【摘要】 目的探讨乙型肝炎病毒(HBV)转染对HepG2细胞表面Toll样受体(TLR)表达的影响及脂多糖(LPS)加重HBV转染致肝细胞损伤是否存在直接作用,进一步探讨乙型肝炎发病及重型化的发生机制。方法以不同浓度的LPS刺激HepG2细胞、HepG2．2．15细胞(转染HBV全基因组的HepG2细胞),并以免疫细胞化学方法检测HepG2细胞及HepG2．2．15细胞表面TLR 2、4蛋白质表达情况,以逆转录聚合酶链反应(RT-PCR)检测TLR2、4 mRNA表达情况,用Abbot试剂检测HepG2．2．15细胞HgsAg和HgeAg表达情况,用流式细胞仪观察细胞生长周期及细胞凋亡情况。结果HepG2、HepG2．2．15细胞膜上均有TLR2、4蛋白质表达,免疫细胞化学染色在未加LPS及各浓度LPS刺激细胞均可见细胞膜有棕褐色着色,且随着LPS浓度增加,其着色强度增加;对0mg/L及10mg/L LPS诱导HepG2、HepG2．2．15细胞RNA进行RT-PCR扩增,其产物行10g/L琼脂糖凝胶电泳均可见特异性条带,且10mg/L LPS诱导细胞TLR2和TLR4与分子量标准品的吸光度值比值明显高于0mg/L LPS诱导细胞扩增产物,分别为306．63±6．18对519．84±9．15和700．54±13．56对1116．62±37．67,F值分别为740．58和426．07,P值分别为0．01和0．02;流式细胞仪检测细胞周期发现HepG2绌胞及未经LPS诱导的HepG2．2．15未发生凋亡,各浓度LPS诱导的HepG2．2．15细胞均有细胞凋亡发生,且细胞凋亡率随着LPS浓度增加上升。结论LPS可能通过其与肝细胞膜上的TLR结合,而参与HBV转染后肝细胞损害及肝脏炎症重型化的发病机制。更多还原

【Abstract】 Objective In order to explore the roles of TLR2 and TLR4 in the hepatocyte dam- age caused hy hepatitis B virus infection,and to find whether LPS can affect the damage of hepato- cytes pre-and pos-HBV infection,we detected the changes of TLR2 and TLR4 expressions in hu- man hepatocyte lines HepG2 cells and 2.2.15 cells.Methods HepG2 ceils are most similar to normal human hepatocytes and 2.2.15 ceils are HepG2 cells infected with HBV.We selected these two cell lines to study the differences of TLR2 and TLR4 expression between HepG2 cells before and after HBV infection.In this research,both HepG2 and 2.2.15 cells were stimulated with 0μg/ml, 1μg/ml,10μg/ml,100μg/ml,1 mg/ml and 10 mg/ml LPS.Then the expression of protein of TLR2 and TLR4 were examined by immuno-histochemistry(IHC).The cnRNA of HepG2 and 2.2.15 ceils stimulated with 0μg/ml and 10 mg/ml LPS were examined by reversal transcription-pol- ymerase chain reaction(RT-PCR).Thereafter,the apoptosis of HepG2 and HepG2.2.15 cells were examined by flow cytometry(FC),and the expressions of HBsAg and HBeAg of HepG2.2.15 cells tested with Abbott kits.Results IHC and RT-PCR analysis revealed that TLR2 and TLR4 expres- sions could he detected in both HepG2 and 2.2.15 cells.Moreover,without immune activation, TLR2 and TLR4 expressions were higher in the presence of higher concentrations of LPS.FC analy sis revealed that no apoptosis detected in HepG2 ceils stimulated with LPS in this research,but apop- tosis could be detected in 2.2.15 cells when treated with the same factors.Furthermore,the apoptosis ratios increased with the increase of LPS concentrations.When concentrations of LPS were 1μg/ml, 10μg/ml,100μg/ml,1 mg/ml and 10 mg/ml,the apoptosis ratios were 1.94%,3.03%,3.50%, 3.72%,5.30%,respectively.Abbott analysis revealed that expressions of HBsAg and HBeAg of 2.2.15 cells stimulated with LPS were lower than those not stimulated with LPS.Conclusion HBV can affect the expressions of TLR2 and TLR4 in HepG2 cell lines.LPS can lead 2.2.15 cells to apop- tosis but not HepG2 cells.Although LPS cannot damage normal hepatocytes,it might aggravate hep- atocytes damage when their microenvironment was changed by HBV infection.更多还原