分化率;Western blot检测心肌特异转录因子NKx2.5、GATA-4和CaSR的蛋白表达。结果:ICA诱导2 d后,可见自发性收缩的细胞簇;随着诱导分化时间的延长,α-actinin和cTnI蛋白的表达逐渐增多;CaSR、NKx2.5和GATA-4蛋白在分化早期表达最多,持续表达至晚期;电镜观察分化细胞中可见连接结构、肌丝;CaSR激动剂新霉素能够增加早期EBs中CaSR、NKx2.5和GATA-4的表达,CaSR抑制剂NPS2390能够阻断上述作用。结论:CaSR在胚胎干细胞分化的心肌细胞中有表达,CaSR活化可通过增加NKx2.5和GATA-4的表达促进心肌细胞的分化。AIM： To study the effect of calcium sensing receptor （CaSR） on icariin （ICA） induced mouse embryonic stem cells （mESCs） to differentiate into cardiomyocytes in vitro. METHODS： mESCs were cultured to embry- oid bodies （EBs） by direct suspension method and the differentiation of EBs into cardiomyocytes was induced by ICA. The expression of cardiac specific proteins α-actinin and cardiac troponin-I （cTnI） was analyzed by Western blot and immuno- fluorescence. The differentiation rate was determined by flow cytometry. The ultrastructure of the derived cardiomyocytes was further characterized by transmission electron microscopy. The expression of cardiac-specific transcription factors Nkx2.5 and GATA-4, as well as CaSR was detected by Western blot. RESULTS： After induction with ICA, the positive characteristics of myocardial cells appeared in the EBs cultured for 2 d. The expression of cardiac-specific sarcomeric pro- tein actinin （α-actinin） and cTnI showed an overall upward trend by Western blot in different phases of ICA induced differ- entiation. The expression of CaSR, Nkx2.5 and GATA-4 was the highest at an early stage of ICA-induced differentiation. Neomycin （ an activator of CaSR） up-regulated CaSR, NKx2.5 and GATA-4 expression in the EBs at early stage of ICA-in- duced differentiation, all of which were reversed by NPS2390 （an inhibitor of CaSR）. CONCLUSION： CaSR is function- ally expressed in mESC-derived cardiomyocytes, and activation of CaSR is involved in the differentiation of mESCs into car- diomyocytes by facilitating the expression of NKx2.5 and GATA-4.

关 键 词：钙敏感受体 淫羊藿苷 细胞分化 心肌细胞 

分 类 号：R363.2[医药卫生—病理学]