中文摘要：

目的构建带有绿色荧光蛋白（GFP）标记并携带人骨形态发生蛋白7（hBMP-7）基因的无致病性重组的腺相关病毒（AAV）,以其作为基因载体感染兔骨髓基质干细胞（BMSCs）,体外检测病毒生物学活性及功能。方法从含有hBMP-7基因的pBluescript KS-hBMP7质粒中扩增出hBMP-7片段,构建重组骨架质粒pAAV-hBMP7-IRES-GFP。将此质粒和AAV包装质粒pAAV-RC、辅助质粒pAAV-Helper共转染AAV-293细胞,通过同源重组产生重组腺相关病毒rAAV-hBMP7-IRES-GFP。感染AAV-HT1080测定病毒滴度,应用该重组病毒感染体外培养的兔BMSCs,荧光细胞计数法检测病毒最佳感染复数,Western blot法检测BMP-7基因的体外表达,成骨诱导实验检测BMP-7的体外生物学活性。结果重组腺相关病毒表达质粒经酶切、测序验证正确。重组腺相关病毒感染滴度约为3.6×1011v.p./mL。重组病毒感染兔BMSCs的最佳感染复数为5×104v.p/细胞。Western blot结果显示重组病毒rAAV-hBMP7-IRES-GFP转染组BMSCs细胞内有BMP表达。成骨诱导实验证实rAAV-hBMP7-IRES-GFP表达的BMP体外具有良好的成骨活性。结论成功制备了rAAV-hBMP7-IRES-GFP病毒,实验获得的病毒载体滴度高、感染性好,可以满足骨组织工程的需要,为进一步体内和体外研究基因治疗促进骨愈合提供了有利的工具。

英文摘要：

Objective To construct the non-pathogenic recombinant adeno-associated virus（AAV） simultaneously carrying human bone morphogenetic protein-7 gene and green fluorescent protein（GFP） label,and assess its biological activity and function by infecting rabbit bone marrow mesenchymal stem cells（BMSCs） in vitro.Methods Plasmid pAAV-hBMP7-IRES-GFP was constructed by hBMP-7 segments amplified from plasmid pBluescript KS-hBMP7 carrying hBMP7 gene.The recombinant expression plasmid pAAV-hBMP7-IRES-GFP was co-transfected into AAV-293 cells with pAAV-Helper and pAAV-RC for recombinant AAV replication and package through homologous recombination.In vitro cultured rabbit BMSCs were infected with this recombinant virus.The virus titer was measured by infecting AAV-HT1080.Through infecting rabbit bone marrow mesenchymal stem cells in vitro,the optimal MOI of BMSCs transfected with rAAV was detected by fluorescent cell counting.The BMP-7 gene expression was detected by Western blot assay,and its biological activity was assessed by osteogenic assay in vitro.Results The hBMP-7 gene was successfully amplified and recombinant pAAV-hBMP7-IRES-GFP was verified by double digestion.The purified recombinant virus had a high titer of 3.6×1011v.p./mL.The optimal MOI of BMSCs transfected with rAAV was 5×104 v.p./cell.Western blot showed that expression of the BMP7 genes in rAAV-hBMP7-IRES-GFP group was higher than that in the rAAV-IRES-GFP group.BMP proteins secreted from BMSC transfected with rAAV-hBMP7-IRES-GFP enhanced osteogenesis in vitro.Conclusion