丙型肝炎病毒核心蛋白上调基质金属蛋白酶组织抑制因子-1的表达
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摘要
目的研究丙型肝炎病毒(HCV)核心蛋白对基质金属蛋白酶组织抑制因子(TIMP)-1基因表达的影响,以探讨HCV的致肝纤维化机制。方法聚合酶链反应(PCR)扩增TIMP-1的启动子DNA片段,克隆至真核报告载体pCAT3-basic中,构建pCAT3-TIMP-1p报告载体;将该质粒转染人肝癌细胞系HepG2细胞,以酶链免疫吸附试验(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性;将构建的pCAT3-TIMP-1p与HCV核心蛋白真核表达载体PcDNA3.1(-)-HCVcore共转染入人肝星状细胞系LX-2细胞,同时平行设立pCAT3-basic阴性对照组、pCAT3-control阳性对照组、pCAT3-TIMP-1p单独转染组,用ELISA法检测各组CAT的表达活性。结果成功获得TIMP-1基因启动子DNA片段,构建的pCAT3-TIMP-1p具有转录活性,与PcDNA3.1(-)-HCVcore共转染LX-2细胞后,ELISA法检测结果显示pCAT3-TIMP-1p吸光度值为0.833±0.040,同期单独转染LX-2细胞的pCAT3-TIMP-1p吸光度值为0.677±0.049,方差分析各组差异有统计学意义(F=132.401,P<0.05)。结论 HCV核心蛋白通过上调TIMP-1启动子的活性,促进TIMP-1基因的表达。
Objective To investigate the influence of hepatitis C virus ( HCV) core protein on tissue inhibitor of metalloproteinases-1 ( TIMP-1) expression via molecular methods,and provide a potential pathogenesis for hepatic fibrosis during chronic HCV infection. Methods Polymerase chain reaction ( PCR) technique was employed to amplify the sequence of TIMP-1 promoter,the product was cloned into pCAT3-basic,the constructed vector was named as pCAT3TIMP-1p. The vector was transfected into HepG2 cells,the activity of Chloramphenycol acethyltransferase ( CAT) was detected by enzyme-linked immunoassay ( ELISA) . CAT analysis was performed on LX-2 cells cotransfected with PcDNA3. 1( ) -HCVcore and pCAT3-TIMP-1p. At the same time,pCAT3-basic was transfected into LX-2 cells served as negative-control,positive-control cells transfected with pCAT3-control,and the control group was transfected by pCAT3TIMP-1p alone. Results The report vector pCAT3-TIMP-1p had been constructed and confirmed by restriction enzyme digestion. After cotransfected into LX-2 cell with PcDNA3. 1( ) -HCVcore and pCAT3-TIMP-1p,the OD of pCAT3TIMP-1p was 0. 833 ± 0. 040. In contrast,the OD of pCAT3-TIMP-1p transfected alone was 0. 677 ± 0. 049. Statistics was performed by ANOVA,and differences were considered to be significant ( F = 132. 401,P < 0. 05) . Conclusion HCV core protein facilitated the expression of TIMP-1 through enhancing TIMP-1 promoter activity.
Objective To investigate the influence of hepatitis C virus ( HCV) core protein on tissue inhibitor of metalloproteinases-1 ( TIMP-1) expression via molecular methods,and provide a potential pathogenesis for hepatic fibrosis during chronic HCV infection. Methods Polymerase chain reaction ( PCR) technique was employed to amplify the sequence of TIMP-1 promoter,the product was cloned into pCAT3-basic,the constructed vector was named as pCAT3TIMP-1p. The vector was transfected into HepG2 cells,the activity of Chloramphenycol acethyltransferase ( CAT) was detected by enzyme-linked immunoassay ( ELISA) . CAT analysis was performed on LX-2 cells cotransfected with PcDNA3. 1( ) -HCVcore and pCAT3-TIMP-1p. At the same time,pCAT3-basic was transfected into LX-2 cells served as negative-control,positive-control cells transfected with pCAT3-control,and the control group was transfected by pCAT3TIMP-1p alone. Results The report vector pCAT3-TIMP-1p had been constructed and confirmed by restriction enzyme digestion. After cotransfected into LX-2 cell with PcDNA3. 1( ) -HCVcore and pCAT3-TIMP-1p,the OD of pCAT3TIMP-1p was 0. 833 ± 0. 040. In contrast,the OD of pCAT3-TIMP-1p transfected alone was 0. 677 ± 0. 049. Statistics was performed by ANOVA,and differences were considered to be significant ( F = 132. 401,P < 0. 05) . Conclusion HCV core protein facilitated the expression of TIMP-1 through enhancing TIMP-1 promoter activity.
引文
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[2]Inoue K,Tsukiyama-Kohara K,Matsuda C,et al.Impairment of inter-feron regulatory factor-3activation by hepatitis C virus core protein bas-ic amino acid region1[J].Biochem Biophys Res Commun,2012,428(4):494-499.
[3]Ohtake N,Nakai Y,Yamamoto M,et al.Separation and isolationmethods for analysis of the active principles of Sho-saiko-to(SST)ori-ental medicine[J].J Chromatogr B Analyt Technol Biomed Life Sci,2004,812(1-2):135-148.
[4]Zhao Q,Qin CY,Zhao ZH,et al.Epigenetic modifications in hepatic stellate cells contribute to liver fibrosis[J].Tohoku J Exp Med,2013,229(1):35-43.
[5]Ramachandran P,Iredale JP.Liver fibrosis:a bidirectional model of fibrogenesis and resolution[J].QJM,2012,105(9):813-817.
[6]Hold GL,Untiveros P,Saunders KA,et al.Role of host genetics in fi-brosis[J].Fibrogenesis Tissue Repair,2009,2(1):6.
[7]Zeisberg M,Kalluri R.Cellular mechanisms of tissue fibrosis.1.Common and organ-specific mechanisms associated with tissue fibrosis[J].Am J Physiol Cell Physiol,2013,304(3):C216-C225.
[8]Attallah AM,Badr El-Din NK,Omran MM,et al.Assessment of ma-trix metalloproteinase-1for marking liver cirrhosis in chronic hepatitis C patients[J].Egypt J Immunol,2011,18(1):33-42.
[9]Liu DP,Lu FM,Wang MF,et al.Rhodiola rosea influence the ex-pression of TIMP-1,Smad4in the liver tissue of liver fibrosis rat[J].Chinese Journal of Gerontology,2012,32(12):2556-2558.刘东璞,卢凤美,王明富,等.红景天对大鼠肝纤维化肝脏组织中TIMP-1、Smad4表达的影响[J].中国老年学杂志,2012,32(12):2556-2558.