人脂肪干细胞向内皮分化最佳诱导体系的建立

Establishment the best method for endothelial differentiation of human adipose stem cells

目的探讨人脂肪干细胞(human Adipose-derived stem cells,hADSCs)体外分化为内皮细胞的最佳诱导体系。方法利用胶原酶消化法和贴壁筛选法从人脂肪组织中分离、培养及扩增hADSCs,分3组分别进行内皮诱导:即纤维连接蛋白(fibronectin,FN)包被组、明胶包被组和未铺组;同时诱导液分为两组:单纯内皮细胞生长培养基(EGM2-MV)组及内皮细胞生长培养基(EGM2-MV)+50ng/ml血管内皮细胞生长因子(VEGF165)组。诱导15d后,通过流式细胞术检测各组内皮细胞特异性表面抗原CD34的表达;通过相差显微镜观察诱导前后细胞的一般形态学特征。结果体外分离、培养出高度同源性的hADSCs,CD29、CD44、CD90、CD105及CD166呈阳性表达,而CD34、CD45及HLA-DR呈阴性表达;诱导后细胞流式结果显示单纯EGM-2MV诱导的3组细胞内皮细胞特异性标志CD34阴性表达,而EGM2-MV+50ng/mlVEGF165诱导的细胞,其中FN包被组CD34表达率为8.4%,明胶包被组为5.4%,未铺组为4.2%;相差显微镜下可见诱导后细胞呈三角形或多边形,融合处呈铺路石样生长。结论FN作为细胞外基质与EGM2-MV+50ng/mlVEGF165组成的诱导液协同作用,构成了hADSCs体外分化为内皮细胞的最佳诱导体系,可为临床上缺血性疾病的自体细胞移植治疗提供大量的种子细胞来源。

Objective To discuss the best method for endothelial differentiation of human adipose-derived stem cells （hADSCs）.Methods hADSCs were isolated from human adipose tissue by collagenase digestion and adherence to flasks,divided into three groups and induced into endothelia respectively,including Fibronectin （FN）-coated,gelatin-coated group and non-coated group.At the same time,inducing medium was divided into pure endothelial cell growth medium （EGM2-MV） and endothelial cell growth medium （EGM2-MV） ＋50 ng/ml vascular endothelial growth factor （VEGF165） groups.After induced 15 d,CD34,which was endothelial cell-specific surface antigen,was detected by flow cytometry.General morphological characteristics of hADSCs and induced hADSCs were observed by phase-contrast microscope.Results A high degree of homology and the CD34 negative hADSCs were succeed to isolate and cultivate.At the end of differentiation,flow results of the three groups induced by simple EGM2-MV all showed CD34 negative expression,but the other three groups induced by EGM2-MV plus 50 ng/ml VEGF165 showed that CD34 expression rate of FN-coated group was 8.4%,gelatin-coated group was 5.6%,not-coated group was 4.2%;Induced hADSCs were triangular or polygonal,paving stone-like growth was observed at the place of local integration.Conclusions Combination of FN-coated and EGM2-MV ＋50 ng/ml VEGF165 is the best method for endothelial differentiation of hADSCs.The results will offer abundant of seeding cells for autologous cell transplantation therapy of ischemic disease.