双顺反子表达人组织因子途径抑制因子和绿色荧光蛋白重组病毒的构建及其在内皮祖细胞中表达

目的 构建双顺反子表达人组织因子途径抑制因子(TFPI)与绿色荧光蛋白(GFP)的逆转录病毒表达载体pMSCV-TFPI-IRES-GFP,并观察其在大鼠内皮祖细胞(EPCs)中的表达,为进一步研究TEPI在预防血管再狭窄中的作用奠定基础.方法 将人全长TFPI cDNA亚克隆到逆转录病毒载体pMSCV-IRES-GFP中,获得定向插入重组子pMSCV-TFPI-IRES-GFP,采用酶切图谱分析和测序进行鉴定.用磷酸钙法经293T细胞包装,收集病毒上清,感染EPCs.用荧光显微镜和流式细胞术观察感染后细胞内GFP表达情况.RT-PCR检测EPCs中TFPI mRNA的表达水平,ELISA法检测EPCs细胞培养上清中TFPI蛋白的含量.结果 经限制性内切酶酶切图谱分析证实重组病毒中含有人TFPI的cDNA片段,基因测序结果与Genebank中TFPI cDNA序列相符.荧光显微镜观察及流式细胞仪检测显示,90%以上的感染细胞中存在GFP表达;RT-PCR分析显示,重组病毒感染细胞中TFPI mRNA水平明显增高;ELISA法检测发现,感染重组病毒的EPCs培养上清中有TFPI蛋白表达.结论 本研究成功构建重组pMSCV-TFPI-IRES-GFP真核表达载体,其在EPCs中能够同时表达TFPI和GFP,为TFPI基因结合血管内皮祖细胞进行血管再狭窄防治研究的进一步深入开展奠定了良好实验基础.

Construction of recombinant retroviral vector containing human tissue factor pathway inhibitor and its expression in endothelial progenitor ceils

Objective To construct the recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor and GFP in rat endothelial progenitor cells (EPCs). Methods Full length TFPI cDNA obtained from pIRES-TFPI by PCR amplification was digested with EcoRI and Xhol restriction enzymes and subsequently inserted into pMSCV-IRES-GFP expression vector to create the recombinant bicistronic retroviral vector pMSCV- TFPI- IRES -GFP encoding both TFPI and GFP . The recombinant plasmid was identified with restrictive endonuclease digestion and DNA sequencing. The recombinant plasmid was transfected into 293T and the supernatant containing packaged recombinant retroviral particles was collected and used to infect the EPCs isolated from rat bone marrow. TFPI mRNA was measured by RT-PCR and the amount of TFPI protein secreted from the transfected cells was determined by ELISA. GFP expression in the infected cells was analyzed by fluorescent microscopy and fluorescence activated cell sorting (FACS). Results Restriction endonuclease mapping and DNA sequencing confirmed the in-frame insertion of TFPI cDNA into the constructed vectorpMSCV-TFPI-IRES-GFP. Both RT-PCR and ELISA analysis demonstrated increased TFPI expression in the EPCs infected with pMSCV-TFPI-IRES-GFP. FACS analysis demonstrated that the transduction efficiency of EPCs with pMSCV-TFPI-IRES-GFP in vitro was over 90%. Conclusion pMSCV-TFPI-IRES-GFP could be effectively expressed in cultured EPCs and may provide a useful tool for further study on the application of TFPI in the prevention of restenosis.