【作者】 叶慧燕；

【导师】 熊思东； 储以微； 徐薇；

【作者基本信息】 复旦大学 ， 免疫学， 2008， 硕士

【摘要】 乙型肝炎病毒(hepatitis B virus,HBV)是一种很小的包膜病毒,属于DNA病毒科,HBV持续感染是导致慢性肝炎后肝硬化、肝细胞性肝癌等疾病的重要原因,严重威胁着人类健康,目前全世界约有3.5亿慢性HBV感染者,约占全球人口的6%,我国作为乙肝的高发流行区,占全球HBV表面抗原总携带率的近50%,且60%的人受过HBV的感染。固有免疫应答是机体抗病毒感染免疫的第一道防线,这一过程的启动需要PRR(pattern recognition receptor,模式识别受体)识别病毒的某些成份,进而活化下游的信号途径。在PRR中除了我们所熟知的Toll受体家族,近年来一类Toll非依赖性PRR也成为固有免疫的研究热点,这其中RIG-Ⅰ(retinoid induced gene-I,维甲酸诱导基因I)因其表达谱广、识别病毒类型多而备受关注。研究证实RIG-I信号途径在dsRNA(double stranded RNA,双链RNA)病毒的固有免疫应答过程中发挥重要作用,其信号的传导主要由RIG-IN端CARD结构域(RIG-IN)介导,RIG-IN被活化后,可通过蛋白-蛋白结合方式,激活下游接头蛋白,进而活化NF-kB和IRF(interferon regulatory factor,干扰素调节因子),最终引起干扰素和炎性因子的大量释放,发挥抗病毒效应。固有免疫应答在HBV的抗感染过程中有着很重要的地位,Chisari等证实TLR及其配体在体内可抑制HBV的复制,且这些TLR配体主要通过诱导I型干扰素的产生而抑制HBV DNA的合成。但目前并不清楚RIG-I信号途径在HBV抗感染免疫中的角色。本研究中,我们利用HBV感染体内外模型研究RIG-IN对HBV复制的影响,探讨RIG-I信号途径在HBV抗感染过程中的作用及其机制。第一部分:pcDNA-RIG-IN质粒鉴定及HBV感染体内外模型的建立RIG-I,又名Ddx58,是近年来比较热门的Toll非依赖性模式识别受体,定位在人染色体10q11-15,猪染色体的10q13。它有925个氨基酸,属于DExD/H家族。RIG-I在N端包含一前一后两个CARD(caspase recruitment domain,半胱天冬酶招募结构域),这一结构域也为其他的胞内识别受体所具有,如NOD1、NOD2,C端有一RNA解旋酶(Helicase)结构域,此外,在RIG-IC端有一抑制性结构域,可负调控RIG-IN介导的信号途径,研究表明RNA病毒一般为RIG-I Helicase结构域识别,其信号由RIG-IN端结构域传导。课题的第一部分是鉴定pcDNA-RIG-IN质粒并建立HBV感染的体内外模型。pcDNA-RIG-IN质粒经NheI和BamHI酶切后显示有一约790bp大小的插入片段,测序结果显示在pcDNA载体中已克隆进RIG-IN端2-229位氨基酸,序列正确且在前面带有myc标签蛋白;为在体外检测RIG-IN对HBV复制的影响,我们首先建立HBV感染的细胞模型,因嗜肝DNA病毒具有组织特异性,因此我们选取了分化成熟的肝癌细胞系HepG2来建模,使用磷酸钙转染将HBV质粒pTHBV导入细胞内,通过检测上清中HBVs抗原(HBsAg)和HBVe抗原(HBeAg)的表达量,证实HBV在这两株细胞系中均可有效复制,从而建立HBV感染细胞模型。体内我们通过高压注射法建立HBV感染小鼠模型,将pTHBV从尾静脉快速注入正常BALB/c小鼠体内,在注射后1、2、4、7、10天收集小鼠血清,用ELISA法检测HBsAg和HBeAg的表达量;结果显示在注射后24小时以内,即产生高水平的HBsAg及HBeAg;从第2天开始,血清中HBsAg及HBeAg的水平下降,至第10天抗原水平已很难测到;以上数据证实高压注射法可建立HBV感染的体内模型。第二部分RIG-IN端结构域对HBV复制的影响在质粒鉴定正确且HBV感染模型成功建立的基础上,课题的第二部分是关注RIG-IN对HBV复制的影响,分为体内和体外两方面的研究。体外我们通过质粒共转的方法,将RIG-IN和pTHBV共转进HepG2细胞中,同时设立pcDNA和pTHBV共转染组为对照组,在转染1、2、3天后收集上清用ELISA法检测HBsng和HBeAg的表达量;用Northern blot法检测HBV转录本水平;用real-time PCR检测转染后HBV复制中间体DNA的水平。结果显示RIG-IN和pTHBV转染组,相比空质粒对照组,上清中抗原表达量明显降低,通过共转GFP,我们未发现空质粒对照组和RIG-IN组在转染效率上有显著区别;而且Northern blot结果显示RIG-IN组的3.5kb及2.4/2.1kb转录本水平均明显降低;进一步为明确RIG-IN对HBV复制的影响,我们从转染后的细胞内提取病毒颗粒,用荧光定量PCR检测cccDNA拷贝数(covalently closed circular DNA copies),结果显示pcDNA转染组HBV-DNA拷贝数为6.1×10~5,相比之下RIG-IN转染组HBV-DNA拷贝数为3.2×10~5,即RIG-IN可下调HBV-DNA拷贝数50%。在体外证实RIG-IN对HBV复制的抑制作用后,那么在体内RIG-IN是否有类似的抑制效应,将正常BALB/c小鼠分为空质粒对照组和RIG-IN质粒组,通过尾静脉快速注入pTHBV和pcDNA或RIG-IN,在注入质粒1、4、7天后比较两组HBV血清学指标,结果显示RIG-IN组HBsAg只有空质粒对照组的41%,HBeAg为空质粒对照组的50%;为进一步证实RIGI-N在体内对HBV的抑制作用,我们用免疫组化的方法检测小鼠肝组织中HBcAg的表达,结果显示转染后1、4、7天,RIG-IN转染组HBcAg阳性细胞的数目相对于pcDNA转染组明显减少,而且在胞浆和胞核内都观察到HBcAg的存在。这部分结果显示RIG-IN可抑制HBV复制中间体DNA的合成,降低HBV转录本水平及病毒蛋白的表达。第三部分RIG-IN端结构域在影响HBV复制中的作用机制已有大量研究表明NF-κB和I型干扰素信号通路的激活可明显抑制HBV的复制和转录,为进一步了解RIG-IN抑制HBV的作用机制,我们以核因子kappa B以及I型干扰素的荧光报告质粒,检测RIG-IN对它们的作用,结果显示相比空质粒对照组,RIG-IN转染组可明显上调NF-κB至2.4倍;IFN-α至2倍;IFN-β至10倍,提示RIG-IN可能是通过活化NF-κB和I型干扰素信号途径而发挥对HBV的抑制效应。为明确RIG-IN对HBV转录抑制的分子机制,我们用荧光报告质粒的方法检测RIG-IN对SpⅠ、SpⅡ、Core、X四种HBV启动子的影响,有趣的是,我们的结果显示RIG-IN可明显下调这四个HBV启动子活性。