Toll样受体7在HBV感染中作用及相关机制的研究
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摘要
研究背景
乙型肝炎病毒(Hepatitis B virus,HBV)感染是一个全球性健康问题。部分人初次感染HBV后不能彻底清除病毒,成为慢性HBV携带者或慢性乙型肝炎患者,甚至发展为肝硬化,肝癌而严重危害人类健康。HBV持续感染与宿主的特异性免疫耐受、干扰素α产生水平低下,T细胞应答不足或浆细胞样树突状细胞减少及功能受损等现象相关,但其确切机制尚有待进一步研究。
Toll样受体(TLR)是近年来发现的一类模式识别受体,通过对病原体相关分子模式(PAMP)的识别,在天然免疫和获得性免疫中起着重要桥梁的作用[1]。TLR7是TLR家族的重要成员之一,与其配体结合可以诱导干扰素、白介素和肿瘤坏死因子等细胞因子的产生,发挥抗病毒作用[2,3]。TLR7的激动剂咪喹莫特(IMQ/R-847))和艾沙托立宾(Loxribine的同类物),已分别用于皮肤癌和慢性丙型肝炎的治疗[4,5]。TLR7的另一配体Resiquimod(R-848)可以通过诱导IFN-α,IFN-β的产生显著抑制HBV的复制[2]。TLR7在抗病毒免疫作用中必不可少,而在慢性乙型肝炎患者的外周血单个核细胞TLR7表达显著降低[6]。然而,目前国内外关于HBV感染对TLR7表达调控影响的研究甚少,TLR7在乙型病毒性肝炎发病中的作用及机理尚不确定。
本研究通过对TLR7介导的体外抗HBV作用和HBV转染对TLR7表达调控影响的研究,阐述TLR7在乙型病毒性肝炎发病中的作用。通过检测TLR7介导的慢性乙肝患者外周血单个核细胞产生IFN-α,IL-12含量的改变,探讨慢性HBV感染对免疫细胞TLR7功能的损伤及意义,阐述TLR7在慢性HBV感染中的作用及可能的机制,以期为TLRs介导的固有免疫的研究提供参考资料,为寻找治疗乙肝的新靶点和新药的开发提供初步实验依据。
研究目的
1.通过TLR7特异性配体Loxoribine刺激HepG2.2.15细胞株的研究,探讨TLR7介导的体外抗HBV作用及机制。
2.通过检测TLR7特异性配体Loxoribine刺激HBV转染前后的HepG2和HepG2.2.15细胞株TLR7的表达,探讨HBV转染对TLR7表达及调控的影响,阐述TLR7在乙型病毒性肝炎发病中的作用。
3.通过检测TLR7介导的慢性乙肝患者外周血单个核细胞产生IFN-α,IL-12含量的改变,探讨慢性HBV感染对免疫细胞TLR7功能的损伤及意义,阐述TLR7在慢性HBV感染中作用及机制。
研究方法
1.选用能稳定分泌HBV颗粒和HBV抗原标志物的HepG2.2.15细胞株为HBV转染的细胞模型,应用ELISA及实时荧光定量PCR法检测TLR7特异性配体Loxoribine对HepG2.2.15细胞HBsAg,HBeAg分泌和HBV DNA复制的抑制作用,探讨TLR7介导的体外抗HBV作用。
2. MTT比色法检测TLR7特异性配体Loxoribine刺激对HepG2.2.15细胞的毒性作用。
3.流式细胞术检测HepG2,HepG2.2.15细胞株胞内TLR7蛋白的表达水平,及特异性配体Loxoribine诱导对其表达调控的影响。
4. ELISA法检测TLR7特异性配体Loxoribine刺激慢性HBV感染患者和健康人外周血单个核细胞产生IFN-α,IL-12含量的改变,评价慢性HBV感染对免疫细胞TLR7功能的影响。
研究结果
1.在TLR7特异性配体Loxoribine作用下,可有效抑制HepG2.2.15细胞HBsAg的分泌及HBV DNA的复制,对HBsAg分泌的抑制作用呈剂量和时间依赖性。对HBeAg的分泌无抑制作用。
2.低浓度Loxoribine对HepG2.2.15细胞生长有一定抑制作用,但随着配体浓度增高,对细胞生长无明显毒性作用。
3. HepG2和HepG2.2.15细胞胞内均有TLR7表达,但转染了HBV全基因组的HepG2.2.15细胞胞内TLR7的表达量明显低于未转染的HepG2细胞。TLR7特异性配体Loxoribine诱导后HepG2和HepG2.2.15细胞胞内TLR7表达量均有增加,但差异无统计学意义。
4.乙肝病毒携带组及慢性乙肝患者组外周血单个核细胞培养上清液中IFN-α水平明显低于健康对照组;慢性乙肝患者组IL-12水平明显高于健康对照组;乙肝病毒携带组和慢性乙肝患者组间,PBMC培养上清液中IFN-α和IL-12水平无明显差异。
5. TLR7特异性配体Loxoribine刺激培养后,健康对照组外周血单个核细胞培养上清液中IFN-α,IL-12水平显著升高,乙肝病毒携带组及慢性乙肝患者组IFN-α,IL-12水平亦均有升高,但差异无统计学意义。
结论
1.特异性配体Loxoribine有较显著的抗HBV作用,其对HBV的抑制可能是TLR7介导作用于病毒复制及蛋白合成等环节。
2. HBV感染抑制了TLR7的表达,特异性配体Loxoribine诱导则可发挥显著的抗HBV作用,即TLR7表达下调可能参与了乙型病毒性肝炎的发病。
3.慢性HBV感染患者外周血单个核细胞TLR7功能受损,INF-α产生水平低下, IL-12调节功能紊乱,这可能是导致HBV持续感染的重要原因。
Background
Hepatitis B virus(HBV) infection is the Global health problem. Following initial infection some individuals fail to resolve their infection and thereby become chronic carriers or chronic hepatitis B(CHB), and evern liver cirrhosis or hepatocellular carcinoma(HCC)patients, Serious harm to human health. Chronie HBV infection is associated with phenomenon sueh as T cell hyporesponsiveness or toleranee,lower interferon-αproduction of host,and decreased plasmaeytoid dendritic cells numbers and Impaired fuctions.However,the mechanism on initiation and persistence of HBV infection still remains to be elucidated.
