【作者】 曲忠花；

【导师】 孙汶生； 陈有海；

【作者基本信息】 山东大学 ， 免疫学， 2010， 博士

【摘要】 第一部分：HBV及其片段HBc、HBx对补体介导的肝细胞杀伤作用的影响研究乙型肝炎病毒(Hepatitis B virus, HBV)在感染肝细胞中极易发生基因片段的整合从而导致病毒在机体内长期存在不易被清除,如今已经成为世界性难题。目前关于乙肝及其所致肝癌的发病机制还不是很明确。本课题组前期实验表明,HBV全基因,HBx, PreS2均可提高肝细胞对TRAIL诱导凋亡的敏感性。另已有研究证实,补体系统在保护机体抵抗病毒感染中发挥重要作用,急性期补体成分C3、C4的胞浆水平与患者疾病的转归有着密切的联系。补体系统由约30种可溶性及膜结合型蛋白组成,其中近90%的胞浆补体成分及可溶性的补体调节蛋白都是在肝脏(主要是肝细胞)中合成的。生理条件下,所有的补体成分处于自发地低水平活化状态,体内的这种活化对机体细胞形成潜在的威胁。然而,机体细胞可表达数种能够抑制自身补体活化从而保护自身的胞浆和膜蛋白,目前已知至少有10种可溶性和膜结合型蛋白。人类肝细胞表达CD59,CD55,CD46和CFH,其中CD55,CD46和CFH都通过限制C3活化发挥作用,而CD59则通过抑制MAC形成起作用；阻断CD59可显著提高补体对肝细胞的杀伤率,而阻断CD55,CD46,CFH作用不明显。因此CD59被认为是肝细胞中最重要的补体调节蛋白。于是我们提出一个问题：HBV对补体杀伤感染肝细胞的活性有何影响?其作用机制是什么?HBV片段HBc、HBx是否参与补体杀伤作用的调节?目的：1.探讨HBV感染对补体介导的细胞杀伤作用的影响及其机制。2.探讨HBV片段HBc、HBx诱导对补体杀伤的影响。方法：1.高通量分析HBV感染对肝细胞基因表达谱的影响1.1 HBV转基因小鼠基因芯片分析提取HBV转基因小鼠及其对照小鼠肝组织总RNA,进行基因芯片分析,比较两组mRNA水平的表达差异,分析补体系统成分的变化情况。1.2 HBV转基因小鼠蛋白质组学分析提取HBV转基因小鼠及其对照小鼠肝组织总蛋白,通过二维电泳分离蛋白,染色后比较并分离出差异表达的蛋白点,进行质谱分析并鉴定相应蛋白,分析其与HBV感染的关系。2.HBV表达对补体介导的肝细胞杀伤的影响2.1表达HBV的肝癌细胞模型的建立将课题组先前构建的pcDNA3-HBV1.1与空载体对照pcDNA3分别利用以阳离子脂质体介导的方式分别稳定转染肝癌细胞BEL7402,HL7702,并分别命名为BEL7402-HBV1.1、BEL7402-pcDNA3, HL7702-HBV1.1、HL7702-pcDNA3细胞。2.2 HBV表达对补体介导的肝细胞杀伤的影响分别以BEL7402和HL7702细胞免疫新西兰大白兔,制备针对两种细胞的多克隆抗体。将该多克隆抗体与BEL7402-HBV1.1、BEL7402-pcDNA3, HL7702-HBV1.1、HL7702-pcDNA3细胞分别作用30min后,加入1／4稀释的正常人血清或对照灭活血清,37℃孵育1h后,以1μg／ml PI染色10min,通过流式细胞术检测细胞杀伤情况；或利用CCK-8试剂盒检测细胞存活状况。2.3 HBV感染对补体调节蛋白CD59表达的影响细胞模型检测：通过RT-PCR,实时定量PCR及Western Blot和流式细胞术的方法,从转录和翻译的水平分别检测BEL7402-HBV1.1、BEL7402-pcDNA3, HL7702-HBV1.1、HL7702-pcDNA3细胞中CD59的表达动物模型检测：HBV转基因与正常小鼠分别肝脏灌注后,提取肝组织RNA,通过RT-PCR及实时定量PCR检测肝细胞中CD59的表达临床标本检测：以来自于肝脏血管瘤或肝破裂的病人肝组织标本作为正常肝标本,与HBV阳性病人肝组织进行免疫组织化学染色检测,比较其CD59蛋白表达水平。2.4 CD59在补体杀伤HBV感染细胞中的作用以特异性CD59抗体阻断CD59的作用,然后进行补体杀伤实验,利用流式细胞术和CCK-8法检测杀伤率。3.HBV片段HBc、HBx表达对补体杀伤的影响3.1表达HBc、HBx的肝癌细胞模型的建立在BEL7402细胞系中稳定转染pcDNA3-HBc、pcDNA3-HBx,经G418筛选后,分别命名为BEL7402-HBc、BEL7402-HBx细胞。3.2 HBc、HBx表达对补体调节蛋白CD59表达的影响通过RT-PCR,实时定量PCR及Western Blot和流式细胞术的方法,从转录和翻译的水平分别检测BEL7402-HBc、BEL7402-HBx及对照BEL7402-pcDNA3细胞中CD59的表达。3.3HBc、HBx表达对补体介导的肝细胞杀伤的影响及机制由经典途径启动补体系统对BEL7402-HBc、BEL7402-HBx及BEL7402-pcDNA3细胞的杀伤作用,通过CCK-8试剂盒检测细胞存活状况。同时设CD59阻断组,检测CD59在其中的作用。3.3 CD59蛋白水平表达的调控机制以5μM MG132阻断细胞内泛素-蛋白酶体降解途径,不同时间点检测CD59的蛋白表达,以探索CD59是否经泛素化降解。结果：1.HBV表达明显改变补体成分的表达基因芯片的结果显示,HBV转基因小鼠中约30%的补体成分表达水平发生显著变化。2.HBV表达对补体介导的肝细胞杀伤的影响2.1 HBV表达上调补体对肝细胞杀伤的敏感性流式细胞术及CCK-8均提示,BEL7402-HBV1.1和HL7702-HBV1.1细胞较对照组细胞的杀伤率均明显升高(P<0.01)。2.2 HBV感染抑制CD59的表达细胞模型检测：半定量RT-PCR及实时定量PCR均显示BEL7402-HBV1.1和HL7702-HBV1.1细胞较对照组细胞CD59 mRNA水平明显下降(P<0.01)；同样,流式细胞术及Western Blot也显示CD59的蛋白水平在BEL7402-HBV1.1和HL7702-HBV1.1细胞中明显下降(P<0.01,P<0.05)。动物模型检测：半定量RT-PCR及实时定量PCR检测HBV转基因小鼠与正常小鼠肝组织中CD59的表达。结果显示HBV转基因小鼠肝组织中CD59明显下降(P<0.01)。临床标本检测：对于12例乙肝病人标本和6例正常肝脏标本进行免疫组化检测,结果显示：乙肝病人肝脏中CD59的表达明显低于正常组(P<0.05)。2.3 CD59在HBV提高肝细胞对补体敏感性中的作用以15ng／ml特异性CD59抗体BRIC229阻断细胞上的CD59的作用后,通过流式细胞术和CCK-8的方法检测补体杀伤率的变化,结果发现：阻断CD59后肝细胞对补体杀伤的敏感性明显增加,但HBV转染细胞与对照细胞间的差异己不显著(P>0.05)。