以上研究结果表明,这部分结果显示RIG-IN可抑制HBV复制中间体DNA的合成,降低HBV转录本水平及病毒蛋白的表达;进一步在体内我们也观察到RIG-IN不仅下调HBV病毒蛋白水平,也可降低HBcAg阳性的肝细胞数,提示RIG-IN在体内也有类似的抑制效应;在这些现象的基础上,我们以NF-κB和I型干扰素的荧光报告质粒,观察RIG-IN对这两种启动子活性的影响,结果显示RIG-IN在所研究的HepG2细胞中可明显上调NF-κB至2.4倍;IFN-α至2倍;IFN-β至10倍,提示RIG-IN对HBV的抑制作用可能与NF-κB和I型干扰素信号途径的活化有关,通过检测HBV四个启动子的荧光素酶活性,发现RIG-IN可抑制HBV启动子活性,提示RIG-IN对HBV转录水平的抑制可能是通过下调HBV启动子活性而实现。综合以上研究,我们的结果显示RIG-IN可抑制HBV复制及转录并下调HBV蛋白表达水平,其机制可能在于RIG-IN活化了NF-KB和I型干扰素信号途径,我们还观察到RIG-IN明显下调HBV四种启动子的活性,这可能与RIG-IN对HBV的转录抑制有关,但因为RIG-I在胞浆内可与其他蛋白结合,目前还不清楚RIG-IN的这种抑制效应是否能通过抑制某些肝富集转录因子转录入核而发挥抑制效应,这一问题仍有待进一步研究证实。更多还原

【Abstract】 Hepatitis B virus(HBV) is a small enveloped virus,belonging to DNA virus.The persistent HBV infection contributes much to posthepatitic cirrhosis,hepatoceUular liver cancer and so on,which threatens human health seriously.At present,there are 350 million chronic HBV infection patients,which is about 6%of world population China is a region with high epidemic rate.50%of the whole HBsAg takers are in China and 60%of Chinese people have infected with HBV.Innate immune response is the first defense during the anti-virus immune process and its start-up needs the recognition of virus by pattern recognition receptor(PRR), subsequently activates the downstream signaling pathway.Among PRR family,Toll receptors are familiar with us.Recently,a group of Toll non-dependent PRR becomes hot point in innate immune system.Retinoid induced gene-I attracts much attention because of its broad expression ranges and recognition of various virus.The research has shown that RIG-I signaling pathway plays an important role during the innate immune response.RIG-I N terminal CARD domain(RIG-IN) is responsible for the signaling transduction.After RIG-IN is activated,it activated the downstream adapter proteins by protein-protein combination pathway,then activated NF-κB and IRF (interferon regulatory factor),finally stimulated the production of interferon and chemokines,which exhibited anti-virus effect.Innate immune response is important during HBV infection and Chisari has proved that TLR and its ligands could inhibit HBV replication in vivo by stimulation of type I interferon,and then inhibited HBV DNA.However,besides TLR,the role of RIG-I signaling pathway during HBV infection remains unclear.In our research,we used in vitro and in vivo HBV infection model to explore the influence of RIG-IN on HBV viral proteins expression,virus transcription and replication and its mechanism.PartⅠ:Confirmation of pcDNA-RIG-IN and establishment of in vitro and in vivo HBV infection modelRIG-I,Ddx58,is a popular Toll independent pattern recognition receptor.It is located at human chromosome 10qll-15,pig chromosome 10q13.It has 925 amino acids and belongs to DExD/H family.RIG-I contains a tandem CARD domains, which is shared with other pattern recognition receptors,such as NOD1,NOD2,and a Helicase domains at C terminal.Recent research proved that RIG-IC contains a repression domain,which could negatively regulate RIG-I mediated signaling pathway.As we know,RNA virus is recognized by RIG-I Helicase domain and the signaling is