【作者】 王蓉；

【导师】 高立芬；

【作者基本信息】 山东大学 ， 医学免疫学， 2013， 硕士

【摘要】 研究背景乙型肝炎病毒(Hepatitis B virus,HBV)简称乙肝病毒,是一种小环型部分双链DNA病毒,其基因组至少包含S(包含S基因、PreS1基因和PreS2基因)、C、X、P等4个开放读码框架,分别编码包膜抗原(HBs)、核心蛋白(HBc)、X蛋白(HBx)和DNA聚合酶。HBV属于嗜肝病毒属,但没有直接的肝细胞毒性,病毒感染后引发的免疫性肝损伤是其致病的重要机制,HBV感染后可进一步导致肝硬化或肝细胞肝癌的发生。据统计,目前全世界HBV感染人数已超过4亿,每年约100余万人死于HBV相关肝病,因HBV相关疾病死亡的人数占全球40%左右,因此,HBV是目前危害人类健康的主要杀手之一,但HBV感染致免疫性肝损伤的作用尚不十分清楚,其致病机制亟待于深入探讨。长期以来,多数学者认为HBV的成功清除与体内较强的特异性细胞免疫应答有关,然而,越来越多的证据表明固有免疫在HBV感染及致病过程中发挥重要作用,明确HBV感染引发的免疫效应及分子机制对于HBV相关肝病的诊断和治疗极为关键。研究发现,HBV感染后是由肝脏非实质细胞识别,主要由枯否氏细胞(Kupffer cell,KC)识别。KC是肝脏中定居的巨噬细胞,占全身巨噬细胞总数的80%-90%,是体内最大的巨噬细胞群,也是肝内主要的炎性反应细胞和细胞因子来源,KC通过产IL-1β、IL-18等炎症因子参与各种慢性肝病过程中肝损伤的发生与发展。IL-1β和IL-18的产生依赖于炎性体的激活。炎性体(也称炎症小体)是Tschopp等于2002年首次发现并报道的,第-个被发现的炎性体是NLRP1炎性体。随着研究的深入,陆续发现的炎性体有NLRP3(或NALP3).NLRC4.AIM2、RIG-I炎性体等,其中,NLRP3炎性体被研究得最多,NLRP3分子通过接头蛋白ASC招募Caspase-1,从而组成多蛋白复合物即NLRP3炎性体。多种病毒感染可介导NLPR3炎性体的活化,NLRP3炎性体即可感应RNA病毒如流感病毒、丙型肝炎病毒(HCV),也可感应DNA病毒如腺病毒、水痘-带状疱疹病毒,由此说明无包膜的小分子DNA病毒或有包膜的大分子DNA病毒都可调节NLRP3炎性体的活化。炎性体的激活可使Caspase-1活化进而对IL-1β或IL-18前体进行剪切,促进IL-1β或IL-18的成熟和释放,引起炎症反应。文献报道,IL-18、IL-1β启动子基因多态性与HBV感染后的疾病进程及预后密切相关,HBx蛋白和HBc蛋白可诱导IL-18的产生,慢乙肝患者血清中IL-1β的水平显著高于正常人,且乙肝患者体内IL-1β的水平与HBV病毒滴度呈正相关。那么HBV如何调节IL-1β和IL-18的产生呢?目前尚未见相关研究的报道,本文首次探讨了HBV对NLRP3炎性体的调节作用。T细胞免疫球蛋白域黏蛋白样蛋白(T-cell immunoglobulin domain and mucin domain, Tim)家族是McIntire于2001年发现并鉴定的与哮喘和过敏性疾病相关的一个新基因家族。小鼠Tim基因家族位于染色体11B1.1,目前共发现8个基因,编码蛋白Tim-l-Tim-4和4个假想基因Tim-5-Tim-8。人类TIM家族只有3个基因,定位于染色体5q33.2,分别编码蛋白TIM-1、TIM-3、TIM-4。Tim蛋白是一类具有共同基序的Ⅰ型跨膜糖蛋白,其结构包括免疫球蛋白(IgV)样区、黏蛋白(Mucin)样区、跨膜区和胞内区。除Tim-4外,Tim-1、Tim-2、Tim-3胞内区均含有酪氨酸激酶磷酸化位点,直接参与胞内信号转导。Tim-4分子在免疫调节及维持机体稳态中具有重要作用。Tim-4是Tim-1的天然配体,主要表达在活化的抗原提呈细胞表面,能维持腹腔巨噬细胞平衡,Tim-4与Tim-1相互作用可为T细胞提供协同刺激信号,Tim-4对T细胞的活化具有双重调控作用,而Tm-4对巨噬细胞具有负调控作用。课题组前期研究发现,Tim-4过表达可抑制LPS诱导的IL-1β产生,在ConA诱导的小鼠肝炎模型中,发现转输Tim-4过表达的巨噬细胞系可抑制IL-1β的产生,提示Tim-4可能抑制炎性体的活化。另外,发现慢乙肝患者PBMCs、HBV转基因小鼠肝单个核细胞中Tim-4的表达水平显著减低,那么HBV如何调节Tim-4的表达,是否通过调节其启动子活性进而影响其表达呢?HBV是否通过调节Tim-4的表达影响NLRP3炎性体的活性呢?本文基于相关问题进行了初步探讨。研究目的明确HBV对NLRP3炎性体活性的影响,并初步探讨其作用机制；构建人TIM-4启动子报告基因载体并鉴定其活性；探讨TIM-4对NLRP3炎性体活性的影响。方法第一部分：HBV对NLRP3炎性体活化的影响1临床标本检测(1)收集慢乙肝患者及健康对照血浆标本收集了56例慢乙肝患者的初诊病人和20例健康对照组的肝素抗凝血，离心取取上清,-20℃保存。(2)ELISA检测血浆中IL-1β的水平利用ELISA试剂盒检测初诊的慢乙肝病人以及健康对照组的血浆中IL-1β的水平。2.动物实验(1)H3V转基因小鼠及正常对照取鉴定为阳性的8-12周龄的HBV转基因小鼠(Balb/c)35只,以及由山东大学动物中心提供的,12只同龄Balb/c小鼠为正常对照。(2)HBV转基因小鼠血清DNA及抗原的检测分批处死HBV转基因小鼠,摘眼球取血，温放置2h,4℃离心收取上清,检测HBV DNA及表面抗原。(3)ELISA检测血清中IL-1β的水平用ELISA试剂合检测HBV转基因小鼠与健康对照鼠血清中的IL-1β(4) Western blot检测小鼠肝组织中Caspase-1的活化随机选取14例HBV转基因小鼠及珔常对照小鼠的肝组织,取米粒大小的肝组织,匀浆并提取蛋白,Western blot检测Caspase-1的活化情况。3.细胞模型(1) Caspase-1、ASC、NLRP3siRNA的筛选与鉴定分别设计并合成了人ASC、caspase-1、NLRP3三个基因的小干扰套餐,在HEK293细胞中用质粒DNA与小干扰用脂质体法共转,转染48h后,收取细胞,提蛋白,用Western blot验证干扰效果,选取效果较好的进行后续实验。同时,利用脂质体转染PMA诱导的THP-1细胞,验证以上基因siRNA对内源性基因的干扰效果。(2) HepG2.2.15或HepG2细胞培养上清刺激THP-1细胞常规培养HepG2或HepG2.215细胞(无G418),待细胞铺满培养瓶即将传代时,收取其上清,1.5ml Ep管分装并冻存于-80℃,避免反复冻融。THP-1细胞以4-5×105/m1的密度接种于24孔板,分别加入200μ1的HepG2或HepG2.215细胞培养上清刺激O h、3h、24h或48h,加上清前吸取取出等量的培养基,以保持相同的培养体系。轻轻晃动细胞板混匀,置于37℃、5%C02培养箱孵育24h,离心收取上清及细胞。(3) HepG2.2.15或HepG2细胞与THP-1细胞共培养利用corning transwell小室,THP-1细胞以4×105/ml的密度接种于12孔板下室,HepG2.2.15/HepG2细胞铺于上室。