EGFP标记的VEGF165重组质粒的构建及其在骨髓间充质干细胞中的表达

Construction of EGFP-labled recombinant plasmid of VEGF165 gene and its expression in bone marrow mesenchymal stem cells

目的 构建pIRES2 EGFP VEGF16 5真核表达质粒 ,并在大鼠骨髓间充质干细胞内表达。方法 应用DNA重组方法构建pIRES2 EGFP VEGF16 5 ,并将其转染到大鼠骨髓间充质干细胞内 ,采用ELISA和MTT法检测转染后细胞培养上清液中VEGF16 5的含量及生物活性。结果 成功构建了pIRES2 EGFP VEGF16 5 ,将其转染骨髓间充质干细胞后 ,VEGF蛋白表达水平明显增高 ,转染后的细胞培养上清具有促使内皮细胞增殖的生物活性。结论 成功构建了真核表达质粒pIRES2 EGFP VEGF16 5 ,将其转染骨髓间充质干细胞后可高水平地表达具有生物学活性的VEGF蛋白。

Objective To construct a EGFP-labled recombinant plasmid of VEGF165 (vascular endothelial growth factor) gene, and to study the transfection and expression of VEGF165 eukaryotic expression plasmid in mesenchymal stem cells (MSCs). Methods pIRES2-EGFP-VEGF165 recombinant plasmid was constructed, which was then transfected into rat MSCs. ELISA and MTT were used to detect the expression level and biological activity of VEGF in the conditioned medium after transfection. Results There was a significant increase in VEGF protein in the MSCs after being transfected with pIRES2-EGFP-VEGF165. The conditioned medium after transfection showed the biological activity of stimulating the proliferation of endothelial cells. Conclusions The pIRES2-EGFP-VEGF165, a eukaryotic expression plasmid for VEGF165 gene, is constructed. High levels of VEGF protein expression can be obtained in the MSCs transfected with pIRES2-EGFP-VEGF165. The expressed protein has the biological activity of VEGF.