中文摘要：

目的探讨乙型肝炎病毒（HBV）X基因编码的蛋白（HBx）导致大鼠系膜细胞（MC）增殖及TNF-α高表达的细胞外蛋白调节激酶（ERK）1/2信号转导机制。方法PCR扩增HBVX基因，然后与真核表达载体PCI-neo质粒连接，构成含有HBVX基因的质粒（PCI-neo—X），采用限制性内切酶法及测序证实。采用脂质体法将PCI-neo—X转染入体外培养的大鼠MC，采用抑制剂U0126阻断ERK1/2通路，观察培养细胞增殖、TNF—α及其mRNA表达，以不加抑制剂作为对照。Western blot检测HBx、ERK1/2及磷酸化ERK（p-ERK）。表达。半定量RT—PCR检测TNF-αmRNA表达，酶联免疫吸附法（ELISA）检测培养上清液TNF—α表达。甲基噻唑基四唑（MTT）法检测MC增殖。结果无论是否加入U0126，在转染PCI-neo-X36、48h后，肾小球系膜细胞（GMC）明显表达HBx，细胞TNF-αmRNA的表达加入U0126较不加入U0126显著下降。不加入U0126组上清液TNF—α水平在转染后36h和48h分别为（189．0±18．1）ng／L和（172．3±24．3）ns／L；加入U0126组上清液中TNF-α水平在转染后36、48h分别为（65．6±11．6）ns／L和（84．0±26．6）ng／L，组间不同时间点比较均有显著性差异（Pa〈0．05）。加入U0126后MC增殖也显著下降。结论HBx可促使MC增殖，细胞TNF—αmRNA及上清液TNF-α表达均增高，这一作用可能是通过HBx激活ERK1/2信号通路实现的。

英文摘要：

Objective To investigate the extracellular regulated protein kinases （ERKs） pathway of hepatitis B virus（HBV） X protein （HBx） on glomerular mesangial cell（GMC） proliferation of rat and tumor necrosis factor（TNF） - α expression. Methods The HBV X gene was amplified by polymerase chain reaction（PCR） ,inserted into the eukaryotic expression vector PCI -neo and confirmed by restrict endonuclease digestion and sequence analysis. PCI -neo contained HBV X gene （PCI -neo -X） was transfected into cultured GMC via liposome. GMC proliferation,TNF -α and its mRNA expression were investigated in the condition of with or without U0126 in the culture media. HBx, ERK1/2 and phosphorynated - ERK1/2 （ p - ERK1/2 ） expression in GMC were assessed by Western blot. TNF -α mRNA expression was assessed by semi - quantitative reverse transcriptase - PCR （ RT - PCR）. TNF - α level in supernatants was measured by enzymelinked immunosorbent assay（ELISA）. GMC proliferation was assessed by methyl thiazolyl tetrazolium （MTT）. Results HBx expression was found in transfected GMC,and became prominent at 36 and 48 hours after transfection whether with or without U0126 in culture media. TNF - α mRNA expression was significantly decreased in U0126 group compared with U0126 - free group. TNF - α levels in supernatants in PCI - neo - X transfection without U0126 group were （ 189.0 ± 18.1 ） ng/L at 36 hours and （ 172.3 ± 24.3） ng/L at 48 hours after transfection, respectively. In contrast,TNF - α levels in supernatants with U0126 were （65.6 ±11.6） ng/L and （ 84.0 ± 24.6 ） ng/L, respectively. The TNF -α levels in the latter groups were significantly lower than the formers （Pa〈 0.05 ）. GMC proliferation was also lower in added U0126 group at 36 and 48 hours after transfection. Conclusions HBx can induce GMC cell proliferation and increase TNF - α mRNA expression and its protein pro- duction. HBx upregnlates TNF - α expression and induced cell proliferation of GMC line partly