THP-1细胞数目固定,设THP-1/HepG2或HepG2.215细胞分别为1：1、1：2和1：4三个比例,共培养48h后收细胞及上与。(4) ELISA检测IL-1β的水平同上检测上清中IL-1β的水平。(5)细胞免疫荧光观察HBV促NLRP3炎性体的形成HepG2或HepG2.215细胞培养上清刺激48h后,收集细胞,以4%多聚甲醛固定,以兔抗人Caspase-1、NLRP3(?)单克隆抗体为-抗、Cy3标记的羊抗兔IgG为二抗进行免疫荧光染色,荧光显微镜观察红色荧光,拍照保存。4.NLRP3炎性体重构实验(1) NLRP3炎性体重构体系的建立参考文献建立NLRP3炎性体重构系统,将HEK293细胞、HepG2、HeLa、Huh7细胞分别接种于24孔培养板,利用Lipofectin2000共转染以下质粒DNA,质粒及其用量分别为：pDsRed-monomer-C1-human ASC5ng、 pDsRed-monomer-C1-human caspase-15ng、 pCR4-TOPO-human NLRP315ng、 pDsRed-monomer-C1-human IL-lbeta150ng,混匀后共转细胞,转染前将细胞的培养基换成无血清培养基,转染5-6h换成含血清的培养基。48h后收取细胞及其上清,ELISA检测上清中IL-1β,提取蛋白Western blot检测Caspase-1的活化情况。(2)HBV质粒DNA与NLRP3炎性体共转在HEK293、HepG2、HeLa、Huh7细胞中分别进行共转染,NLRPP3炎性体组分质粒DNA(?)用量同上,分别同时转入pcDNA3.0或pcDNA3.0-HBV1.1质粒DNA600ng,转染48h后收取细胞及其上清。(3) ELISA检测IL-1β的水平收取细胞上清,用eBioscience公司的human IL-1β ELISA试剂盒检测IL-1β。(4) Western blot检测Caspase-1的活化24孔板转染细胞24h后,收细胞,提取取蛋白,Western blot(?)检测Caspase-1的活化情况。(5)ASC表达载体的构建及过表达稳筛细胞系的建立a.构建真核载体pc3-hASC-HA,以pDsRed-monomer-C1-hASC载体为模板,以加了HA标签的下游引物来扩增hASC-HA基因片段,双酶切后连入pcDNA3.0载体。经测序并比对无误后的的重组载体经Western Blot验证。b. HEK293细胞最佳G418的筛选浓度：将HEK293细胞稀释到1000个细胞/ml,分别加入G418,使其终浓度为400μg/ml、600μg/ml、800μ g/ml、1000μ g/ml,隔1-2天换含相同G418浓度的培养基,两至三周后观察结果,HEK293细胞全部死亡的最低G418浓度即为最佳筛选浓度。建立ASC过表达稳筛细胞系：HEK293细胞铺板后,脂质体法转染pDsRed-monomer-C1-human ASC,转染48h后换成含800μ g/ml G418的培养基,隔1-2天换液。稳筛两周后,荧光显微镜观察,提取蛋白Western blot检测ASC的表达。(6)共培养体系验证HBV可能通过Racl激活NLRP3炎性体THP-1细胞以4×105/ml的密度种板,用320nMPMA(12-16h)诱导成巨噬细胞后,Racl小干扰干扰24h后,再与HepG2.2.15细胞共培养48h后ELISA检测培养上清中的IL-1β水平。第二部分：人TIM-4启动子报告基因载体的构建及检测(1)人TIM-4启动子系列载体的构建利用软件预测并分析了人TIM-4基因的启动子-1887/+65,并根据转录因子结合位点设计系列截短的启动子。以健康人外周血PBMC基因组DNA为模板,利用高保真酶PCR扩增出人TIM-4基因启动子区域-1887至+65(转录起始位点为+1)片段,胶回收PCR产物连T载体,蓝白斑筛选等鉴定阳性克隆,经Sac Ⅰ、Xho Ⅰ双酶切连入经相同酶切的pGL3载体,转化、涂板、双酶切、测序鉴定阳性克隆。以载体pGL3-human PTIM4(-1887/+65)为模板,构建-1682/+65、-1307/+65、-1162/+65、-887A65、-662/+B5、-392/+65、-162/+65、-42/+65等截短的启动子。(2)启动子活性检测双荧光素酶法检测分析报告质粒人TIM-4启动子的活性。A549细胞以2×10。个/ml的密度铺于48孔板,以聃动子质粒DNA500ng/孔、pGL-TK10ng/孔利用脂质体法转染细胞,转染后48h,吸掉培养基,PBS洗三遍,以1×PBL细胞裂解液彻底裂解细胞,用荧光计数仪检测启动子的荧光素酶活性与内参pGL-TKRenilla荧光素酶活性,比较其ratio值。每次实验设3复孔,取取平均值。第三部分：TIM-4对NLRP3炎性体活性的影响(1) NLRP3炎性体重构同上重构NLRP3炎性体,利用脂质体法分别同时转入pcDNA3.0或pcDNA3.0-TIM-4600ng,转染48h后收细胞及其上清,检测IL-1p的水平。(2)小鼠TIM-4系列表达载体的构建与鉴定构建小鼠TIM-4全长及不同结构域缺失的系列载体：pEGFP-Mtim-4(full length)、pEGFP-mTIM-4(ΔC)(胞内区缺失)、pEGFP-mTIM-4(ΔTC)(跨膜区和胞内区缺失)、pEGFP-mTIM-4(ΔTC)-DAF(跨膜区以人DAF膜区代替),分别以PCR、酶切、测序鉴定序列的正确性。转染CHO细胞,荧光显微镜观察荧光表达情况。结果第一部分：HBV对NLRP3炎性体活化的影响1.HBV感染导致IL-1β升高慢乙肝患者(初诊)及HBV转基因小鼠外周血中IL-1β水平显著高于正常对照(p<0.05),但并未发现其与HBV DNA及抗原的相关性。2.HBV促单核/巨噬细胞产生IL-1β共培养实验结果表明随着HepG2.2.15细胞数目的减少,IL-1β的产生也随之降低。HepG2细胞或HepG2.2.15上清处理THP-1细胞,结果发现含HBV上清呈时间依赖性刺激IL-1β的产生,HepG2.2.15细胞上清也可显著刺激RAW264.7细胞产生IL-1β。3.HBV促单核/巨噬细胞产生IL-1β依赖于Caspase-1THP-1细胞用Caspase-1抑制剂处1-1.5h后,进行IIcpG2.2.15上清处理48h,结果发现Caspase-1抑制剂处理组IL-1β水平显著低于DMSO对照组。4.HBV促IL-1β产生是通过NLRP3炎性体发挥作用4.1验证小干扰效果利用质粒DNA或siRNA共转染HEK293细胞、siRNA转染THP-1细胞,Western blot检测验证Caspase-1、ASC、NLRP3siRNA的干扰效果,结果显示siNLRP3-978、2545可显著抑制NLRP3蛋白的表达；siASC-217、397、499均具有干扰效果；siCaspase-1-788、1094于扰效果较好。