3.HBV片段HBc和HBx对补体介导的肝细胞杀伤作用的影响3.1 HBc和HBx转染对CD59表达的影响半定量RT-PCR及实时定量PCR均显示BEL7402-HBc和BEL7402-HBx细胞较对照组细胞CD59 mRNA水平无明显变化(P>0.05)；然而,流式细胞术及Western Blot显示BEL7402-HBc中CD59的蛋白水平明显低于对照组(P<0.01),而BEL7402-HBx细胞与对照组无显著差异(P>0.05)。3.2 HBc和HBx对补体杀伤肝细胞作用的影响以25%的正常人血清循经典途径进行补体介导的肝细胞杀伤实验,CCK-8结果显示,BEL7402-HBc细胞对补体杀伤的敏感性显著高于对照组(P<0.01)；而BEL7402-HBx细胞与对照组差异不明显(P>0.05)。以阻断性抗体阻断CD59作用后,补体对BEL7402-HBc/HBx的杀伤率与对照组无显著差异(P>0.05)。3.3 HBc表达对CD59表达的调控机制以MG132阻断BEL7402细胞中蛋白酶体降解途径后,检测CD59的蛋白水平。流式细胞术结果显示,CD59的水平并没有明显变化(P>0.05),说明CD59不经泛素化降解,也不会是HBc调节其表达的机制。MicroRNA芯片显示,针对CD59的microRNA水平上调,可能参与HBc对CD59的调节。结论：1.HBV表达可明显改变补体系统的表达谱,对补体系统的调控可能是HBV致病的机制之一。2.HBV表达可提高肝细胞对补体杀伤的敏感性,证实HBV具有促进补体杀伤的生物学活性。3.HBV感染可在RNA和蛋白水平抑制肝细胞内CD59的表达。4.HBV表达提高肝细胞杀伤敏感性这一作用主要是通过下调CD59而发挥的。5.HBV片段HBc可在蛋白水平抑制肝细胞内CD59表达并进而提高细胞对补体杀伤的敏感性,可能是HBV发挥作用的一个关键片段。6.HBx对CD59和补体杀伤活性均无显著影响。创新点及意义：1.本研究首次结合高通量分析的方法对HBV影响的基因表达谱尤其是补体基因表达谱进行了分析,有利于从总体上理解HBV的作用。2.本研究通过HBV表达质粒转染不同肝细胞系,首次发现HBV表达可提高肝细胞对补体杀伤的敏感性,为进一步阐明HBV感染相关疾病的发病机制提供实验依据。3.本研究分别利用HBV感染的细胞模型、动物模型和临床标本检测了CD59的表达水平,首次发现HBV感染可下调CD59表达,为研究补体调节蛋白在HBV感染中的作用奠定了基础。4.应用抗体阻断CD59作用的方法,首次证实HBV提高补体杀伤敏感性的作用是通过下调CD59表达发挥的。阐明了HBV影响补体作用的机制,为临床治疗提供新的靶点。5.在研究HBV全基因对补体杀伤功能影响的基础上,本研究又深入探讨了HBV片段HBc和HBx对补体功能的作用,初步筛选了HBV影响补体杀伤的关键片段。第二部分：LPS预处理对胞内DNA免疫的影响固有免疫系统通过模式识别受体来识别病原体感染,从而启动免疫应答。病原体刺激主要分为两类：一类是病原体特异性产物,如LPS,LTA等；第二类包括病原体核酸。已知部分TLRs可以识别核酸,如TLR3识别单链RNA,TLR9识别CpG。最近研究发现胞浆双链DNA可以不依赖于TLR而引起机体的免疫应答,其受体尚不明确(可能为DAI或其他分子),进入细胞后可以激活TANK结合激酶(TBK1),进而活化IRF3,促进I型干扰素基因的转录,产生并分泌大量干扰素。LPS是革兰氏阴性菌表面的一种脂多糖,能被固有免疫细胞表面的TLR4识别,分别经MyD88依赖途径活化NF-κB,经MyD88非依赖途径即TRIF途径活化IRF3和NF-κB,从而产生大量细胞因子和I型干扰素。当给予细胞两次LPS后,其分泌的细胞因子和干扰素显著降低,称为LPS耐受。在LPS耐受时,并非所有基因均被抑制,部分基因甚至表达更强。应答受抑制的现象也出现在当LPS与其他TLR的刺激物先后作用时,称为交叉耐受。那么LPS对DNA免疫的影响又是如何呢??作用机制又是什么?其作用是否与LPS耐受一致从这几个问题出发,我们研究了LPS对DNA免疫的影响,并取得了一定的研究成果。目的：探讨LPS预处理对细胞内DNA免疫的影响及其机制。方法：1.LPS预处理对胞内B-DNA免疫产生的I型干扰素的影响1.1 LPS预处理对化学合成DNA转染诱导的I型干扰素产生的影响LPS预处理对DNA转染效率的影响：以100ng／ml的LPS刺激RAW264.724h后,用PBS洗两次,再加入新鲜培养基孵育1h,然后用阳离子脂质体Lipofectamine LTX转染pEGFP质粒,24h后流式细胞术检测GFP阳性细胞。DNA转染RAW264.7或BMDM：以100ng／ml的LPS刺激RAW264.7/BMDM24h后转染DNA,4h后提取RNA,实时定量PCR检测Ifnb1和Ifna4的表达。1.2 LPS预处理对细菌DNA感染诱导的I型干扰素产生的影响1)LPS预处理对细菌转染效率的影响实时定量PCR:LPS预处理RAW264.7后24h后,按照MOI为6：1加入LM,4h后提取基因组DNA,实时定量PCR检测细菌基因组与细胞基因组的相对量。流式细胞术：LPS预处理RAW264.7后24h后,按照MOI为6：1加入GFP-LM,4h后充分洗涤细胞,流式细胞仪检测GFP阳性细胞比例。2)LM感染RAW264.7按照MOI为6：1感染经或不经LPS预处理的RAW264.7细胞4h后,提取RNA,实时定量PCR检测Ifnb1的表达。2.LPS预处理影响B-DNA免疫的机制2.1基因转录水平建立荧光报告系统,将含有Ifnb1启动子的荧光报告载体转染HEK293-TLR4细胞,24h后,以LPS刺激该细胞,再24h后,转染10μg／ml DNA,20h后双荧光报告系统检测荧光素酶活性。2.2细胞通路水平以LPS刺激RAW264.7细胞24h后,再转染DNA,于不同时间点分别提取蛋白,Western Blot检测IκB,cJun, IRF3磷酸化情况。以cJun上游分子JNK的特异性抑制物SP600125和IκB通路的抑制物BAY7082阻断cJun和NF-κB活化,实时定量PCR检测Ifnb1表达。3.LPS预处理对DNA诱导基因表达谱的影响以LPS刺激BMDM细胞24h后,再转染DNA,4h后提取RNA,质检合格后进行基因芯片检测,对芯片结果进行分析验证及机制探讨。结果：1.LPS预处理对胞内DNA产生的I型干扰素的影响1.1 LPS预处理对细胞DNA转染率和细菌感染率无明显影响转染pEGFP质粒后,通过流式检测阳性细胞,发现LPS预处理与否对转染率无明显影响(P>0.05)。同样,感染细菌后,实时定量PCR和流式细胞术也都显示LPS预处理不影响细菌的感染效率(P>0.05)。1.2 LPS预处理抑制胞内DNA引起的I型干扰素的表达分别以RAW264.7及BMDM作为细胞来源,以化学合成的DNA片段和LM作为DNA来源,发现LPS预处理均可明显抑制细胞中Ifna4和Ifnb1产生(P<0.01)。2.LPS预处理抑制I型干扰素产生的机制2.1 LPS预处理抑制DNA诱导的Ifnb1启动子活性以HEK293-TLR4为报告细胞,转染Ifnb1的启动子。