IM To establish a nonradioactive assay for 2′5′ oligoadenylate synthetase (25 AS) and to measure the 25AS in peripheral blood mononuclear cell (PBMC) extracts of patients with chronic hepatitis C before IFNα injec...IM To establish a nonradioactive assay for 2′5′ oligoadenylate synthetase (25 AS) and to measure the 25AS in peripheral blood mononuclear cell (PBMC) extracts of patients with chronic hepatitis C before IFNα injection, 24 hours and one month after the first injection.METHODS 25AS in cell extracts of PBMCs from 10 normal persons and 15 chronic hepatitis C patients were determined with PEI cellulose thinlayer chromatography.RESULTS The assay of 25AS in human PBMC was found to be rapid, sensitive, specific and reliable. The 25AS activity of PBMC in normal persons was in a quite low level (20%), and it was increased about tenfolds after stimulation of IFN (197%), (P<001). In 15 chronic hepatitic C patients, the basal levels of 25AS before IFN treatment were higher than those of normal persons, being much higher in the group showing poor response to IFN treatment, but 24h after the first injection of IFNα the 25AS level showed a more rapid and much greater rise in those patients with a good response.CONCLUSION 25AS may be a useful parameter of biological response during the IFN therapy..展开更多
AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected wit...AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. I^ESULTS： L02/core cell line stably expressing HCV- core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION： HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.展开更多
The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a turnour suppressor gene product.Catalytically inactive mutants of PKR give rise to a tumourigenic phenotype...The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a turnour suppressor gene product.Catalytically inactive mutants of PKR give rise to a tumourigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another dotlblestranded RNA-dependent enzyme,the 40kDa form of 2'5'oligoadenylate synthetase. Inhibition of both wile-type PKR or 2'5' oligoadenylate synthetase is reversed by adding higher concentrations of double-stranded RNA. These results suggest competition between PKR K296R and wild-type PKR or 2'5' oligoadenylate synthetase for limiting amounts of dublestranded RNA. Moreover,the data imply that the tumourigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double-strandeed RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself.展开更多
目的:研究HH胶囊体外抗乙型肝炎病毒的作用及其对抗病毒蛋白2'5'-寡腺苷酸合成酶(2'5'-Oligoadenylate Synthetase,2'5'-OAS)、RAN依赖蛋白激酶(RAN-dependent protein kinase,PKR)的影响.方法:HepG2.2.15是目...目的:研究HH胶囊体外抗乙型肝炎病毒的作用及其对抗病毒蛋白2'5'-寡腺苷酸合成酶(2'5'-Oligoadenylate Synthetase,2'5'-OAS)、RAN依赖蛋白激酶(RAN-dependent protein kinase,PKR)的影响.方法:HepG2.2.15是目前最常用的乙型肝炎病毒感染的体外实验模型,将细胞随机分为空白对照组、阳性对照组(3TC)、不同浓度的HH胶囊组.用CCK-8检测药物细胞毒性,酶联免疫法检测乙型肝炎病毒表面抗原(HBsAg)和乙型肝炎病毒e抗原(HBeAg);荧光定量聚合酶链反应法检测乙型肝炎病毒脱氧核糖核酸(HBVDNA);用Westernblot、荧光定量PCR方法分别检测细胞内抗病毒蛋白2'5'-OAS、PKR及其mRNA水平.结果:HH胶囊的TC50是2.11g/L、312.00mg/L、156.00mg/L、78.00mg/L、39.00mg/LHH胶囊均可以降低细胞外HBeAg(0.285±0.007,0.462±0.008,0.565±0.009,0.733±0.008vs1.334±0.007)和HbsAg(0.834±0.008,1.021±0.011,1.347±0.017,1.548±0.015vs2.593±0.008)水平,312.00mg/L、156.00mg/LHH胶囊均可以减低细胞内乙型肝炎病毒DNA[(3.423±0.110)×109copies/L,(3.640±0.082)×109copies/Lvs(6.857±0.060)×109copies/L]、细胞外乙型肝炎病毒DNA(6547±87、7710±62vs24300±200),312.00mg/LHH胶囊可以增加细胞内OASmRNA(0.885±0.038vs0.688±0.068)、PKRmRNA(0.139±0.06vs0.058±0.005)表达及其OAS、PKR蛋白水平.结论:HH胶囊具有良好的抗乙型肝炎病毒作用,推测可能与增加细胞内OAS、PKR及其mRNA水平有关.展开更多
