人LIM矿化蛋白1基因腺病毒重组表达载体构建及在犬骨髓间充质干细胞中的表达  

Construction of human LIM mineralization protein-1 gene recombinant adenovirus and its expression in dog bone marrow mesenchymal stem cells

作　　者：蒲超[1] 倪卫东[1] 高仕长[1] 邱宇[1] 

机构地区：[1]重庆医科大学附属第一医院骨科,重庆市神经病学重点实验室,重庆市400016

出　　处：《中国组织工程研究与临床康复》2011年第19期3433-3437,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：重庆市医学科技计划项目~~

摘　　要：背景:研究表明LIM矿化蛋白1在体内和体外都可促使骨发生。目的:应用Adeasy-1腺病毒系统构建含人LIM矿化蛋白1基因的腺病毒重组体,并感染犬骨髓间充质干细胞,检测其体外表达及诱导成骨作用。方法:构建重组腺病毒质粒pAd-LMP-1,经人胚胎肾293细胞包装、扩增后得到复制缺陷重组腺病毒Ad-LMP-1,以最佳感染复数值体外感染犬骨髓间充质干细胞,行RT-PCR及Western Blot检测LIM矿化蛋白1、骨形态发生蛋白7基因的表达。重组Ad-LMP-1和(或)外源性重组人骨形态发生蛋白7处理犬骨髓间充质干细胞,21d后行矿化(钙)结节茜素红染色分析LIM矿化蛋白1基因的诱导成骨作用。结果与结论:①将Ad-LMP-1以感染复数值100感染犬骨髓间充质干细胞可获得最佳感染效率,感染后骨髓间充质干细胞能在基因和蛋白水平表达LIM矿化蛋白1,未引起骨形态发生蛋白7基因的表达。②Ad-LMP-1或重组人骨形态发生蛋白7均不能单独促使骨髓间充质干细胞向成骨细胞转化,两者联合可促使骨髓间充质干细胞向成骨细胞转化。③实验成功构建重组腺病毒载体Ad-LMP-1,并实现其在犬骨髓间充质干细胞中表达,证实了LIM矿化蛋白1在诱导成骨作用中表现为与外源性重组人骨形态发生蛋白7的协同效应。BACKGROUND： Studies shows that the LIM mineralization protein 1 （LMP-1） can stimulate bone formation in vivo and in vitro.OBJECTIVE： To construct the recombinant adenovirus vector containing LMP-1 gene by using the Ad-Easy system,then to detect the gene expression and study the mechanism of bone formation induced by LMP-1 in infected dog bone marrow mesenchymal stem cells （BMSCs）.METHODS： The recombinant plasmid pAd-LMP-1 was constructed.The replication-defective recombinant adenovirus Ad-LMP-1 was packaged and amplified in the human embryonic kidney 293 cells （HEK293）.Dog BMSCs were infected by Ad-LMP-1 in the best value of MOI in vitro.The gene expression of LMP-1and BMP-7 was detected by RT-PCR and Western Blot.Dogs BMSCs were treated with Ad-LMP-1 and recombinant human bone morphogenetic protein 7 （rhBMP-7） to study the mechanism of bone formation of LMP-1 undergoing mineralization （Ca） nodules at 21 days by alizarin red staining.RESULTS AND CONCLUSION： ①Ad-LMP-1 was successfully constructed and obtained titers in the packaging about 1×1011 efu/mL after amplified in HEK293.②The best efficiency could be obtained at the most appropriate MOI 100 after Ad-LMP-1 infected BMSCs.Infected BMSCs can express mRNA and protein of LMP-1,but not caused expression of BMP-7.③ Dog BMSCs were treated with Ad-LMP-1 and （or） rhBMP-7 for 21 days.We found that Ad-LMP-1 with exogenous rhBMP-7 promote transformation into osteoblasts together.Neither Ad-LMP-1 nor rhBMP-7 alone induced any transformation on day 21.The recombinant adenoviral vector containing hLMP-1 gene was successfully constructed and expressed effectively in dogs BMSCs.It is confirmed that the mechanism of bone formation induced by LMP-1 is synergistic effect with the performance of exogenous rhBMP-7.

关 键 词：重组人骨形态发生蛋白7 骨髓间充质干细胞 LIM矿化蛋白 腺病毒 骨组织工程 

分 类 号：R394.2[医药卫生—医学遗传学]