血管内皮生长因子基因在兔骨髓间充质干细胞中的转染和表达

目的：探讨应用人血管内皮生长因子165 (hVEGF₁₆₅)进行基因修饰的可行性，为血管化组织工程组织的构建及缺血性疾病的治疗奠定实验基础。方法：构建pcDNA3.0-VEGF₁₆₅真核表达载体，利用脂质体介导转染兔骨髓间充质干细胞（MSCs），ELISA方法检测转基因MSCs的hVEGF蛋白表达情况，MTT法检测转基因MSCs表达物对血管内皮细胞增殖活性的影响，设单纯培养MSCs及pcDNA3.0转染MSCs组为对照组。结果：成功构建hVEGF真核表达载体，并成功地将其转入MSCs中；MSCs细胞在转染 pcDNA3.0-VEGF₁₆₅ 质粒24、48和72 h后，其培养上清中hVEGF蛋白表达量均较对照组显著升高（P<0.05），至G418筛选后12代，转基因MSCs培养上清中仍有hVEGF蛋白的表达，含2%、4%、8%、16%和32%转基因细胞培养上清的培养液均明显增加血管内皮细胞的增殖率，与对照组比较，差异均具有显著性（P<0.05）。 结论： hVEGF₁₆₅基因成功地转染至MSCs中，并可进行有效地表达。

Transfection of VEGF gene in rabbit mesenchymal stem cells and its expression

Objective To explore the possibility of human vascular endothelial growth factor 165(hVEGF165) to transfect rabbit mesenchymal stem cells(MSCs),and establish the experimental foundation of angiogenesis tissue engineering organization and the treatment of ischemic disorders.Methods pcDNA3.0-VEGF165 eukaryotic expression vector was constructed,the vector was used directedly to transfect MSCs.The supernatant then was collected and the soluble protein of hVEGF gene expression was analysed with ELISA method.The proliferation capability of human umbilical vein endothelial cells(HUVEC) stimulated by the supernatant was measured with MTT methods,untreated MSCs and transfected MSCs were used as control groups.Results hVEGF eukaryotic expression vector was successfully constructed and transfected into the MSCs.Compared with the control group,concentration of hVEGF protein in the supernatant of the cultured cells increased significantly after the MSCs were tranfected with pcDNA3.0-VEGF165 for 24,48,72 h(P<0.05),hVEGF protein was still found till the 12th progeny.The proliferation rate of HUVEC cells in the culture containing 2% supernatant of transfected cells was obviously higher than that of the control group(P<0.05).Conclusion hVEGF gene could be successfully transfected into MSCs and could express effectively.