Toll like receptors(TLR),a kind of pattern recognition receptors, was discovered in recent years. It playes an important role in the bridge of natural immunity and acquired immunity through pathogen-associated molecular patterns (PAMP) recognition. TLR7 is an important member of the TLR family,which play a central role of antiiviral immuner responses by inducing interferon, interleukin, tumor necrosis factor and other cytokines when binding with its ligand. As the agonist of TLR7,Imiquimod (IMQ/R-847)) and isatoribine((loxribine similar goods) have beening used in skin cancer and chronic hepatitis C treatment. Another ligand of TLR7 Resiquimod (R-848) can significantly inhibit the replication of HBV by inducing IFN-α, IFN-β.TLR7 playes an essential role in antiviral immunity, but in chronic hepatitis B(CHB) patients’peripheral blood mononuclear cells(PBMC),the expression of TLR7 was markedly reduced . However, There's only little study about how the expression and regulation of TLR7 impacted by hepatitis B virus infection in the world now,the role of TLR7 in the pathogenesis and mechanism of hepatitis B infection is uncertain yet.
This study in order to described the role of TLR7 in the pathogenesis of hepatitis B infection by researching on the TLR7-mediated anti-HBV effect in vitro, and the expression and regulation signal transduction pathway of TLR7 affected by HBV transfection. To discuss the impairment significance of TLR7 in immune cells after chronic HBV infection by detecting IFN-α,IL-12 content changes which produced by CHB patients’PBMCs on the TLR7-mediated, describe the role and the mechanism of TLR7 in chronic HBV infection.The main aim of this study was to provides credible informations for the study of TLRs-mediated innate immunity and its signal transduction, as well as to provide a theoretical basis for find a new targe for hepatitis B treatment and the development of new drugs.
Objective
1. To discuss TLR7-mediated anti-HBV effects and the mechanism through stimulating the HepG2.2.15 cell line by TLR7 specific ligand loxoribine in vitro.
2. To discuss the effect on expression and regulation signal pathway of TLR7 with the HBV transfected and describe the role of TLR7 in the pathogenesis of hepatitis B ,by detecting the expression of TLR7 after using TLR7 specific ligand loxoribine to stimulate the HepG2 and HepG2.2.15 cell lines which are before and after HBV transfected.
3. To study the impairment of TLR7 in immune cells and the significance with chronic HBV infection, by detecting IFN-α, IL-12 content changes which produced by CHB patients’PBMC by the TLR7-mediated, in order to describe the role and the mechanism of TLR7 in chronic HBV infection.
Methods
1. Selecting HepG2.2.15 cell lines which can synthesis and secrete HBV and HBV marks as a cell model of HBV infection. ELISA and Fluorescent quantificative was used to assay the inhibition of HepG2.2.15 cells secretion HBsAg, HBeAg and the HBV DNA replicationg after TLR7 specific ligand loxoribine stimulated,and dicuss TLR7-mediated anti-HBV effects in vitro.
2. Using MTT colorimetric to observe the toxic effects of TLR7 specific ligand loxoribine on HepG2.2.15 cells.
3. Flow cytometry was used to detect expression levels and regulation of TLR7 protein of HepG2, HepG2.2.15 cell lines intracellular before and after specifice ligand loxoribine induced.
4. ELISA was used to assay IFN-α, IL-12 content changes which are produced by chronic HBV infection patients and healthy human peripheral blood mononuclear cells after stimulated by TLR7 specific ligand loxoribine, evaluation the function of TLR7 after chronic HBV infection.
Results
1. TLR7 specific ligand Loxoribine could inhibit HBsAg secretion and HBV DNA replication effectively in HepG2.2.15 cells, the inhibition of HBsAg secretion was a dose and time-dependent. There is no effect on HBeAg.
2. Low concentration of Loxoribine inhibited the growth of HepG2.2.15 cell at a certain extent ,but as the drug concentration increased, toxic effects of cell growth was apparent.
3. HepG2 and HepG2.2.15 cells both express TLR7 intracellular, but the expression of TLR7 in HepG2.2.15 transfected by HBV intracellular significantly lower than HepG2 cells. TLR7 expression in HepG2 and HepG2. 2.15 cells increased a little after loxoribine induction, but there was no statistically significant.
4. The level of IFN-αin PBMC culture supernatants of Hepatitis B virus carriers and CHB patients’were significantly lower than healthy people; level of IL-12 in PBMC culture supernatants of CHB patients were significantly higher than healthy people; there was no significant difference of IFN-αand IL-12 level in PBMC culture supernatants between hepatitis B virus carriers and CHB patients .
