HBcAg和TBK1对dsRNA诱导肝细胞表达IFN-β的调节作用
摘要
乙型肝炎病毒(hepatitis B virus,HBV)常引起持续性感染,导致慢性肝炎,并引起肝硬化和肝细胞癌。HBV感染引起慢性化的机制尚不清楚,但病毒编码的蛋白对机体免疫应答的调节作用是其中之一,随着固有免疫应答的深入研究,HBV对固有免疫的调节作用也日益受到重视,同时通过诱导固有免疫应答有望在慢性HBV的治疗中发挥一定的作用。
固有性免疫(innate immunity)应答是机体快速抵抗微生物病原体的重要机制。表达于免疫细胞的模式识别受体(pattern recognition receptor,PRR)通过识别病原相关分子模式(pathogen-associated molecular pattern,PAMP),启动一系列信号转导途径,诱导保护性细胞因子如Ⅰ型干扰素(IFN-α/β)和前炎症细胞因子的表达,发挥抗病毒固有免疫作用,并调节适应性免疫(adaptive immunity)应答。以Toll样受体(Toll-like receptor,TLR)为代表的PRR是抗病毒固有免疫的关键性识别结构,TLR3,TLR7,TLR8和TLR9主要与病毒的识别有关,如TLR3识别病毒双链RNA(double stranded RNA,dsRNA)类似物聚肌苷-聚胞嘧啶核苷酸(polyinosinic-poly cytosine nucleotide,polyⅠ:C)。TLRs与特异性配体结合后,通过MyD88依赖性和TRIF依赖性途径,激活NF-κB和干扰素调节因子3(interferonregulatory factor3,IRF3),诱导抗病毒免疫分子的表达。此外,胞浆内的PRR如视黄酸诱导基因Ⅰ(retinoic acid-inducible gene I,RIG-I)和黑素瘤分化相关基因5(melanoma differentiation-associated gene 5,MDA5)等也能识别病毒PAMP如dsRNA,诱导IFN-β的表达。在TLRs和RIG-I样受体(RIG-I like receptors,RLRs)介导的固有免疫应答信号通路中均能激活TANK结合激酶1(TANK-bindingkinase-1,TBK1),表明其在机体固有免疫应答中起着至关重要的作用。通过激活TBK1,活化的TBK1能诱导IRF3二聚化并转入核内,从而增强Ⅰ型干扰素的表达,调节机体的固有免疫应答,并影响适应性免疫,有可能在慢性病毒感染如HBV和HCV感染的治疗中发挥一定的作用。
目的:
本研究以肝癌细胞株HepG2和整合HBV的肝癌细胞株HepG2.2.15为细胞模型,研究dsRNA诱导肝细胞表达IFN-β的信号转导特点;HBcAg真核表达质粒体外转染及其重组蛋白处理HepG2细胞,观察HBcAg对dsRNA诱导肝细胞表达IFN-β的调节作用;TBK1真核表达质粒体外转染HepG2细胞和HepG2.2.15细胞,观察TBK1的抗HBV作用。本研究以期为阐明肝细胞内抗病毒免疫机制和HBV慢性感染的分子机制,同时也为研制新型慢性乙肝治疗药物提供实验和理论依据。
方法:
1.HBcAg对dsRNA诱导肝细胞表达IFN-β的调节作用:
1.1 pcDNA3-HBc质粒的构建:以HBV ayw亚型全长质粒pCP10为模板,PCR扩增编码HBcAg的基因,将其插入真核表达质粒载体pcDNA3。
1.2 polyⅠ:C刺激HepG2细胞诱导IFN-β的表达:取对数生长期HepG2细胞,polyⅠ:C以0μg/ml、25μg/ml、50μg/ml、100μg/ml刺激细胞12h、24h,刺激后提取细胞总RNA,荧光定量PCR检测IFN-βmRNA表达。
1.3 pcDNA3-HBc转染HepG2细胞对polyⅠ:C诱导表达IFN-β的作用:取对数生长期HepG2细胞,将质粒pcDNA3-HBc以1.2μg转染细胞,培养24h后,100μg/ml polyⅠ:C刺激细胞24h,定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度。
1.4 HBcAg重组蛋白对polyⅠ:C诱导肝细胞表达IFN-β的调节作用:取对数生长期HepG2细胞,HBcAg重组蛋白以0μg/ml、10μg/ml处理细胞48h后,100μg/ml polyⅠ:C刺激细胞24h。定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度。
2 TBK1对dsRNA诱导肝细胞表达IFN-β的调节作用:
2.1 pcDNA3-hTBK1质粒的构建:以hTBK1 DNA为模板,PCR扩增目的基因,将其插入真核表达质粒载体pcDNA3。
2.2 pcDNA3-hTBK1转染Hela细胞对polyⅠ:C诱导表达IFN-β的作用:取对数生长期Hela细胞,将质粒pcDNA3-hTBK1以0μg、0.6μg、1.2μg、1.8μg转染细胞,培养24h后,100μg/ml polyⅠ:C刺激细胞24h,定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度。
2.3 pcDNA3-hTBK1转染HepG2细胞对polyⅠ:C诱导表达IFN-β的作用:取对数生长期HepG2细胞,将质粒pcDNA3-hTBK1以不同浓度转染细胞,培养24h后,100μg/ml polyⅠ:C刺激细胞24h,定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度。
2.4 polyⅠ:C刺激HepG2.2.15细胞诱导IFN-β的表达:取对数生长期HepG2.2.15细胞,polyⅠ:C以0μg/ml、25μg/ml、50μg/ml、100μg/ml刺激细胞24h,定量PCR检测IFN-β的表达;ELISA检测细胞上清中IFN-β浓度;时间分辨荧光免疫测定技术(TRFIA)检测细胞上清中HBsAg和HBeAg含量;定量PCR检测细胞上清和细胞中HBV DNA拷贝数。
2.5 pcDNA3-hTBK1转染HepG2.2.15细胞对polyⅠ:C诱导抗病毒作用的调节作用:取对数生长期HepG2.2.15细胞,将质粒pcDNA3-hTBK1以1.8μg转染细胞,培养24h后,polyⅠ:C以0μg/ml、25μg/ml、50μg/ml、100μg/ml刺激细胞24h,定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度;同时检测细胞上清中HBsAg和HBeAg含量及上清和细胞中HBV DNA拷贝数。
结果:
1.HBcAg对dsRNA诱导肝细胞表达IFN-β的调节作用:
1.1经EcoRⅠ和BamHⅠ酶切电泳鉴定,以及重组体插入片段的DNA序列测定,证实成功地构建了含C基因的真核表达质粒pcDNA3-HBc。
1.2 polyⅠ:C刺激HepG2细胞诱导IFN-β表达:polyⅠ:C刺激细胞12h,与对照组相比,刺激组IFN-β表达显著增高(P＜0.05),并随浓度增高而表达升高,各刺激组有显著差异(P＜0.05),同时polyⅠ:C刺激细胞24h较刺激12h组IFN-β表达显著增高(P＜0.05)。
1.3 pcDNA3-HBc转染HepG2细胞对polyⅠ:C诱导表达IFN-β的作用:与对照组相比,1.2μg转染组IFN-β表达有所降低,但无统计学差异(P＞0.05);与对照组相比,细胞上清中IFN-β浓度显著减少(P＜0.05)。
1.4 HBcAg重组蛋白对polyⅠ:C诱导肝细胞表达IFN-β的调节作用:与对照组相比,HBcAg重组蛋白处理组IFN-β表达和浓度均显著降低(P＜0.05)。
2.TBK1对dsRNA诱导肝细胞表达IFN-β的调节作用:
2.1经XbaⅠ和BamHⅠ酶切电泳鉴定,以及重组体插入片段的DNA序列测定,证实成功地构建了含hTBK1的真核表达质粒pcDNA3-hTBK1。
2.2 pcDNA3-hTBK1转染Hela细胞对polyⅠ:C诱导表达IFN-β的作用:与对照组和其他转染组相比,1.8μg转染组IFN-β表达显著增加;与对照组和0.6μg转染组相比,1.2μg转染组IFN-β表达也显著增加(P＜0.05);与对照组相比,各转染组IFN-β浓度均有增加,但无统计学差异(P＞0.05)。
2.3 pcDNA3-hTBK1转染HepG2细胞对polyⅠ:C诱导表达IFN-β的作用:随着转染量的增加,IFN-β表达显著增加,与对照组和0.6μg转染组相比,1.2μg、1.8μg转染组IFN-β表达显著增加,不同浓度转染组有显著差异(P＜0.05);与对照组相比,各转染组细胞上清IFN-β浓度均无显著差异(P＞0.05)。
2.4 polyⅠ:C刺激HepG2.2.15细胞诱导IFN-β的表达:与对照组相比,不同浓度刺激组IFN-β表达无显著差异(P＞0.05)。随着polyⅠ:C刺激浓度的增加,上清中IFN-β浓度呈梯度显著增加,不同浓度刺激组也存在显著差异(P＜0.05)。与对照组相比,不同浓度刺激组上清中HBsAg和HBeAg含量、及上清和细胞中HBV DNA拷贝数均无显著差异(P＞0.05)。
