中国组织工程研究与临床康复2008，Vol.12Issue(34)：6795-6798,4.
构建含人胰岛素样生长因子基因腺病毒载体及在兔骨髓间充质干细胞的表达
Construction of adenovirus vectors containing human insulin-like growth factor-1 gene and its expression in rabbit mesenchymal stem cells
丁幸坡 1金先庆 2罗小辑 2邱林 2刘伟2
作者信息
- 1. 河南省正骨研究院小儿骨科,河南省洛阳市,471000
- 2. 重庆医科大学儿科研究所,重庆市,400014
- 折叠
摘要
Abstract
BACKGROUND: Compared to those original viruses systems, adeasy adenovirus, a recombinant adenoviral system widely used in recent years, based on viruses with a deletion of both El and E3, reported by T.C. He in 1998, is an improved one. It simplifies the generation and production of such viruses and expedite the process of generating and testing recombinant adenoviruses using homologous recombination in bacteria rather than in eukaryotic cells. Moreover, it can be conveniently followed with the aid of green fluorescent protein encoded by EGFP gene incorporated into the viral backbone.OBJECTIVE: To construct the recombinant adenovirus and to evaluate them by transfect them to mesenchymal stem cells (MSCs)and detect the expression of target gene hlGF-I at gene and protein levels.DESIGN: Repetitive measurement wail.SETTING: The Institute of Pediatric Research, Chongqing University of Medical Science.METHODS: The study was performed at the Institute of Pediatric Research, Chongqing University of Medical Science from November 2004 to March 2005. After the amplification of truncated hlGF-1 gene from pcDNA3.l-hlGF-I by polymerase chain reaction (PCR), the gene fragment was inserted into the shuttle plasmid pAdtrack-CMV for homologous recombination with backbone plasmid pAdeasy-I in bacteria BJ5183 to get adenovirus.Ad-hlGF-1. The high titer adenovirus supernatant was obtained by repeated transducing of HEK 293 cells by adenovirus harvested after confirmation of the adenovirus structure. As target cells,MSCs were infected with adenovirus earned target gene, hIGF-1, to determine the expression of hlGF-1 gene.MAIN OUTCOME MEASURES: ① The construction of recombinant adenovirus vector;② the expression of target gene hIGF-1 in HEK 293 cells and the proper multiplicity of infection (MOI); ③ hIGF-1 gene expression in MSCs.RESULTS: The adenovirus vector based on adeasy system was constructed successfully and the Ad-hlGF transducing was successfully or efficiently expressed in MSCs cells. The ideal expression of harvested recombinant adenovirus in MSCs was detected by fluorescence microscope, RT-PCR, immunocytochemistry, and Western Blot.CONCLUSION: Adenovirus vector is an effective vector tools for gene expression and wansfection of MSCs. MSCs transduced with Ad-hIGF-1 maybe another option to gene-modified seed cells for articular cartilage tissue engineering.关键词
人胰岛素样生长因子-1/腺病毒载体/骨髓间充质干细胞分类
医药卫生引用本文复制引用
丁幸坡,金先庆,罗小辑,邱林,刘伟..构建含人胰岛素样生长因子基因腺病毒载体及在兔骨髓间充质干细胞的表达[J].中国组织工程研究与临床康复,2008,12(34):6795-6798,4.
