人骨形成蛋白7重组腺病毒载体的构建及其在牙周膜细胞中的表达  

Construction of recombinant adenoviruse vector containing bone human morphogenetic protein-7 and its expression in human periodontal ligament cells

作　　者：秦伟[1] 林正梅[1] 高红飞[2] 张念华[2] 宋智[1] 凌均柴[1] 

机构地区：[1]中山大学口腔医学研究所中山大学光华口腔医学院附属口腔医院牙体牙髓病科,广州510055 [2]中山大学肿瘤防治中心华南肿瘤学国家重点实验室

出　　处：《中华生物医学工程杂志》2010年第4期328-331,共4页Chinese Journal of Biomedical Engineering

基　　金：基金项目：广东省科技计划项目（2007803000002）

摘　　要：目的研究腺病毒介导人骨形成蛋白7（BMP-7）基因转染人牙周膜细胞（PDLC）后目的基因的表达及对人PDLC增殖的影响。方法通过PCR扩增，获得BMP-7基因片段，酶切亚克隆到穿梭质粒pShuttle—CMV-BMP-7上，在293细胞内和质粒pBHGE3同源重组得到腺病毒载体质粒Ad—BMP-7，PCR扩增目的基因鉴定重组腺病毒，扩增纯化后测定滴度。体外培养人PDLC，Ad—BMP-7转染人PDLC，免疫印迹法（Westernblot）检测BMP-7的表达，噻唑蓝（MTY）比色法检测转基因细胞的增殖情况。结果PCR显示BMP-7基因重组腺病毒载体构建成功，病毒滴度为1．785×10^12pfu／ml。Ad—BMP-7转染人PDLC后可检测到BMP－7表达。转染BMP－7对人PDLC增殖没有明显影响。结论构建的BMP．7腺病毒表达载体可有效转染人PDLC。并在体外高效表达。Objective To investigate the expression of adenovirus-transfected human bone morphogenetic protein-7 （BMP-7） in human periodontal ligament cells （PDLCs） and its effect on PDLCs proliferation after transfection. Methods The BMP- 7 fragment was amplified by PCR according to the pCMV-SPORT6-BMP-7, and the fragment was cloned into pShuttle-CMV-BMP-7, then it was homogenously recombined with pBHGE3 in 293 cells to obtain adenovirus expression vector containing BMP-7 （Ad-BMP- 7）. Ad-BMP-7 was identified and the titer of virus was measured after amplification and purification. Ad- BMP-7 transfected human PDLCs in vitro. The BMP-7 protein expression and proliferation of transfected PDLCs were detected by Western blot and MTT assay respectively. Results PCR analysis confirmed that the human BMP-7 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.785x1012 pfu/ml. Expression of BMP-7 protein was detected in PDLCs transfected with Ad- BMP-7. The MTr test showed no significant difference between PDLCs transfected with or without Ad-BMP- 7. Conclusions Adenovirus expression vector containing BMP-7 can transfect human PDLCs successfully with high expresion of BMP-7 in PDLCs in vitro.

关 键 词：转染 骨形态发生蛋白质类 基因疗法 牙周膜细胞 腺病毒载体 

分 类 号：R78[医药卫生—口腔医学]