5. After TLR7 specific ligand Loxoribine stimulating, levels of IFN-α,IL-12 in PBMC cultured supernatants of healthy human were significantly increased; levels of IFN-α, IL-12 in PBMC cultured supernatants of hepatitis B virus carriers and CHB patients were also elevated ,but there was no statistically significant.
Conclusion
1. Specific ligands loxoribine showed remarkably inhibitory effect on anti-HBV effects, its inhibition of HBV probably affected on the stages of viral replication and/or protein synthesis and other links mediated by TLR7.
2. HBV infection inhibits TLR7 expression,TLR7 specific ligand Loxoribine induced show a remarkably anti-HBV effect, in other words, decreased expre ssion of TLR7 may involve in the pathogenesis of hepatitis B virus.
3. Dysfunction of TLR7 in PBMC of chronic HBV infection, lower production of IFN-α, indiscriminate regulation of IL-12, all of this may be an important reason leading to HBV persistent infection.
乙型肝炎病毒(Hepatitis B virus,HBV)感染是一个全球性健康问题。部分人初次感染HBV后不能彻底清除病毒,成为慢性HBV携带者或慢性乙型肝炎患者,甚至发展为肝硬化,肝癌而严重危害人类健康。HBV持续感染与宿主的特异性免疫耐受、干扰素α产生水平低下,T细胞应答不足或浆细胞样树突状细胞减少及功能受损等现象相关,但其确切机制尚有待进一步研究。
Toll样受体(TLR)是近年来发现的一类模式识别受体,通过对病原体相关分子模式(PAMP)的识别,在天然免疫和获得性免疫中起着重要桥梁的作用[1]。TLR7是TLR家族的重要成员之一,与其配体结合可以诱导干扰素、白介素和肿瘤坏死因子等细胞因子的产生,发挥抗病毒作用[2,3]。TLR7的激动剂咪喹莫特(IMQ/R-847))和艾沙托立宾(Loxribine的同类物),已分别用于皮肤癌和慢性丙型肝炎的治疗[4,5]。TLR7的另一配体Resiquimod(R-848)可以通过诱导IFN-α,IFN-β的产生显著抑制HBV的复制[2]。TLR7在抗病毒免疫作用中必不可少,而在慢性乙型肝炎患者的外周血单个核细胞TLR7表达显著降低[6]。然而,目前国内外关于HBV感染对TLR7表达调控影响的研究甚少,TLR7在乙型病毒性肝炎发病中的作用及机理尚不确定。
本研究通过对TLR7介导的体外抗HBV作用和HBV转染对TLR7表达调控影响的研究,阐述TLR7在乙型病毒性肝炎发病中的作用。通过检测TLR7介导的慢性乙肝患者外周血单个核细胞产生IFN-α,IL-12含量的改变,探讨慢性HBV感染对免疫细胞TLR7功能的损伤及意义,阐述TLR7在慢性HBV感染中的作用及可能的机制,以期为TLRs介导的固有免疫的研究提供参考资料,为寻找治疗乙肝的新靶点和新药的开发提供初步实验依据。
研究目的
1.通过TLR7特异性配体Loxoribine刺激HepG2.2.15细胞株的研究,探讨TLR7介导的体外抗HBV作用及机制。
2.通过检测TLR7特异性配体Loxoribine刺激HBV转染前后的HepG2和HepG2.2.15细胞株TLR7的表达,探讨HBV转染对TLR7表达及调控的影响,阐述TLR7在乙型病毒性肝炎发病中的作用。
3.通过检测TLR7介导的慢性乙肝患者外周血单个核细胞产生IFN-α,IL-12含量的改变,探讨慢性HBV感染对免疫细胞TLR7功能的损伤及意义,阐述TLR7在慢性HBV感染中作用及机制。
研究方法
1.选用能稳定分泌HBV颗粒和HBV抗原标志物的HepG2.2.15细胞株为HBV转染的细胞模型,应用ELISA及实时荧光定量PCR法检测TLR7特异性配体Loxoribine对HepG2.2.15细胞HBsAg,HBeAg分泌和HBV DNA复制的抑制作用,探讨TLR7介导的体外抗HBV作用。
2. MTT比色法检测TLR7特异性配体Loxoribine刺激对HepG2.2.15细胞的毒性作用。
3.流式细胞术检测HepG2,HepG2.2.15细胞株胞内TLR7蛋白的表达水平,及特异性配体Loxoribine诱导对其表达调控的影响。
4. ELISA法检测TLR7特异性配体Loxoribine刺激慢性HBV感染患者和健康人外周血单个核细胞产生IFN-α,IL-12含量的改变,评价慢性HBV感染对免疫细胞TLR7功能的影响。
研究结果
1.在TLR7特异性配体Loxoribine作用下,可有效抑制HepG2.2.15细胞HBsAg的分泌及HBV DNA的复制,对HBsAg分泌的抑制作用呈剂量和时间依赖性。对HBeAg的分泌无抑制作用。
2.低浓度Loxoribine对HepG2.2.15细胞生长有一定抑制作用,但随着配体浓度增高,对细胞生长无明显毒性作用。
3. HepG2和HepG2.2.15细胞胞内均有TLR7表达,但转染了HBV全基因组的HepG2.2.15细胞胞内TLR7的表达量明显低于未转染的HepG2细胞。TLR7特异性配体Loxoribine诱导后HepG2和HepG2.2.15细胞胞内TLR7表达量均有增加,但差异无统计学意义。
4.乙肝病毒携带组及慢性乙肝患者组外周血单个核细胞培养上清液中IFN-α水平明显低于健康对照组;慢性乙肝患者组IL-12水平明显高于健康对照组;乙肝病毒携带组和慢性乙肝患者组间,PBMC培养上清液中IFN-α和IL-12水平无明显差异。
5. TLR7特异性配体Loxoribine刺激培养后,健康对照组外周血单个核细胞培养上清液中IFN-α,IL-12水平显著升高,乙肝病毒携带组及慢性乙肝患者组IFN-α,IL-12水平亦均有升高,但差异无统计学意义。
结论
1.特异性配体Loxoribine有较显著的抗HBV作用,其对HBV的抑制可能是TLR7介导作用于病毒复制及蛋白合成等环节。
2. HBV感染抑制了TLR7的表达,特异性配体Loxoribine诱导则可发挥显著的抗HBV作用,即TLR7表达下调可能参与了乙型病毒性肝炎的发病。
3.慢性HBV感染患者外周血单个核细胞TLR7功能受损,INF-α产生水平低下, IL-12调节功能紊乱,这可能是导致HBV持续感染的重要原因。
Background
Hepatitis B virus(HBV) infection is the Global health problem. Following initial infection some individuals fail to resolve their infection and thereby become chronic carriers or chronic hepatitis B(CHB), and evern liver cirrhosis or hepatocellular carcinoma(HCC)patients, Serious harm to human health. Chronie HBV infection is associated with phenomenon sueh as T cell hyporesponsiveness or toleranee,lower interferon-αproduction of host,and decreased plasmaeytoid dendritic cells numbers and Impaired fuctions.However,the mechanism on initiation and persistence of HBV infection still remains to be elucidated.