2.5 pcDNA3-hTBK1转染HepG2.2.15细胞对polyⅠ:C诱导抗病毒作用的调节作用:与对照组和25μg/ml刺激组相比,100μg/ml polyⅠ:C刺激组IFN-β表达显著增加(P＜0.05);与对照组相比,各刺激组上清中IFN-β浓度均无显著差异(P＞0.05)。与对照组相比,50μg/ml和100μg/ml polyⅠ:C刺激组HBsAg表达显著降低(P＜0.05),不同浓度刺激组HBeAg表达无显著差异(P＞0.05);与对照组相比,不同浓度刺激组上清HBV DNA拷贝数均有显著下降(P＜0.05),各组则无显著差异(P＞0.05);不同浓度刺激组细胞HBV DNA拷贝数无显著差异(P＞0.05)。
结论:
polyⅠ:C刺激HepG2和HepG2.2.15细胞可诱导IFN-β表达,提示肝细胞可能在抗病毒固有性免疫应答中起重要的作用。HBcAg真核表达质粒体外转染及其重组蛋白处理可抑制polyⅠ:C诱导HepG2细胞表达IFN-β,提示HBcAg具有拮抗机体抗病毒固有免疫的作用。hTBK1真核表达质粒转染HepG2细胞和HepG2.2.15细胞,可促进polyⅠ:C诱导HepG2细胞和HepG2.2.15细胞表达IFN-β,同时一定浓度的hTBK1转染可有效降低HepG2.2.15细胞分泌HBsAg及HBV DNA拷贝数,提示TBK1可能通过增强固有免疫信号转导途径,增加细胞因子如Ⅰ型干扰素(IFN-β)的表达,进而对HBV起到一定抑制作用。结果提示,病毒感染时肝细胞可产生一定的固有免疫应答,发挥抗病毒作用,而HBV可通过表达病毒蛋白HBcAg拮抗机体的抗病毒固有免疫应答,同时促进TBK1的表达和激活能提高肝细胞的固有免疫应答。研究结果有助于了解肝细胞在抗HBV感染固有免疫应答中的作用以及HBV慢性感染的分子机制,同时也为研制新的慢性乙肝治疗药物提供新的思路和策略。
Hepatitis B viruses frequently establish persistent infection and cause chronic hepatitis and its serious complications,including cirrhosis and hepatocellular carcinoma. The mechanism involved in the pathogenesis of the chronic hepatitis is not elucidated and one of it is down-regulation of the immune response by viral protein encoded by HBV.With the recently advances in the research of the innate immune,the innate immunity initiated by HBV infection and the down-regulation of HBV protein received increasing attention.The innate immunity agonist may be applicated in treatment of chronic HBV infection.
The innate immune system plays an important role in viral infection.Pattern recognition receptor(PRR) molecule that expressed on immune cells senses invading viral pathogens by recognition of pathogen-associated molecular pattern(PAMP) and initiates a series of signaling pathways leading to the induction of protective cellular genes,including typeⅠinterferon(IFN-α/β) and proinflammatory cytokines,then plays the antiviral innate immunity effect and also help to shape subsequent adaptive immune responses.Toll-like receptors(TLRs),as the representative of the PRR,such as TLR3,TLR7,TLR8 and TLR9 have been shown to detect infection by viruses.TLR3 recognize viral double-stranded RNA analogue polyinosinic-poly cytosine nucleotide (poly I:C).After binding with the ligand specifically,TLRs signal through two pathways, MyD88-dependent and TRIF-dependent pathway activate NF-κB and IRF-3,and induce the expression of antiviral immune effectors.In addition,the cytoplasm of the PRR, such as RIG-I and MDA5 were also able to identify the viral PAMP,such as dsRNA, and then induce the expression of IFN-β.The innate immune response signaling pathways mediated by TLRs and RLRs both activate the TANK binding kinase 1 (TBK1),which plays an important role in the innate immunity response.After activation of TBK1,IRF3 is activated and transferred to the nucleus,thereby enhancing the expression of typeⅠinterferon to regulate the innate immune response,and influence the adaptive immune.TBK1 may acts as potential agent in therapeutic application in chronic viral infection such as HBV and HCV.
Objective:
To investigate the regulation of HBcAg and TBK1 on innate immunity of hepatocytes,the expression of IFN-βinduced by dsRNA in hepatoma cell line HepG2 and HBV replication cell line HepG2.2.15 was tested.To investigate the regulation of HBcAg,HepG2 cells were transfected with eukaryotic plasmid expressing HBcAg and treated with HBcAg recombinant protein.To investigate the regulation of TBK1,we transfect eukaryotic plasmid expressing TBK1 into HepG2 and HepG2.2.15 cells.The results would be helpful to clarify the antiviral immunity in hepatocytes,and unraveling the mechanism of chronic HBV infection,and might provide new ideas and strategies for the development of treatments for chronic hepatitis.
Methods:
1.The regulation of HBcAg in production of IFN-βinduced by dsRNA in hepatocytes.
1.1 Construction of pcDNA3-HBc plasmid:the plasmid pCP10 containing the HBV ayw subtype full-length as template,the gene encoding HBcAg was obtained by PCR and was inserted into plasmid pcDNA3.
1.2 The expression of IFN-βinduced by poly I:C in HepG2 cells:HepG2 cells were stimulated by poly I:C at 0μg/ml,25μg/ml,50μg/ml,100μg/ml for 12 hours,24 hours, then the expression of IFN-βmRNA was assayed by real time PCR.
