【摘要】 目的比较乙型肝炎病毒(hepatitis B virus,HBV)基因组稳定转染细胞株HepG2.2.15和肝癌细胞株HepG2中TLR7表达,分析HepG2.2.15细胞中TLR7激活后诱导炎症因子表达。方法通过实时荧光定量(Real-time)PCR分析HepG2和HepG2.2.15中TLR7受体、IL-6和TNF-αmRNA水平表达。TLR7配体Gardiquimod刺激20min后,Western blot分析激活的信号通路;刺激6h,Real-timePCR分析TLR7激活后IL-6和TNF-αmRNA水平表达变化;刺激48h,ELISA法分析IL-6和TNF-α蛋白质水平表达变化。结果HepG2.2.15细胞株中TLR7mRNA水平表达比HepG2中的表达高约30倍(P<0.01)。在没有任何刺激的条件下,HepG2.2.15细胞株中IL-6和TNF-αmRNA水平表达比HepG2中的表达分别高约7.5和9.3倍;Gardiquimod刺激6h后,HepG2.2.15细胞株中IL-6和TNF-αmRNA表达水平明显升高,并具有剂量依赖关系(P<0.01),而HepG2细胞在相同的刺激下IL-6和TNF-αmRNA表达水平升高并不明显(P<0.05);刺激48h后,IL-6和TNF-α蛋白质表达水平变化与其mRNA水平变化一致(P<0.01)。HepG2.2.15细胞株经Gardiquimod刺激后NF-κBp65亚单位进入细胞核中量显著增加。结论TLR7激活后能够上调HepG2.2.15细胞中IL-6和TNF-α的表达,提示TLR7可能在乙型病毒性肝炎的病理生理过程中发挥作用。更多还原

【Abstract】 Objective To explore the different expression of Toll-like receptor 7 (TLR7) between HepG2.2.15 cells,which were stably transfected HBV gene,and HepG2 cells,and then analyze the expressions of the TLR7-induced inflammation factors in HepG2.2.15 cell line.Methods Real-time PCR was used to analyze the different expressions of TLR7,IL-6 and TNF-α between in HepG2 and HepG2.2.15 cells.After the cells were treated with Gardiquimod,a TLR ligand,at 0,1,3 and 10 μg/ml for 6 h,the messenger ribonucleic acid (mRNA) expressions of IL-6 and TNF-α were measured by real-time PCR,and the protein secretion of the 2 cytokines was determined by ELISA after 48 hours’ treatment.Western blot analysis was used to analyze the activation of NF-κB in HepG2.2.15 cells after Gardiquimod stimulation for 20 min.Results The expression of TLR7 in HepG2.2.15 cells was about 30-fold higher than that in the HepG2 cells.The mRNA levels of IL-6 and TNF-α were higher in HepG2.2.15 cells than in HepG2 cells (about 7.5 and 9.3 folds respectively),even without Gardiquimod stimulation.After the cells were stimulated by Gardiquimod for 6 h,although the mRNA of IL-6 and TNF-α in HepG2 cells were slightly increased (compared with the control,P<0.05),the increase of IL-6 and TNF-α mRNA levels was more significant in HepG2.2.15 cells than in HepG2 cells (compared with the control,P<0.01,and with HepG2 cells,P<0.01).The cells were treated with Gardiquimod for 48 h,the induced expressions of the 2 cytokines protein levels had a consistent changes with the mRNAs level at a dose-dependent manner (compared with control P<0.01).While HepG2.2.15 cells were stimulated by Gardiquimod for 20 min,the nuclear immigration of NF-κB p65 subunit was significantly increased.Conclusion The activation of TLR7 upregulates the expressions of the IL-6 and TNF-α,suggesting that the TLR7 might be involved in the pathogenesis of the viral hepatitis.更多还原