Toll like receptors(TLR),a kind of pattern recognition receptors, was discovered in recent years. It playes an important role in the bridge of natural immunity and acquired immunity through pathogen-associated molecular patterns (PAMP) recognition. TLR7 is an important member of the TLR family,which play a central role of antiiviral immuner responses by inducing interferon, interleukin, tumor necrosis factor and other cytokines when binding with its ligand. As the agonist of TLR7,Imiquimod (IMQ/R-847)) and isatoribine((loxribine similar goods) have beening used in skin cancer and chronic hepatitis C treatment. Another ligand of TLR7 Resiquimod (R-848) can significantly inhibit the replication of HBV by inducing IFN-α, IFN-β.TLR7 playes an essential role in antiviral immunity, but in chronic hepatitis B(CHB) patients’peripheral blood mononuclear cells(PBMC),the expression of TLR7 was markedly reduced . However, There's only little study about how the expression and regulation of TLR7 impacted by hepatitis B virus infection in the world now,the role of TLR7 in the pathogenesis and mechanism of hepatitis B infection is uncertain yet.
This study in order to described the role of TLR7 in the pathogenesis of hepatitis B infection by researching on the TLR7-mediated anti-HBV effect in vitro, and the expression and regulation signal transduction pathway of TLR7 affected by HBV transfection. To discuss the impairment significance of TLR7 in immune cells after chronic HBV infection by detecting IFN-α,IL-12 content changes which produced by CHB patients’PBMCs on the TLR7-mediated, describe the role and the mechanism of TLR7 in chronic HBV infection.The main aim of this study was to provides credible informations for the study of TLRs-mediated innate immunity and its signal transduction, as well as to provide a theoretical basis for find a new targe for hepatitis B treatment and the development of new drugs.
Objective
1. To discuss TLR7-mediated anti-HBV effects and the mechanism through stimulating the HepG2.2.15 cell line by TLR7 specific ligand loxoribine in vitro.
2. To discuss the effect on expression and regulation signal pathway of TLR7 with the HBV transfected and describe the role of TLR7 in the pathogenesis of hepatitis B ,by detecting the expression of TLR7 after using TLR7 specific ligand loxoribine to stimulate the HepG2 and HepG2.2.15 cell lines which are before and after HBV transfected.
3. To study the impairment of TLR7 in immune cells and the significance with chronic HBV infection, by detecting IFN-α, IL-12 content changes which produced by CHB patients’PBMC by the TLR7-mediated, in order to describe the role and the mechanism of TLR7 in chronic HBV infection.
Methods
1. Selecting HepG2.2.15 cell lines which can synthesis and secrete HBV and HBV marks as a cell model of HBV infection. ELISA and Fluorescent quantificative was used to assay the inhibition of HepG2.2.15 cells secretion HBsAg, HBeAg and the HBV DNA replicationg after TLR7 specific ligand loxoribine stimulated,and dicuss TLR7-mediated anti-HBV effects in vitro.
2. Using MTT colorimetric to observe the toxic effects of TLR7 specific ligand loxoribine on HepG2.2.15 cells.
3. Flow cytometry was used to detect expression levels and regulation of TLR7 protein of HepG2, HepG2.2.15 cell lines intracellular before and after specifice ligand loxoribine induced.
4. ELISA was used to assay IFN-α, IL-12 content changes which are produced by chronic HBV infection patients and healthy human peripheral blood mononuclear cells after stimulated by TLR7 specific ligand loxoribine, evaluation the function of TLR7 after chronic HBV infection.