4.2NLRP3在HBV诱导的IL-1β产生中发挥重要作用THP-1细胞(5×105ml)接种于12孔培养板,以终浓度为320nmol/ml的PMA诱导12-16h贴壁,转染前换成无血清的培养基,用Lipo2000转siNLRP3-9785μ1/孔,6-8h换成含10%血清的培养基,干扰24h后,加入HepG2.2.15细胞与之共培养48h收细胞及其上清,结果表明：与对照组相比,干扰NLRP3后HBV诱导的IL-1β水平显著降低。5. HBV促进NLRP3炎症小体组装5.1NLRP3炎性体重构：参考相关文献,利用K293/HepG2/Hela/Huh7细胞重构NLRP3炎症小体。实验结果显示：共转染pcDNA3-HBV1.1可显著促进IL-1β的产生,与对照组相比,Western blot检测结果显示HBV可促进Caspase-1的活化。5.2HBV促进NLRP3炎性体的形成：含HBV上清刺激THP-1细胞48h,免疫荧光检测NLRP3、Caspase-1分子的表达,利用荧光显微镜观察,结果显显HBV刺激组表达水平上调。5.3ASC过表达稳筛细胞系的建立：利用HEK293细胞确定G418的有效作用浓度为800μg/ml, pDsRed-express-C1-ASC及空载体pDsRed-express-C1分别转染HEK293细胞,并利用含800μg/mlG418培养基进行筛选,待细胞克隆形成后进行传代,利用荧光显微镜观察发现筛选的细胞呈现红色荧光,Vestern blot可检测到ASC蛋白的表达。5.4真核表达载体pcDNA3.0-human ASC-HA的构建：阳性克隆分别利用PCR及酶切、测序鉴定,PCR可扩增ASC特异性片段,经HindⅢ、Xbal酶切鉴定,测序结果经blast分析序列与genebank提供的人ASC基因序列完全一致。pcDNA3.0-human ASC-HA质粒DNA转染HEK293细胞后,利用HA抗体可检测到其蛋白的表达。6.HBV通过Racl激活NLRP3炎性体THP-1细胞经PMA诱导贴壁,转染Racl siRNA及对照24h,再用含HBV的HepG2.2.15细胞与之共培养48h,ELISA检测结果显示Racl干扰组IL-1β水平低于对照组。第二部分：人TIM-4启动子报告基因载体的构建及检测成功构建人TIM-4启动子报告基因的全长及系列截短载体,启动子活性待验证。第三部分：TIM-4对NLRP3炎性体活性的影响NLRP3炎性体重构体系中,过表达TIM-4可显著抑制IL-1β的产生。成功构建了小鼠TIM-4的不同功能域缺失的载体pEGFP-mTIM4(full length)、 pEGFP-mTIM4(ΔC)、 pEGFP-mTIM4(ΔTC)、 pEGFP-mTIM4(ΔTC)-DAF,转染CHO细胞后,于荧光显微镜下可观察到绿色荧光。结论1.利用慢乙肝病人及HBV转基因小鼠证实HBV可促进IL-1β的产生,提示其产生与炎性体激活有关。2.利用THP-1细胞证实HBV诱导IL-1β的产生依赖于Caspase-1。3.利用THP-1细胞及重构实验证实HBV通过激活NLRP3炎性体诱导IL-1β的产生。4. HBV通过Rac1通路激活NLRP3炎性体。5.TIM-4抑制NLRP3炎性体的活化。创新点及意义本研究首次发现HBV通过激活NLRP3炎性体诱导Caspase-1的活化及IL-1p产生,并发现HBV通过Racl通路激活NLRP3炎性体,另外新型免疫分子TIM-4可抑制NLRP3炎性体的活化。本文研究结果提示HBV感染可能通过激活NLRP3炎性体诱导免疫性肝损伤,为HBV相关肝病的发病机制研究提供了新的思路,并为其临床治疗提供了理论基础。更多还原

【Abstract】 BackgroundThe hepatitis B virus (HBV) genome is a3.2-kb uncomplicated double stranded DNA. The HBV genome has4overlapping open reading frames (ORFs) coding coated antigen (HBs),core protein (HBc), X protein (HBx) and DNA polymerase respectively. HBV is a hepatotropic virus. HBV infection could induce chronic hepatitis, even progress to liver fibrosis, cirrhosis, and eventually to hepatocellular carcinoma (HCC). It is estimated that over400million people are chronically infected with HBV worldwide.And there are more than100million people died of liver disease every year. HBV-related liver disease and its complications result in approximately40%deaths out of the world. Therefore, HBV is harmful to human health and is one of the major killers to people. However, immunological liver injury induced by HBV infection is not very clear so far, its pathogenic mechanism is urgent to be discussed.For a long time, most scholars believed that the success of HBV clearance was associated with strong specificity of cellular immune responses, however, a growing evidence suggests that innate immunity plays a significant role in the process of HBV infection and pathogenesis.It is found that HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells). KCs are macrophages residing in the liver. KCs occupy80%to90%out of the total number of macrophages, which is the largest group of macrophages in the body.KCs are the major cells responsible for the inflammatory reaction and cytokines in the liver and involved in a variety