内皮祖细胞条件培养基对间充质干细胞增殖和成骨分化的影响及其机制

目的:探讨小鼠骨髓来源的内皮祖细胞(EPCs)条件培养基(CM) 对同源间充质干细胞(MSCs)增殖和成骨分化功能的影响,并阐明其作用机制。方法:小鼠骨髓细胞经差速贴壁法结合专用培养基分别培养扩增MSCs和EPCs,采用流式细胞术(FCM)检测MSCs和EPCs表面标记物,以成骨、成脂和成软骨诱导分化对MSCs进行功能鉴定,以成血管对EPCs进行功能鉴定。将MSCs分为0% EPCs-CM组(对照组,采用LG-DMEM培养)、50% EPCs-CM组(采用50% EPCs-CM+50% LG-DMEM培养)和100% EPCs-CM 组(采用100% EPCs-CM培养)。采用MTT比色法检测各组MSCs的增殖活性;采用茜素红染色检测各组MSCs的成骨分化能力。结果:FCM法检测,第3代MSCs高表达Sca-1、CD29, 低表达CD45、CD11b;经诱导可向成骨、成软骨和成脂方向分化。第3代EPCs 高表达CD34、CD133和VEGFR2;在铺有基质胶的96孔培养板中可形成血管样结构。与对照组比较,50% EPCs-CM组和100% EPCs-CM组MSCs增殖活性明显增加,且呈浓度依赖性(P<0.05)。茜草色素红染色法检测,培养21 d后50%和100% EPCs-CM组MSCs的钙结节数量和钙盐沉积均高于对照组(P<0.05)。结论:利用差速贴壁法可同时分离MSCs和EPCs,EPCs-CM能促进MSCs 的增殖和成骨分化。

Effects of endothelial progenitor cells conditioned medium on proliferation and osteogenic differentiation of mesenchymal stem cells and their mechanisms

Objective To investigate the effects of bone marrow-derived endothelial progenitor cells(EPCs) conditioned medium(EPCs-CM) on the proliferation and osteogenic differentiation of mesenchymal stem cells(MSCs),and to clarify the mechanisms.Methods The EPCs and MSCs were isolated from bone marrow of the mice using differential adhesion method.The surface markers of EPCs and MSCs were identified by flow cytometry (FCM).The osteogenic,chondrogenic,and adipogenic induction differentiation abilities of the MSCs were identified.The function of EPCs was identified by tube formation experiment.The MSCs were divided into 0% EPCs-CM group(control group,cultured with LG-DMEM),50% EPCs-CM group(cultured with 50% EPGs-CM and 50% LG-DMEM),and 100% EPCs-CM group(cultured with 100% EPCs-CM).The proliferation activities of the MSCs in various groups were detected by MTT method;the osteogenic differentiation abilities of the MSCs in various groups were detected by Alizarin red staining.Results The FCM results showed that the third passage MSCs were strongly positive for Sca-1,CD29 and negative for CD45,CD11b and could be induced to complete differentiation process into osteoblasts and adipocytes and chondrocytes.The third passage EPCs cultured on Matrigel showed tube-like structures and highly expressed CD34,CD133 and VEGFR2.Compared with control group,the proliferation activities of the MSCs in 50% EPCs-CM group and 100% EPCs-CM group were increased(P<0.05),which presented a dose-dependent manner.The Alizarin red staining results showed the number of mineralized nodules and calcium deposition of the MSCs in 50% EPCs-CM group and 100% EPCs-CM group were higher than those in control group after cultured for 21 d(P<0.05).Conclusion The method of differential adhesion can simultaneously isolate the MSCs and EPCs.EPCs-CM can promote the proliferation and osteogenic differentiation of the MSCs.