1.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-HBc:HepG2 cells were transfected with 1.2μg pcDNA3-HBc for 24 hours, poly I:C was added to the medium at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.
1.4 The regulation of HBcAg recombinant protein in production of IFN-βinduced by poly I:C in hepatocytes:HepG2 cells were treated with HBcAg recombinant protein at 0μg/ml,10μg/ml for 48 hours and poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.
2.The regulation of TBK1 in production of IFN-βinduced by dsRNA in hepatocytes.
2.1 Construction of pcDNA3-hTBK1 plasmid:The hTBK1 DNA as template,the gene encoding the human TBK1 was amplified by PCR and was inserted into plasmid pcDNA3.
2.2 The expression of IFN-βinduced by poly I:C in Hela cells transfected with pcDNA3-hTBK1:Hela cells were transfected with pcDNA3-hTBK at 10μg,0.6μg, 1.2μg,1.8μg for 24 hours,poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.
2.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-hTBK1:HepG2 cells were transfected with different concentrations of pcDNA3-hTBK1 for 24 hours,poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.
2.4 The expression of IFN-βinduced by poly I:C in HepG2.2.15 cells:HepG2.2.15 cells were stimulated by poly I:C at 0μg/ml,25μg/ml,50μg,/ml,100μg/ml for 24 hours,and the expression of IFN-βwas assayed by real time PCR and ELISA,the production of HBsAg,HBeAg and HBV DNA was detected by time-resolved fluoroimmunoassay (TRFIA) and real time PCR respectively.
2.5 The regulation of antiviral effect induced by poly I:C in HepG2.2.15 cells transfected with pcDNA3-hTBK1:HepG2.2.15 cells were transfected with 1.8μg pcDNA3-hTBK1 for 24 hours,and was stimulated by poly I:C at 0μg/ml,25μg/ml, 50μg/ml,100μg/ml for 24 hours,the expression of IFN-βwas assayed by real time PCR and ELISA,the production of HBsAg,HBeAg and HBV DNA was detected by TRFIA and real time PCR respectively.
Result:
1.The regulation of HBcAg in production of IFN-βinduced by dsRNA in hepatocytes.
1.1 Plasmid expressing HBcAg pcDNA3-HBc was identified by Agarose gel electrophoresis of the plasmid after enzyme EcoRⅠand BamHⅠdigested,and sequencing.
1.2 Compared with the control,the expression of IFN-βinduced by poly I:C in HepG2 cells was significantly increased by poly I:C stimulation for 12 hours.With the increased concentration,the expression increased and there are significantly difference among different groups(P＜0.05).The expression of IFN-βinducted by poly I:C for 24 hours was significantly higher than 12 hours(P＜0.05).
1.3 Compared with the control,there is no significantly difference in the expression of IFN-βinduced by poly I:C in HepG2 cells transfected with 1.2μg pcDNA3-HBc(P＞0.05),while the concentration of IFN-βwere significantly decreased(P＜0.05).
1.4 Compared with the control,the expression and concentration of IFN-βtreated with HBcAg recombinant protein were significantly decreased(P＜0.05).
2.The regulation of TBK1 in production of IFN-βinduced by dsRNA in hepatocytes.
2.1 Plasmid expressing HBcAg pcDNA3-hTBK1was identified by Agarose gel electrophoresis of the plasmid after enzyme XbaⅠand BamHⅠdigested,and sequencing.
2.2 Compared with the control and other groups,the expression of IFN-βinduced by poly I:C in Hela cells transfected with 1.8μg pcDNA3-hTBK1 increased significantly. Compared with the control and cells transfected with 0.6μg pcDNA3-hTBK1,the expression also increased significantly in cells transfected with 1.2μg pcDNA3-hTBK1 (P＜0.05).There is no significantly difference in the concentration of IFN-βin the different group(P＞0.05).
2.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-hTBK1 was significantly increased with the increased transfection except the control and low concentration(0.6μg).Compared with the control,there is no significantly difference among different groups in the concentration of IFN-βin supernatant(P＞0.05).
2.4 The expression of IFN-βinduced by poly I:C in HepG2.2.15 cells,compared with the control,there was no significantly difference among different groups(P＞0.05) while the concentration of IFN-βin supernatant was significantly increased among different groups(P＜0.05).Compared with the control,there is no significantly difference among different groups in the production of HBsAg and HBeAg,and HBV DNA in supertanant and cells(P＞0.05).
2.5 Compared with the control and 25μg/ml stimulation group,the expression of IFN-βincreased significantly by poly I:C stimulated as 100μg/ml(P＜0.05);compared with the control,there are no significantly difference among different groups in the concentration of IFN-β(P＞0.05).Compared with the control,the production of HBsAg significantly decreased in supernatant of cells stimulated by polyI:C at 50μg/ml and 100μg/ml(P＜0.05) while there are no significantly difference among different groups in the expression of HBeAg(P＞0.05).Compared with the control,HBV DNA in supernatant decreased significantly with poly I:C stimulation(P＜0.05),while there are no significantly difference among different groups(P＞0.05);and there is no significantly difference of HBV DNA in cells among different groups(P＞0.05).
Conclusion:
Poly I:C induces the expression of IFN-βin HepG2,HepG2.2.15 cells,suggesting that hepatocytes may play an important role in antiviral innate immune response. HepG2 cells transfected with eukaryotic plasmid expressing HBcAg,and directly treated with HBcAg recombinant protein,the expression of IFN-βinduced by poly I:C is down-regulated,suggesting that HBcAg is an antagonist antiviral innate immune response.HepG2 and HepG2.2.15 cells transfected with eukaryotic plasmid expressing hTBK1,the expression of IFN-βinduced by poly I:C is increased in HepG2 and HepG2.2.15 cells,at the same time,a certain concentration of hTBK1 can effectively inhibit the expression of virus protein HBsAg and the viral loads of HBV DNA in HepG2.2.15 cells,suggesting that TBK1 may inhibit the HBV replication by increasing the expression cytokines such as typeⅠinterferon(IFN-β) and other effectors.The results show that hepatocytes play a role in innate immune response during viral infection,while HBV can anti-host innate immune response by expressing viral protein HBcAg.Innate immunity effector signaling molecule such as TBK1 may enhance innate immune response.The result is helpful in unraveling the role of hepatocytes in innate immune response against HBV infection and the pathogenesis of persistent HBV infection,also might lead to design of novel therapeutic interventions in future.
