CAJ | 학술논문

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)+50 ng/ml血管内皮细胞生长因子(VEGF_(165))组.诱导15 d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+50 ng/ml VEGF_(165)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+50 ng/ml VEGF_(165)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.
목적 탐토인지방간세포(human Adipose-derived stem cells,hADSCs)체외분화위내피세포적최가유도체계.방법 이용효원매소화법화첩벽사선법종인지방조직중분리、배양급확증hADSCs,분3조분별진행내피유도:즉섬유련접단백(fibronectin,FN)포피조、명효포피조화미포조;동시유도액분위량조:단순내피세포생장배양기(EGM2-MV)조급내피세포생장배양기(EGM2-MV)+50 ng/ml혈관내피세포생장인자(VEGF_(165))조.유도15 d후,통과류식세포술검측각조내피세포특이성표면항원CD34적표체;통과상차현미경관찰유도전후세포적일반형태학특정.결과 체외분리、배양출고도동원성적hADSCs,CD29、CD44、CD90、CD105급CD166정양성표체,이CD34、CD45급HLA-DR정음성표체;유도후세포류식결과현시단순EGM-2MV유도적3조세포내피세포특이성표지CD34음성표체,이EGM2-MV+50 ng/ml VEGF_(165)유도적세포,기중FN포피조CD34표체솔위8.4%,명효포피조위5.4%,미포조위4.2%;상차현미경하가견유도후세포정삼각형혹다변형,융합처정포로석양생장.결론 FN작위세포외기질여EGM2-MV+50 ng/ml VEGF_(165)조성적유도액협동작용,구성료hADSCs체외분화위내피세포적최가유도체계,가위림상상결혈성질병적자체세포이식치료제공대량적충자세포래원.
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 (VEGF_(165)) 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 VEGF_(165) 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 VEGF_(165) 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.