【摘要】　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 lt;0.05），但E组与C组的差异无统计学意义（P gt;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 lt;0.05），且E组与D组差异也有统计学意义（P lt;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。
【Abstract】　Objective　 To study the expression of LIM mineralization protein (LMP)-1 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.

Citation： TANG Chunhui,TANG Xudong,LAI Tieying. Cotransfection and Expression of LIM mineralization protein-1 and -3 in Mesenchymal Stem Cells in vitro. West China Medical Journal, 2011, 26(9): 1331-1335. doi: Copy