携人血栓调节蛋白基因慢病毒载体的构建及其在内皮祖细胞中的表达

Construction of lentiviral vector containing human thrombomodulin gene and its expression in endothelial progenitor cells of rabbits

目的 构建携带人血栓调节蛋白（hTM）基因的慢病毒表达载体（plenti6.3-hTM-IRES-EGFP）,体外转染兔外周血内皮祖细胞（EPCs）,并观察其在EPCs中的表达及对其功能的影响.方法 采用DNA重组技术构建plenti6.3-hTM-IRES-EGFP慢病毒表达载体；密度梯度离心法分离兔外周血EPCs; EPCs分为实验组、病毒对照组及空白对照组；3组细胞分别转染plenti6.3-hTM-IRES-EGFP、pLenti6.3-MCS-IRES-EGFP及磷酸盐缓冲液（PBS）；流式细胞仪检测转染效率,转染72 h后激光共聚焦显微镜观察增强型绿色荧光蛋白表达,定量聚合酶链反应（Q-PCR）及Western blot检测细胞中hTM mRNA及蛋白表达；炭花青荧光燃料标记的乙酰化低密度脂蛋白（Dil-ac-LDL）和异硫氰酸荧光素标记的荆豆凝集素-1（FITC-UEA-1）双荧光法鉴定EPCs；噻唑蓝（MTT）法及Tr answell小室法检测各组细胞增殖及迁移活性.结果 成功构建plenti6.3-hTM-IRES-EGFP,转染EPCs 72 h后,绿色荧光蛋白表达率超过90.0％,流式细胞仪测定hTM阳性EPCs达92.9％,Q-PCR及Western blot显示转染组hTM mRNA表达明显高于对照组.3组EPCs的增殖及迁移活性比较差异无统计学意义（P＞0.05）.结论 plenti6.3-hTM-IRES-EGFP慢病毒表达载体在EPCs中高效表达且不影响其生物学功能,为进一步研究hTM在动脉血栓疾病中的作用奠定了物质基础.

Objective To construct the lentiviral vector containing human thrombomodulin （hTM） gene,and examine the expression of hTM in rabbit peripheral endothelial progenitor cells （EPCs）after transduction and its effect on the biological functions of EPCs.Methods The lentivirus plenti6.3-hTM-IRES-EGFP was reconstructed by polymerase chain reaction （PCR）.EPCs were isolated from fresh blood obtained from the heart of a rabbit by density-gradient centrifugation and then were transduced with above lentiviral vector.To evaluate the transduction efficiency of plenti6.3-hTM-IRES-EGFP,quantitativepolymerase chain reaction （Q-PCR）,Western blotting and facial action coding system（FACS） were performed.Acetylated low density lipoprotein （DiI-ac-LDL） and fluorescein isothiocyanate （FITC）-UEA-1 double fluorescent labeling was used for the identification of EPCs.Methyl thiazol tetrazolium （MTT） and transwell assays were carried out to examine the proliferation and migration of EPCs in the presence or absence of hTM.Results The recombinant plenti6.3-hTM-IRES-EGFP was confirmed by the evidence of DNA sequence analysis and Western blotting.The transduced EPCs were found overexpressing hTM by Q-PCR,Western blotting and FACS,suggesting the recombinant lentivirus system was successfully constructed.There were no changes in the biological functions of EPCs overexpressing hTM.Conclusion The plenti6.3-hTM-IRES-EGFP has been successfully constructed and hTM can be efficiently and highly expressed in EPCs.All of these provide us experimental evidence for gene-cell combined therapy and further study of TM on inhibiting thrombotic restenosis of arterial occlusive diseases after percutaneous transluminal angioplasty （PTA） treatment.