of chronic liver disease in the process of the occurrence and development of the liver damage by producing inflammatory factors such as IL-1β, IL-18.The maturation of IL-1β and IL-18depends on the activation of inflammasome. Tschopp group discovered and reported inflammasome for the first time in2002. The first identified inflammasome is NLRP1inflammasome. The inflammasome is a molecular platform for IL-1β and IL-18production by triggering activation of inflammatory caspase-1.IL-1β is synthesized as pro-IL-1β which is further processed to its mature form by activated caspase-1. So far, several inflammasome complexes have been identified, including NLRP3inflammasome, NLRP1inflammasome, NLRC4inflammasome, AIM2inflammasome, RIG-I inflammasome et al. The NLR family, and pyrin domain-containing3(NLRP3) inflammasome is the best-characterized. The components of the NLRP3inflammasome are involved in sensing multiple danger signals. Recent studies have indicated that both DNA viruses such as adenovirus, varicella-zoster virus and RNA viruses such as influenza virus, hepatitis c virus (HCV) can stimulate the NLRP3inflammasome.It is reported that small DNA virus without or with a coating of macromolecular DNA viruses can activate NLRP3inflammasome.The activation of NLRP3inflammasome involves proteolytic activation of caspase-1, promoting mature and release of IL-1β or IL-18, leading to inflammation.It is reported that the promoter polymorphism of IL-18, IL-1β gene and HBV infection and prognosis is closely related. HBx and HBc protein can induce the generation of IL-18. IL-1β level is increased in patients with chronic hepatitis B, and IL-1β levels are positively correlate with HBV DNA. However, related reports about the mechanism of HBV regulating production of IL-1β and IL-18have not been found.This paper aims to evaluate the regulatory effect of HBV on NLRP3inflammasome for the first time.Tim (T-cell immunoglobulin domain and mucin domain) family was firstly discovered and identificated by McIntire in2001, which was a gene familiy associated with asthma and allergic disease. Murine Tim genes are located on chromosome11B1.1. There are eight members in mouse Tim family, which includes genes encoding Tim-1to Tim-4proteins and Tim-5to Tim-8hypothetical genes. Human Tim family includes only three genes, located on chromosome5q33.2, coding Tim-1, Tim-3, Tim-4protein respectively. Tim protein is a type I transmembrane glycoprotein with a common motif containing immunoglobulin (IgV) domain, mucins (Mucin) domain, transmembrane region and intracellular region. Except for Tim-4, intracellular regions of Tim-1to Tim-3molecules contain tyrosine kinase phosphorylation site, so these molecues are involved in the intracellular signal transduction directly. Tim-4plays an important role in immune regulation and maintaining homeostasis. Tim-4is a natural ligand of Tim-1, mainly expressed on the surface of activated antigen presenting cells, especially on macrophages and dendritic cells.Tim-4and Tim-1can provide costimulatory