relayed by protein-protein interaction between CARD domains.The first part is the confirmation of pcDNA-RIG-IN plasmid and establishment of in vitro and in vivo HBV infection model.The enzyme digestion results of pcDNA-RIG-IN plasmid by NheI and BamHI showed a 790bp fragment inserted into vector.The DNA sequencing result confirmed there were 2-229 AA cloned into vector and it had a myc tag protein.Then the plasmid was collected for the further use.In order to analyze whether RIG-IN could inhibit HBV replication in vitro.First of all, we establish HBV infection cell model.As DNA virus own organ specificity,we chosed the hepatoma cell line HepG2 with mature differentiation.Firstly,the cells were transfected with pTHBV plasmid by calcium phosphate.Then HBsAg and HBeAg were analyzed by ELISA to confirm whether HBV could replicate in HepG2 cells.To conduct the in vivo experiments,we established HBV infection mouse model by high hydrodynamic injection.We injected pTHBV plasmid into BAIB/c through vail van,then collected mouse serum after 1,2,4,7,10 days and analysed HBsAg and HBeAg in the serum.The results showed that after injection for 24 hours, HBsAg and HBeAg level was high in the serum.After 2 days,the HBV antigens level was decreased and nearly undetectable after 10 days.The previous results proved the establishment of HBV infection model in vivo. PartⅡThe influence of RIG-I N terminal domain on HBV replicationBased on the plasmid confirmation and establishment of HBV infection models, the second part of the research focused on the influence of RIG-IN on HBV replication,which is divided into in vitro and in vivo part.In vitro,HepG2 cells were transfected with RIG-IN and pTHBV or pcDNA and pTHBV as control.After transfection for 1,2,3 days,we collected culture supertanant to analyze HBsAg and HBeAg by ELISA,HBV transcription level by Northern blot,HBV DNA copies by real-time PCR.The results showed that antigens levels were decreased obviously in RIG-IN and pTHBV co-transfection group by contrast with vector control.The GFP results did not show any difference between transfection efficiency.As we know,the decrease of HBV viral antige expression is because of the down-regulation of HBV transcription level.Then,we analyze HBV transcription level by Northern blot.The results showed that 3.5 kb and 2.4/2.1kb transcription levels were decreased obviously in RIG-IN group.The results showed that RIG-IN could inhibit HBV transcription.To further investigate the effect on HBV replication of RIG-IN,we exacted HBV viral particles from the cell and then analyzed HBV-DNA by real-time PCR to reflect virus replication.The results showed that HBV-DNA copies is 6.1×10~5 in the pcDNA group.However,the HBV-DNA copies decreased to 3.2×10~5.In the other words, RIG-IN down-regulated HBV-DNA copies 50%.After confirming the inhibitory effect of RIG-IN on HBV replication,we wonder whether RIG-IN could inhibit HBV replication in vitro.We divided BALB/c mice into two groups:vector and RIG-IN.After