[1]来珊珊,陈卫波,徐康,等.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(04):83-88.
 Lai Shanshan,Chen Weibo,Xu Kang,et al.Construction of Recombinant Adenovirus Vector Containing Human ggpps Gene and Its Effect on Activation of HSCs[J].Journal of Nanjing Normal University(Natural Science Edition),2011,34(04):83-88.
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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()
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《南京师大学报(自然科学版)》[ISSN:1001-4616/CN:32-1239/N]

卷:
第34卷
期数:
2011年04期
页码:
83-88
栏目:
生命科学
出版日期:
2011-12-20

文章信息/Info

Title:
Construction of Recombinant Adenovirus Vector Containing Human ggpps Gene and Its Effect on Activation of HSCs
作者:
来珊珊23陈卫波1徐康23丁尧23沈宁1潘飞燕23李朝军14薛斌1
( 1. 南京大学医学院,江苏南京210093) ( 2. 南京师范大学生命科学学院,江苏南京210046) ( 3. 分子医学生物技术江苏省重点实验室,江苏南京210046) ( 4. 江苏省医学分子技术实验室,江苏南京210093)
Author(s):
Lai Shanshan23Chen Weibo1Xu Kang23Ding Yao23Shen Ning1 Pan Feiyan23Li Chaojun14Xue Bin14
1.School of Medicine,Nanjing University,Nanjing 210093,China
关键词:
GGPPS重组腺病毒肝星状细胞
Keywords:
GGPPSrecombinant-adenovirusHSCs
分类号:
R346
摘要:
目的:构建携带人源ggpps基因的重组腺病毒并用其研究GGPPS对人肝星状细胞的活化作用.方法:利用PCR方法扩增ggpps基因片段,并亚克隆至腺病毒穿梭质粒pAdTrack-CMV中,形成穿梭质粒pAdTrack-CMV-GGPPS.测序正确后,经PmeI单酶切线性化的pAdTrack-CMV-GGPPS与腺病毒骨架质粒pAdEasy-1用电穿孔法共转化大肠杆菌BJ5183,进行同源重组.经PacI酶切鉴定正确后,用磷酸钙法转染QBI-293A细胞,包装成重组体腺病毒Ad-GGPPS颗粒.利用穿梭质粒p
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.

参考文献/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.

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备注/Memo

备注/Memo:
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) .通讯联系人: 薛斌,讲师,博士,研究方向: 细胞生物学. E-mail: : xuebin@ nju. edu. Cn
更新日期/Last Update: 2013-03-21