|Table of Contents|

Construction of Recombinant Adenovirus Vector Containing Human ggpps Gene and Its Effect on Activation of HSCs(PDF)

《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

Issue:
2011年04期
Page:
83-88
Research Field:
生命科学
Publishing date:

Info

Title:
Construction of Recombinant Adenovirus Vector Containing Human ggpps Gene and Its Effect on Activation of HSCs
Author(s):
Lai Shanshan2; 3; Chen Weibo1; Xu Kang2; 3; Ding Yao2; 3; Shen Ning1;  Pan Feiyan2; 3; Li Chaojun1; 4; Xue Bin1; 4
1.School of Medicine,Nanjing University,Nanjing 210093,China
Keywords:
GGPPS; recombinant-adenovirus; HSCs
PACS:
R346
DOI:
-
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 plasmid of pAdTrack-CMV to form the shuttle vector of pAdTrack-CMV-GGPPS． After sequencing，it was linearized with PmeI and co-transfected into E． coli BJ5183 cells with adenovirus genomic plasmid of pAdEasy-1 to achieve homologous recombination． The DNA of identified recombinant plasmid was digested with PacI and then transfected into QBI-293A cells to package adenovirus carrying GGPPS． Generation of recombinant adenovirus was monitored by the expression of green 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 α-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．

References:

［1］ KMC C，Hudock M P，Zhang Y，et al． Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates: a crystallographic and computational investigation［J］． J Med Chem， 2008，51( 18) : 5 594-5 607．
［2］ Casey P J，Seabra M C． Protein prenyltransferases［J］． Journal of Biological Chemistry，1996，271( 10) : 5 289-5 292．
［3］ Ericsson J，Greene J M，Carter K C，et al． Human geranylgeranyl diphosphate synthase: isolation of the cDNA，chromosomal mapping and tissue expression［J］． J Lipid Res，1998，39( 9) : 1 731-1 739．
［4］ Shen N，Gong T，Wang J D，et al． Cigarette smoke-induced pulmonary inflammatory responses are mediated by EGR-1 /GGPPS /MAPK signaling［J］． American Journal of Pathology，2011，178( 1) : 110-118．
［5］ Choi K L，Aldrich R W，Yellen G． Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K + channels［J］． Proc Natl Acad Sci，1991，88( 12) : 5 092-5 095．
［6］ Friedman S L． Hepatic stellate cells: Protean，multifunctional，and enigmatic cells of the liver［J］． Physiological Reviews， 2008，88( 1) : 125-172．
［7］ Xu L，Hui A Y，Albanis E，et al． Human hepatic stellate cell lines，LX － 1 and LX － 2: new tools for analysis of hepatic fibrosis ［J］． Gut，2005，54( 1) : 142-151．
［8］ Boguski M S，Mccormick F． Proteins regulating ras and its relatives［J］． Nature，1993，366( 6456) : 643-654．
［9］ 赵寿元，赵余龙，高洁，等． 人牻牛儿基牻牛儿基焦磷酸合成酶的cDNA 克隆基因定位及组织表达谱分析［J］． 中国科学C 辑， 2000，30( 5) : 467-474．
［10］ Yoshida T，Asanuma M，Grossmann L，et al． Geranylgeranyl-pyrophosphate ( GGPP) synthase is down-regulated during differentiation of osteoblastic cell line MC3T3-E1［J］． Febs Letters，2006，580( 22) : 5 203-5 207．
［11］ 何金生，王健伟，涛洪． 腺病毒载体构建原理与方法的研究进展［J］． 中华实验和临床病毒学，2001，15( 4) : 399- 400．
［12］ 卢大儒，邱信芳． 腺病毒载体、腺病毒相关病毒载体与基因治疗［J］． 国外医学: 遗传学，1995，18( 2) : 61-64．
［13］ Povero D，Busletta C，Novo E，et al． Liver fibrosis: a dynamic and potentially reversible process［J］． Histol Histopathol， 2010，25( 8) : 1 075-1 091．
［14］ Margadant C，Sonnenberg A． Integrin-TGF-beta crosstalk in fibrosis，cancer and wound healing［J］． EMBO Rep，2010，11 ( 2) : 97-105．
［15］ Jiao J，Friedman S L，Aloman C． Hepatic fibrosis［J］． Curr Opin Gastroenterol，2009，25( 3) : 223-229．
［16］ Breitkopf K，Roeyen C，Sawitza I，et al． Expression patterns of PDGF-A，-B，-C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells ( HSC) ［J］． Cytokine，2005，31( 5) : 349-357．
［17］ Kitamura K，Tada S，Nakamoto N，et al． Rho /Rho kinase is a key enzyme system involved in the angiotensin II signaling pathway of liver fibrosis and steatosis［J］． Journal of Gastroenterology and Hepatology，2007，22( 1) : 2 022-2 033．
［18］ Murata T，Arii S，Mori A，et al． Therapeutic significance of Y-27632，a Rho-kinase inhibitor，on the established liver fibrosis ［J］． Journal of Surgical Research，2003，114( 1) : 64-71．
［19］ Tada S，Iwamoto H，Nakamuta M，et al． A selective ROCK inhibitor，Y27632，prevents dimethylnitrosamine-induced hepatic fibrosis in rats［J］． Journal of Hepatology，2001，34( 4) : 529-536．
［20］ Wettschureck N，Offermanns S． Rho /Rho-kinase mediated signaling in physiology and pathophysiology［J］． Journal of Molecular Medicine，2002，80( 10) : 629-638．

Memo

Memo:
-
Last Update: 2013-03-21