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目的探讨LIM矿化蛋白(LIM mineralization protein,LMP)-1和LMP-3双基因共转染骨髓间充质干细胞(bone mesenchymal stem cells,BMSC)的表达情况。方法采用人工设计合成人LMP-1和LMP-3基因片段,分别与质粒pEGFP-N2连接,经酶切、测序鉴定后。分离培养新西兰兔BMSC,用脂质体包裹转染BMSC,按转染情况分为5组:未转染组(A组)、转染空载体组(B组)、转染LMP-1基因组(C组)、转染LMP-3基因组(D组)、LMP-1与LMP-3双基因共转染组(E组)。采用实时聚合酶链反应(real-time polymerase chain reaction,RT-PCR)和蛋白质印迹法检测LMP-1和LMP-3的表达。结果酶切及测序表明真核表达质粒pEGFP-N2-LMP-1和pEGFP-N2-LMP-3构建成功。E组可同时较高水平表达LMP-1和LMP-3分子。对RT-PCR及蛋白质印迹法检测结果行灰度值测量并行统计学分析显示:LMP-1 mRNA及蛋白水平的表达,5组间差异有统计学意义(P<0.05),但E组与C组的差异无统计学意义(P>0.05);LMP-3 mRNA及蛋白水平的表达,5组间差异有统计学意义(P<0.05),且E组与D组差异也有统计学意义(P<0.05)。结论双基因共转染的BMSC能在体外同时表达LMP-1与LMP-3,为基因修复骨缺损带来新思路。
Objective To investigate the expression of LIM mineralization protein (LMP) -1 and LMP-3 double gene in bone marrow mesenchymal stem cells (BMSC). Methods The artificial LMP-1 and LMP-3 gene fragments were synthesized and ligated with plasmid pEGFP-N2, respectively. After digestion and sequencing, they were identified. BMSCs were isolated from New Zealand rabbits and transfected into BMSCs by lipofectamine. The transfected BMSCs were divided into 5 groups according to the transfection conditions: untransfected group (A group), empty vector group (B group), LMP-1 genome (Group C), transfected with LMP-3 genome (group D), LMP-1 and LMP-3 double gene co-transfected group (group E). Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of LMP-1 and LMP-3. Results Enzyme digestion and sequencing showed that the eukaryotic expression plasmids pEGFP-N2-LMP-1 and pEGFP-N2-LMP-3 were successfully constructed. E group could express LMP-1 and LMP-3 molecules at the same time. The results of RT-PCR and Western blotting showed that the expression of LMP-1 mRNA and protein were statistically significant (P <0.05), but the difference between group E and group C (P> 0.05). The expression of LMP-3 mRNA and protein had statistical significance between the 5 groups (P <0.05), and the difference between the E group and the D group was also statistically significant (P <0.05). Conclusion BMSCs co-transfected with double genes can express LMP-1 and LMP-3 simultaneously in vitro, which brings new ideas for gene repair of bone defects.