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  

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

作　　者：夏利龙[1] 朱成楚[1] 陈保富[1] 张波[1] 陈宇[1] 胡炳川 张健[1] 

机构地区：[1]温州医科大学附属台州医院,临海317000

出　　处：《医学研究杂志》2014年第2期117-120,共4页Journal of Medical Research

基　　金：浙江省公益技术应用研究基金资助项目(2011C37083)

摘　　要：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,rea1-time PCR法测定病毒效价。Western blot检测重组rAAV2-hVEGF165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-hVEGF165经双酶切和测序鉴定正确,rea1-time PCR法测定病毒效价为5×1012vg/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-hVEGF165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。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.

关 键 词：血管内皮生长因子腺相关病毒 心肌缺血 骨髓间充质干细胞 

分 类 号：R541[医药卫生—心血管疾病]