LMP-1基因慢病毒重组体对大鼠骨髓间充质干细胞的增殖影响及其表达

目的：探讨LMP-1重组慢病毒载体体外转染大鼠骨髓间充质干细胞（BMSC）的方法，并检测LMp-1基因对BMSC增殖能力的影响及表达。方法：选取清洁级4周龄SD大鼠6只（雌雄不限），无菌条件下提取骨髓间充质干细胞并培养至第3代，设立空白对照组（未经特殊处理，只有第3代骨髓间充质干细胞）、慢病毒载体转染组（在未经特殊处理的第3代骨髓间充质干细胞中加入PGC-FU-GFP及转染试剂Polybrene）和重组基因转染组（在未经特殊处理的第3代骨髓间充质干细胞中加入PGC-FU-LMP．1-GFP及转染试剂Polybrene）进行转染。转染48h后，通过免疫荧光显微镜观察荧光表达，流式细胞仪检测慢病毒的转染效率，采用MTY法评价慢病毒转染对BMSC增殖的影响；WesternBlot检测转染后基因的表达情况。结果：①成功培养出第3代SD大鼠BMSC，以100的感染复数（MOI）转染，48h后免疫荧光显微镜下可见大量绿色荧光蛋白表达，转染效率达67％；②不同时间点慢病毒载体转染组和重组基因转染组，与空白对照组细胞增殖比较差异无统计学意义；③WestemBlot检测示重组基因转染组72kDa处有条特征带，其大小与LMP－l融合蛋白（～50kDa＋28kDa=78kDa）基本吻合。结论：经LMp-1基因慢病毒重组体转染大鼠骨髓间充质干细胞对其增殖活力没有影响并可有效表达LMP－1。

Effects of recombinant gene lentivirus containing LIM mineralization protein-1 on proliferation effect and expression of bone marrow mesenchymal stem cells in rats

To explore method of recombinant gene lentivirus containing LIM mineralization protein-1 （LMP-1） in transfecting bone marrow mesenchymal stem cells （BMSC）, and to observe the effect of gene LMP-1 on proliferation effect and expression of BMSC. Methods： Six clean SD rats aged 4 weeks were selected, bone marrow mesenchymal stem cells were extracted under sterile conditions and cultured to the third generation, then divided into three groups：control group （the third generation of BMSC ） ,lentiviral vector transfection group （PGC-FU-GFP and Polybrene were injected into the third generation of BMSC ） and recombinant gene transfection group （ PGC-FU-LMP-1-GFP and Polybrene transfection were injected into the third generation of BMSC ）. After 48 hours＇ transfection, fluorescent expression were detected under immunofluorescence mi croscopy ; lentiviral transfection efficiency were detected by flow cytometry ; effect of lentiviral transfection on BMSC were eval- uated by MTF; gene expression of transfected cells were determined by Western Blot. Results： ①The third generation of BM- SC was cultured successfully, and transfected with MOI： 100. After 48 hours, green fluorescent expression were detected and transfection efficiency was 67% under immuno-fluorescence microscopy; ②Compared to control group ,there were no statisti cal differences between control group and other two groups; ③Western blot teast showed that 72KDa specific band was ob- served in recombinant gene transfection group and its sizewas similar to LMP-1 fusion protein（50 kDa＋28 kDa=78 kDa）. Con clusion：There is no effect of recombinant gene lentivirus containing LIM on BMSC, and can effectively influence the expres sion of LMP-1.