双荧光报告系统显示LPS预处理组Ifnb1启动子活性明显低于未处理组(P<0.01),说明LPS预处理可抑制DNA免疫诱导的Ifnb1启动子活性。2.2 LPS预处理抑制DNA诱导的Ifnb1转录因子活化的影响对调控I型干扰素的通路分子进行Western Blot检测。结果显示,调控Ifnb1表达的主要转录因子IRF3, IκB和cJun活化均下调。2.3 IKB和cJun通路在I型干扰素产生中的作用分别以化学阻断剂阻断IκB和cJun通路,检测对DNA免疫的影响。发现阻断IκB通路Ifnb1的产生不降低,而抑制cJun通路后则明显减少。3.LPS预处理对DNA诱导基因表达谱的影响基因芯片结果显示：几种I型干扰素在LPS预处理组的表达均明显低于无预处理组。另外,综合分析表明DNA诱导表达的754个基因中,有276个可被LPS预处理抑制(Tolerizalbe genes, T),有92个不被抑制或反而表达更强(Non-tolerizalbe genes, NT)。与已发表的LPS耐受的耐受基因和非耐受基因谱并不完全一致。可抑制基因中富含调控分子stat1。Western Blot显示LPS预处理组Stat1活化降低,而负性调控蛋白SOCS1表达明显升高。结论：1.LPS预处理可显著的I型干扰素的产生,说明TLR4通路的改变会影响到DNA免疫的水平。2.LPS可通过抑制DNA诱导的Ifnb1的启动子活性而抑制其表达,从通路上主要是抑制转录因子IRF3和cJun而非IκB的磷酸化而实现的。3.LPS预处理并非抑制DNA免疫诱导的所有基因表达,部分基因甚至表达明显上调,这与LPS-LPS耐受类似,但其表达谱又存在差别,说明其调控机制仍存在差别。4.LPS-DNA免疫所诱导的耐受基因受到IRF、Statl等的调控,负性调控因子SOCS1的上调也是原因之一创新点及意义：1.本研究首次探讨了LPS对细胞内DNA免疫的影响,有助于解释临床复合性感染的发病机制,并对于感染性疾病的治疗,DNA疫苗的应用等均具有重要意义。2.首次从启动子活性,转录因子水平及信号通路上解释了LPS抑制DNA免疫的机制,提出LPS可能是通过上调SOCS1并抑制IRF3和cJun的磷酸化,从而抑制DNA诱导的I型干扰素表达。3.率先利用基因芯片分析了LPS对DNA免疫诱导的基因表达谱的影响,并对其进行了分析,为进一步的深入研究奠定基础。更多还原

【Abstract】 Part I:The effect of HBV and its fragments HBc and HBx on CDCHBV is hard to be cleared entirely because HBV DNA often integrating into host DNA, which results in persistent virus infection--a major worldwide health problem. Till now, the pathogenesis for HBV infection and HCC development is still not well-known. The involvement of some immune factors, such as T and NK cells, was documented by other groups before. Our previous studies showed that both the whole HBV genome and its fragments HBx and PreS2 enhanced TRAIL (TNF related apoptosis inducing ligand)-induced hepatocyte apoptosis, while HBc inhibited TRAIL induced apoptosis.Some other studies have already shown that complement system plays a crucial role in HBV infection. The presence of C3 and C4 in the liver parenchyma is abnormal and may be helpful in histological evaluation in chronic hepatitis. The complement system consists of about 30 soluble and membrane bound proteins,90% of plasma complement components and their soluble regulators is synthesized in the liver (mainly hepatocytes). All complement components are activated spontaneous low rate in plasma under physical condition, which is a potential threat to the body. However, Hepatocytes gain their effective complement resistance by expressing multiple regulatory proteins. Human primary hepatocytes express CD46, CD55, CD59 and factor H. CD46,CD55, factor H function by inhibiting C3 activation, while CD59 by restricting MAC formation. Blocking CD59, but not CD46, CD55, or factor H leads to higher hepatocyte lysis rate. Thus, CD59 is considered to play a decisive role in protecting hepatocytes against complement lysis. Then what’s the role of complement system in HBV infection? What’s the mechanism? How about its fragments HBc and HBx?OBJECTIVES:1. To explore the effect of HBV infection on complement-dependent cytotoxicity (CDC) and its