人血管内皮生长因子165基因转染兔骨髓间充质干细胞的蛋白表达

Protein expression of bone marrow mesenchymal stem cells after human vascular endothelial growth factor 165 gene transfection

背景:骨髓间充质干细胞是最好的组织工程种子细胞来源,含有血管内皮生长因子165(vascular endothelial growth factor 165,VEGF165)不仅对血管再生和启动成骨修复有重要意义,其持续稳定的释放还能够提高新生骨的矿化程度,增强修复组织的力学性能。目的:观察hVEG F165基因转染的兔骨髓间充质干细胞分泌血管内皮生长因子的蛋白功能。方法:体外分离、培养兔骨髓间充质干细胞,纯化并鉴定兔骨髓间充质干细胞;免疫荧光法检测细胞表面标志;传代培养后的骨髓间充质干细胞以pcDNA3.1-VEGF165质粒和脂质体1:3比例的混合液转染,并分为3组:转染组应用pcDNA3.1-VEGF165转染细胞,空载体转染组应用pcDNA3.1-空载体转染,未转染组不处理。通过ELISA和Western-blot检测转染后细胞中外源性血管内皮生长因子的表达。结果与结论:转染组与其他两组比较,VEGF165蛋白含量显著增高,差异有显著性意义(P<0.05),但空载体转染组与未转染组之间差异无显著性意义(P>0.05),转染组不同时间点之间VEGF165蛋白含量差异均有显著性意义(P<0.05),hVEGF165基因转染的骨髓间充质干细胞能成功分泌VEGF165蛋白。提示采用基因转染技术可将hVEGF165基因转染到骨髓间充质干细胞中并可有效表达具有生物活性的VEGF165。

BACKGROUND：Bone marrow mesenchymal stem cells（BMSCs） is the best source of seed cells for tissue engineering,which containing vascular endothelial growth factor 165（VEGF165） not only has an important significance in angiogenesis and the start of bone repair,but also its sustained release can increase the degree of mineralization of new bone,and enhance the mechanical properties of repair tissue.OBJECTIVE：To observe the hVEGF165 protein function expressed by the gene transfected BMSCs.METHODS：BMSCs from rabbits were isolated and cultured in vitro,and which were purified and identified.Cell surface makers were detected by immunofluorescence assay.And the cultured BMSCs were transfected with the composite of PcDNA3.1-hVEGF165 plasmid and lipofectamine in the ratio of 1/3 mixed liquor,and which were divided into 3 groups：transfection group,empty vector transfection group,and non-transfection group.PcDNA3.1-hVEGF165 plasmid was used to transfect cells in transfection group;pcDNA3.1-empty vector transfection was applied in empty vector transfection group;no treatment was performed in non-transfection group.The expression of exogenous hVEGF165 was detected by ELISA and Western-blot.RESULTS AND CONCLUSION：Compared with empty vector transfection group and non-transfection group,the protein level of VEGF165 in transfection group was significantly higher（P 0.05）,however,there was no significant difference between empty vector transfection group and non-transfection group（P 0.05）.There were significant differences of the protein level of VEGF165 in transfection group（P 0.05） at different time points.The transfected BMSCs by hVEGF165 can successfully secret the protein of VEGF165.It is indicated that hVEGF165 can be transfected to BMSCs and efficiently express VEGF165 protein of biological activity by using of gene transfection technology.