固有性免疫(innate immunity)应答是机体快速抵抗微生物病原体的重要机制。表达于免疫细胞的模式识别受体(pattern recognition receptor,PRR)通过识别病原相关分子模式(pathogen-associated molecular pattern,PAMP),启动一系列信号转导途径,诱导保护性细胞因子如Ⅰ型干扰素(IFN-α/β)和前炎症细胞因子的表达,发挥抗病毒固有免疫作用,并调节适应性免疫(adaptive immunity)应答。以Toll样受体(Toll-like receptor,TLR)为代表的PRR是抗病毒固有免疫的关键性识别结构,TLR3,TLR7,TLR8和TLR9主要与病毒的识别有关,如TLR3识别病毒双链RNA(double stranded RNA,dsRNA)类似物聚肌苷-聚胞嘧啶核苷酸(polyinosinic-poly cytosine nucleotide,polyⅠ:C)。TLRs与特异性配体结合后,通过MyD88依赖性和TRIF依赖性途径,激活NF-κB和干扰素调节因子3(interferonregulatory factor3,IRF3),诱导抗病毒免疫分子的表达。此外,胞浆内的PRR如视黄酸诱导基因Ⅰ(retinoic acid-inducible gene I,RIG-I)和黑素瘤分化相关基因5(melanoma differentiation-associated gene 5,MDA5)等也能识别病毒PAMP如dsRNA,诱导IFN-β的表达。在TLRs和RIG-I样受体(RIG-I like receptors,RLRs)介导的固有免疫应答信号通路中均能激活TANK结合激酶1(TANK-bindingkinase-1,TBK1),表明其在机体固有免疫应答中起着至关重要的作用。通过激活TBK1,活化的TBK1能诱导IRF3二聚化并转入核内,从而增强Ⅰ型干扰素的表达,调节机体的固有免疫应答,并影响适应性免疫,有可能在慢性病毒感染如HBV和HCV感染的治疗中发挥一定的作用。
目的:
本研究以肝癌细胞株HepG2和整合HBV的肝癌细胞株HepG2.2.15为细胞模型,研究dsRNA诱导肝细胞表达IFN-β的信号转导特点;HBcAg真核表达质粒体外转染及其重组蛋白处理HepG2细胞,观察HBcAg对dsRNA诱导肝细胞表达IFN-β的调节作用;TBK1真核表达质粒体外转染HepG2细胞和HepG2.2.15细胞,观察TBK1的抗HBV作用。本研究以期为阐明肝细胞内抗病毒免疫机制和HBV慢性感染的分子机制,同时也为研制新型慢性乙肝治疗药物提供实验和理论依据。
方法:
1.HBcAg对dsRNA诱导肝细胞表达IFN-β的调节作用:
1.1 pcDNA3-HBc质粒的构建:以HBV ayw亚型全长质粒pCP10为模板,PCR扩增编码HBcAg的基因,将其插入真核表达质粒载体pcDNA3。
1.2 polyⅠ:C刺激HepG2细胞诱导IFN-β的表达:取对数生长期HepG2细胞,polyⅠ:C以0μg/ml、25μg/ml、50μg/ml、100μg/ml刺激细胞12h、24h,刺激后提取细胞总RNA,荧光定量PCR检测IFN-βmRNA表达。
1.3 pcDNA3-HBc转染HepG2细胞对polyⅠ:C诱导表达IFN-β的作用:取对数生长期HepG2细胞,将质粒pcDNA3-HBc以1.2μg转染细胞,培养24h后,100μg/ml polyⅠ:C刺激细胞24h,定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度。
1.4 HBcAg重组蛋白对polyⅠ:C诱导肝细胞表达IFN-β的调节作用:取对数生长期HepG2细胞,HBcAg重组蛋白以0μg/ml、10μg/ml处理细胞48h后,100μg/ml polyⅠ:C刺激细胞24h。定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度。
2 TBK1对dsRNA诱导肝细胞表达IFN-β的调节作用:
2.1 pcDNA3-hTBK1质粒的构建:以hTBK1 DNA为模板,PCR扩增目的基因,将其插入真核表达质粒载体pcDNA3。
2.2 pcDNA3-hTBK1转染Hela细胞对polyⅠ:C诱导表达IFN-β的作用:取对数生长期Hela细胞,将质粒pcDNA3-hTBK1以0μg、0.6μg、1.2μg、1.8μg转染细胞,培养24h后,100μg/ml polyⅠ:C刺激细胞24h,定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度。
2.3 pcDNA3-hTBK1转染HepG2细胞对polyⅠ:C诱导表达IFN-β的作用:取对数生长期HepG2细胞,将质粒pcDNA3-hTBK1以不同浓度转染细胞,培养24h后,100μg/ml polyⅠ:C刺激细胞24h,定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度。
2.4 polyⅠ:C刺激HepG2.2.15细胞诱导IFN-β的表达:取对数生长期HepG2.2.15细胞,polyⅠ:C以0μg/ml、25μg/ml、50μg/ml、100μg/ml刺激细胞24h,定量PCR检测IFN-β的表达;ELISA检测细胞上清中IFN-β浓度;时间分辨荧光免疫测定技术(TRFIA)检测细胞上清中HBsAg和HBeAg含量;定量PCR检测细胞上清和细胞中HBV DNA拷贝数。
2.5 pcDNA3-hTBK1转染HepG2.2.15细胞对polyⅠ:C诱导抗病毒作用的调节作用:取对数生长期HepG2.2.15细胞,将质粒pcDNA3-hTBK1以1.8μg转染细胞,培养24h后,polyⅠ:C以0μg/ml、25μg/ml、50μg/ml、100μg/ml刺激细胞24h,定量PCR检测IFN-βmRNA表达;ELISA检测细胞上清中IFN-β浓度;同时检测细胞上清中HBsAg和HBeAg含量及上清和细胞中HBV DNA拷贝数。
结果:
1.HBcAg对dsRNA诱导肝细胞表达IFN-β的调节作用:
1.1经EcoRⅠ和BamHⅠ酶切电泳鉴定,以及重组体插入片段的DNA序列测定,证实成功地构建了含C基因的真核表达质粒pcDNA3-HBc。
1.2 polyⅠ:C刺激HepG2细胞诱导IFN-β表达:polyⅠ:C刺激细胞12h,与对照组相比,刺激组IFN-β表达显著增高(P＜0.05),并随浓度增高而表达升高,各刺激组有显著差异(P＜0.05),同时polyⅠ:C刺激细胞24h较刺激12h组IFN-β表达显著增高(P＜0.05)。
1.3 pcDNA3-HBc转染HepG2细胞对polyⅠ:C诱导表达IFN-β的作用:与对照组相比,1.2μg转染组IFN-β表达有所降低,但无统计学差异(P＞0.05);与对照组相比,细胞上清中IFN-β浓度显著减少(P＜0.05)。
1.4 HBcAg重组蛋白对polyⅠ:C诱导肝细胞表达IFN-β的调节作用:与对照组相比,HBcAg重组蛋白处理组IFN-β表达和浓度均显著降低(P＜0.05)。
2.TBK1对dsRNA诱导肝细胞表达IFN-β的调节作用:
2.1经XbaⅠ和BamHⅠ酶切电泳鉴定,以及重组体插入片段的DNA序列测定,证实成功地构建了含hTBK1的真核表达质粒pcDNA3-hTBK1。
2.2 pcDNA3-hTBK1转染Hela细胞对polyⅠ:C诱导表达IFN-β的作用:与对照组和其他转染组相比,1.8μg转染组IFN-β表达显著增加;与对照组和0.6μg转染组相比,1.2μg转染组IFN-β表达也显著增加(P＜0.05);与对照组相比,各转染组IFN-β浓度均有增加,但无统计学差异(P＞0.05)。
2.3 pcDNA3-hTBK1转染HepG2细胞对polyⅠ:C诱导表达IFN-β的作用:随着转染量的增加,IFN-β表达显著增加,与对照组和0.6μg转染组相比,1.2μg、1.8μg转染组IFN-β表达显著增加,不同浓度转染组有显著差异(P＜0.05);与对照组相比,各转染组细胞上清IFN-β浓度均无显著差异(P＞0.05)。
2.4 polyⅠ:C刺激HepG2.2.15细胞诱导IFN-β的表达:与对照组相比,不同浓度刺激组IFN-β表达无显著差异(P＞0.05)。随着polyⅠ:C刺激浓度的增加,上清中IFN-β浓度呈梯度显著增加,不同浓度刺激组也存在显著差异(P＜0.05)。与对照组相比,不同浓度刺激组上清中HBsAg和HBeAg含量、及上清和细胞中HBV DNA拷贝数均无显著差异(P＞0.05)。
2.5 pcDNA3-hTBK1转染HepG2.2.15细胞对polyⅠ:C诱导抗病毒作用的调节作用:与对照组和25μg/ml刺激组相比,100μg/ml polyⅠ:C刺激组IFN-β表达显著增加(P＜0.05);与对照组相比,各刺激组上清中IFN-β浓度均无显著差异(P＞0.05)。与对照组相比,50μg/ml和100μg/ml polyⅠ:C刺激组HBsAg表达显著降低(P＜0.05),不同浓度刺激组HBeAg表达无显著差异(P＞0.05);与对照组相比,不同浓度刺激组上清HBV DNA拷贝数均有显著下降(P＜0.05),各组则无显著差异(P＞0.05);不同浓度刺激组细胞HBV DNA拷贝数无显著差异(P＞0.05)。
结论:
polyⅠ:C刺激HepG2和HepG2.2.15细胞可诱导IFN-β表达,提示肝细胞可能在抗病毒固有性免疫应答中起重要的作用。HBcAg真核表达质粒体外转染及其重组蛋白处理可抑制polyⅠ:C诱导HepG2细胞表达IFN-β,提示HBcAg具有拮抗机体抗病毒固有免疫的作用。hTBK1真核表达质粒转染HepG2细胞和HepG2.2.15细胞,可促进polyⅠ:C诱导HepG2细胞和HepG2.2.15细胞表达IFN-β,同时一定浓度的hTBK1转染可有效降低HepG2.2.15细胞分泌HBsAg及HBV DNA拷贝数,提示TBK1可能通过增强固有免疫信号转导途径,增加细胞因子如Ⅰ型干扰素(IFN-β)的表达,进而对HBV起到一定抑制作用。结果提示,病毒感染时肝细胞可产生一定的固有免疫应答,发挥抗病毒作用,而HBV可通过表达病毒蛋白HBcAg拮抗机体的抗病毒固有免疫应答,同时促进TBK1的表达和激活能提高肝细胞的固有免疫应答。研究结果有助于了解肝细胞在抗HBV感染固有免疫应答中的作用以及HBV慢性感染的分子机制,同时也为研制新的慢性乙肝治疗药物提供新的思路和策略。
