LIM矿化蛋白-1与LIM矿化蛋白-3共转染骨髓间充质干细胞的基因表达

Cotransfection and Expression of LIM mineralization protein-1 and-3 in Mesenchymal Stem Cells in vitro

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 study the expression of LIM mineralization protein （LMP）-I and LMP-3 genes after cotransfecting them into bone mesenchymal stem cells （BMSC） of rabbit in vitro. Methods Fragments of LMP-1 gene and LMP-3 gene were gained through artificial synthesis, and were constructed respectively into the plasmid vector pEGFP-N2. The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes. The plasmids carrying LMP-1 and LMP-3 genes were cotransfected into chondrocytes by liposome method. According to the transfected situation, the BMSC were divided into 5 groups： the non-transfected group （Group A）, the group transfected by empty vector （Group B）, the group transfected by LMP-1 （Group C）, the group transfected by LMP- 3 （Group D） and the group transfected by both LMP-1 and LMP-3 （Group E）. The expressions of LMP-1 and LMP- 3 were detected by RT-PCR and western bloting technique. Results The plasmid pEGFP-N2-LMP-1 and pEGFP-N2- LMP-1 were obtained successfully by cloning technique and verified by nucleotide sequencing and enzymes. The LMP- 1 and LMP-3 molecules were both expressed at a high level in Group E. The results of RT-PCR and western bloting were measured with the grey value. For the expression of LMP-1 mRNA and protein of LMP-1, the differences between groups A, B and groups C, D, E were significant （P〈0. 05）, while the difference between groups C and E was not significant （P〉0. 05） For the expression of LMP-3 mRNA and protein of LMP-3, the differences between groups A, B and groups C, D, E were significant （P〈0. 05）, and the difference between groups D and E was also significant（P〈0. 05）. Conclusion LMP-1 and LMP-3 genes can be expressed effectively after being cotransfected into BMSC, which provides a basis for gene therapy for treating bone defects.