Results
1. TLR7 specific ligand Loxoribine could inhibit HBsAg secretion and HBV DNA replication effectively in HepG2.2.15 cells, the inhibition of HBsAg secretion was a dose and time-dependent. There is no effect on HBeAg.
2. Low concentration of Loxoribine inhibited the growth of HepG2.2.15 cell at a certain extent ,but as the drug concentration increased, toxic effects of cell growth was apparent.
3. HepG2 and HepG2.2.15 cells both express TLR7 intracellular, but the expression of TLR7 in HepG2.2.15 transfected by HBV intracellular significantly lower than HepG2 cells. TLR7 expression in HepG2 and HepG2. 2.15 cells increased a little after loxoribine induction, but there was no statistically significant.
4. The level of IFN-αin PBMC culture supernatants of Hepatitis B virus carriers and CHB patients’were significantly lower than healthy people; level of IL-12 in PBMC culture supernatants of CHB patients were significantly higher than healthy people; there was no significant difference of IFN-αand IL-12 level in PBMC culture supernatants between hepatitis B virus carriers and CHB patients .
5. After TLR7 specific ligand Loxoribine stimulating, levels of IFN-α,IL-12 in PBMC cultured supernatants of healthy human were significantly increased; levels of IFN-α, IL-12 in PBMC cultured supernatants of hepatitis B virus carriers and CHB patients were also elevated ,but there was no statistically significant.
Conclusion
1. Specific ligands loxoribine showed remarkably inhibitory effect on anti-HBV effects, its inhibition of HBV probably affected on the stages of viral replication and/or protein synthesis and other links mediated by TLR7.
2. HBV infection inhibits TLR7 expression,TLR7 specific ligand Loxoribine induced show a remarkably anti-HBV effect, in other words, decreased expre ssion of TLR7 may involve in the pathogenesis of hepatitis B virus.
3. Dysfunction of TLR7 in PBMC of chronic HBV infection, lower production of IFN-α, indiscriminate regulation of IL-12, all of this may be an important reason leading to HBV persistent infection.
引文
1 Duan X Z,Zhuang H,Wang M,et al.Decreased numbers and impaired funetion of circulating dendritic cell subsets in patients with chronic hepatitis B infection. Gastroe nterol Hepalol,2005,20(2):234- 242.
2 Masanori Isogawa, Michael D.Robek,Yoshihiro Furuichi, et al. Toll-Like Receptor Signaling Inhibits Hepatitis B Virus Replication In Vivo.Journal of Virology, 2005:7269–7272.
3 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): 1769-1777.
4 Sch?n MP,Sch?n M.TLR7 and TLR8 as targets in cancer therapy. Oncogene, 2008, 27 (2): 190-199.
5 Horsmans Y,Berg T,Desager J,et al.Isatoribine,an agonist of TLR7,reduces plasma virus concentration in chronic hepatitis C infection. Hepatology, 2005, 42(3): 724-731.
6 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.
7 Hoofoagle J H.Hepatitis B-Preventable and now treatable.N Engl J Med 2006, 354(10):1074-1076.
8 Chinese Soeiety of Hepatology and Chinese Soeiety of lnfectious Diseases CMA:Th Guideline of Prevention and treatment for chronic hepatitis B.Chin J Hepatol 2005,13 (12): 881-890.
9 Thimme R,Wieland S ,Steiger C,et al:CD8 (+)T cells mediate viral clearance and disease Pathogenesis during acute hepatitis B virus infeetion. Virol, 2003,77(1):66-76.
10 F.V.C.LueaGGuidotti,IMMUNOBIOLOGY AND PATHOGENESIS OF VIRAL HEPATITIS, Annual Review of Pathology:Meehanisms of Diseasel,2006,23(5):23-61.
11 MedzhidovR,PrestonHP,JanewayCA.A human homologue of the Drosophila Toll protein signal sactivation of adaptive immunity.Nature,1997;3886640:394.
12 Constantinos Brikos,Luke A.J.O’Neill.singnaling of Toll-like recaptor. Toll - like Receptor(TLRs)and Innate Imnuity,2007,183:21-50.
13 Satoshi Uematsu,Shizuo Akira.Toll-like receptors and innate immunity. Springer-Verlag, 2006,84:712–725.
14 Wen-Wei Chang,Ih-Jen Su,Ming-Derg Lai, et al. Toll-like receptor 4 plays an anti-HBV role in a murine model of acute hepatitis B virus expression.World J Gastroenterol, 2005,11(42): 6631-6637.
15 Satoshi Uematsu, Shizuo Akira. PRRs in pathogen recognition. CEJB,2006, 1(3): 299–313.
16 Zong Liang.Role of Toll-like receptors in regulatory functions of T and B cells. Chinese Science Bulletin,2008,53(8):1121-1127.
17 Erika Schlaepfer and Roberto F.Speck.Anti-HIV Activity Mediated by Natural Killer and CD8+ Cells after Toll-Like Receptor 7/8 Triggering.PLoS ONE,2008, 375(44): 91-99.
18 Heather J.Martin,Jae Myun Lee, et al.Manipulation of the Toll -Like Receptor 7 Signaling Pathway by Epstein-Barr Virus. JOURNAL OF VIROLOGY, 2007,45 (5): 9748–9758.
19 Lund J M,Alexopoulou L,Sato A,et al:Reeognition of single-stranded RNA viruses by Toll- liker receptor7.Proc Natl Acad Sci USA,2004,101(15):5598-5603.