Hepatitis B viruses frequently establish persistent infection and cause chronic hepatitis and its serious complications,including cirrhosis and hepatocellular carcinoma. The mechanism involved in the pathogenesis of the chronic hepatitis is not elucidated and one of it is down-regulation of the immune response by viral protein encoded by HBV.With the recently advances in the research of the innate immune,the innate immunity initiated by HBV infection and the down-regulation of HBV protein received increasing attention.The innate immunity agonist may be applicated in treatment of chronic HBV infection.
The innate immune system plays an important role in viral infection.Pattern recognition receptor(PRR) molecule that expressed on immune cells senses invading viral pathogens by recognition of pathogen-associated molecular pattern(PAMP) and initiates a series of signaling pathways leading to the induction of protective cellular genes,including typeⅠinterferon(IFN-α/β) and proinflammatory cytokines,then plays the antiviral innate immunity effect and also help to shape subsequent adaptive immune responses.Toll-like receptors(TLRs),as the representative of the PRR,such as TLR3,TLR7,TLR8 and TLR9 have been shown to detect infection by viruses.TLR3 recognize viral double-stranded RNA analogue polyinosinic-poly cytosine nucleotide (poly I:C).After binding with the ligand specifically,TLRs signal through two pathways, MyD88-dependent and TRIF-dependent pathway activate NF-κB and IRF-3,and induce the expression of antiviral immune effectors.In addition,the cytoplasm of the PRR, such as RIG-I and MDA5 were also able to identify the viral PAMP,such as dsRNA, and then induce the expression of IFN-β.The innate immune response signaling pathways mediated by TLRs and RLRs both activate the TANK binding kinase 1 (TBK1),which plays an important role in the innate immunity response.After activation of TBK1,IRF3 is activated and transferred to the nucleus,thereby enhancing the expression of typeⅠinterferon to regulate the innate immune response,and influence the adaptive immune.TBK1 may acts as potential agent in therapeutic application in chronic viral infection such as HBV and HCV.
Objective:
To investigate the regulation of HBcAg and TBK1 on innate immunity of hepatocytes,the expression of IFN-βinduced by dsRNA in hepatoma cell line HepG2 and HBV replication cell line HepG2.2.15 was tested.To investigate the regulation of HBcAg,HepG2 cells were transfected with eukaryotic plasmid expressing HBcAg and treated with HBcAg recombinant protein.To investigate the regulation of TBK1,we transfect eukaryotic plasmid expressing TBK1 into HepG2 and HepG2.2.15 cells.The results would be helpful to clarify the antiviral immunity in hepatocytes,and unraveling the mechanism of chronic HBV infection,and might provide new ideas and strategies for the development of treatments for chronic hepatitis.
Methods:
1.The regulation of HBcAg in production of IFN-βinduced by dsRNA in hepatocytes.
1.1 Construction of pcDNA3-HBc plasmid:the plasmid pCP10 containing the HBV ayw subtype full-length as template,the gene encoding HBcAg was obtained by PCR and was inserted into plasmid pcDNA3.
1.2 The expression of IFN-βinduced by poly I:C in HepG2 cells:HepG2 cells were stimulated by poly I:C at 0μg/ml,25μg/ml,50μg/ml,100μg/ml for 12 hours,24 hours, then the expression of IFN-βmRNA was assayed by real time PCR.
1.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-HBc:HepG2 cells were transfected with 1.2μg pcDNA3-HBc for 24 hours, poly I:C was added to the medium at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.
1.4 The regulation of HBcAg recombinant protein in production of IFN-βinduced by poly I:C in hepatocytes:HepG2 cells were treated with HBcAg recombinant protein at 0μg/ml,10μg/ml for 48 hours and poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.
2.The regulation of TBK1 in production of IFN-βinduced by dsRNA in hepatocytes.
2.1 Construction of pcDNA3-hTBK1 plasmid:The hTBK1 DNA as template,the gene encoding the human TBK1 was amplified by PCR and was inserted into plasmid pcDNA3.
2.2 The expression of IFN-βinduced by poly I:C in Hela cells transfected with pcDNA3-hTBK1:Hela cells were transfected with pcDNA3-hTBK at 10μg,0.6μg, 1.2μg,1.8μg for 24 hours,poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.
2.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-hTBK1:HepG2 cells were transfected with different concentrations of pcDNA3-hTBK1 for 24 hours,poly I:C was added at 100μg/ml for 24 hours,then the expression of IFN-βwas assayed by real time PCR and ELISA.
2.4 The expression of IFN-βinduced by poly I:C in HepG2.2.15 cells:HepG2.2.15 cells were stimulated by poly I:C at 0μg/ml,25μg/ml,50μg,/ml,100μg/ml for 24 hours,and the expression of IFN-βwas assayed by real time PCR and ELISA,the production of HBsAg,HBeAg and HBV DNA was detected by time-resolved fluoroimmunoassay (TRFIA) and real time PCR respectively.