mechanism.2. To investigate the role of HBc and HBx on CDC.METHODS:1. Effects of HBV1.1 expression on gene profile by high throughout screening.1.1 Microarray analysis of HBV transgenic miceTotal liver RNA from HBV transgenic mice and control mice was extracted and hybridized on gene chip. Significant differentially expressed genes were analyzed by bioinformatics approach and some differentially expressed complement components were verified.1.2 Proteomic analysis of HBV transgenic miceTotal liver protein from HBV transgenic mice and control mice was extracted and then resolved by 2D electrophoresis. Identify the differentially expressed protein by mass spectrum and bioinformatics approach.2. The effect of HBV expression on CDC2.1 Establishment of hepatoma cell line stably-expressing HBV.Plasmid pcDNA3-HBV1.1, constructed in our lab before, or pcDNA3.0 was transfected into hepatoma cell line BEL7402 and HL7702 mediated by lipofectamine 2000, and the positive cell clones were obtained by selection with G418 and termed as BEL7402-HBV1.1, BEL7402-pcDNA3 and HL7702-HBV1.1, HL7702-pcDNA3 cells respectively.2.2 The effect of HBV on complement-dependent cytotoxicityPolyclonal anti-sera were prepared by immunizing New Zealand White Rabbit with BEL7402 or HL7702 lysates three times. BEL7402-HBV1.1, BEL7402-pcDNA3 and HL7702-HBV1.1, HL7702-pcDNA3 cells were incubated with anti-sera for 30min and then incubated with normal human serum(NHS) or heat-inactivated NHS(HI-NHS) for 30min or 1h, washed twice; then labeled by incubation with 1μg/ml propidium iodide (PI) for 10 min at room temperature and detected by flow cytometry. The CCK-8 kit (Dojindo Inc.) was also used to determine the cell viability and the percentage of dead cells.2.3 The effect of HBV on CD59 expressionHepatocyte cell line model:mRNA expression level of CD59 on BEL7402-pcDNA3、BEL7402-HBV1.1; HL7702-pcDNA3、HL7702-HBV1.1 cells were detected by semi-quantitative RT-PCR and real-time PCR, protein expression level by Western blot and flow cytometry.Mouse model:Extract mRNA of HBV transgenic mice and normal control mice after liver perfusion, CD59 expression is detected by semi-quantitative RT-PCR and real-time PCR.Clinical samples:Liver tissue samples from twelve chronic hepatitis B patients were obtained by liver centesis. Six normal liver tissue samples were obtained from patients with liver hemangioma or hepatorrhexis. Expression of CD59 was determined by immunohistochemistry.2.4 Contribution of CD59 on CDC of HBV-infected hepatocytes.After blocking CD59 function by specific blocking antibody, CDC assay was carried on and cytotoxicity rate was determined by FCM and CCK-8.3. Role of HBV fragments HBc on CDC.3.1 Establishment of hepatoma cell line stably-expressing HBc or HBxPlasmid pcDNA3-HBc or pcDNA3-HBx constructed in our lab before, was transfected into hepatoma cell line BEL7402 mediated by LF 2000, and the positive cell clones were obtained by selection with G418 and termed as BEL7402-HBc, and BEL7402-HBx, cells respectively.3.2 The effect of HBV on CD59 expressionThe mRNA level of CD59 on BEL7402-pcDNA3、pcDNA3-HBc or pcDNA3-HBx cells were detected by semi-quantitative RT-PCR and real-time PCR; The protein level of CD59 was detected by Western blot and flow cytometry. 