小鼠卵黄囊间充质干细胞培养及诱导成血管内皮细胞

Culture and differentiation of mouse yolk sac mesenchymal stem cells into vascular endothelial cells

目的:成体间充质干细胞发育分化接近终末状态后经过一段时间的体外培养,易衰老凋亡,而来自于早期胚胎的卵黄囊间充质干细胞具有更大的增殖潜能和可塑性。那么体外分离培养的小鼠卵黄囊间充质干细胞能否定向诱导分化为血管内皮细胞呢?方法:实验于2006-03/2007-01在首都医科大学组织学与胚胎学实验室完成。①实验方法:显微分离妊娠10d的ICR小鼠胚胎卵黄囊,经Ⅰ型胶原酶消化得到卵黄囊细胞,取贴壁细胞培养,于接近90%融合时按1∶2进行传代。②实验评估:取生长状态良好的第1代细胞,透射电镜观察细胞器超微结构,流式细胞仪检测其表面标志的表达;以血管内皮生长因子和碱性成纤维细胞生长因子进行体外诱导,观察诱导后细胞形态及超微结构变化,免疫荧光法鉴定诱导后内皮细胞标志物的表达。结果:①体外分离培养可获得小鼠卵黄囊间充质干细胞,大多数呈梭形。透射电镜下卵黄囊间充质干细胞表面有微绒毛,胞浆中细胞器丰富,核大,形态不规则。流式细胞仪检测第1代卵黄囊间充质干细胞CD44、CD105均呈阳性,CD34亦有少量表达。②诱导后的细胞形态明显改变,透射电镜下可见内皮细胞特征性Weible-Palade小体,胞质中有大量吞饮小泡,同时内皮细胞标志物FLK1染色呈阳性。结论:体外可分离培养出小鼠卵黄囊间充质干细胞,并能够成功定向诱导分化为血管内皮细胞。

Adult mesenchymal stem cells, which develop into the end condition, are easy to fade and apoptosis after in vitro culture, yolk sac mesenchymal stem cells （YS-MSCs） from early embryo have greater proliferation potentiality and ductility. Can the mouse YS-MSCs that are cultured in vitro be directionally induced into vascular endothelial cells？ METHODS： The experiment was conducted at the Department of Histology and Embryology in Capital Medical University from March 2006 to January 2007. ①Empiricel method： Mouse YS were harvested on day 10 post-coitus by microisolation, YS cells were obtained after YS were digested by type Ⅰ collagenase, then the adherent cells were cultured in medium and passaged according to 1：2 when they were confluence to 90%. ②Empidcal appraisal： The first generation of YS-MSCs which grew well were obtained, the ultrastructure of YS-MSCs was observed by transmission electron microscope; the surface markers of YS-MSCs were detected by flow cytometry; After in vitro inductions with vascular endothelial growth factor and basic fibroblast growth factor, the shape and ultrastructure of the induced cells were observed, and the expression of endothelial markers in the induced cells were identified by immunofluorescence staining method. RESULTS： ①YS-MSCs cultured in vitro were of spindle shape. Under transmission electron microscope, there were abundant organella and microvilli, the cell nucleus of YS-MSCs was big, and the shape of cell nucleus was irregular. The analysis of flow cytometry suggested that the first generation of YS-MSCs were positive for CD44 and CD105, while the expression of CD34 was minor. ②The morphology of induced cells was obviously changed, Weible-Palade corpuscles and swallow vesicles were observed in differentiated cells under transmission electron microscope, and the confluent cells were positive for endothelial-specific markers FLKI. CONCLUSION： Mouse YS-MSCs can be cultured in vitro and directionally induced into vascular endothelial cells successfully.