2.5 The regulation of antiviral effect induced by poly I:C in HepG2.2.15 cells transfected with pcDNA3-hTBK1:HepG2.2.15 cells were transfected with 1.8μg pcDNA3-hTBK1 for 24 hours,and was stimulated by poly I:C at 0μg/ml,25μg/ml, 50μg/ml,100μg/ml for 24 hours,the expression of IFN-βwas assayed by real time PCR and ELISA,the production of HBsAg,HBeAg and HBV DNA was detected by TRFIA and real time PCR respectively.
Result:
1.The regulation of HBcAg in production of IFN-βinduced by dsRNA in hepatocytes.
1.1 Plasmid expressing HBcAg pcDNA3-HBc was identified by Agarose gel electrophoresis of the plasmid after enzyme EcoRⅠand BamHⅠdigested,and sequencing.
1.2 Compared with the control,the expression of IFN-βinduced by poly I:C in HepG2 cells was significantly increased by poly I:C stimulation for 12 hours.With the increased concentration,the expression increased and there are significantly difference among different groups(P＜0.05).The expression of IFN-βinducted by poly I:C for 24 hours was significantly higher than 12 hours(P＜0.05).
1.3 Compared with the control,there is no significantly difference in the expression of IFN-βinduced by poly I:C in HepG2 cells transfected with 1.2μg pcDNA3-HBc(P＞0.05),while the concentration of IFN-βwere significantly decreased(P＜0.05).
1.4 Compared with the control,the expression and concentration of IFN-βtreated with HBcAg recombinant protein were significantly decreased(P＜0.05).
2.The regulation of TBK1 in production of IFN-βinduced by dsRNA in hepatocytes.
2.1 Plasmid expressing HBcAg pcDNA3-hTBK1was identified by Agarose gel electrophoresis of the plasmid after enzyme XbaⅠand BamHⅠdigested,and sequencing.
2.2 Compared with the control and other groups,the expression of IFN-βinduced by poly I:C in Hela cells transfected with 1.8μg pcDNA3-hTBK1 increased significantly. Compared with the control and cells transfected with 0.6μg pcDNA3-hTBK1,the expression also increased significantly in cells transfected with 1.2μg pcDNA3-hTBK1 (P＜0.05).There is no significantly difference in the concentration of IFN-βin the different group(P＞0.05).
2.3 The expression of IFN-βinduced by poly I:C in HepG2 cells transfected with pcDNA3-hTBK1 was significantly increased with the increased transfection except the control and low concentration(0.6μg).Compared with the control,there is no significantly difference among different groups in the concentration of IFN-βin supernatant(P＞0.05).
2.4 The expression of IFN-βinduced by poly I:C in HepG2.2.15 cells,compared with the control,there was no significantly difference among different groups(P＞0.05) while the concentration of IFN-βin supernatant was significantly increased among different groups(P＜0.05).Compared with the control,there is no significantly difference among different groups in the production of HBsAg and HBeAg,and HBV DNA in supertanant and cells(P＞0.05).
2.5 Compared with the control and 25μg/ml stimulation group,the expression of IFN-βincreased significantly by poly I:C stimulated as 100μg/ml(P＜0.05);compared with the control,there are no significantly difference among different groups in the concentration of IFN-β(P＞0.05).Compared with the control,the production of HBsAg significantly decreased in supernatant of cells stimulated by polyI:C at 50μg/ml and 100μg/ml(P＜0.05) while there are no significantly difference among different groups in the expression of HBeAg(P＞0.05).Compared with the control,HBV DNA in supernatant decreased significantly with poly I:C stimulation(P＜0.05),while there are no significantly difference among different groups(P＞0.05);and there is no significantly difference of HBV DNA in cells among different groups(P＞0.05).
Conclusion:
Poly I:C induces the expression of IFN-βin HepG2,HepG2.2.15 cells,suggesting that hepatocytes may play an important role in antiviral innate immune response. HepG2 cells transfected with eukaryotic plasmid expressing HBcAg,and directly treated with HBcAg recombinant protein,the expression of IFN-βinduced by poly I:C is down-regulated,suggesting that HBcAg is an antagonist antiviral innate immune response.HepG2 and HepG2.2.15 cells transfected with eukaryotic plasmid expressing hTBK1,the expression of IFN-βinduced by poly I:C is increased in HepG2 and HepG2.2.15 cells,at the same time,a certain concentration of hTBK1 can effectively inhibit the expression of virus protein HBsAg and the viral loads of HBV DNA in HepG2.2.15 cells,suggesting that TBK1 may inhibit the HBV replication by increasing the expression cytokines such as typeⅠinterferon(IFN-β) and other effectors.The results show that hepatocytes play a role in innate immune response during viral infection,while HBV can anti-host innate immune response by expressing viral protein HBcAg.Innate immunity effector signaling molecule such as TBK1 may enhance innate immune response.The result is helpful in unraveling the role of hepatocytes in innate immune response against HBV infection and the pathogenesis of persistent HBV infection,also might lead to design of novel therapeutic interventions in future.
引文
[1] Ganem D, Prince AM. Hepatitis B virus infection-natural history and clinical consequences. N Engl J Med, 2004, 350(11):1118-1129.
[2] Parker GA, Picut CA. Liver Immunobiology. Toxi Path, 2005, 33:52-62.
[3] Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol, 2005, 5:215-229.
[4] Crispe IN. Hepatic T cells and liver tolerance. Nat Rev Immunol, 2003, 3:51-62.
[5] Livngston BD, Crimi C, Fikes J, et al. Immunization with the HBV core 18-27 epitope elicits CTL responses in humans expressing different HLA-A2 supertype molecules. Hum Immunol, 1999, 60:1013-1017.
[6] Lazdina U, Cao T, Steinbergs J, et al. Molecular basis for the interaction of the hepatitis B virus core antigen with the surface immunoglobulin receptor on naive B cells. J Virol, 2001, 75:6367-6374.
[7] Twu JS, Schloemer RN. Transciption of human beta interferon gene is inhibitied by hepatitis B virus. J Virol, 1989, 63:3065-3071.
[8] Whitten TM, Quets AT, Schloemer RH. Identification of hepatitis B virus factor that inhibits expression of the beta interferon gene. J Virol, 1991, 65:4699-4704.
[9] Janssens S, Beyaert R. Role of toll-like receptors in pathogen recognition. Clin Microbiol, 2003,16:637-646.
[10] Alexopoulou L, Holt AC, Medzhitov R, et al. Recognition of double-stranded RNA and activation of NF-kappaB by Toll like receptor 3. Nature, 2001,413(6857):732-738.
[11] Diebold SS, Kaisho T, Hemmi H, et al. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science, 2004, 303(5663): 1529-1531.
[12] Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 2004, 303(5663):1526-1529.
[13]Ahmad-Nejad P,Hacker H,Rutz M,et al.Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments.Eur J Immunol,2004,32:1958-1968.
[14]Kawei T,Akira S.TLR signaling.Cell Death Differ,2006,13(5):816-825.
[15]Akira S,Takeda K.Toll-like receptor signalling.Nat Rev Immunol,2004,4(7):499-511.
[16]Meylan E,Bums K,Hofmann K,et al.RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation.Nat Immunol,2004,5(5):503-507.
