干扰素信号传导相关蛋白和基因检测方法的初步建立和应用

A method for detection of proteins and genes related to IFN-α signal transduction

目的:建立α鄄干扰素(IFN鄄α)信号传导蛋白和基因检测的方法。方法:收集慢性乙型病毒性肝炎、自愈性乙型肝炎病毒感染者、健康对照者的外周静脉血并分离外周血单核细胞(PBMC),建立EB病毒(EBV)转化的B淋巴细胞系。体外分别用不同浓度的IFN鄄α(0、100、1000、10000IU/ml)刺激细胞,用凝胶电泳迁移率试验(EMSA)检测IFN活化的干扰素刺激基因因子3(ISGF鄄3)和γ鄄干扰素活化因子(GAF);设计特异的引物和探针,用实时荧光定量聚合酶链反应(PCR)检测IFN诱导抗病毒蛋白MxA、OAS1、PKR的相对表达。结果:成功建立了EBV转化的B淋巴细胞系。建立了EMSA检测ISGF鄄3和GAF转录蛋白的方法,用实时荧光定量PCR能够准确地检测样本中MxA、OAS1和PKR的表达。结论:建立了IFN信号传导蛋白和基因检测的方法,为进一步研究IFN治疗慢性乙型病毒性肝炎的疗效提供依据。

Objective To establish a method for detection of prot ei ns and genes related to IFN-ásignal transduction,and to study the relation bet ween the effectiveness of IFN-áon treatment of chronic hepatitis B(CHB)and I FN-ásignal transduction.Methods The peripheral bloods were obtained from chron ic hepatitis B patients,self-limited HBV infected patients and healthy control and their peripheral blood mononuclear cells(PBMCs)were isolated.Then EBV was us ed to transform and immortalize the B lymphocytes,which were then stimulated wit h different concentration of IFN-á(IFN-á2a,0,100,1000,10000IU/ml).EMSA(elect rophoretic mobility shift assay)was used to semi-quantify the important nucleus transcription factors(ISGF-3and GAF)on the IFN-ásignal pathway.Specific pr imers and probes were designed and syn-thesized for real-time PCR,which was th en applied to quantify the MxA,OAS1,PKR expression in the RNA preparations extra cted from cells.Results EBV-transformed B cell lines were successfully construc ted,among them were37from CHB patients,12from self-limited hepatitis,8from heal thy controls.The EMSA were successfully established for detect-ing the ISRE/GAF DNA binding activity,and real-time PCR accurately quantified MxA,OAS1,PKR mRNA expression of IFN-ástimulated genes.Conclusion A method for detecting protein s and genes on the interferon signal transduction is successfully established,wh ich could be used for further research on interferon therapy.