signal for T cell activation, which is important for T cells activation. However, the regulation of Tim-4on T cell activation is a two-edged sword. Tim-4shows negative regulation on macrophages. Our previous results showed that overexpress of Tim-4can inhibit IL-1β production from macrophages induced by LPS. In mice hepatitis model induced by ConA, transfer macrophages with Tim-4overexpression inhibited the production of IL-1β, indicating that Tim-4may inhibit the activation of inflammasome. In addition, it is found that Tim-4expression in PBMCs from the patients with chronic hepatitis B or macrophages from HBV transgenic mice were significantly lower than those of controls. Whether HBV regulates the expression of Tim-4by affecting the promoter activity of Tim-4is not clear. It should be clarified that whether HBV affects NLRP3inflammasome through regulating Tim-4expression. This study is performed to solve these related questions.AimsTo investigate the effects of HBV on NLRP3inflammasome activity and explore the related mechanisms. To construct the report vector of Tim-4gene promoter and identify the promoter activity. To explore the effects of Tim-4on NLRP3inflammasome activity.MethodsPartⅠ:The effects of HBV on NLRP3inflammasome1. Clinical samples(1)Collection of plasma from CHB patients and healthy controlsHeparin anticoagulant whole blood from56patients with chronic hepatitis B without treatment and20healthy controls were collected. Plasma were collected after centrifugation and saved at-20℃. (2)Detection of IL-1β by ELISAThe plasma IL-1β levels were measured by ELISA kits according to the instructions.2. Animal experiments(1) HBV transgenic mice and normal controls35HBV transgenic mice with Balb/c background and12Balb/c mice were included in this study.(2) Detection the HBV DNA and antigenThe whole blood was taken from HBV transgenic mice.The serum was separated and used to detect HBV DNA copies by real time PCR, and HBsAg by ELISA.(3)Detection of IL-1β by ELISAIL-1β levels in the serum were detected using ELISA kits.(4) Detection of Caspase-1activation in liver tissue by Western blotWe chose14cases of HBV transgenic mice, and liver proteins were extracted to measure Caspase-1by western blot.2. Cell models(1) Screen and identification of the Caspase-1, ASC, NLRP3siRNASmall interfering RNA targeting human ASC, caspase-1and NLRP3were designed and synthesized respectively.In HEK293cells, co-transfection with siRNA and plasmid DNA was performed.48h later, western blot was used to verify the interference effect. The effect of siRNA was also assayed with THP-1cells induced by PMA.(2) THP1cells were stimulated with200μl culture supernatants of HepG2.2.15or HepG2cells, and supernatants were collected at indicated time points for IL-1β detection.(3) HepG2.2.15cells or HepG2cells were cocultured with THP1cells in transwell Chambers. THP1cells were plated in12culture plates at the density of4×105/ml, and HepG2.2.15/HepG2cells were plated in the upper chamber with different ratio.48hours later, THP-1cells and supernatants were collected for detection.