[17]Yoneyama M,Kikuchi M,Natsukawa T,et al.The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat Immunol,2004,5:730-737.
[18]Beignon AS,Skoberne M,Bhardwaj N.Type Ⅰ interferons promote cross-priming:more functions for old cytokines.Nat Immunol,2003,4:939-941.
[19]Iwasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune responses.Nat Immunol,2004,5:987-995.
[20]Hoebe K,Janssen E,Beutler B.The interface between innate and adaptive immunity.Nat Immunol,2004,5:971-974.
[21]Wu J,Lu M,Meng Z,et al.Toll-like receptor-mediated control of HBV replication by nonparenchymal liver cells in mice.Hepatology,2007,46(6):1769-1778.
[22]Isogawa M,Robek MD,Furuich Y,et al.Toll-like receptor signaling inhibits hepatitis B virus replication in vivo.J Virol,2005,79(11):7269-7272.
[23]Xia C,Lu M,Zhang Z,et al.TLRs antiviral effect on hepatitis B virus in HepG2cells.J Appl Microbiol,2008,105(5):1720-1727.
[24]Murray JM,Wieland SF,Purcell RH,Chisari FV.Dynamics of hepatitis B virus clearance in chimpanzees.Proe Natl Acad Sci USA 2005;102:17780-17785.
[25]Bigger CB,Brasky KM & Lanford RE.DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection.J Virol 2001;75:7059-7066.
[26]Li K,Chen Z,Kato N,et al.Distinct poly(I:C) and virus-activated signaling path-ways leading to interferon-beta production in hepatocytes.J Biol Chem,2005,280(17):16739-16747.
[27]Preiss S,Thompson A,Chen X,et al.Characterization of the innate immune signalling pathways in hepatocyte cell lines.J Viral Hepat,2008,15(12):888-900.
[28]陈皴琼.病毒PAMP诱导肝细胞表达IFN-β信号转导途径的研究.浙江大学硕士学位论文,2007年5月.
[29]Wu J,Meng J,Jiang M,et al.Hepatitis B Virus suppresses Toll-like receptor-Mediated innate immune responses in murine parenchymal and nonparenchymal liver cells.Hepatology,2009,49(4):1132-1140.
[30]Makkouk A,Abdelnoor Am.The potential use of TLR agonists and antagonists as prophylactic and/or therapeutic agents.Immunopharmocol immunotoxicol,2009,31(3):331-338.
[1]Kumagai Y,Takeuchi O,Akira S.Pathogen recognition by innate receptors.J Infect Chemother,2008,14(2):86-92.
[2]Hoshino K,Takeuchi O,Kawai T,et al.Cutting edge:Toll-like receptor 4(TLR4)-deficient mice are hyporesponsive to lipopolysaccharide:evidence for TLR4 as the Lps gene product.J Immunol,1999,162(7):3749-3752.
[3]Alexopoulou L,Holt AC,Medzhitov R,et al.Recognition of double-stranded RNA and activation of NF-kappaB by Toll like receptor 3.Nature,2001,413(6857):732-738.
[4]Diebold SS,Kaisho T,Hemmi H,et al.Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.Science,2004,303(5663):1529-1531.
[5]Heil F,Hemmi H,Hochrein H,et al.Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.Science,2004,303(5663):1526-1529.
[6]Kawei T,Akira S.TLR signaling.Cell Death Differ,2006,13(5):816-825.
[7]Akira S,Takeda K.Toll-like receptor signalling.Nat Rev Immunol,2004,4(7):499-511.
[8]Meylan E,Bums K,Hofmann K,et al.RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation.Nat Immunol,2004,5(5):503-507.
[9]Nembrini C,Abel B,KopfM,et al.Strong TCR signaling,TLR ligands,and cytokine redundancies ensure robust development of type 1 effector T cells.J Immunol,2006,176(12):7180-7188.
[10]Isogawa M,Robek MD,Furuich Y,et al.Toll-like receptor signaling inhibits hepatitis B virus replication in vivo.J Virol,2005,79(11):7269-7272.
[11]Wu J,Lu M,Meng Z,et al.Toll-like receptor-mediated control of HBV replication by nonparenchymal liver cells in mice.Hepatology,2007,46(6):1769-1778.
[12]Preiss S,Thompson A,Chen X,et al.Characterization of the innate immune signalling pathways in hepatocyte cell lines.J Viral Hepat,2008,15(12):888-900.
[13] Li K, Chen Z, Kato N, et al. Distinct poly (I:C) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes. J Biol Chem, 2005, 280(17):16739-16747.
[14] Matsumura T, Hayashi H, Takii T, et al. TGF-beta down-regulates IL-1 alpha-induced TLR2 expression in murine hepatocytes. J Leukoc Biol, 2004, 75:1056-1061.
[15] Riordan SM, Skinner N, Kurtovic J, et al. Reduced expression of Toll-like receptor 2 on peripheral monocytes in patients with chronic hepatitis B. Clin Vaccine Immunol, 2006, 13(8):972-974.
[16] Visvanathan K, Skinner NA, Thompson AJ, et al. Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology, 2007, 45(1):102-110.
[17] Guo YW, Wei XQ, Yang SJ. A close relationship between viral hepatitis B and Toll-like receptor 2. Chin J Hepatol, 2007,15(7):485-488.
[18] Li YW, Guo YW, Zhu MF, et al. Expression of TLR2 and TLR4 strain transfected in hepatits B virus. China Tropical Medicine, 2008, 8(3):367-369.
[19] Zhou LP, Zou DW, Fan X, et al. Correlation between the TLR4 mRNA levels in peripheral blood mononuclear cells and the disease progression of Hepatitis B. Journal of Microbiology, 2008,28(1):32-35.
[20] Xu N, Yao HP, Sun Z, et al. Toll-like receptor 7 and 9 expression in peripheral blood mononuclear cells from patients with chronic hepatitis B and related hepatocellular carcinoma. Acta Pharmacol Sin, 2008, 29(2):239-244.
[21] Xia C, Lu M, Zhang Z, et al. TLRs antiviral effect on hepatitis B virus in HepG2 cells. JAppl Microbiol, 2008, 105(5):1720-1727.
[22] Wu J, Meng J, Jiang M, et al. Hepatitis B Virus suppresses Toll-like receptor-Mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology, 2009, 49(4): 1132-1140.
[2] Parker GA, Picut CA. Liver Immunobiology. Toxi Path, 2005, 33:52-62.
[3] Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol, 2005, 5:215-229.
[4] Crispe IN. Hepatic T cells and liver tolerance. Nat Rev Immunol, 2003, 3:51-62.
[5] Livngston BD, Crimi C, Fikes J, et al. Immunization with the HBV core 18-27 epitope elicits CTL responses in humans expressing different HLA-A2 supertype molecules. Hum Immunol, 1999, 60:1013-1017.
[6] Lazdina U, Cao T, Steinbergs J, et al. Molecular basis for the interaction of the hepatitis B virus core antigen with the surface immunoglobulin receptor on naive B cells. J Virol, 2001, 75:6367-6374.
[7] Twu JS, Schloemer RN. Transciption of human beta interferon gene is inhibitied by hepatitis B virus. J Virol, 1989, 63:3065-3071.
[8] Whitten TM, Quets AT, Schloemer RH. Identification of hepatitis B virus factor that inhibits expression of the beta interferon gene. J Virol, 1991, 65:4699-4704.
