南京师大学报（自然科学版）2011，Vol.34Issue(4)：83-88,6.
重组GGPPS腺病毒载体的构建及其调节肝星状细胞活化的研究
Construction of Recombinant Adenovirus Vector Containing Human ggpps Gene and Its Effect on Activation of HSCs
摘要
Abstract
Objective: To construct the recombinant adenovirus vector containing human ggpps gene and study its effect on activation of HSCs. Methods: ggpps gene was obtained by PCR amplification and subcloned into adenovirus shuttle plas-mid of pAdTrack-CMV to form the shuttle vector of pAdTrack-CMV-GGPPS. After sequencing, it was linearized with Pmel and co-transfected into E. Coli BJ5183 cells with adenovirus genomtc plasmid of pAdEasy-1 to achieve homologous recombination. The DNA of identified recombinant plasmid was digested with Pacl and then transfected into QBI-293A cells to package adenovirus carrying GGPPS. Generation of recombinant adenovirus was monitored by the expression ofgreen fluorescent protein. The titer of viral stock was determined by TCID50 assay. The expression of GGPPS in human hepatocyte after Ad-GGPPS infection was detected by Western Blotting and Real-time PCR. Activation of HSCs and synthesis of ECM were checked by Real-time PCR after Ad-GGPPS infection in LX -2. Results; The Ad-GGPPS was successfully constructed and the infective titer is 2. 5 × 109 PFU/mL. The expression of GGPPS in human hepatocyte infected by Ad-GGPPS was efficient. Over-expressed GGPPS in LX - 2 cell can promote the expression of ot-SMA, a reliable marker of stellate cell activation, and Fibronectin, the composition of ECM is significantly increased too. Conclusion; GGPPS promotes the activation of HSCs.关键词
GGPPS/重组腺病毒/肝星状细胞Key words
GGPPS/recombinant-adenovirus/HSCs分类
生物科学引用本文复制引用
来珊珊,陈卫波,徐康,丁尧,沈宁,潘飞燕,李朝军,薛斌..重组GGPPS腺病毒载体的构建及其调节肝星状细胞活化的研究[J].南京师大学报（自然科学版）,2011,34(4):83-88,6.基金项目
国家自然科学基金(30700394)、江苏省分子医学生物技术重点实验室开放基金(NMB09KF03)、中央高校基本科研业务费专项资金(1116021402). （30700394）