(4) Detection of IL-1β:Same as above (5) Identification of NLRP3infammasome:THP1cells were stimulated with HepG2.2.15or HepG2cell culture supernatants.48h later, stimulated THP-1cells were collected and fixed with4%paraformaldehyde. Rabbit anti-human Caspase-1and rabbit anti-human NLRP3monoclonal antibodies were used to do IFA, and the fluorescence was observed under fluorescence microscopy.4. NLRP3inflammsome reconstitution(1) Reconstitution of NLRP3inflammasome system According to the references, NLRP3inflammasome system was reconstituted in HEK293cells by cotransfection with pDsRed-monomer-Cl-hASC, pDsRed-monomer-Cl-hCaspase-1,pCR4-TOPO-h-NLRP3, pDsRed-monomer-Cl-hIL-1β.48hours after transfection, the supernatants were collected to detect IL-1β by ELISA.(2)Co-transfection of HBV and NLRP3inflammasome As above, NLRP3inflammasome was reconstituted together with pcDNA3.0-HBV1.1or control vector in HEK293, HepG2, HeLa or Huh7cells.48h later, the cells and supernatants were collected.(3) Detection of IL-1β by ELISA IL-1β was detected using ELISA kits.(4) Detection of Caspase-1activation by Western blot After cotransfection, HEK293cell protein was extracted to detect Caspase-1activation by western blot.5. Set up the stable cell line with ASC overexpressionOptimal concentration of G418was screened with HEK293cells. HEK293cells were set up at1000cells/ml, and G418was added with final concentration at400μg/ml,600ug/ml,800μg/ml or1000μg/ml. Culture supernatants were changed with fresh mediun containing the same concentration of G418every1to2days. After two to three weeks, the lowest concentration of G418leading to HEK293cell death was used for screening.pDsRed-monomer-Cl-hASC or control vecter plasmid DNA were transfected into HEK293cells using liposome.48h after transfection,800μg/ml of G418was added to transfected HEK293cells, and fresh medium was changed every1-2days. After two weeks, cells were observed under fluorescence microscopy. Western blot was used to assay ASC expression.PartⅡ:Constuction of report vector of TIM-4promoter and identification(1) Construction of the report vector of TIM-4promoterAfter analysis with software, the human TIM-4gene promoter region was located at-1887/+65, and a series of truncated promoters were designed according to the binding site of transcription factors.Human PBMCs genome DNA was used as template to amplify TIM-4gene promoter fragment by PCR amplification.TIM-4gene promoter fragment was linked to T-esay vector by T4Iigase, and positive clone was screened by blue white spot screening. Then the plasmid was digested with Sac Ⅰ and Xho Ⅰ and linked to pGL3. After transformation, positive recombinant plasmid was identified by PCR, enzyme digestion and sequencing.pGL3-TIM-4(-1887/+65) plasmid DNA was used as the template, other truncated promoters including pGL3-TIM-4(1682/+65),-1307/+65,-1162/+65,-887/+65,-662/+65,-392/+65,-162/+65,-42/+65were constructed as above.