20 Gray D,Gray M,Barr T.Inniate responses of B cells.Eur J Immunol.2007,37(12): 3304- 3310.
21 Barr TA,Brown S,Ryan G, etal.TLR-mediated stimulation of APC:Distinet cytokine Responses of B cells and dendritic cells.Eur J Immunol.2007,37(11):3040-3053.
22 Hanten J A,Vasi1akos JP,Rter CL etal. Comparison of human B cell activation by TLR7 and TLR9 agonists.BMC Immunol.2008,24(9):32-39.
23 Mansson A,Adner M,Hekerfelt Ueral.A distinct Toll- like receptor in human tonsillar B cells,directly activated by PamCSK,R-837 and CPG-2006 stimulation. Immunology. 2006, 118(4):539-548.
24 Ito T,Wang YH,LiuYJ.Plasmacytoid dendritic cell preeursors/typel interferon- producing cells sense viral infeotion by Toll-like receptor(TLR)7andTLR9.Exp Med,2007,204(3): 667-680.
25 Hornung V, Schlender J,Guenthner-Biller M, etal.Replieation-dependent potent IFN- alpha induction in human plasmacytoid dendritic cells by a single- stranded RNA virus.J lmmunol,2004,173(10):5935-5943.
26 UCHIDA T, SCUMPIA P O,MURASKO D M, et al.Variable requirement of dendritic cells for recruitment of NK and T cells to different TLR agonists.J Immunol,2007, 178 (6): 3886-3892.
27 Hardy,A.W.et al.HIV turns plasmacytoid dendritic cells(Pdc) into TRAIL- exressing killer pDC and down-regulates HIV coreceptors by Toll-like receptor 7-induced IFN-alpha.Proe Natl Aead Sei USA,2007,104(44):17453-17458.
28 Liu Y J.IPC:Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.Annu Rev Immunol,2005,23:275-306.
29 Guidueei C,et al.PI3K is critical for the nuelear transloeation of IRF-7 and I-IFN production by human Plasmacytoid predendritic cells in response to TLR activation.J.Exp,Med,2008.205(2):315-322.
30 Horsmans Y,Berg T,Desager J P,et al:Isatoribine,an agonist of TLR7,reduees plasma virus concentration in chroniche patitis C infection. Hepatology, 2005,42(3):724-731.
31 Weeratna R D,Makinen S R,McCluskie M J,etal.TLR agonists as vaceine adjuvants:Comparison of CpGODN and Resiquimod(R-848).Vaccine,2005,23(45):5263-5270.
32 Wa ng Y,Abel K,Lant Z K,et al.The Toll- like receptor 7(TLR7) agonist,imiquimod, and the TLR gagonist, CpGODN,induce antiviral cytokines and chemokines but do not prevent vaginal transmission of simian immunodeficiency virus when applied intravaginally to thesus macaques.J Virol,2005,79(22):14355-14370.
33 Wasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune response. Nat Immunol,2004,5(10):987-995.
34 Arancibia SA,Belt rán CJ,Aguirre IM,et al.Toll-like receptors are key participants in innate immune responses.Biol Res,2007,40(2):97-112.
35 Heil F,Ahmad-Nejad P,Hemmi H,et a1.The Toil-like receptor 7(TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7,8 and 9 subfamily. European Journal of Immunology,2003,33(9):2987-2997.
36 Oodman MG.Cellular and biochemical studies of substituted guanine ribonucleoside immunostimulants. Immunopharmacol,1991,484(7):21-51.
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1 R.Thimme,5.Wieland,C.Steiger,et al.CDS(+)T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infeetion. Virol,2003 (77):68-76.
2 Medzhidov R,Preston HP,Janeway CA.A human homologue of the Drosophila Toll protein signal sactivation of adaptive immunity.Nature,1997,3886640:394.
3 Constantinos Brikos,Luke A.J.O’Neill.singnaling of Toll-like recaptor.Toll - like Receptor(TLRs)and Innate Imnuity,2007,183:21-50.
4 Satoshi Uematsu,Shizuo Akira.Toll-like receptors and innate immunity. Spri nger-Verlag, 2006,84:712–725.
5 Wen-Wei Chang,Ih-Jen Su,Ming-Derg Lai.Toll-like receptor 4 plays an anti-HBV role in a murine model of acute hepatitis B virus expression.World J Gastroenterol,2005, 11(42): 6631-6637.
6 Zong Liang.Role of Toll-like receptors in regulatory functions of T and B cells. Chinese Science Bulletin,2008,53(8):1121-1127.
7 Satoshi Uematsu, Shizuo Akira. PRRs in pathogen recognition. CEJB,2006,1(3): 299–313.
8 Thomas Müller,Svetlana Hamm,Stefan Bauer.TLR9-Mediated Recognition of DNA. To ll-like Receptor(TLRs)and Innate Imnuity,2007,183:51-70.
9 Matsumura T,Hayashi H,Takii T.TGF-beta down-regulates IL-1 alpha-induced TLR2 expression in murine.hepatocytes.J Leukoc Biol,2004,75:1056-1061.
10 Guo YW,Wei XQ,Yang SJ.A close relationship between viral hepatitis B and Toll- like receptor 2.ZhongHua Gan Zang Bing Za Zhi,2007,15(7):485-8.(in Chinese )
11 Dilip Ratnam,Kumar Visvanathan.New concepts in the immunopathogenesis of chron ic hepatitis B:the importance of the innate immune response. Hepatology Inte rnational,2008 Feb.
