TFR和VEGF基因双顺反子慢病毒感染骨髓内皮祖细胞的研究

目的:研究转铁蛋白受体 (transferrin receptor,TFR)和血管内皮生长因子(vascular endothelial growth factor，VEGF)的双基因共表达慢病毒载体是否能介导目的基因在中华小型猪骨髓内皮祖细胞（endothelial progenitor cells,EPCs）中的有效表达。方法:通过分子克隆技术构建双顺反子慢病毒表达载体pLenti-GFP-TIR；分离培养中华小型猪骨髓EPCs，用流式细胞仪(FCM)检测细胞表面抗原CD31和Flk-1的表达；并利用Lipofectin 2000将含目的基因的转移质粒与pRsv-REV、pMDlg-pRRE及pMD2G共转染293T细胞并进行慢病毒包装。72 h后，收集病毒上清，感染中华小型猪骨髓EPCs，并通过RT-PCR法检测TFR和VEGF基因的表达。结果:①成功地构建了双顺反子慢病毒表达载体pLenti-GFP-TIR；②FCM检测证实，分离的细胞为骨髓EPCs；包装好的慢病毒颗粒可成功地感染中华小型猪骨髓EPCs；③RT-PCR法检测表明，TFR和VEGF呈高水平的表达。结论:TFR和VEGF基因双顺反子慢病毒载体Lenti-GFP-TIR可有效地转移目的基因至中华小型猪骨髓EPCs中，并成功地表达目的基因，为进一步探讨移植细胞分子成像奠定了基础。

Construction and expression of bicistronic lentiviral vector containing TFR and VEGF genes in endothelial progenitor cells

AIM: To investigate whether bicistronic lentiviral vectors containing TFR and VEGF genes can mediate the expression of TFR and VEGF genes in endothelial progenitor cells(EPCs) of Chinese mini-swine.METHODS: TFR,IRES and VEGF genes were cloned into pLenti-GFP-Neo to generate the lentiviral vector pLenti-GFP-TIR.We sucessfully cultured EPCs of Chinese mini-swine where we identified the expression of cell surface CD31 and Flk-1 by flow cytometry(FCM).The four-plasmid lentiviral vector system(pRsv-REV,pMDlg-pRRE,pMD2G and pLenti-GFP-TIR) was cotransfected into human embryonic kidney 293T cells using the Lipofectin 2000 method.The packaged virus was harvested 72 h later and EPCs were infected by lentivirus carrying TFR and VEGF genes.RT-PCR was used to detect expression of TFR and VEGF genes in the viral-infected EPCs.RESULTS: EPCs were successfully infected by the recombinant lentivirus.TFR and VEGF genes were detected in the infected EPCs by RT-PCR.CONCLUSION: The bicistronic lentiviral vector containing TFR and VEGF genes can effectively deliver genes into EPCs and express target genes,providing the basis for future research on cardiac molecular imaging of cell transplantation.