骨形态发生蛋白4和骨形态发生蛋白4/7基因转染对骨髓基质干细胞成骨活性差异的比较

背景：有文献报道骨形态发生蛋白（bone morphogenetic proteins,BMPs）异源二聚体比同源二聚体的活性高,目前国内外尚无BMP-4/7异源二聚体与BMP-4同源二聚体间诱导成骨活性比较的报道。目的：观察不同基因BMP-4、BMP-4/7对骨髓基质干细胞（bone marrow mesenchymal stem cells,BMSCs）成骨活性的差异,探讨融合基因BMP-4/7的生物学活性。方法：经脂质体转染分离培养的兔BMSCs,G418筛选出稳定表达株,利用RT-PCR法证明目的基因在BMSCs中的表达情况;以BMP-4和BMP4/7融合基因修饰的AAV载体,转染兔BMSCs,MTT法检测不同基因转染细胞的增殖能力;以BMP-4单基因和BMP-4/7融合基因修饰的AAV载体转染兔BMSCs7d后,测定两组细胞内碱性磷酸酶及骨钙素水平,观察成骨活性的变化。结果与结论：实验成功构建高滴度的分别携带BMP-4单基因、BMP-4/7融合基因的重组腺相关病毒载体AAV-BMP-4、AAV-BMP-4/7;RT-PCR结果证明目的基因能够在BMSCs中得到稳定表达。AAV-BMP-4及AAV-BMP-4/7载体转染效率分别为68.20%、72.18%;转染BMSCs后,细胞增殖能力均明显提高,BMP-4/7融合基因的细胞增殖能力活性高于BMP-4单基因。以BMP-4和BMP-4/7融合基因修饰的AAV转染细胞7d后,细胞内碱性磷酸酶活性明显上调,骨钙素水平升高,成骨活性均增强;两种基因比较,BMP-4/7融合基因成骨活性强于BMP-4单基因（P〈0.01）。提示BMP-4、BMP-4/7修饰的AAV载体转染效率高,对BMSCs均有成骨活性,其中BMP-4/7融合基因成骨活性强于BMP-4单基因。

Osteogenous differentiation of bone marrow mesenchymal stem cells transfected with bone morphogenetic protein 4 and bone morphogenetic proteins 4/7

BACKGROUND：Heterodimer of bone morphogenetic protein（BMP） has higher activity than BMP homodimer.Currently,there are no reports addressing comparison between BMP-4/7 heterodimer and BMP4 homodimer to induce osteogenic activity.OBJECTIVE：To compare the difference in osteogenous differentiation of bone marrow mesenchymal stem cells（BMSCs） transfected with BMP4 and BMP-4/7,and to investigate biology activity of BMP-4/7 fusion gene.METHODS：The mature peptide of BMP-4 and BMP-7 were gained by one-step RT-PCR from the human palcenta,and the BMP-4/7 fusion gene was gained through gene recombinant techniques and then transferred to pGEM plasmid.The BMP4 gene and BMP-4/7 fusion gene was cut down from the pGEM plasmid and the recombination was successfully completed in colibacillus and recombinant adeno-assosiated was produced in 293 cells.The BMSCs cell lines which could express each of the target protein w ere selected out by G418 and RT-PCR were used to certificate the expression of exogenous gene in BMSCs.Rabbit BMSCs were transfected with the recombinant adeno-assosiated virus vectors carrying BMP4 and BMP-4/7 fusion gene and cell reproductive activity detected by MTT method.The ossification of cells was evaluated by investigating the shape change of the cell ability of alkaline phosphatase and osteocalcin after transfection for 7 days.RESULTS AND CONCLUSION：We successfully constructed the recombinant adeno-assosiated virus with BMP-4 gene and BMP-4/7 fusion gene;RT-PCR results certificated the confirm expression of the exogenous gene in BMSCs.The transfection efficiency of AAV-BMP4 and AV-BMP-4/7 were 68.20% and 72.18%.MTT showed that almost all of the BMSCs which had been transfected with exogenous gene had more strong proliferative potential than the untransfected group.The cell multiplication of BMP-4 fusion gene was stronger than BMP4.There was significantly higher alkaline phosphatase and osteocalcin in AAV-BMP4 and AAV-BMP-4/7 transfection groups than untransfection group.BMP-4/7 group had a stronger osteogenous differentiation than the BMP4 group（P〈0.01）.The BMP4 and AAV-BMP-4/7 fusion gene can transfect rabbit BMSCs cultured in vitro at high transfection rate,and have significant ossification of activity.The BMP-4/7 fusion gene has a stronger osteogenous differentiation than the BMP4 single gene.