成人骨髓间充质干细胞分化为血管内皮细胞的研究

EXPERIMENTAL STUDY ON DIFFERENTIATION OF ADULT MARROW MESENCHYMAL STEM CELLS INTO VASCULAR ENDOTHELIAL CELLS IN VITRO

目的研究成人骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)体外分化为血管内皮细胞的能力并探讨其诱导条件。方法分离成人骨髓单个核细胞,经贴壁法获取MSCs。所获细胞分4组进行诱导组1、2以5×103/cm2细胞密度接分别接种于含2%和15%胎牛血清的L-DMEM培养基;组3、4以5×104/cm2细胞密度分别接种于上述两种血清浓度的培养基,各组细胞经血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)辅以牛脑提取物的诱导,对照组为正常传代培养的MSCs。在诱导的第14、21天分别测定表面分子CD34、VEGFR-2、CD31和因子相关抗原(factor-relatedantigen,又称vonWillebrandfactor,vWF)以及体外成血管实验对分化细胞进行鉴定,同时测定不同诱导条件下的细胞增殖指数(proliferationindex,PI)。结果组3细胞经诱导后,内皮系表面分子CD34、VEGFR-2、CD31、vWF于第14天均有不同程度表达,分别为8.5%、12.0%、40.0%、30.0%,其中CD34、VEGFR-2在第21天表达上调,为15.5%、20.0%,余各诱导组细胞未表达上述表面分子;诱导后的组3细胞呈低增殖状态(PI值约为10.4%),在半固体培养基中还可形成血管腔样结构。结论从成人骨髓中分离培养的MSCs,在较高细胞接种密度和低增殖状态下,经VEGF、牛脑垂体提取物诱导后,可分化为血管内皮细胞,可作为治疗性血管生成及组织工程理想的种子细胞。

Objective To study the differenation of adult marrow mesenchymal stem cells（MSCs） into vascular endothelial cells in vitro and to explore inducing conditions. Methods MSCs were isolated from adult marrow mononuclear cells by attaching growth. MSCs were divided into 4 groups to induce ： the cells seeded at a density of 5 × 10^3/cm^2 in 2% and 15% FCS L-DMEM respectively （group 1 and group 2）, at a density of 5× 10^4/cm^2 in 2% and 15% FCS L-DMEM respectively （group 3 and group 4）; vascular endothelial growth factor （VEGF） supplemented with Bovine pituitary extract was used to induce the cell differentiation. The differentiated cells were identified by measuring surface marks （CD34, VEGFR-2, CD31 arid vWF ） on the 14th day and 21st day and performed angiogenesis in vitro on the 21st day. The cell proliferation index（PI）of different inducing conditions were measured. Results After induced in VEGF supplemented with Bovine pituitary extract, the cells of group 3 expressed the surface marks CD34, VEGFR-2, CD31 and vWF on the 14th day, the positive rates were 8.5%, 12.0%, 40. 0% and 30. 0% respectively, and on the 21st day the positive rates of CD34 and VEGFR-2 increased to 15. 5% and 20. 0%, while the other groups did not express these marks; the induced cells of group 3 showed low proliferating state（PI was 10. 4% ） and formed capillarylike structure in semisolid medium. Conclusion Adult MSCs can differentiate into vascular endothelial cells after induced by VEGF and Bovine pituitary extract at high cell densities and low proliferating conditions,suggesting that adult MSCs will he ideal seed cells for the therapeutic neovascularization and tissue engineering.