中国组织工程研究与临床康复2011，Vol.15Issue(19)：3433-3437,5.DOI:10.3969/j.issn.1673-8225.2011.19.003
人LIM矿化蛋白1基因腺病毒重组表达载体构建及在犬骨髓间充质干细胞中的表达
Construction of human LIM mineralization protein-1 gene recombinant adenovirus and its expression in dog bone marrow mesenchymal stem cells
摘要
Abstract
BACKGROUND: Studies shows that the LIM mineralization protein 1 (LMP-1) can stimulate bone formation in vivo and in vitro.OBJECTIVE: To construct the recombinant adenovirus vector containing LMP-1 gene by using the Ad-Easy system, then to detect the gene expression and study the mechanism of bone formation induced by LMP-1 in infected dog bone marrow mesenchymal stem cells (BMSCs).METHODS: The recombinant plasmid pAd-LMP-1 was constructed. The replication-defective recombinant adenovirus Ad-LMP-1was packaged and amplified in the human embryonic kidney 293 cells (HEK293). Dog BMSCs were infected by Ad-LMP-1 in the best value of MOI in vitro. The gene expression of LMP-1and BMP-7 was detected by RT-PCR and Western Blot. Dogs BMSCs were treated with Ad-LMP-1 and recombinant human bone morphogenetic protein 7 (rhBMP-7) to study the mechanism of bone formation of LMP-1 undergoing mineralization (Ca) nodules at 21 days by alizarin red staining.RESULTS AND CONCLUSION : ①Ad-LMP-1 was successfully constructed and obtained titers in the packaging about 1×1011 efu/mL after amplified in HEK293. ②The best efficiency could be obtained at the most appropriate MOI 100 after Ad-LMP-1 infected BMSCs. Infected BMSCs can express mRNA and protein of LMP-1, but not caused expression of BMP-7. ③Dog BMSCs were treated with Ad-LMP-1 and (or) thBMP-7 for 21 days. We found that Ad-LMP-1 with exogenous thBMP-7promote transformation into osteoblasts together. Neither Ad-LMP-1 nor thBMP-7 alone induced any transformation on day 21 .The recombinant adenoviral vector containing hLMP-1 gene was successfully constructed and expressed effectively in dogs BMSCs. It is confirmed that the mechanism of bone formation induced by LMP-1 is synergistic effect with the performance of exogenous rhBMP-7.关键词
重组人骨形态发生蛋白7/骨髓间充质干细胞/LIM矿化蛋白/腺病毒/骨组织工程分类
医药卫生引用本文复制引用
蒲超,倪卫东,高仕长,邱宇..人LIM矿化蛋白1基因腺病毒重组表达载体构建及在犬骨髓间充质干细胞中的表达[J].中国组织工程研究与临床康复,2011,15(19):3433-3437,5.基金项目
重庆市医学科技计划项目. （）