文摘IM To establish a nonradioactive assay for 2′5′ oligoadenylate synthetase (25 AS) and to measure the 25AS in peripheral blood mononuclear cell (PBMC) extracts of patients with chronic hepatitis C before IFNα injection, 24 hours and one month after the first injection.METHODS 25AS in cell extracts of PBMCs from 10 normal persons and 15 chronic hepatitis C patients were determined with PEI cellulose thinlayer chromatography.RESULTS The assay of 25AS in human PBMC was found to be rapid, sensitive, specific and reliable. The 25AS activity of PBMC in normal persons was in a quite low level (20%), and it was increased about tenfolds after stimulation of IFN (197%), (P<001). In 15 chronic hepatitic C patients, the basal levels of 25AS before IFN treatment were higher than those of normal persons, being much higher in the group showing poor response to IFN treatment, but 24h after the first injection of IFNα the 25AS level showed a more rapid and much greater rise in those patients with a good response.CONCLUSION 25AS may be a useful parameter of biological response during the IFN therapy..
基金Supported by National Natural Science Foundation of China,No.30671846
文摘AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. I^ESULTS： L02/core cell line stably expressing HCV- core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION： HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.
文摘The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a turnour suppressor gene product.Catalytically inactive mutants of PKR give rise to a tumourigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another dotlblestranded RNA-dependent enzyme,the 40kDa form of 2'5'oligoadenylate synthetase. Inhibition of both wile-type PKR or 2'5' oligoadenylate synthetase is reversed by adding higher concentrations of double-stranded RNA. These results suggest competition between PKR K296R and wild-type PKR or 2'5' oligoadenylate synthetase for limiting amounts of dublestranded RNA. Moreover,the data imply that the tumourigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double-strandeed RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself.
文摘目的:研究HH胶囊体外抗乙型肝炎病毒的作用及其对抗病毒蛋白2'5'-寡腺苷酸合成酶(2'5'-Oligoadenylate Synthetase,2'5'-OAS)、RAN依赖蛋白激酶(RAN-dependent protein kinase,PKR)的影响.方法:HepG2.2.15是目前最常用的乙型肝炎病毒感染的体外实验模型,将细胞随机分为空白对照组、阳性对照组(3TC)、不同浓度的HH胶囊组.用CCK-8检测药物细胞毒性,酶联免疫法检测乙型肝炎病毒表面抗原(HBsAg)和乙型肝炎病毒e抗原(HBeAg);荧光定量聚合酶链反应法检测乙型肝炎病毒脱氧核糖核酸(HBVDNA);用Westernblot、荧光定量PCR方法分别检测细胞内抗病毒蛋白2'5'-OAS、PKR及其mRNA水平.结果:HH胶囊的TC50是2.11g/L、312.00mg/L、156.00mg/L、78.00mg/L、39.00mg/LHH胶囊均可以降低细胞外HBeAg(0.285±0.007,0.462±0.008,0.565±0.009,0.733±0.008vs1.334±0.007)和HbsAg(0.834±0.008,1.021±0.011,1.347±0.017,1.548±0.015vs2.593±0.008)水平,312.00mg/L、156.00mg/LHH胶囊均可以减低细胞内乙型肝炎病毒DNA[(3.423±0.110)×109copies/L,(3.640±0.082)×109copies/Lvs(6.857±0.060)×109copies/L]、细胞外乙型肝炎病毒DNA(6547±87、7710±62vs24300±200),312.00mg/LHH胶囊可以增加细胞内OASmRNA(0.885±0.038vs0.688±0.068)、PKRmRNA(0.139±0.06vs0.058±0.005)表达及其OAS、PKR蛋白水平.结论:HH胶囊具有良好的抗乙型肝炎病毒作用,推测可能与增加细胞内OAS、PKR及其mRNA水平有关.
