自体内皮祖细胞促进组织工程骨血管化的体内外实验研究

Autologous endothelial progenitor cells promote the neovascularization of tissue engineering bone in vitro and vivo

目的探讨自体内皮祖细胞(EPCs)在体内、外促进组织工程骨血管化的能力。方法将兔自体外周血EPCs及骨髓间充质干细胞(BMSCs)按联合培养时细胞增殖率最大时配比(EPCs∶BMSCs=1∶2)体外培养(联合培养组),在体外采用实时定量PCR方法检测成骨相关细胞因子Osteonectin、Osteopotin、Col-1及成血管相关细胞因子血管内皮生长因子(VEGF)表达,于培养3、7、14d观察表达量的变化并与单纯EPCs及BMSCs组比较;将单纯EPCs、BMSCs及联合培养组的组织工程骨移植到兔四肢肌袋内,于移植后2、4、8周观察组织工程骨生长情况,同时制成组织切片行CD34、CD105、ZO-1免疫组织化学染色,采用Image-Pro plus 6.0图像分析软件,测量光密度值,比较3组工程骨表达量变化。结果3、7、14dOsteonectin、Osteopotin、Col-1、VEGF各组表达均逐渐增高,其中联合培养组增高最明显,且各时间点表达量最高(P<0.01);2、4、8周兔四肢肌袋内的组织工程骨中联合培养组细胞复合的工程骨随时间延长成骨增加最明显,新生血管长入最多,免疫组织化学显示CD34、CD105、ZO-1表达最明显(P<0.01)。结论自体EPCs与BMSCs相互作用,在体内、体外均可促进组织工程骨血管化。

Objective Investigate the ability of autologous endothelial progenitor cells （EPCs） in promoting the neovascular- ization of tissue engineering bone in vitro and vivo. Methods Co-culture EPCs drived from autologous peripheral blood and mesenchymal stem cells drived from bone marrow （BMSCs） at the best proportion of 1 ： 2 which was the largest cell proliferation rate in vitro,osteogenesis related cytokines Osteonectin, Osteopotin, Collagen Type 1 （Col-1） and angiogenesis related cytokine VEGF in vitro by using real-time quantitative polymerase chain reaction（PCR） ,and compared with pure EPCs and BMSCs groups at 3rd, 7th and 14th day; the tissue engineering bone seeded with EPCs,BMSCs and co-culture cells （EPCs ： BMSCs= 1 ： 2） were transplanted into rabbit limbs muscle,the growing states of tissue engineering bone were observed at 2,4 and 8 weeks after transplantation, at the same time the expression of CD34,CD105 and ZO-1 were detected with immunohistochemistry staining. Results The mRNA expression of Osteonectin,Osteopotin,Col-1 and VEGF were gradually increased when detected at 3rd, 7th and 14th day with real- time PCR,and the co-culture cells group increased most obviously in the three groups in vitro at the same period time（P〈0.01）; The microvascularization of the engineering biological bone were observed in vivo with immunohistochemistry,and neovascularization of co-culture cells group was also the most obvious group in three groups,immunohistochemical showed that CD34 ,CD105 and ZO-1 was also higher than the other two groups（P〈0.01）. Conclusion Autologous EPCs interact with BMSCs could promote vascularization of tissue engineering bone both in vivo and in Vitro.