(2)Detection of promoter activityThe activity of the report plasmid TIM-4promoter was assay by double luciferase method. A549cells were planted in48well plate at the density of2×105/ml,500ng of promoter plasmid DNA and10ng of pGL-TK were cotransfected using liposome.48h later, cells were washed with PBS for three times, then PBL lysis buffer was used to lyse cells completely. Fluorescent counting instrument was used to detect promoter luciferase activity and internal pGL-TK. Renilla luciferase activity.PartⅢ:The effect of TIM-4on NLRP3inflammasome(1) Reconstitution of NLRP3inflammasome systemAs above, NLRP3inflammasome was reconstituted together with pcDNA3.0-hTIM-4or control vector in HEK293or HeLa cells.48h later, the supernatants were collected for IL-1β assay.(2) Construction the expression vector of murine Tim-4Recombinant plasmids containing murine Tim-4with different domain deletion were constructed.These plasmids included pEGFP-mTim4(full length), pEGFP-mTim4(ΔC), pEGFP-mTim4(ΔTC), pEGFP-mTim4(ΔTC)-DAF. The positive recombinant plasmids were identified by PCR, enzyme digestion and sequencing. Then plasmids were transfected into CHO cells, and the fluorescence was observed under fluorescence microscope.ResultsPartⅠ:The effect of HBV on NLRP3inflammasome1. Enhanced IL-1β in chronic hepatitis B patients or HBV transgenic miceIL-1β levels in plasma from patients with chronic hepatitis B or serum from HBV transgenic mice were significantly higher than those of normal controls. However, the levels of IL-1β were relatively low, no significant correlation of IL-1β with HBV DNA or antigen was found.2. Increased IL-1β in HBV stimulated monocyte/macrophagesTHP-1cells were cocultured with different ratio of HepG2.2.15cells for48hours. The supernatants were used to detect IL-1β. The results showed that IL-1β levels were increased in THP-1cells after coculture with HepG2.2.15cells. Supernatants from HepG2.2.15cells also stimulated THP-1or RAW264.7cells to produce IL-1β.3. HBV promoted IL-1β production in a Caspase-1dependent wayELISA results showed that HBV induced IL-1β production in THP-1cells treatment with Caspase-1inhibitor was significantly lower than those of the control group.4. NLRP3played a role in HBV stimulated IL-1β production4.1The effect of siRNAAfter cotransfection with plasmid DNA and siRNA in HEK293cells,the effect of siRNA was assayed by western blot.The results showed that siNLRP3-978,2545could inhibit NLRP3expression. For ASC,the three siRNAs all took effect, while siCaspase-1-788,1094showed better effect of knock down.4.2HBV promoted IL-1β production through activating NLRP3inflammasome The results showed that HBV induced IL-1β production in NLRP3siRNA transfected THP-1cells was significantly lower than that of siRNA control group,indicating that NLRP3inflammasome is responsible for HBV stimulated IL-1β secretion.4.3HBV promoted the NLRP3inflammasome assemblyIn order to further identify the role of NLRP3inflammasome in HBV induced IL-1β production,NLRP3inflammasome system was reconstituted successfully by IL-1β assay and RT-PCR