含hIGF-1基因腺病毒的构建及在兔骨髓间充质干细胞的表达

目的用Adeasy系统构建荧光蛋白基因标记的腺病毒载体Ad-hIGF-1,然后转染兔骨髓间充质干细胞并检测其表达.方法根据GenBank公布的hIGF-1序列设计PCR引物,从质粒pcDNA3.1-hIGF-1中扩增出截短型hIGF-1,亚克隆至pMD-T载体,鉴定后酶切出目的基因插入pAdtrack-CMV中构建成腺病毒穿梭质粒pAdtrack-hIGF-1,经Pme Ⅰ线性化后采用电穿孔法转化至已包含有腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌中,挑选同源重组质粒线性化后转染HEK293细胞收获腺病毒,然后转染靶细胞兔骨髓间充质干细胞,通过绿色荧光蛋白表达情况以及流式细胞仪、免疫细胞化学等方法检测hIGF-1的表达.结果成功构建了绿色荧光蛋白基因标记的腺病毒Ad-hIGF-1,并在兔骨髓间充质干细胞得以高效表达.结论Adeasy腺病毒系统是基因转染的高效载体系统.腺病毒Ad-hIGF-1转染的骨髓间充质干细胞可作为基因修饰的软骨组织工程种子细胞.

Construction of adenovirus vectors contained human insulin-like growth factor-1 gene and expression in rabbit mesenchymal stem cells

Objective To construct adenovirus vectors containing truncated human insulin-like growth factor-1 gene and green fluorescent protein gene,transfect them into mesenchymal stem cells and detect the expression of hIGF-1 gene.Methods After the amplification of truncated human insulin-like growth factor-1(hIGF-1) gene from pcDNA3.1-hIGF-1 by polymerase chain reaction(PCR),the gene fragment was inserted to shuttle plasmid pAdtrack-CMV for homologous recombination with backbone plasmid pAd-easy-1 in bacteria BJ5183,followed by transfection of restructured adenovirus plasmid to HEK 293 cells to get adenovirus Ad-hIGF-1.The high titer adenovirus supernatant was gotten by repeated infection of HEK 293 cells by adenovirus harvested after confirmation of the adenovirus structure.As target cells,mesenchymal stem cells were infected to determine the expression of hIGF-1 gene by inspection of fluorescence,flow cytometry,immuocytochemistry and Western blotting.Results The adenovirus vector was constructed successfully and high efficient expression of hIGF-1 was detected in mesenchymal stem cells.Conclusion Adenovirus vector is effective for gene expression and transfection into MSCs.MSCs transfected with Ad-hIGF-1 may be another option to gene-motified seed cells for articular cartilage tissue engineering.