3.3 The effect of HBc and HBx on complement-dependent cytotoxicity and its mechanismComplement-dependent lysis of BEL7402-HBc, BEL7402-HBx and BEL7402-pcDNA3 was performed through the classical pathway. The CCK-8 kit was also to determine the cell viability and calculate the percentage of dead cells.。Meantime, examine the role of CD59 in this, BRIC229 was used to block CD59 function.3.4 The regulation of CD59expression in protein level.The ubiquitin-proteasome activity in BEL7402 was blocked by 5μM MG132, and CD59 protein was detected by FCM. at different time point.RESULTS:1. Expression of HBV in hepatocytes changed the level of complement components greatly.As shown by gene microarray, the expression of about 30%of complement components changed in HBV transgenic mice.2. The effect of HBV expression on CDC2.1 HBV expression upregulated hepatocytes sensitivity to CDC.Both FCM and CCK-8 assay showed, the lysis rate of complement on BEL7402-HBV1.1 and HL7702-HBV1.1 increased significantly compared with the control cells (P<0.01)2.2 HBV expression in hepatocytes downregulated CD59 expression.Hepatoma cell model:By RT-PCR and real-time PCR, we found that the RNA level of CD59 after HBV transfection was reduced in both BEL7402 (P< 0.01) and HL7702 (P< 0.05). In addition, we also tested the CD59 expression by FCM and Western blot analysis. The protein level of CD59 was also downregulated by the HBV transfection (P< 0.05).Mouse model:RT-PCR and real-time PCR data showed, the level of CD59 mRNA decreased in HBV transgenic mice (P< 0.05).Clinical Samples:Compared with normal liver tissue, CD59 expression was significantly reduced in the livers of HBV patients by immunochemistry.2.3 Role of CD59 on CDC of HBV-transfected hepatocytes.FCM and CCK-8 assay showed, blocking CD59 by 15ng/ml specific antibody BRIC229 significantly increased cytotoxicity rate in all cell lines, but the difference between HBV transfected cells and control cells diminished (P> 0.05)3. The impact of HBV fragments HBc and HBx on CDC3.1 The effect of HBc and HBx transfection on CD59 expression.Both semi-quantitative PCR and real-time PCR data showed no significant difference of CD59 mRNA level between BEL7402-HBc or BEL7402-HBx and control cells (P> 0.05). However, the protein level of CD59 on BEL7402-HBc was much lower than control (P< 0.01). There was still no difference on CD59 protein level between BEL7402-HBx and control cells (P> 0.05).3.2 Role of HBc and HBx on lysis of hepatocytes by complementCDC assay was applied with 25%of NHS by activating the classical pathway. CCK-8 data showed, the lysis rate of BEL7402-HBc was much higher than control group(P< 0.01), while the lysis rate of BEL7402-HBx was similar with control group (P>0.05)。