detection of NLRP3or other gene mRNA.Then we reconstituted NLRP3inflammasome together with pcDNA3-HBV1.1in HEK293, HepG2, HeLa, or Huh7cells, and the results showed that cotransfection with plasmid containing HBV could promote IL-1β production, and Caspase-1activation was also increased significantly. In addition, we found that NLRP3inflammasome activation could inhibit the production of HBV antigen.4.4We detected the expression of NLRP3and Caspase-1in THP-1cells stimulated with supernatants containing HBV. IFA results showed that expression of NLRP3and Caspase-1in THP-1cells treated with supernatants containing HBV was higher than that of control group.5. Establish of ASC overexpression cell line and construction of pcDNA3-hASC-HA5.1After G418screening, the cells could expression the red fluorescence fusion protein which could be observed under fluorescence microscopy. In addition, ASC protein could also be detected in HEK293cells with ASC overexpression, indicating that the stable cell line with ASC overexpression was established successfully.5.2pcDNA3.0-hASC-HA was contructed regularly. After identification with PCR, digestion and sequencing, the recombinant plasmid was verifed correctly.6. HBV activated NLRP3inflammasome through RaclThe results showed that Racl siRNA treatment could reduce IL-1β production in HBV stimulated THP-1cells, indicating that Racl signal pathway was important for HBV induced NLRP3inflammasome activation.PartⅡ:Constuction of the report vector of TIM-4promoterA series of report vectors of full length or trancated TIM-4promoter were constructed and verified by PCR, digestion or sequencing.The activity of these TIM-4promoters were assayed in A549cells, however the activity was relatively low.ParⅢ:The effect of TIM-4on NLRP3inflammasomeIn the reconstituteion system, cotransfection with plasmid containing TIM-4could inhibit IL-1β production significantly.Then a series of vectors of murine Tim-4with different domain deleteon were constructed successfully.The recombinant plasmids were transfected into CHO cells, and the green fluorescence could be observed under the fluorescence microscope.Conclusion1. HBV could promote IL-1β production, which was verified with chronic hepatitis B patients and HBV transgenic mice.These data indicate that HBV infection might be involved in inflammasome activation.2. HBV induces IL-1β production in a Caspase-1dependent manner.4. Rac1is involved in activation of NLRP3inflammasome induced by HBV.5. TIM-4reduces the activation of NLRP3inflammasome.Innovation points and significanceIn the current study, we found that HBV activated NLRP3inflammasome and TIM-4inhibited NLRP3inflammasome activation for the first time. In addition, we found Rac1was involved in activation of NLRP3inflammasome by HBV infection. These data indicate that HBV infection leads to liver injury partially through activating NLRP3inflammasome, which would provide novel theory for the pathogenesis of hepatitis B and provide new targets for intervene of HBV related liver diseases.更多还原