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

【】　目的　探讨LIM矿化蛋白（LIMmineralizationprotein，LMP）-1和LMP-3双基因共转染骨髓间充质干细胞（bonemesenchymalstemcells，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-timepolymerasechainreaction，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-1mRNA及蛋白水平的表达，5组间差异有统计学意义（P0.05）；LMP-3mRNA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。【Abstract】　Objective　TostudytheexpressionofLIMmineralizationprotein(LMP)-1andLMP-3genesaftercotransfectingthemintobonemesenchymalstemcells(BMSC)ofrabbitinvitro.　Methods　FragmentsofLMP-1geneandLMP-3geneweregainedthroughartificialsynthesis,andwereconstructedrespectivelyintotheplasmidvectorpEGFP-N2.Theinsertedtargetgenesinplasmidwereverifiedbynucleotidesequencingandenzymes.TheplasmidscarryingLMP-1andLMP-3geneswerecotransfectedintochondrocytesbyliposomemethod.Accordingtothetransfectedsituation,theBMSCweredividedinto5groupsthenon-transfectedgroup(GroupA),thegrouptransfectedbyemptyvector(GroupB),thegrouptransfectedbyLMP-1(GroupC),thegrouptransfectedbyLMP-3(GroupD)andthegrouptransfectedbybothLMP-1andLMP-3(GroupE).TheexpressionsofLMP-1andLMP-3weredetectedbyRT-PCRandwesternblotingtechnique.　Results　TheplasmidpEGFP-N2-LMP-1andpEGFP-N2-LMP-1wereobtainedsuccessfullybycloningtechniqueandverifiedbynucleotidesequencingandenzymes.TheLMP-1andLMP-3moleculeswerebothexpressedatahighlevelinGroupE.TheresultsofRT-PCRandwesternblotingweremeasuredwiththegreyvalue.FortheexpressionofLMP-1mRNAandproteinofLMP-1,thedifferencesbetweengroupsA,BandgroupsC,D,Eweresignificant(P＜0.05),whilethedifferencebetweengroupsCandEwasnotsignificant(P＞0.05);FortheexpressionofLMP-3mRNAandproteinofLMP-3,thedifferencesbetweengroupsA,BandgroupsC,D,Eweresignificant(P＜0.05),andthedifferencebetweengroupsDandEwasalsosignificant（P＜0.05).　Conclusion　LMP-1andLMP-3genescanbeexpressedeffectivelyafterbeingcotransfectedintoBMSC,whichprovidesabasisforgenetherapyfortreatingbonedefects.