12 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):102-10.
13郭云蔚,尉秀清,李永伟,等.Toll样受体4在乙型肝炎组织及细胞株HepG2/HepG2.2. 15内的表达特征.中山大学学报(医学科学版),2007,28(3).
14金生,张大志,陈压西,等.乙型肝炎病毒转染及脂多糖刺激对HepG2细胞Toll样受体2和4表达的影响.中华传染病杂志.2006,24(4):230- 234.
15姚景宏,贺永文.TLR4在急性肝衰竭模型大鼠中的动态变化及意义.中国组织化学与细胞化学杂志,2007,16(2):164-167.
16晏春根,谢青.N-乙酰半胱氨酸对肝缺血再灌注损伤大鼠Toll样受体4表达的影响.中国新药与临床杂志,2006,25(7):481-484.
17 Khvalevsky E, Rivkin L, Rachmilewitz J, Galun E, Giladi H. TLR3 signaling in a hepatoma cell line is skewed towards apoptosis. J Cell Biochem,2007,100 (5): 1301-1312.
18李永伟,朱明芬,郭云蔚,等.乙型肝炎病毒对HepG2细胞表达TLR3的影响.广东医学, 2007,28(12):1905-1907.
19 Stephen M. Riordan, Narelle Skinner, et al. Reduced Expression of Toll-Like Receptor 2 on Peripheral Monocytes in Patients with Chronic Hepatitis B. Clinical and Vaccine Immunology, 2006: 972–974.
20 Wei XQ, Wen ZF, Zheng FP, et al. Changes of Toll-like receptor (TLR) 2 and TLR4 on the peripheral blood mononuclear cells in patients with chronic hepatitis B and chronic severe hepatitis B. Zhonghua Gan Zang Bing Za Zhi,2007,15 (5): 354-357.(in Chinese)
21 Hermoso MA,Matsuguchi T,Smoak K,et al.Glucocorticoids and tumornecrosis factor alpha cooperatively regulate toll-like receptor 2 gene expression. Mol Cell Biol, 2004,24 (11):4743-4756.
22 Tamandl D, Bahrami M, Wessner B, et al. Modulation of toll - like recep tor 4 exp ression on human monocytes by tumor necrosis factor and interleukin - 6: tumor necrosis factor evokes lipopolysaccharide hyporesponsiveness,whereas interl eukin-6 enhances lipopolysaccharide activity.Shock,2003,20(3):224-229.
23 Riordan SM, SkinnerN, Nagree A, et al. Peripheral blood mononuclear cell exp ression of toll-like receptors and relation to cytokine levels in cirrhosis. Hepatology, 2003,37(5): 1154-1164.
24 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):239-44.
25 An BY, Xie Q, Lin LY, et al. Expression of Toll-like receptor 3 on peripheral blood dendritic cells in HBeAg positive patients with chronic hepatitis B.Zhonghua Gan Zang Bing Za Zhi, 2007,15 (10): 729-733.(in Chinese)
26李军,韩亚萍,孔练花,等.慢性乙型肝炎患者单核细胞来源树突状细胞Toll样受体7的表达低下.南京医科大学学报,2007,27(9):907-910.
27徐宁.Toll样受体7,9在慢性乙型肝炎发病机制中作用的研究.浙江大学优秀博士论文,2007.
28 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):1769-7.
29 Xiong W,Wang X,Liu X, et al.Interferon-inducible MyD88 protein inhibits hepatitis B virus replication. Virology,2004,319(2):306-14.
30 Masanori Isogawa, Michael D.Robek,Yoshihiro Furuichi, et al. Tol l-Like Receptor Signaling Inhibits Hepatitis B Virus Replication In Vivo.Journal of Virology, 2005:7269–7272.
31 T. Xing,L.Li,H.Cao, et al.Altered immune function of monocytes in different stages of patients with acute on chronic liver failure. Clinical and Experimental Immunology, 2006, 147:184-188.
32杨卫兵,易继林,杨志芳,等.TLR4和DcR3在肝细胞癌中的表达及其意义.华中医学杂志,2007,31(1):41-42.
2 Masanori Isogawa, Michael D.Robek,Yoshihiro Furuichi, et al. Toll-Like Receptor Signaling Inhibits Hepatitis B Virus Replication In Vivo.Journal of Virology, 2005:7269–7272.
3 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): 1769-1777.
4 Sch?n MP,Sch?n M.TLR7 and TLR8 as targets in cancer therapy. Oncogene, 2008, 27 (2): 190-199.
5 Horsmans Y,Berg T,Desager J,et al.Isatoribine,an agonist of TLR7,reduces plasma virus concentration in chronic hepatitis C infection. Hepatology, 2005, 42(3): 724-731.
6 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.
7 Hoofoagle J H.Hepatitis B-Preventable and now treatable.N Engl J Med 2006, 354(10):1074-1076.
8 Chinese Soeiety of Hepatology and Chinese Soeiety of lnfectious Diseases CMA:Th Guideline of Prevention and treatment for chronic hepatitis B.Chin J Hepatol 2005,13 (12): 881-890.
9 Thimme R,Wieland S ,Steiger C,et al:CD8 (+)T cells mediate viral clearance and disease Pathogenesis during acute hepatitis B virus infeetion. Virol, 2003,77(1):66-76.
