期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

重组Akt腺病毒的构建及其在肝硬化大鼠肝脏中的表达 被引量：5

Construction of recombinant adenovirus containing Akt and its expression in the liver of cirrhotic rats
暂未订购
导出
摘要 目的探讨持续活化Akt且带有HA标签(myr-HA-Akt)基因的重组腺病毒在肝硬化大鼠肝脏中的表达特性。方法酶切正向插入目的基因的真核表达载体pcDNA3.1-myr-HA-Akt,获得myr-HA-Akt cDNA后,将其定向克隆到穿梭质粒pDC316中,然后将重组质粒与病毒骨架质粒pBHGloxΔE1,3Cre共转染293细胞,获得复制缺陷型重组腺病毒Ad-myr-HA-Akt,并进行扩增、纯化。通过观察腺病毒感染293细胞后是否出现细胞病变效应;PCR和基因测序方法对所构建病毒进行鉴定,并采用TCID50法检测病毒滴度。自尾静脉注射重组腺病毒Ad-myr-HA-Akt感染肝硬化模型大鼠。免疫印迹法检测大鼠肝组织内Akt和p-Akt蛋白的表达。结果感染的293细胞出现明显的细胞病变效应,PCR产物电泳证实重组腺病毒的存在,基因测序证实myr-HA-Akt的cDNA正确插入穿梭质粒且与pBHGloxΔE1,3Cre正确同源重组;病毒滴度为5.5×1011vp/mL。从蛋白水平证实感染病毒的肝硬化模型大鼠有外源性Akt基因的表达。结论构建的重组腺病毒Ad-myr-HA-Akt能有效地感染肝硬化模型大鼠,并可在模型大鼠中正确转录和翻译,为进一步研究腺病毒介导的Akt基因对肝硬化的治疗奠定了实验基础。 Objective To study the expression of the adenoviral vector containing myr-HA-Akt gene in the liver of cirrhotic rats in vivo. Methods myr-HA-Akt eDNA obtained from the plasmid pcDNA3. 1-myr-HA-Akt was cloned into the plasmid pDC316. Then, pDC316-myr-HA-Akt was cotransferred with adenoviral backbone vector into 293 cells. The recombinant adenovirus was reproduced and purified. The recombinant adenovirus was identified by the cytopathic effect of 293 cells, polymerase chain reaction （PCR） , and gene sequencing for myr-HA-Akt eDNA. TCID50 assay was performed to determine the titer of virus. After the adenovirus infected the hepatic cirrhosis rat models via the tail vein, protein and phosphorylation status of Akt were examined by Western blotting. Results Infected 293 cells showed significant cytopathic effect. Products of PCR confirmed the presence of recombinant adenovirus. The identification result by DNA sequence analysis showed that myr-HA-Akt eDNA was cloned to pDC316 correctly and homologously recombinated with pBHGloxAE 1 , and 3 Cre and Ad-myr-HA-Akts were packaged successfully. The titer of virus was 5.5 × 10^11 vp/ml. The expression of Akt in the liver of cirrhotic rats was verified by Western blotting. Conclusions The recombinant adenoviral vector containing myr-HA-Akt was constructed and the transgene of hepatic cirrhosis rats expressed Akt gene in vivo successfully. It provides a basis for the further study of treatment for hepatic cirrhosis by Akt gene.
作者 邓刚 黄飞舟 刘浔阳 罗成群 郭立武
机构地区 中南大学湘雅三医院普外一科 中南大学湘雅三医院烧伤外科 美国纽约州立大学Buffloa分校
出处 《中国普通外科杂志》 CAS CSCD 2008年第7期687-691,共5页 China Journal of General Surgery
关键词 肝硬化 重组腺病毒载体 AKT 转染 基因表达 Liver Cirrhosis Recombinant Adenovirus Vector Akt Transfection Gene Expression
分类号 R657.3 [医药卫生—外科学]
  • 相关文献

