重组转化生长因子β1腺相关病毒感染骨髓基质干细胞的活性

Effect of recombinant adeno-associated virus transforming growth factor-beta 1 on the infection activity of bone marrow mesenchymal stem cells

背景:目前常用基因载体具有一定缺陷性,无法直接在体内应用。应用腺相关病毒作为转化生长因子β1载体促进软骨修复的研究报道较少。目的:构建重组转化生长因子β1腺相关病毒,测定病毒滴度,并测定重组病毒对骨髓基质干细胞的感染活性。方法:PCR方法扩增转化生长因子β1基因,构建重组骨架质粒pAAV-转化生长因子β1-绿色荧光蛋白,与包装质粒pAAV-RC、辅助质粒pAAV-Helper共转染AAV-293细胞包装重组腺相关病毒rAAV-转化生长因子β1-绿色荧光蛋白。应用该重组病毒感染AAV-HT1080细胞测定病毒滴度并鉴定。并感染体外培养的兔骨髓基质干细胞,检测重组病毒感染骨髓基质干细胞的效率及活性。结果与结论:转化生长因子β1扩增产物测序结果正确,重组骨架质粒pAAV-转化生长因子β1-绿色荧光蛋白双酶切后可见位于1.3Kb附近转化生长因子β1条带。收获病毒滴度5.2×1011v.g/mL。鉴定重组病毒rAAV-转化生长因子β1-绿色荧光蛋白包装成功。重组病毒感染骨髓基质干细胞后可于荧光显微镜下观测到绿色荧光蛋白的表达,感染效率达42%。结果证实,成功构建重组腺相关病毒rAAV-转化生长因子β1-绿色荧光蛋白,能够高效感染兔骨髓基质干细胞。

BACKGROUND：The commonly used genetic carrier has a certain shortcoming,such as cannot be used directly in vivo,and using recombinant adeno-associated virus（AAV） as a vector carrying transforming growth factor-β1（TGF-β1） to improve cartilage renovation has not been reported.OBJECTIVE：To construct recombinant adeno-associated virus expressing TGF-β1,and assess the virus titer,and to identify the effect of recombinant adeno-associated virus on the infection activity of bone marrow mesenchymal stem cells.METHODS：The TGF-β1 gene was amplified by PCR and directly cloned into the plasmid pAAV-TGF-β1-hrgreen fluorescent protein.The recombinant pAAV-TGF-β1-GFP was co-transfected into AAV-293 cells with pAAV-RC and pAAV-Helper for AAV-2 replication and packaging through homologous recombination.The virus titer was measured through infecting AAV-HT1080,and the recombinant virus was verified by PCR of the exogenous interest gene.Through infecting rabbit bone marrow mesenchymal stem cells in vitro,the infection efficiency and activity was detected.RESULTS AND CONCLUSION：The TGF-β1 gene was successfully amplified and recombinant pAAV-TGF-β1IRES-GFP was verified by double digestion and there was a 1.3 Kb target strap in accordance with TGF-β1.The system provided a high packing ratio and the purified recombinant virus has a high titer of 5.2×10 11 v.g/mL.The recombinant virus was confirmed by exogenous human VEGF165 gene PCR.The rAAV-TGF-β1-GFP could infect rabbit bone marrow mesenchymal stem cells at a ratio of 42% and express green fluorescence under fluorescent microscope.The rAAV-TGF-β1-GFP was successfully constructed with a satisfying biological ability of infecting bone marrow mesenchymal stem cells,which may offer the basement of gene therapy for cartilage repairation.