VEGF165基因重组腺相关病毒构建及转染猪骨髓间充质干细胞的研究

目的构建携带人血管内皮细胞生长因子（hVEGF）的重组腺相关病毒载体，在体外转染猪骨髓间充质干细胞并观察其表达。方法细胞AAV包装质粒pAAV-IRES-ZsGreen及含hVEGF的质粒经EcoRI＋BamHI双酶切、连接，构建pAAV-hVEGF165-IRES-ZsGreen载体，采用磷酸钙法转染293AAV细胞系，获得rAAV2-hVEGF165及对照重组腺相关病毒，real-timePCR法测定病毒效价。Westernblot检测重组rAAV2-hVEGFl65在猪骨髓间充质干细胞的表达，MTS法检测VEGF蛋白活性。结果成功构建rAAV2-hVEGF165重组腺相关病毒载体，rAAV2-hVEGFl65经双酶切和测序鉴定正确，real-timePCR法测定病毒效价为5×10 12vg／ml。转染rAAV2-hVEGF165的猪骨髓干细胞能够有效表达VEGF蛋白，并能促进血管内皮细胞的增殖。结论成功构建rAAV2-hVEGFl65重组腺相关病毒载体，并能在猪骨髓间充质干细胞中有效转染和表达，为进一步探索rAAV2-hVEGF165对治疗缺血性心肌疾病的研究提供实验基础。

Construction of Recombinant Adeno - associated Viruse Vector Carrying Human Vascular Endothelial Growth Factor Gene and Its Expres-sion in Porcine Mesenchymal Stem Cells.

Objective To construct recombinant adeno - associated viruse vector carrying human vascular endothelial growth factor gene, and assess its expression in porcine mesenchymal stem cells in vitro. Methods AAV packaging plasmid pAAV - IRES - ZsGreenand plasmid containing hVEGF were digested by EcoR I ＋ BamH I ~ The digestion products were connected. And pAAV - hVEGF165 - IRES- ZsGreen vector was generated. After transfecting into 293AAV cells by calcium phosphate method, rAAV2 -hVEGF165 and the control recombinant adeno - associated virus were obtained, viral titer was determined by real - time PCR. The expression of recombinant rAAV2 - hVEGF165 in porcine mesenchymal stem ceils was detected by western blot and MTS assay. Results rAAV2 - hVEGF165 re- combinant adeno - associated virus vector was successfully constructed and was verified by double digestion and DNA sequencing. The pu- rified recombinant virus had a high titer of 5 ~ 1012vg/ml. VEGF can be efficiently expressed and the proliferation of vascular endothelial cell was promoted after porcine mesenchymal stem cells transfected with AAV2 - hVEGF165. Conclusion rAAV2 - hVEGF165 was con- structed successfully,which had an effectively transfection and high expression in porcine mesenchymal stem cells. It provided an experi- mental basis to further explore the effect of rAAV2 - hVEGF165 in the therapy of ischaemic heart disease.