黑色素瘤细胞诱导骨髓间充质干细胞向血管内皮细胞分化的初步研究

Pilot study of differentiation of bone marrow derived mesenchymal stem cells into endothelial cells induced by B16 melanoma cells in vitro

目的 体外观察和分析小鼠黑色素瘤细胞B16诱导小鼠骨髓间充质干细胞（BMSC）向血管内皮细胞分化的现象和过程，初步分析肿瘤细胞分泌血管生长因子（VEGF）-a在该诱导过程中的时序关系。方法利用Transwell系统进行BMSC与B16细胞共培养，利用免疫荧光、蛋白印迹、透射电镜等技术观察BMSC经B16诱导后向内皮细胞分化的过程，利用ELISA检测培养液内VEGF-a的水平。结果BMSC表型鉴定为CD44^＋／CD73^＋／CD90^＋／CD105^＋／CD166^＋／CD34^-／CD45^-／CD133^-，经B16诱导后出现内皮细胞标志物VEGFR-1、VEGFR-2和第八因子（FⅧ）的表达并随时间延长而增强，共培养48h后VEGFR-1、VEGFR-2及FⅧ均可见明显表达，72h时VEGFR-2表达量较48h时增加了1倍，FⅧ表达量增加了约3倍。共培养体系培养基中可检测到由B16细胞分泌的VEGF-a量随时间增多。结论B16细胞可能通过分泌VEGF-a来诱导BMSC具备血管内皮细胞表型，提示BMSC在肿瘤血管生成中起了重要的作用。

Objection Bone-marrow derived mesenchymal stem cells （BMSC） have the potential to differentiate into endothelial cells. The aim of the study was to investigate the induction process of BMSC by B16 melanoma cells in vitro and to analyze the role of VEGF-a in the process. Methods A co-culture system containing BMSC and B16 melanoma cells based on transwell indirect model was established, and the induction process of BMSC by B16 melanoma cells was studied in vitro. Results BMSC were isolated from the bone marrow of C57 mice. BMSC expressed CD105, CD90, CD73, CD44 and CD166, and acquired expressin of endothelial phenotype markers including VEGFR-1, VEGFR-2 and Factor Ⅷ after co-culture with B16 melanoma cells for 48 hours. The expression level of VEGFR-2 would be double and Factor Ⅶ threefold more by extending the co-culture time to 72 hours. In the co-culture system, B16 melanoma cells also up-regulated the expression of VEGF-a. Conclusions VEGF-a plays a significant role in the differentiation of BMSC into cells of endothelial phenotype, therefore, is important to tumor angiogenesis.