3.3 Regulation of CD59 by HBcThe ubiquitin-proteasome activity in BEL7402 was blocked by MG132, and CD59 protein was detected by FCM. The result displayed no change on CD59 protein level (P> 0.05), demonstrated that CD59 wasn’t degraded by ubiquitinization。Our MicroRNA chip showed, some microRNAs targeting CD59 was upregulated in BEL7402-HBc, which might participate in the regulation of CD59 by HBc.CONCLUSIONS:1. HBV expression changes gene profile of hepatocytes greatly, the regulation of complement system might be one of the mechanism of HBV-related diseases.2. HBV expression can significantly sensitize hepatocytes to complement-dependent cytotoxicity. The further mechanism study demonstrates HBV functions by downregulating CD59 expression. 3. HBV fragment HBc can dramatically increase the sensitivity of hepatocytes to CDC by downregulating the protein level of CD59.4. HBx has effect on neither CD59 expression nor CDC of hepatocytes.INNOVATIONS AND SIGNIFICANCES:1. It’s the first time to compare the gene profile influenced by HBV with HBV transgenic mice by both microarray and proteomic methods, which will help to understand the overall effect of HBV.2. Through establishing of HBV expressed hepatoma cell lines, the present study demonstrates that the effects of HBV on CDC and firstly reports the upregulation of CDC by HBV. This is helpful to disclose the mechanism of HBV infection.3. It’s the first time to prove downregulation of CD59 expression in HBV transfected hepatocytes, HBV transgenic mice and HBV patients.4. By blocking CD59 function, we firstly show the increased sensitivity to complement of HBV expressed cells is mediated by downregulating CD59, CD59 downregulation is the key factor, which can be a new target for the treatment of HBV infection.5. Based on the study of HBV on CDC, we further report HBc can downregulte CD59 and enhance CDC, while HBx has no effect.Keywords:HBV; CD59; complement; HBc; HBxPart II:The effect of LPS pretreatment on DNA immunityThe innate immune system uses pattern recognition to sense infection, and it initiates an immune response upon pathogen detection. Recent advances have defined two broad categories of microbial stimuli. The first class includes products unique to microbes, such as lipopolysaccharide (LPS) and lipotechoic acid. The second class encompasses nucleic acids derived from pathogens, particularly viruses. Some known TLRs can detect nucleic acids, for example, TLR3 can detect double-stranded RNA, TLR9 can sense methylated CpG motifs. Recent studies show almost all cytosolic double-stranded B-form DNA can trigger immune response without activating TLRs. The sensor for it is still not clear, maybe DAI or others. Cytosolic DNA can activate TBK1 and then IRF3, promote the transcription of type I IFN.LPS is a