10 F.V.C.LueaGGuidotti,IMMUNOBIOLOGY AND PATHOGENESIS OF VIRAL HEPATITIS, Annual Review of Pathology:Meehanisms of Diseasel,2006,23(5):23-61.
11 MedzhidovR,PrestonHP,JanewayCA.A human homologue of the Drosophila Toll protein signal sactivation of adaptive immunity.Nature,1997;3886640:394.
12 Constantinos Brikos,Luke A.J.O’Neill.singnaling of Toll-like recaptor. Toll - like Receptor(TLRs)and Innate Imnuity,2007,183:21-50.
13 Satoshi Uematsu,Shizuo Akira.Toll-like receptors and innate immunity. Springer-Verlag, 2006,84:712–725.
14 Wen-Wei Chang,Ih-Jen Su,Ming-Derg Lai, et al. Toll-like receptor 4 plays an anti-HBV role in a murine model of acute hepatitis B virus expression.World J Gastroenterol, 2005,11(42): 6631-6637.
15 Satoshi Uematsu, Shizuo Akira. PRRs in pathogen recognition. CEJB,2006, 1(3): 299–313.
16 Zong Liang.Role of Toll-like receptors in regulatory functions of T and B cells. Chinese Science Bulletin,2008,53(8):1121-1127.
17 Erika Schlaepfer and Roberto F.Speck.Anti-HIV Activity Mediated by Natural Killer and CD8+ Cells after Toll-Like Receptor 7/8 Triggering.PLoS ONE,2008, 375(44): 91-99.
18 Heather J.Martin,Jae Myun Lee, et al.Manipulation of the Toll -Like Receptor 7 Signaling Pathway by Epstein-Barr Virus. JOURNAL OF VIROLOGY, 2007,45 (5): 9748–9758.
19 Lund J M,Alexopoulou L,Sato A,et al:Reeognition of single-stranded RNA viruses by Toll- liker receptor7.Proc Natl Acad Sci USA,2004,101(15):5598-5603.
20 Gray D,Gray M,Barr T.Inniate responses of B cells.Eur J Immunol.2007,37(12): 3304- 3310.
21 Barr TA,Brown S,Ryan G, etal.TLR-mediated stimulation of APC:Distinet cytokine Responses of B cells and dendritic cells.Eur J Immunol.2007,37(11):3040-3053.
22 Hanten J A,Vasi1akos JP,Rter CL etal. Comparison of human B cell activation by TLR7 and TLR9 agonists.BMC Immunol.2008,24(9):32-39.
23 Mansson A,Adner M,Hekerfelt Ueral.A distinct Toll- like receptor in human tonsillar B cells,directly activated by PamCSK,R-837 and CPG-2006 stimulation. Immunology. 2006, 118(4):539-548.
24 Ito T,Wang YH,LiuYJ.Plasmacytoid dendritic cell preeursors/typel interferon- producing cells sense viral infeotion by Toll-like receptor(TLR)7andTLR9.Exp Med,2007,204(3): 667-680.
25 Hornung V, Schlender J,Guenthner-Biller M, etal.Replieation-dependent potent IFN- alpha induction in human plasmacytoid dendritic cells by a single- stranded RNA virus.J lmmunol,2004,173(10):5935-5943.
26 UCHIDA T, SCUMPIA P O,MURASKO D M, et al.Variable requirement of dendritic cells for recruitment of NK and T cells to different TLR agonists.J Immunol,2007, 178 (6): 3886-3892.
27 Hardy,A.W.et al.HIV turns plasmacytoid dendritic cells(Pdc) into TRAIL- exressing killer pDC and down-regulates HIV coreceptors by Toll-like receptor 7-induced IFN-alpha.Proe Natl Aead Sei USA,2007,104(44):17453-17458.
28 Liu Y J.IPC:Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.Annu Rev Immunol,2005,23:275-306.
29 Guidueei C,et al.PI3K is critical for the nuelear transloeation of IRF-7 and I-IFN production by human Plasmacytoid predendritic cells in response to TLR activation.J.Exp,Med,2008.205(2):315-322.
30 Horsmans Y,Berg T,Desager J P,et al:Isatoribine,an agonist of TLR7,reduees plasma virus concentration in chroniche patitis C infection. Hepatology, 2005,42(3):724-731.
31 Weeratna R D,Makinen S R,McCluskie M J,etal.TLR agonists as vaceine adjuvants:Comparison of CpGODN and Resiquimod(R-848).Vaccine,2005,23(45):5263-5270.
32 Wa ng Y,Abel K,Lant Z K,et al.The Toll- like receptor 7(TLR7) agonist,imiquimod, and the TLR gagonist, CpGODN,induce antiviral cytokines and chemokines but do not prevent vaginal transmission of simian immunodeficiency virus when applied intravaginally to thesus macaques.J Virol,2005,79(22):14355-14370.
33 Wasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune response. Nat Immunol,2004,5(10):987-995.
34 Arancibia SA,Belt rán CJ,Aguirre IM,et al.Toll-like receptors are key participants in innate immune responses.Biol Res,2007,40(2):97-112.
35 Heil F,Ahmad-Nejad P,Hemmi H,et a1.The Toil-like receptor 7(TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7,8 and 9 subfamily. European Journal of Immunology,2003,33(9):2987-2997.
36 Oodman MG.Cellular and biochemical studies of substituted guanine ribonucleoside immunostimulants. Immunopharmacol,1991,484(7):21-51.
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