大鼠骨髓间充质干细胞诱导血管内皮细胞的实验研究

Experimental study of rat bone marrow mesenchymal stem cells in the induction of vascular endothelial cells

目的探讨诱导大鼠骨髓间充质干细胞(BMSCs)向血管内皮细胞(VEC)分化的方法和VEC的生物学特性。方法采用贴壁筛选法收集SD大鼠股骨及胫骨骨髓细胞中的BMSCs,孵育至第三代细胞汇合90%后分为2组:实验组在含10%胎牛血清(FBS)的M199培养液中添加诱导剂血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF);对照组加入含10%FBS的M199培养液,继续培养、孵育。观察2组细胞生长形态特征,应用CCK8试剂盒检测细胞增殖,绘制细胞生长曲线。采用流式细胞仪检测2组细胞表面特异性标记物CD29、CD44、CD90、CD31、CD34和血管性假血友病因子(vWF),免疫组织荧光法检测2组vWF。结果 P3代细胞表达CD29、CD44、CD90的阳性率分别为74.8%、84.4%、95.5%,提示在P3代时已可以得到相对较纯的MSCs。实验组细胞表面特异性标记物CD31、CD34、vWF分别为0.1%、0.4%、6.8%,对照组细胞表面特异性标记物CD31、CD34、vWF分别为3.3%、0.1%、5.5%。实验组免疫荧光染色vWF阴性。结论在无菌环境下SD大鼠全骨髓细胞通过换液、传代培养得到相对较纯的P3代细胞,方法简单易行,但大鼠BMSCs添加VEGF和bFGF诱导后未能分化为VEC。

Objective To discuss the methods of inducing the differentiation of rat bone marrow mesenchy-mal stem cells to vascular endothelial cells and biological characteristics of vascular endothelial cells. Meth-ods Rat bone marrow mesenchymal stem cells were collected from the femoral and tibial bone marrow of SD rats by adherence method, which were passaged into the third passage whose cell confluence were a-round 90% , then divided into two groups. Experimental group was added with VEGF and bFGF into M199 medium with 10% FBS; Control group where M199 medium containing 10% FBS were subject to incuba-tion. Morphological characteristics of cells was observed; growth was monitored using CCK8 kit and inci-dentally, growth curve was plotted, flow cytometry was used to detect specific markers, including CD29, CD44, CD90,CD31, CD34 and vWF on cell＇s surface. While immunohistochemical fluorescence was used for detection of vWF expression. Results The cells at third passage was shown to be able to express CD29, CD44 and CD90 whose positive rate was 74.8 % , 84.4%, 95.5% respectively, suggesting that MSCs could be obtained at the third passage.