融合基因骨形态发生蛋白-4／7重组腺相关病毒载体转染对骨髓基质干细胞成骨活性的作用

Osteogenesis of recombinant adeno-associated-BMP-4/7 fusion gene

目的应用高效细菌内同源重组系统pAd-Easy制备融合基因骨形态发生蛋白-4／7（BMP-4／7）基因的重组腺相关病毒（AAV），转染兔骨髓基质干细胞（BMSCs），检测BMP-4／7的成骨活性。方法采用RT-PCR一步法和基因重组法，从胎盘组织中克隆BMP．4和BMP-7成熟肽cDNA，获取BMP-4／7融合基因，克隆到pGEM质粒中；从pGEM-BMP-4／7质粒中切取BMP-4／7融合基因克隆到穿梭载体，在大肠杆菌内重组，在293细胞中构建AAV—BMP-4／7重组腺相关病毒载体；采用SDS—PAGE电泳检测BM-4／7融合基因蛋白在大肠杆菌的表达；采用ELISA法检测融合基因BMP-4／7在BMSCs中的表达。AAV-BMP4／7转染兔BMSCs细胞7、14d后，行碱性磷酸酶及骨钙素含量测定，观察成骨活性，以非转染组作为对照。结果成功构建高滴度的携带BMP4／7融合基因的重组腺相关病毒AAV-BMP4／7。AAV-BMP-4／7重组质粒载体转化大肠杆菌进行诱导表达，SDS—PAGE电泳可见29～30KD蛋白带，大多数蛋白存在于包涵体内。ELISA检测显示经AAV转染的BMSCs中BMP-4／7蛋白有表达，随着转染MOI值的增加，BMP4／7蛋白的表达增高（P〈0．01）。AAV．BMP-4／7转染细胞后，碱性磷酸酶及骨钙素含量均明显增高，并与转染后诱导培养的时间呈正相关，与对照组比较差异有统计学意义（P〈0．01）。结论融合基因BMP4／7可提高BMPs表达水平，有成骨活性，通过AAV可稳定表达。

Objective To study the osteogenesis of BMP-4/7 in rabbit bone marrow stem cells （BMSCs） transfected by the recombinant adeno-associated virus （AAV） with morphogenefic protein-4/7 of fusion gene. Methods The mature peptides of BMP-4 and BMP-7 were gained by One-Step RT-PCR from the human placenta, and the BMP-4/7 fusion gene was gained by gene recombination techniques and then transferred to pGEM plasmid. The BMP-4/7 fusion gene was cut down from the pGEM-BMP-4/7 and the recombination was successfully completed in colibacillus. The recombinant adeno-associated virus was pro- duced in 293 cells. The expression of BMP-4/7 fusion gene was detected in the coliform. The expression of fusion gene BMP-4/7 protein was determined by ELISA method. The ossification of cells was evaluated by investigating the contents of ALP and OC after transfection for 7 and 14 days. Results The recombinant adeno-associated virus with BMP-4/7 fusion gene was successfully constructed. The 29 ~ 30 KD protein tap was shown by SDS-PAGE electrophoresis after transfection of AAV-BMP-4/7 in coliform and the expression mostly existed in bodies. The exploration of the protein of fusion gene BMP-4/7 in BMSCs transferred by AAV showed positive correlation with various MOI values. There were significantly more ALP and OC in AAV-BMP-4/7 transfection groups than in AAV-EGFP groups （ P 〈 0.01）. Conclusion By transfect- ing rabbit BMSCs cultured in vitro, the fusion gene BMP-4/7 can enhance the expression of BMPs and have significant osteogenesis through AAV.