[9] Janssens S, Beyaert R. Role of toll-like receptors in pathogen recognition. Clin Microbiol, 2003,16:637-646.
[10] Alexopoulou L, Holt AC, Medzhitov R, et al. Recognition of double-stranded RNA and activation of NF-kappaB by Toll like receptor 3. Nature, 2001,413(6857):732-738.
[11] Diebold SS, Kaisho T, Hemmi H, et al. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science, 2004, 303(5663): 1529-1531.
[12] Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 2004, 303(5663):1526-1529.
[13]Ahmad-Nejad P,Hacker H,Rutz M,et al.Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments.Eur J Immunol,2004,32:1958-1968.
[14]Kawei T,Akira S.TLR signaling.Cell Death Differ,2006,13(5):816-825.
[15]Akira S,Takeda K.Toll-like receptor signalling.Nat Rev Immunol,2004,4(7):499-511.
[16]Meylan E,Bums K,Hofmann K,et al.RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation.Nat Immunol,2004,5(5):503-507.
[17]Yoneyama M,Kikuchi M,Natsukawa T,et al.The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat Immunol,2004,5:730-737.
[18]Beignon AS,Skoberne M,Bhardwaj N.Type Ⅰ interferons promote cross-priming:more functions for old cytokines.Nat Immunol,2003,4:939-941.
[19]Iwasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune responses.Nat Immunol,2004,5:987-995.
[20]Hoebe K,Janssen E,Beutler B.The interface between innate and adaptive immunity.Nat Immunol,2004,5:971-974.
[21]Wu J,Lu M,Meng Z,et al.Toll-like receptor-mediated control of HBV replication by nonparenchymal liver cells in mice.Hepatology,2007,46(6):1769-1778.
[22]Isogawa M,Robek MD,Furuich Y,et al.Toll-like receptor signaling inhibits hepatitis B virus replication in vivo.J Virol,2005,79(11):7269-7272.
[23]Xia C,Lu M,Zhang Z,et al.TLRs antiviral effect on hepatitis B virus in HepG2cells.J Appl Microbiol,2008,105(5):1720-1727.
[24]Murray JM,Wieland SF,Purcell RH,Chisari FV.Dynamics of hepatitis B virus clearance in chimpanzees.Proe Natl Acad Sci USA 2005;102:17780-17785.
[25]Bigger CB,Brasky KM & Lanford RE.DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection.J Virol 2001;75:7059-7066.
[26]Li K,Chen Z,Kato N,et al.Distinct poly(I:C) and virus-activated signaling path-ways leading to interferon-beta production in hepatocytes.J Biol Chem,2005,280(17):16739-16747.
[27]Preiss S,Thompson A,Chen X,et al.Characterization of the innate immune signalling pathways in hepatocyte cell lines.J Viral Hepat,2008,15(12):888-900.
[28]陈皴琼.病毒PAMP诱导肝细胞表达IFN-β信号转导途径的研究.浙江大学硕士学位论文,2007年5月.
[29]Wu J,Meng J,Jiang M,et al.Hepatitis B Virus suppresses Toll-like receptor-Mediated innate immune responses in murine parenchymal and nonparenchymal liver cells.Hepatology,2009,49(4):1132-1140.
[30]Makkouk A,Abdelnoor Am.The potential use of TLR agonists and antagonists as prophylactic and/or therapeutic agents.Immunopharmocol immunotoxicol,2009,31(3):331-338.
[1]Kumagai Y,Takeuchi O,Akira S.Pathogen recognition by innate receptors.J Infect Chemother,2008,14(2):86-92.
[2]Hoshino K,Takeuchi O,Kawai T,et al.Cutting edge:Toll-like receptor 4(TLR4)-deficient mice are hyporesponsive to lipopolysaccharide:evidence for TLR4 as the Lps gene product.J Immunol,1999,162(7):3749-3752.
[3]Alexopoulou L,Holt AC,Medzhitov R,et al.Recognition of double-stranded RNA and activation of NF-kappaB by Toll like receptor 3.Nature,2001,413(6857):732-738.
[4]Diebold SS,Kaisho T,Hemmi H,et al.Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.Science,2004,303(5663):1529-1531.
[5]Heil F,Hemmi H,Hochrein H,et al.Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.Science,2004,303(5663):1526-1529.
[6]Kawei T,Akira S.TLR signaling.Cell Death Differ,2006,13(5):816-825.
[7]Akira S,Takeda K.Toll-like receptor signalling.Nat Rev Immunol,2004,4(7):499-511.
[8]Meylan E,Bums K,Hofmann K,et al.RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation.Nat Immunol,2004,5(5):503-507.
[9]Nembrini C,Abel B,KopfM,et al.Strong TCR signaling,TLR ligands,and cytokine redundancies ensure robust development of type 1 effector T cells.J Immunol,2006,176(12):7180-7188.
[10]Isogawa M,Robek MD,Furuich Y,et al.Toll-like receptor signaling inhibits hepatitis B virus replication in vivo.J Virol,2005,79(11):7269-7272.
[11]Wu J,Lu M,Meng Z,et al.Toll-like receptor-mediated control of HBV replication by nonparenchymal liver cells in mice.Hepatology,2007,46(6):1769-1778.
[12]Preiss S,Thompson A,Chen X,et al.Characterization of the innate immune signalling pathways in hepatocyte cell lines.J Viral Hepat,2008,15(12):888-900.
[13] Li K, Chen Z, Kato N, et al. Distinct poly (I:C) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes. J Biol Chem, 2005, 280(17):16739-16747.
[14] Matsumura T, Hayashi H, Takii T, et al. TGF-beta down-regulates IL-1 alpha-induced TLR2 expression in murine hepatocytes. J Leukoc Biol, 2004, 75:1056-1061.
[15] Riordan SM, Skinner N, Kurtovic J, et al. Reduced expression of Toll-like receptor 2 on peripheral monocytes in patients with chronic hepatitis B. Clin Vaccine Immunol, 2006, 13(8):972-974.
[16] Visvanathan K, Skinner NA, Thompson AJ, et al. Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology, 2007, 45(1):102-110.
[17] Guo YW, Wei XQ, Yang SJ. A close relationship between viral hepatitis B and Toll-like receptor 2. Chin J Hepatol, 2007,15(7):485-488.
[18] Li YW, Guo YW, Zhu MF, et al. Expression of TLR2 and TLR4 strain transfected in hepatits B virus. China Tropical Medicine, 2008, 8(3):367-369.
[19] Zhou LP, Zou DW, Fan X, et al. Correlation between the TLR4 mRNA levels in peripheral blood mononuclear cells and the disease progression of Hepatitis B. Journal of Microbiology, 2008,28(1):32-35.
[20] Xu N, Yao HP, Sun Z, et al. Toll-like receptor 7 and 9 expression in peripheral blood mononuclear cells from patients with chronic hepatitis B and related hepatocellular carcinoma. Acta Pharmacol Sin, 2008, 29(2):239-244.
[21] Xia C, Lu M, Zhang Z, et al. TLRs antiviral effect on hepatitis B virus in HepG2 cells. JAppl Microbiol, 2008, 105(5):1720-1727.
[22] Wu J, Meng J, Jiang M, et al. Hepatitis B Virus suppresses Toll-like receptor-Mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology, 2009, 49(4): 1132-1140.