component on Gram negative bacteria, and can be sensed by TLR4. It can induce cytokine production by activating NF-κB through MyD88-dependent and independent pathway, also induce type I IFN production by activating IRF3 through MyD88-independent pathway. When cells are stimulated with LPS twice subsequently, the induction of cytokines is mostly decreased, this phenomenon is called LPS tolerance or endotoxin tolerance. Similar phenomenon also shows up when cells are stimulated with LPS and other TLR simulators subsequently, which is called cross-tolerance. Then, what’s the impact of LPS on cytosolic DNA induced immunity? What’s the mechanism? Is that similar to LPS tolerance? Based on these questions, we focused on the impact of LPS on DNA immunity and have gained some periodic outcome.OBJECTIVES:To probe the impact of LPS on DNA immunity and its mechanismMETHODS:1. The effect of LPS pretreatment on DNA induced type I IFN expression.1.1 The effect of LPS pretreatment on chemically synthesized DNA induced type I IFN expressionThe impact LPS pretreatment on transfection efficiency:Stimulate RAW264.7 with 100ng/ml LPS for 24h, wash twice, incubate with fresh medium for 1h, then transfect the cells with pEGFP plasmid using Lipofectamine LTX,24h later, check the GFP positive signal by FCM. Transfection of DNA into RAW264.7 or BMDM:Pretreat RAW264.7 with LPS then transfect with DNA,4h later, extract mRNA and detect Ifnb1 and Ifna4 expression by real-time PCR.1.2 The effect of LPS pretreatment on bacteria DNA induced type I IFN expression1) Impact of LPS pretreatment on infection rate of LMReal-time PCR:Pretreat RAW264.7 with LPS for 24h, then add LM (MOI 6:1), 4h later, extract genomic DNA, detect the ratio of bacteria genome and cell genome by real-time PCR.FCM:Pretreat RAW264.7 with LPS for 24h, then add GFP-LM (MOI 6:1),4h later, detect the proportion of GFP positive cells.2) Ifnbl induction by infection of LM into RAW264.7Infect RAW264.7 cells pretreated with or without LPS with LM,4h later, detect expression of Inb1 and Ifna4 by real-time PCR.2. The mechanism for the effect of LPS pretreatment on DNA induced type I IFN production.2.1 Transciptional level Tranfect HEK293-TLR4 with luciferase plasmid containing Ifnb1 promoter for 24h, then stimulate with LPS and DNA subsequently as described above,20h later, the cells were harvested and the luciferase assay was used to detect the luciferase activity.2.2 Cellular pathwayExtract protein at different time point after DNA transfection with or without LPS pretreatment. Detect the phosphorylation of IκB, IRF3, cJun. Examine Ifnb1 expression after blocking cJun or IκB activation.3. The impact of DNA induced gene profile by LPS pretreatment.BMDM were stimulated with 100ng/mL LPS for 24h, then transfected with lug/ml DNA,4h later, extract total RNA, and analyzed by microarray.RESULTS:1. The effect of LPS pretreatment on DNA induced type I IFN expression. 