小鼠骨髓间充质干细胞定向诱导分化血管内皮细胞的实验研究

目的探讨体外小鼠骨髓间充质干细胞(mBMMSCs)的分离培养对其定向诱导分化为血管内皮细胞(VECs)的可行性。方法采用全骨髓贴壁法,分离骨髓间充质干细胞(MSCs)体外纯化培养并传代,倒置显微镜观察原代细胞形态变化,采用生长曲线法及3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑蓝(MTT)法观察传代细胞生长特性。采用第3代mBMMSCs在内皮细胞生长培养基(EBM-2)中以血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)进行定向诱导,观察细胞形态学变化;流式细胞仪分析比较第3代mBMMSCs及诱导后细胞DNA周期、细胞免疫表型的变化;免疫细胞荧光技术分别检测CD31、vWF和CD34表达及摄取Di-lac-LDL结合FITC-UEA-1的功能特点;体外血管形成实验检测血管内皮细胞的功能。结果 mBMMSCs原代培养呈长梭形、漩涡状排列生长,P1、P2、P3细胞生长曲线及MTT法显示呈潜伏期、对数生长期及平台期生长。诱导后的部分细胞形态可见类似血管内皮样改变,呈多角形、短梭形、铺路石样排列生长;细胞周期分析显示,第3代mBMMSCs G0/G1期为86%,诱导分化后的细胞G0/G1期为92%,细胞DNA周期无明显差异;第3代mBMMSCs免疫表型结果显示为CD105、CD90、CD73、CD44阳性表达,而CD34、CD45、VEGF(CD309)、CD14阴性表达;诱导分化后的细胞VEGF(CD309)、CD34弱阳性表达,而CD105、CD90、CD73、CD44、CD45、CD14阴性表达;细胞免疫荧光技术检测第3代mBMMSCs表面CD31、vWF和CD34阴性表达,不具有摄取Dil-ac-LDL、结合FITC-UEA-1的功能特点及形成血管管腔样结构;诱导后的细胞表达VECs特异性表面标志CD31、vWF和CD34,具有摄取Di-lac-LDL、结合FITC-UEA-1的功能特点及体外可形成血管管腔样结构。结论采用全骨髓贴壁法培养的mBMMSCs在体外具有定向诱导分化为VECs的潜能,成为血管组织工程理想的细胞来源。

Experimental studies of mouse bone marrow mesenchymal stem cells differentiate into vascular endothelioid cells in vitro

Objective To study the separation culture of mouse bone marrow mesenchymal stem cells （mBMMSCs） and the feasibility of mBMMSCs differentiate into vascular endothelioid cells （VECs） in vitro. Methods The mBMMSCs were isolated by whole bone marrow adherence method and then were cultured and passaged. The morphological changes of primary cell were observed by inverted microscope; the growth curve and 3-（4,5-dimethyl-2-thiahiazo）-2,5-diphenyhetrazolium bromide （MTI＇） method were used to observe the growth characteristics of passage cells. Using the third-generation mBMMSCs which were seeded in endothelial cell growth medium（EBM-2） to differentiate into endothehoid cells with vascular endothelial cell growth factor（ VEGF） and basic fibroblast growth factor（bFGF） for directional induction. The morphological changes of cells were observed. The changes of DNA cycle and cellular immune phenotype of third-generation mBMMSCs and induced cell were analyzed by flow cytometry. The expression of CD31, vWF and CD34, and the function of intaking of Dil-ac-LDL and binding of FITC-UEA were tested by immune cell fluorescence technology. VECs functions were detected via angiogenesis studies in vitro. Results The primary mBMMSCs were long spindle and vortex shape to grow. P1, P2 and P3 cell growth curve and MTr method displayed that the cells were the incubation, logarithmic and plateau growth. After induction, part of the cells morphous was similar to the VECs;the cells were polygonal, short spindle, paving stone shape arrange to grow. The third generation of mBMMSCs G0/G1 phase was 86% ,after differentiation,the cells in G0/G1 phase was 92% ,there were not obvious difference of cell DNA cycle. The expression of CD105 ,CD90,CD73 ,CD44 of the third generation mBMMSCs were positive,while the ex- pression of CD34, CD45, VEGF （CD309） and CD 14 were negative. The expression of VEGF （ CD309）, CD34 were weakly positive and the expression of CD105, CD90, CD73, CD44, CD45, CD14 were negative in differentiation cells. The expression of CD31,vWF and CD34 in the third-generation mBMMSCs were negative, without the functions and characteristics of intaking Dil-ac-LDL, combining with FITC-UEA-1 and forming of vascular lumen structure. After induction, the expression of VECs specific surface marker CD31, CD34 and vWF were positive, with the function of intaking Dil-ac-LDL and combined with the FITC-UEA-1 and could form vascular lumen sample structure in vitro. Conclusion The mBMMSCs which were culture by whole bone marrow adherent have the potential to directly differentiate into VECs in vitro ,which become an ideal cell source for tissue engineering blood vessels.