CAJ | 학술논문

目的探讨Toll样受体4(TLR4)配体脂多糖(LPS)抑制人滋养层细胞Bewo中乙型肝炎病毒( HBV)复制的作用机制,为防治HBV宫内感染提供依据.方法首先将2μg 1.3倍HBV全基因重组质粒pcDNA3.1(+)-HBV1.3转染Bewo细胞12h后,以TLR4配体LPS处理3d.尔后用LPS处理Bewo细胞,观察IFN-β、TNF-α表达的动力学、NF-κB拮抗剂二硫代氨基甲酸吡咯烷( PDTC)对LPS诱导Bewo细胞产生细胞因子的作用.采用微粒子酶免疫分析法(MEIA)和荧光定量PCR法分别检测HBsAg、HBeAg和HBV DNA水平,并以ELISA和RT-PCR分别检测IFN-β、TNF-α水平及TIR结构域的转接蛋白(TRIF)、髓样分化蛋白(MyD88)表达.结果与对照组比较,LPS可显著抑制Bewo细胞中HBV复制(P＜0.01),且LPS可显著诱导Bewo细胞产生TNF-α(P＜0.05),呈时间和剂量依赖性.PDTC可抑制LPS诱导细胞产生TNF-α,显著低于对照组(P＜0.01),但对IFN-β无显著作用(P＞0.05).与对照组比较,LPS可诱导HBV重组质粒转染的Bewo细胞表达MyD88(P＜0.01).结论通过MyD88/NF-κB信号途径诱导Bewo细胞产生TNF-α,TLR4配体LPS可显著抑制HBV复制.
목적 탐토Toll양수체4(TLR4)배체지다당(LPS)억제인자양층세포Bewo중을형간염병독( HBV)복제적작용궤제,위방치HBV궁내감염제공의거.방법 수선장2μg 1.3배HBV전기인중조질립pcDNA3.1(+)-HBV1.3전염Bewo세포12h후,이TLR4배체LPS처리3d.이후용LPS처리Bewo세포,관찰IFN-β、TNF-α표체적동역학、NF-κB길항제이류대안기갑산필각완( PDTC)대LPS유도Bewo세포산생세포인자적작용.채용미입자매면역분석법(MEIA)화형광정량PCR법분별검측HBsAg、HBeAg화HBV DNA수평,병이ELISA화RT-PCR분별검측IFN-β、TNF-α수평급TIR결구역적전접단백(TRIF)、수양분화단백(MyD88)표체.결과 여대조조비교,LPS가현저억제Bewo세포중HBV복제(P＜0.01),차LPS가현저유도Bewo세포산생TNF-α(P＜0.05),정시간화제량의뢰성.PDTC가억제LPS유도세포산생TNF-α,현저저우대조조(P＜0.01),단대IFN-β무현저작용(P＞0.05).여대조조비교,LPS가유도HBV중조질립전염적Bewo세포표체MyD88(P＜0.01).결론 통과MyD88/NF-κB신호도경유도Bewo세포산생TNF-α,TLR4배체LPS가현저억제HBV복제.
Objective To explore the effect and mechanism of Toll-like receptor 4(TLR4) ligand LPS-mediated inhibition hepatitis B virus (HBV) replication in Bewo cells.Methods First of all,2 μg 1.3-fold HBV recombinant vector pcDNA3.1 ( + )-HBV1.3 were transfected into Bewo cells,after 12 h,the cells were treated with LPS for 3 d.To observe the kinetics of IFN-β and TNF-α expression in Bewo cells,the Bewo cells were exposed to TLR4 ligand LPS.And the effect of pyrrolidine dithiocarbamate ( PDTC),an inhibitor of NF-κB,on LPS-induced cytokines was also observed.The HBsAg,HBeAg and HBV DNA level in the culture supernatant were detected by Microparticle Enzyme Immunoassay (MEIA) and fluorescence quantitative PCR,respectively,and the expression of IFN-β,TNF-α,TRIF and MyD88 was detected by ELISA and RT-PCR,respectively.Results Compared with control group,LPS could significantly suppress HBV replication in Bewo cells ( P ＜0.01 ),and it could induce the production of TNFα in Bewo cells ( P ＜ 0.05 ),in time-and dose-dependent manners.PDTC strongly inhibited LPS and induced TNF-α production,but had no much effect on IFN-β in Bewo cells ( P ＜ 0.001 ).Compared with control group,the mRNA levels of MyD88 were significantly induced by LPS in the Bewo cells transfected with this recombinant vector( P ＜ 0.001 ).Conclusions TLR4 ligand LPS could significantly suppress HBV replication by inducing TNF-α production in Bewo cells mainly via the MyD88/ NF-κB signal pathway.