1.1 LPS pretreatment has no obvious impact on transfection efficiency.After transfected with pEGFP plasmid for 24h, GFP positive RAW264.7 cells were determined by FCM, and the data showed no obvious difference with or without LPS pretreatment(P> 0.05). Similarly, there’s no big difference of infection efficiency between the two groups.1.2 Blunted Ifna4 and Ifnbl expression induced by DNA after LPS pretreatmentAfter stimulating both RAW264.7 and BMDM with either synthesized DNA transfection or LM infection, the induction of Ifna4 or Ifnb1 decreased in LPS pretreated cells (P< 0.01).2. Mechanism for the effect of LPS pretreatment on DNA induced IFN production.2.1 LPS pretreatment inhibited the Ifnbl promoter activity induced by DNA.Dual-luciferase reporter assay showed LPS pretreatment significantly inhibited Ifnb1 promoter activity(P<0.01).2.2 Effect of LPS pretreatment on the transcription factors of Ifnbl promoterWestern Blot assay showed, the activation of IRF3, IκB, cJun decreased.2.3 Role of NF-κB和cJun pathway on Ifnbl inductionBlocking cJun pathway by JNK inhibitor inhibited Ifnb1 induction by DNA, but blocking NF-κB pathway by IκB inhibitor didn’t change Ifnb1 level.3. The impact of gene profile induced by DNA by LPS pretreatment.Microarray data showed, almost all type I IFNs induction decreased upon LPS pretreatment.754 genes were induced by DNA, of which 276 were downregulated over 2 folds by LPS pretreatment, and 92 showed no change or even higer response. The downregulated genes were rich in transcription factor IRFs and Statl. Western Blot showed the activation of Statl decreased and SOCS1 increased in LPS pretreated group.CONCLUSIONS:1. LPS pretreatment can inhibit cytosolic DNA induced type I IFNs induction, indicates TLR4 pathway can influence DNA immunity. 2. LPS can inhibit Ifnbl expression by repressing its promoter activity. The activation of two Ifnbl transcription factors, IRF3 and cJun, are tolerized by LPS.3. DNA induced genes are not all tolerized by LPS, some are even primed. This is similar to LPS tolerance, but the profile is not the same, which means the mechanism might be different.4. The tolerizable genes in LPS-DNA immunity is largely mediated by tolerized IRFs and Statl. The increased level of negative regulator SOCS1 is also a key factor.INNOVATIONS AND SIGNIFICANCES:1. It’s the first time to investigate the effect of LPS on DNA immunity. This study will help to explain mechanism of combined clinic infection, and is important for the cure of infectious disease and instruction of DNA vaccine.2. The present study firstly reports LPS pretreatment upregulates the level of SOCS1 and inhibit the phosphorylation of IRF3, cJun and Stat1, result in decreased expression of type I IFN and other tolerizable genes.3. We are the first to study the changed gene profile in DNA immunity by LPS pretreatment through microarray, which is the basic for further study on its mechanism.更多还原