人LMP-1基因腺病毒重组体构建及其在骨髓间充质干细胞的表达

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

背景：LIM矿化蛋白1（LIM Mineralization protein，LMP—1）是一种细胞内非分泌蛋白，主要在骨的钙化阶段起作用。目前发现LMP—1可以促进骨形态发生蛋白2和转化生长因子β1等多种蛋白质合成增加，提示其可能通过招募众多的成骨因子共同参与成骨细胞的分化。目的：应用AdEasy腺病毒载体系统构建人LMP-1基因的腺病毒重组体，并检测感染病毒的兔骨髓间充质干细胞LMP-1的表达。设计、时间及地点：开放性实验，于2006—03／2007—02在解放军第三军医大学西南医院中心实验室完成。材料：纯种新西兰大白兔3只，用于分离制备骨髓间充质干细胞。携带人LMP-1基因的pIRES2-EGFP-LMP-1质粒由重庆医科大学附二院骨科保存；AdEasy腺病毒由美国Tong-ChuanHe博士惠赠：人胚肾293细胞由重庆医科大学检验系王宏兵硕士惠赠。方法：以pIRES2-EGFP—LMP—1质粒为模板进行PCR扩增，在下游引物中加入特定序列，使扩增出的LMP—1基因带有编码6个组氨酸（即His标签）的碱基序列，将其作为目的基因，TA克隆后测序。双酶切后插入至腺病毒穿梭质粒pAdtrack—CMV内，经PmeⅠ线性化，在BJ5183菌内与骨架质粒pAdeasy—1的同源重组，构建重组腺病毒质粒pAd-LMP—1。通过脂质体介导在HEK293细胞内包装出复制缺陷的重组腺病毒Ad—LMP—1，扩增纯化后测定滴度，以最佳MOI值体外感染兔骨髓间充质干细胞，行RT—PCR及Western Blot法检测LMP—1表达。主要观察指标：重组腺病毒质粒pAd—LMP-1的鉴定，检测其滴度及感染效率。RT—PCR、Western Blot法检测感染细胞LMP—1 mRNA及蛋白的表达。结果：①测序鉴定携带His标签的LMP—1基因的重组质粒pMD18-T—LMP—1构建成功，包装扩增后获得滴度约3．5×10^9efu／mL的腺病毒重组体Ad—LMP—1。以150的MOI值感染兔骨髓间充质干细胞可以获得最佳感染效率，感染3d后达50％-70％。②病毒感染后的骨髓间充质干细表达LMP—1 mRNA及蛋白。结论：成功构建携带His标签的人LMP—1基因的腺病毒重组体，证实感染重组病毒的骨髓间充质干细胞可有效表达LMP—1。

BACKGROUND： LIM Mineralization protein 1 （LMP-1）, an intracellular non-secretory protein, plays roles in bone calcification. Presently, it is found that LMP-1 can promote an increase in bone morphogenetic protein 2 and transforming growth factor β1. This indicates that LMP- 1 may recruit a mass of ossified factors to participate in the differentiation of osteoblasts. OBJECTIVE： To construct human LMP-1 gene adenovirus recombinant with AdEasy adenovirus vector system, and to detect LMP-1 expression in infected rabbit bone marrow mesenchymal stem cells （BMSCs）. DESIGN, TIME AND SETTING： An opening experiment was performed at the Central Laboratory of South West Hospital, Third Military Medical University of Chinese PLA from March 2006 to February 2007. MATERIALS： Three New Zealand rabbits were used to isolate bone marrow mesenchymal stem cells. Plasmid pIRES2-EGFP-LMP-1 carrying human LMP-1 gene was kept in Department of Orthopedics, Second Hospital Affiliated to Chongqing Medical University. AdEasy was presented by Dr. Tong-Chuan He from USA. Human embryo kidney 293 cells were gifted by Wang from Department of Clinical Laboratory of Chongqing Medical University. METHODS： LMP-1 gene with a sequence encoding His-tag was amplified by using pIRES2-EGFP-LMP-1 plasmid as a template for polymerase chain reaction （PCR） with a specially designed downstream primer. The target gene was cloned to the pMD 18-T vector for sequencing. Once verified, the gene was cut out by double endonucleases, connected to the shuttle vector pAdTrack-CMV. The newly constructed vector was linearized by Pine Ⅰ following efficient homologous recombination with the backbone vector pAdEasy-1 in B J5183. The correct recombinant pAd-LMP-1 was linearized with Pac Ⅰ and transfected to HEK293 cell by means of mediated Lipofectamine. The titer of virus was measured after amplification and purification. The mRNA and protein expression of LMP-1 was detected in BMSCs, which were infected with Ad-LMP-1 at the most appropriate MOI, were detected by reverse transcription （RT）-PCR and Western blot, respectively. MAIN OUTCOME MEASURES： Plasmid pAd-LMP-1 identification, its titre and efficiency of infection, mRNA and protein expression of LMP- 1 were detected by RT-PCR and Western blot, respectively. RESULTS： The recombinant plasmid pMDI8-T-LMP-1 carrying LMP-1 gene with His-tag was successfully constructed. After packaging and amplification of the recombinant adenovirus, the 3.5×10^9 efu/ml titer of Ad-LMP-1 was obtained by CsCl gradient purification. The optimal efficiency infection was 50%-70%, which was get after Ad-LMP-1 infected BMSCs for 3 days at the most appropriate MOI 150. The mRNA and protein expression of LMP-1 in infected BMSCs had been proved. CONCLUSION： The recombinant adenovirus containing human LMP-1 gene with His-tag is successfully constructed. The BMSCs infected with recombinant adenovirus Ad-LMP- 1 can effectively express LMP- I.