人参皂甙Rg1对体外微环境诱导人骨髓间充质干细胞成血管内皮样细胞分化的影响

Effect of Ginsenoside Rg1 on the Microenvironment Dependent Differentiation of Human Bone Marrow Mesenchymal Stem Cell to Vaso-endothelioid Formative Cells in Vitro

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中文关键词: 人参皂甙Rg1 骨髓间充质干细胞分化内皮细胞共培养

英文关键词:ginsenoside Rg1 bone marrow mesenchymal stem cells differentiation endothelial cell co-culture

基金项目:

作者	单位
何威	广东省人民医院,广东省医学科学院中山大学中山医学院
杨旭辉	中山大学干细胞与组织工程中心
林秋雄	广东省人民医院,广东省医学科学院
余伟华	中山大学干细胞与组织工程中心
吴伟康	中山大学中西医结合研究所

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中文摘要:

目的探讨人参皂甙Rg1在体外微环境诱导分化人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)成血管内皮样细胞的影响。方法采用模拟体内微环境的半透膜隔离非接触共培养的方法体外诱导hMSCs向内皮细胞表型分化,通过RT-PCR检测内皮细胞标志物血小板内皮黏附分子-1(CD31)、血管性血友病因子(vWF)、血管内皮钙黏蛋白(VE-cadherin)mRNA的表达,免疫荧光染色检测CD31蛋白和血管内皮黏附分子-1(VCAM1)的表达。透射电镜观察诱导后细胞的超微结构鉴定诱导细胞的特征,并通过流式细胞仪检测诱导细胞CD31表达百分比。结果与成熟内皮共培养的骨髓基质干细胞具有微环境依赖性向内皮分化的能力。细胞培养第2周,开始出现形态学改变,胞体回缩,呈多角形改变。细胞培养第3周,细胞增殖速度明显加快,形态学上呈卵圆形或"铺路石"样改变;在基因水平上,RT-PCR显示经典内皮细胞标志物CD31、vWF、VE-cadherin mRNA的高表达;在蛋白水平上,免疫荧光染色CD31和VCAM1表达阳性;透射电镜下诱导后细胞显示内皮细胞特有的Weibel-Palade小体;随人参皂甙Rg1剂量的增加,诱导hMSCs表达CD31百分比增加(组间比较,P<0.05)。结论 hMSCs在共培养的微环境中,可以分化成在形态上、基因和蛋白表达、超微结构等方面均显示出血管内皮细胞特征的表型。人参皂甙Rg1可促进微环境依赖性诱导hMSCs向内皮细胞系的分化。

英文摘要:

Objective To investigate the effect of ginsenoside Rg1 on the microenvironment dependent differentiation of human mesenchymal stem cells(hMSCs)to vaso-endothelioid cells(VECs) in vitro.Methods The in vitro differentiation of hMSCs to VECs were established adopting the in vivo environment simulated semi-permeable membrane separated non-contact co-culturing method.The mRNA expressions of endothelial markers,such as platelet endothelial adhesive factor-1(CD31),vascular hemophillia factor(vWF) and vascular endothelial cadherin(VE-cadherin) were analyzed by RT-PCR;the protein expressions of CD31 and vascular endothelial adhesive factor-1(VCAM1) were detected by fluorescence immunohistochemistry;structural identification for the endothelial characteristics of differentiated hMSCs were made under electron microscopy;and the percentage of CD31 expression in differentiated hMSCs was determined by flow cytometry to explore the effect of ginsenoside Rg1 on the differentiation.Results The bone marrow mesenchymal stem cells co-cultured with mature endothelial membrane showed a microenvironment dependent capacity for differentiating to endothelium,with the morphological changes revealed starting from the 2nd week,showing cell body contraction,polygonal-shaped change;and at the 3rd week,the markedly speedily cell proliferation with elliptic or slabstone-like change of cells.High levels of classic endothelial cell markers,such as mRNA expressions of CD31,vWF,VE-cadherin,and protein expressions of CD31 and VCAM1,were shown;the typical weibel-palade body of endothelial cell was found in the differentiated cells.Moreover,percentage of CD31 expression in the differentiated hMSCs was increased after Rg1 treatment dose-dependently.Conclusion Under the microenvironment of co-culture,hMSCs could differentiate into cells presenting the characteristics of endothelial cell in aspects of the morphology and ultrastructure of cells,as well as the gene and protein expressions of cell markers;ginsenoside Rg1 can promote the microenvironment dependent differentiation of hMSCs to VECs system in vitro.

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