人骨髓间充质干细胞分离培养鉴定及VEGF基因的转染

目的：探讨人骨髓间充质干细胞（hMSC）分离培养、鉴定方法及人血管内皮生长因子（hVEGF）165基因转染hMSC后表达情况,拟构建血管化组织工程皮肤.方法：用密度梯度离心法分离骨髓间充质干细胞,筛选贴壁细胞培养并传代.观察细胞形态,生长情况.检验细胞CD44、CD29、CD14、CI45等表面标记.利用基因重组技术,构建pCDN3.1＋VEGF表达质粒,并将其转染第3代hMSC.经G418抗性筛选后扩大培养并用Western blot实验检测其蛋白产物.结果：骨髓密度梯度离心法分离hMSC,接种培养瓶后26 h呈对数生长,6d后达到高峰.hMSC表达CD29,CD44,不表达造血细胞标记物CD14和CD45.通过酶切鉴定验证成功构建pCDN3.1＋ VEGF表达质粒.Western blotting实验证实转染后hMSCs表达VEGF增加.结论：成功建立hMSC分离培养鉴定体系.利用基因重组技术可成功将hVEGF基因导入hMSC并产生预期效应.

Human bone marrow mesenchymal stem cells isolated and cultured and VEGF gene transfection

Objective：human bone marrow mesenchymal stem ceils （hMSC） were isolated and cultured, identification methods and human vascular endothelial growth factor （hVEGF） 165 gene expression of hMSCs after,to be build vascularized tissue-engineered skin. Methods： density gradient centrifugation of bone marrow mesenchymal stem cells, screening adherent cells cultured and passaged. Observation of cell morphology and growth. Test cell CD44, CD29, CD14, CD45 and other surface markers. Using recombinant DNA technology to construct pCDN3.1 VEGF expression plasmid and transfected into the third generation of hMSC. After expanding culture of G418 resistance screening experiments with Western blot to detect the protein product. Results：density gradient centrifugation of bone marrow hMSCs ,26 hours after inoculation flasks logarithmic growth and reached a peak after six days. hMSCs expressed CD29, CD44 ,no expression of hematopoietic cell markers CD14 and CD45. Verified by restriction enzyme digestion pCDN3. 1 VEGF expression plasmid was successfully constructed. Western blot experiments confirmed the expression of VEGF transfected hMSCs increased. Conclusion： hMSC isolated and cultured successfully established system. Using recombinant DNA technology can be successfully VEGF gene into hMSC and produce the desired effect.