目的 探讨乙型肝炎病毒(HBV)X基因转染大鼠系膜细胞肿瘤坏死因子-α(TNF-α)高表达的细胞外蛋白调节激酶(ERKs)、核因子κB(NF-κB)、P38 MAPK信号传导机制.方法 将含有HBV X基因的质粒pCI-neo-X导入体外培养的大鼠系膜细胞,分别采用特异性抑制剂U0126阻断ERK1/2通路,Lactacystin阻断NF-κB通路和SB203580阻断P38 MAPK通路,观察培养细胞TNF-α及其mRNA表达,以不加抑制剂作为对照.RT-PCR检测TNF-α mRNA表达,ELISA检测培养上清液中TNF-α表达.Western blot检测乙型肝炎病毒X蛋白(HBx)表达.结果 转染pCI-neo-X后,系膜细胞TNF-α mRNA及上清液TNF-α表达均增高,通过阻断ERK1/2或NF-κB通路,细胞TNF-α mRNA及上清液TNF-α表达均下降.而阻断P38 MAPK通路对系膜细胞TNF-α mRNA及上清液TNF-α表达无明显影响.结论 HBx蛋白通过激活ERKs和NF-κB信号通路使系膜细胞高表达TNF-α,而与P38 MAPK通路无关.%Objective To investigate the signal transduction pathway of hepatitis B virus X protein (HBx) in glomerular mesangial cell(GMC) TNF-α expression.Methods pCI-neo-X was transfected into glomerular mesangial cells.TNF-α and its mRNA expression of GMC were compared in animals treated or untreated with ERKs inhibitor U0126, NF-κB inhibitor lactacystin and P38 inhibitor SB203580, respectively.HBx expression in glomerular mesangial cells was assessed by Western blot.TNF-α mRNA expression was assessed by semi-quantitative RT-PCR.TNF-α levels in supematants were measured by ELISA.Results HBx expression was found in transfected glomerular mesangial cells, and became prominent at 36 and 48 hours after transfection whatever with or without inhibitors in culture media.TNF - α mRNA expression significantly decreased in U0126 or lactacytin group compared with U0126-free or lactacytin-free group.TNF-α levels in supematants in pCI-neo-X transfection was also partially inhibited by ERKs inhibitor U0126 or NF-κB inhibitor lactacystin, but no effect was observed in P38 inhibitor SB203580 treated group at 36 and 48 h after transfection.Conclusion HBx upregulated TNF-α expression of GMC line partly through ERK1/2 and NF-κB signal transduction pathway, but not P38 MAPK pathway.
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