参考文献13

  • 1孙敬方.动物实验方法学[M].北京:人民卫生出版社,2005:474-475.
  • 2Garcia-Banuelos J, Siller-Lopez F, Miranda A, et al. Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors. Evidence of cirrhosis reversion[J]. Gene Ther,2002,9(2) : 127 -134.
  • 3Guicciardi ME, Gores GJ. Apoptosis : a mechanism of acute and chronic liver injury[ J] . Gut, 2005,54 ( 7 ) : 1024 - 1033.
  • 4Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis [ J ]. Nature Med, 2003,9(3) :347 -351.
  • 5Akazawa Y, Gores GJ. Death receptor-mediated liver injury [J]. Semin Liver Dis,2007,27(4) :327 -338.
  • 6Schattenberg JM, Galle PR, Schuchmann M. Apoptosis in liver disease [ J ] . Liver Int ,2006,26 ( 8 ) :904 - 911 .
  • 7Eichhorst ST. Modulation of apoptosis as a target for liver disease[J]. Expert Opin Ther Targets,2005,9 (1) :83 - 99.
  • 8Su CC, Lin YP, Cheng YJ, et al. Phosphatidylinositol 3- kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells [ J ]. J Immunol, 2007,179(7) :4589 -4597.
  • 9Franke TF, Homik CP, Segev L, et al. PI3 K/Akt and apoptosis: size matters [ J]. Oncogene, 2003, 22 (56) : 8983 -8998.
  • 10Chen Z, Guo X, Ge X, et al. Preparation of Monoclonal Antibodies Against Pseudorabies Virus Glycoprotein gC by Adenovirus Immunization Alone or as a Boost Following DNA Priming[J]. Hybridoma (Larchmt) , 2008,27(1) :36- 42.

二级参考文献13

共引文献20

同被引文献45

  • 1叶媛,但自力.All-trans Retinoic Acid Diminishes Collagen Production in a Hepatic Stellate Cell Line via Suppression of Active Protein-1 and c-Jun N-terminal Kinase Signal[J].Journal of Huazhong University of Science and Technology(Medical Sciences),2010,30(6):726-733. 被引量：8
  • 2周陶友,陈敏,李卫华,赵连三,何芳,刘丽,唐红.携带丙型肝炎病毒非结构蛋白3基因的重组腺病毒构建研究[J].中华医院感染学杂志,2005,15(9):973-976. 被引量：2
  • 3Bosch J, Garcia-Pagan JC. Complications of cirrhosis : Ⅰ. Portal hypertension. J Hepatol,2000,32 : 141-156.
  • 4Garcia-Banuelos J,Siller-Lopez F, Miranda A, et al. Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinicalgrade adenoviral vectors. Evidence of cirrhosis reversion. Gene Ther,2002,9 : 127-134.
  • 5Ludwig D,Schwarting K, Cornelia M, et al. The postprandial portal flow is related to the severity of portal hypertension and liver cirrhosis. Hepatology, 1998,28:631-638.
  • 6Gupta TK,Toruner M, Chung MK, et al. Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats. Hepatology, 1998,28:926-931.
  • 7Rockey DC, Chung JJ. Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension. Gastroenterology, 1998,114 : 344-351.
  • 8Uruno A, Sugawara A, Kanatsuka H, et al. Upregulation of nitric oxide production in vascular endothelial cells by all-trans retinoic acid through the phosphoinositide 3-kinase/Akt pathway. Circulation ,2005,112:727-736.
  • 9Sun Y, Sumi D, Kumagai Y. Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells. Toxicol Appl Pharmacol, 2006,214 : 55 -60.
  • 10Guicciardi ME, Gores GJ. Apoptosis: a mechanism of acute and chronic liver injury. Gut, 2005,54 : 1024-1033.

引证文献5

二级引证文献4

中国普通外科杂志
2008年 第7期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部