重组BMP7腺病毒的构建及转染骨髓基质干细胞后的表达  

Construction of BMP7 Adenovirus and Expression of BMP7 in Transfected BMSCs

作　　者：祝联[1] 刘伟[1] 刘德莉[1] 吴娟娟[1] 王丹茹[1] 崔磊[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学附属第九人民医院整形外科上海市组织工程研究中心,上海200011

出　　处：《中国生物医学工程学报》2005年第2期245-253,共9页Chinese Journal of Biomedical Engineering

基　　金：国家"973"组织工程基本科学问题资助项目 (G19990 5 43 0 4);国家"863"组织工程化骨构建技术与产品开发资助项目 (2 0 0 2AA2 0 5 11)。

摘　　要：根据Clonetech公司的腺病毒载体构建流程 ,原核表达载体pBlue- BMP7经一系列特定的限制性内切酶酶切 ,重组成BMP7的腺病毒载体 ,并转染 2 93细胞进行大量扩增、滴度测定。重组Adeno- BMP7经限制性内切酶PI SceⅠ和Ⅰ CeuⅠ双酶切获得BMP7基因片段 ,PCR扩增得到特异的扩增片段 ,表明重组的Adeno-BMP7载体构建成功 ,其中携带有目的基因BMP7cDNA片段。Adeno- Lac- Z的体外转染骨髓基质干细胞 (BMSCs) ,染色表明转染率可达90 %以上 ;RT- PCR、免疫组化证明Adeno -BMP7转染BMSCs后BMP7在基因和蛋白水平上均有表达 ;转染Adeno- BMP7后的BMSCs细胞悬液裸鼠肌肉内注射后 ,x光片和组织学观察都表明BMP7转染可明显促进新骨的形成 ,与没转染Adeno -BMP7的BMSCs有显著差异。BMP7 adenovirus was constructed by gene cloning(Clontech),BMP7 cDNA was subcloned into the adenovirus genome after a series of restrictive enzyme treatment, large scale viral preparation was preformed by using HEK 293 cells and viral titer was determined. BMP7 could be cut by the restrictive enzyme of PI SceⅠ and Ⅰ CeuⅠ from Adenovirus BMP7 constructed,BMP7 mRNA expression was observed in transfected BMSCs by PCR,so it come true we constructed real Adenovirus BMP7 with BMP7 cDNA. The infection frequency population detected by analyzing β galactosidase expression in Adeno Lac Z infected BMSCs was up 90%,BMP7 expression at BMP7 mRNA and protein levels in Adeno BMP7 infected BMSCs was manifested by RT PCR and immunocytochemistry,with no expression in control. In vivo, new bone of formiation in nude muscle injection with cells was stimulated with Adeno BMP7 infected by X ray and histology with difference from the normal BMSCs.

关 键 词：重组腺病毒 骨髓基质干细胞 骨形成蛋白7 

分 类 号：R318.04[医药卫生—生物医学工程]