应用5-杂氮脱氧胞苷诱导人羊水来源间充质干细胞向成肌细胞样细胞分化的体外研究  

Myogenic differentiation of human amnotic fluid-derived mesenchymal stem cells induced by 5-Aza-2＇-deoxycytidine in vitro

作　　者：马晓荣[1] 张胜利[2] 尚亚峰[2] 吴齐全[2] 高同斌[2] 王雪[1] 周君梅[1] 陈方[1] 

机构地区：[1]上海交通大学附属上海市儿童医院小儿秘尿外科,200040 [2]上海交通大学医学院附属新华医院泌尿外科

出　　处：《中华小儿外科杂志》2010年第11期848-853,共6页Chinese Journal of Pediatric Surgery

基　　金：上海市科学技术委员会基金（08JC1416000）

摘　　要：目的 探讨5-氮杂脱氧胞苷（5-AzaC）体外诱导人羊水来源间充质干细胞向成肌细胞样细胞分化的可行性.方法 B超引导下穿刺抽得孕中期羊水,体外分离、培养得到羊水来源间充质干细胞,传代后计数细胞并绘制生长曲线.体外成骨、成脂诱导观察人羊水来源间充质干细胞多向分化能力.采用5-AzaC对羊水来源间充质干细胞成肌诱导2周,观察细胞形态学变化,RT-PCR、免疫荧光染色方法鉴定成肌细胞特异mRNA及蛋白表达.结果 人羊水来源间充质干细胞体外培养后迅速进入对数生长期,培养7 d未达到平台期.茜素红和油红O染色证实人羊水来源间充质干细胞可诱导分化为成骨、成脂细胞样细胞.人羊水来源间充质干细胞经5-AzaC诱导2周逐渐变长梭形.而对照组细胞呈扁平多角形.免疫荧光染色及RT-PCR结果提示实验组细胞表达肌结蛋白（Desmin）、肌钙蛋白I（Tn I）、横纹肌辅肌动蛋白（α-Actinin）及mRNA;对照组呈阴性.结论 人羊水来源间充质干细胞体外增殖能力强,能分化为成骨、成脂细胞样细胞.5-AzaC能在体外诱导人羊水来源间充质干细胞向成肌细胞样细胞分化.Objective To investigate the efficacy of 5-AzaC to induce the myogenic differentiation of human amniotic fluid-derived mesenchymal stem cells （HAFDMSCs） in vitro. Methods HAFDMSCs were isolated from second trimester amniotic fluid and cultured in vitro. Osteogenic and adipogenic differentiation was carried out to study multipotent potential of HAFDMSCs. HAFDMSCs were cultured in myogenic differentiation medium temporarily supplemented by 10 ummol/L 5-AzaC for 24hours or complete medium as experimental and control groups, respectively. The differentiated cells were identified morphologically and by the expressions of myocyte specific proteins after induction for 2 weeks. Results HAFDMSCs grew well in vitro and didn＇t reach the plateau phase even after being cultured for 7 days. Adipogenic and osteogenic differentiation potential of HAFDMSCs was confirmed by Alizarin red and oil red O staining. After 5-AzaC induction, HAFDMSCs appeared elongated shape.Immunofluorescence staining and RT-PCR confirmed the expression of myocyte specific phenotypes like Desmin,Tn I and α-Actinin. Conclusions 5-AzaC can induce the HAFDMSCs to differentiate into myoblast-like cells in vitro.

关 键 词：干细胞 间充质 细胞分化 骨骼肌 

分 类 号：R686[医药卫生—骨科学]