过表达CXCR4基因的小鼠骨髓间充质干细胞建立及其功能评价

本研究旨在建立稳定表达小鼠CXC型趋化因子受体4（CXC chemokine receptor type 4,CXCR4）基因的小鼠骨髓间充质干细胞（mesenchymal stem cell,MSCs）并研究其功能.采用脂质体转染法将重组慢病毒表达载体LV-CXCR4-IRES-EGFP与包装质粒pMD2.G及包膜蛋白质粒pSPAX2共转染293FT包装细胞,应用超速离心法浓缩病毒颗粒;通过调整感染复数（MOI）、感染时间、促进病毒吸附等方法优化慢病毒感染小鼠MSC的条件,建立过表达CXCR4的MSC,采用荧光显微镜观察EGFP表达,流式细胞术检测细胞表面CXCR4表达,绘制细胞生长曲线检测增殖能力,Annexin-V和7-AAD双染后应用流式细胞术检测细胞凋亡,Transwell实验检测迁移能力.结果发现,通过优化感染条件,重组慢病毒载体对MSC可获得较高感染效率,感染后72 h在荧光显微镜下可观察到较强EGFP表达,流式细胞术检测到MSC表面CXCR4的表达明显增加.细胞生长曲线和凋亡实验显示,过表达CXCR4不会显著影响MSC增殖和凋亡.Transwell实验显示过表达CXCR4可增强MSC迁移能力.结论：慢病毒载体可介导外源性基因CXCR4在小鼠MSC高效表达.

Establishment of Mouse Mesenchymal Stem Cells Overexpressing CXCR4 Gene and Evaluation of Their Functions

This study was purposed to establish the mesenchymal stem cells （MSCs） stably overexpressing mouse CXC chemokine receptor type 4 （CXCR4） gene and to explore their function.The recombinant lentiviral vector LVCXCR4-IRES-EGFP with packaging plasmid pSPAX2 and envelope plasmid pMD.2G were co-transfected into 293FT packaging cell line using lipofectamine 2000 to produce the recombinant lentiviral vectors.The recombinant viruses were harvested and concentrated by using ultracentrifugation.Mouse bone marrow MSC were infected with the viral supematants.Variable methods were used to optimize the transduction condition.EGFP expression was visualized using fluorescence microscope and efficiency of infection was determined by flow cytometry （FCM）.Proliferation and apoptosis were detected by proliferation curve and FCM,respectively.Migration capacity was assessed by a chemotaxis assay using transwell.Expression of EGFP were detected by fluorescence microscopy in MSCs after infection.The results showed that through optimization of infection condition,the recombination lentiviral vectors had higher infection efficacy; after infection for 72 h,the higher expression of EGFP could be observed under fluorescence microscope; the expression of CXCR4 protein on MSC surface in CXCR4-MSC group significantly increased compared with those in the control group.Meanwhile,over-expression of CXCR4 had no effect on their capacity of proliferation and did not induce apoptosis.Moreover,CXCR4 enhanced the migration of cells in the transwell induced by SDF-1 gradient compared with the EGFP control group.It is concluded that the lentiviral vector can not only infect mouse MSCs efficiently,but also can make CXCR4 express stably in MSC.