体外诱导胎盘间充质干细胞分化为内皮细胞的可能性

Possibility of in vitro induced differentiation of embryonic mesenchymal stem cells into vascular endothelial cells

目的:观察体外诱导胎盘间充质干细胞分化为血管内皮细胞的可行性。方法:实验于2004-05/-2005-10在无锡第三人民医院细胞实验室完成。①分离培养胎盘间充质干细胞,鉴定其表面抗原,分别加入CD34-FITC,CD45-PE,CD19-FITC,CD29-FITC,CD44-PE,CD105-FITC,CD106-FITC,HLA-DR-FITC鼠抗人单克隆抗体,进行流式细胞仪分析。②培养细胞诱导分化:取培养2代细胞,用培养基为DMEM/F12+体积分数为0.02的胎牛血清+50μg/L血管内皮细胞生长因子的条件培养基诱导。③诱导细胞免疫组织化学染色分析:诱导后细胞通过内皮细胞标志物KDR,FLT-1以及v-WF染色鉴定。结果:①胎盘间充质干细胞的原代培养结果:随着细胞密度的增加,胞体变得细长,形态类似成纤维细胞。原代细胞生长较慢,传代细胞在3～5d左右可增殖1倍。培养的细胞可以稳定生长传代。②胎盘间充质干细胞的表面抗原测定结果:胎盘间充质干细胞表面抗原CD29,CD44,CD105阳性;CD34,CD45,CD19,CD106以及HLA-DR阴性。③胎盘间充质干细胞的诱导分化及染色结果:诱导后细胞形态明显改变,胞体逐步回缩,立体感增强,内皮细胞标志物KDR,FLT-1以及v-WF染色结果阳性。结论:胎盘间充质干细胞在体外具有分化为血管内皮细胞的能力,可以作为干细胞研究的一种有效来源。

AIM： To investigate the feasibility of in vitro induction of embryo mesenchymal stem cells （MSCs） differentiate into vascular endothelial cells. METHODS： The experiment was conducted in the Cell Laboratory of Wuxi Third People＇s Hospital between May 2004 and October 2005.① Embryonic MSCs were isolated and cultured to identify its surface antigen, in which CD34-FITC, CD45-PE ,CD19-FITC,CD29-FITC,CD44-PE, CD105-FITC, CD106-FITC, HLA-DR-FITC rat-anti-human monoclonal antibody were added respectively to analyze with flow cytometry.②Induced differentiation of cultured cells： Cultured cells of the second cells were induced by nutrient medium of confluent cells in the presence of 2% fetal calf serum and 50 μg/L vascular endothelial growth factor. ③Analysis of induced cells by immunohistochemical staining： induced cells were identified by endothelial cell markers KDR, RLT-1 as well as v-WF staining. RESULTS： ①Primary culture of embryonic MSCs： With the density of cells increasing, the cell body became slender with the shape similar to fibroblast. The primary cells grew slowly, and the passage cells could proliferated one time within 3-5 days. Cultured cells could stably grew and passage.②Determination of surface antigen of embryonic MSCs： the surface antigens of MSCs were positive for the markers CD105, CD29 and CD44, while were negative for CD34, CD45, CD19, CD106 and HLA-DR.③Induced differentiation and immunohistochemical stain analysis of embryonic MSCs： the morphous of induced cells were obviously changed with cell body gradually condensed and reinforced stereological appearance, while markers of KDR, FLT-1 as well as v-WF were positive after staining. CONCLUSION： The embryonic MSC is endowed with the capability to differentiate into vascular endothelial cell in vitro, which can be taken as an effective source for research on stem cells.