血管内皮生长因子基因稳定转染兔骨髓基质干细胞的研究

Experimental study of stable transfection of VEGF gene into rabbit mesenchymal stem cells

目的检测人血管内皮生长因子（VEGF）基因稳定转染对兔骨髓基质干细胞（BM—SC）VEGF表达的影响。方法分离并培养兔骨髓基质干细胞，利用脂质体介导pcDNA3．1-VEGF165转染兔BMSC，G418筛选稳定表达pcDNA3．1-VEGF165的细胞克隆，RT—PCR、Westernblot法检测转染前后细胞中VEGFl65mRNA和蛋白的表达。结果通过筛选获得了稳定表达pcDNA3．1-VEGF165的细胞克隆。逆转录-聚合酶链反应（RT-PCR）、Western blot结果显示：稳定转染组BMSC中可检测到VEGF165mRNA和蛋白的表达，空白和空载体组未见VEGF表达。结论pcDNA3．1-VEGF165载体转染的兔BMSC可稳定表达外源性VEGF165mRNA和蛋白，可作为治疗骨缺损和骨缺血性坏死研究的种子细胞。

Objective To detect the expression of VEGF mRNA and protein in rabbit mesenchyreal stem cells which stablely transferred by VEGF gene and provide a potential method for treatment of defect and ischemic necrosis of bone. Methods BMSCs were isolated from rabbit bone marrow and cultured in vitro. The vector pcDNA3.1-VEGF165 was transfected into the mesenchymal stem cells by liposome mediated. The stable transferred clone was selected by G418. The mRNA and protein of VEGF165 gene in the transferred cells were analyzed by RT-PCR and Western blot. Results pcDNA3.1-VEGF165 vector was successfully transferred into BMSC and the stable transferred clone was selected. RT-PCR and Western blot showed that there were VEGFI65 mRNA and protein expression in the stable transferred cells, but there was no VEGF165 mRNA and protein expression in the other groups. Conclusion pcDNA3.1-VEGF165 vector transfection can obtain the stable expression of exogenous VEGF165 mRNA and protein in rabbit mesenchymal stem cells, and can be used as the seed cells for the treatment of defect and ischemic necrosis of bone.