injection for 1,4,7 days,we collected mice supernatant and compared HBV serum index.The results showed that HBsAg in RIG-IN group is 41%by comparison with vector group,HBeAg is 50%.To further confirm the inhibitory effect on HBV in vivo,we assayed HBcAg expression in the hepotacytes by immunohistochemistry staining.The results showed the HBcAg positive cells were decreased in RIG-IN transfection group and HBcAg particles were detected in both the cytoplasm and nucleus. The results in this part showed that RIG-IN could inhibit HBV DNA copies, decrease HBV transcription levels and HBV protein synthesis.PartⅢ:The mechanisms involved in the effect on HBV replication by RIG-INA lot of researches have proved that activation of NF-κB and type I interferon signaling pathway inhibited HBV replication and transcription obviously.Then we used NF-κB and type I interferon luciferase reporter genes and detected the influence of RIG-IN on their activities.The results showed that RIG-IN over-expression up-regulate NF-κB promoter activity to 2.4 folds,IFN-αto 2 folds and IFN-βto 10 folds separately.These results suggest that RIG-IN displayed inhibitory effect on HBV by activating NF-κB and type I interferon signaling pathway.To identify the molecular mechanism on HBV transcription inhibition by RIG-IN,we used luciferase reporter to analyze the influence on SpⅠ,SPⅡ,Core,X four promoters.Interestingly, we found that RIG-IN down-regulated HBV promoter activities obviously.The previous data have shown that HBV transcription level and HBV DNA copies decreased obviously when co-transfected with RIG-IN and pTHBV.Moreover, we found that RIG-IN down-regulated HBV viral protein levels and HBcAg positive hepatocytes.Then we wonder whether RIG-IN inhibited HBV replication by activating NF-κB and type I interferon pathway.These results showed that RIG-IN over-expression up-regulate NF-κB promoter activity to 2.4 folds,IFN-αto 2 folds and IFN-βto 10 folds separately.Then we focused on the effect on HBV promoter activities of RIG-IN by using luciferase activity assay.The results showed that RIG-IN obviously suppressed the activity of HBV four promoters.The research showed that RIG-IN not only inhibited HBV proteins expression but also down-regulated HBV transcription level and HBV DNA copies.By assaying the luciferase activity of HBV four promoters,we found that RIG-IN inhibited HBV promoter activities,which might contribute to HBV transcription inhibition of RIG-IN.In summary,we found that RIG-IN inhibited HBV antigen expression and replication.The mechanism was most likely that RIG-IN regulated HBV four promoter activities.The luciferase results showed that RIG-IN could activate type I IFN and NF-κB promoter activities in HepG2 cells.Additionally,the obvious down-regulation on HBV four promoter activities was also observered.However,it is still unclear whether RIG-IN inhibited HBV replication by inhibiting the transcription of other liver transcription factors.These questions deserve further research.更多还原