【作者】 资捷；

【导师】 王前；

【作者基本信息】 南方医科大学 ， 临床检验诊断学， 2010， 博士

【摘要】 研究背景和目的乙型肝炎病毒(Hepatitis B virus, HBV)是一种肝向性DNA病毒,严重威胁人类的健康。孕妇感染HBV后,可能经胎盘垂直传播给胎儿,引起宫内感染,是我国人群HBV传播的重要途径。乙型肝炎病毒e抗原(HBeAg)是HBV的一种辅助蛋白,并不参与病毒的复制和组装。HBeAg阳性慢性乙型肝炎(CHB)孕妇常持续感染HBV,并导致宫内感染。研究发现,HBeAg能抑制CHB患者的肝细胞、外周血单核细胞和Kupffer细胞Toll样受体2(TLR2)表达及其介导的天然免疫反应,相似的是,在HBeAg阳性CHB患者的外周血树突状细胞Toll样受体3(TLR3)表达减少。此提示,HBeAg在病毒感染过程中可能起免疫调节作用,促进病毒持续感染。Toll样受体(Toll-like receptors, TLRs)是一种近年来新发现的模式识别受体,可识别和应答病原体相关分子模式(PAMPs),在抵御各种病原微生物的免疫应答中发挥重要作用。天然免疫系统是机体抗病原体感染的第一道防线,TLRs家族是其中重要的组成部分。TLRs可广泛表达于哺乳动物的组织和细胞中。至今,已发现和鉴别了10种人属TLRs,在人胎盘组织均可表达,TLR3、TLR4主要表达于胎盘滋养层细胞。TLRs信号传导包括2条途径：MyD88依赖性和非依赖性途径,TLR3信号传导主要通过MyD88非依赖的TRIF途径,而TLR4主要通过MyD88依赖性或非依赖性途径传导信号。TLR3、TLR4可介导抑制肝脏细胞、外周血树突状细胞中HBV复制。TLR3可识别病毒双链RNA(dsRNA)并发生免疫应答,滋养层细胞在TLR3的配体多聚肌胞(polyI:C)处理后可产生Ⅰ型干扰素(IFN-α／β),而IFN-β是由TLR3诱导细胞产生的主要细胞因子；以TLR4的配体脂多糖(LPS)刺激绒毛滋养层细胞可产生前炎症细胞因子(如TNF-α)和趋化因子(如IL-8)。此提示,胎盘滋养层细胞可能通过TLR3、TLR4识别病毒相关分子模式并触发抗病毒反应。胎盘作为一种特殊的物理和免疫屏障,在防御不同微生物的侵袭中起重要作用。胎盘屏障的第一层细胞是滋养层细胞,该层细胞直接与母亲血液接触,可识别病原微生物并对其作出相应的免疫应答。因此,滋养层细胞在防御HBV宫内感染中可能发挥重要的作用。但是,关于HBeAg阳性CHB孕妇持续感染HBV并导致宫内感染的机制尚不清楚。Bewo细胞是一种人胎盘绒毛癌细胞系,现已被广泛用于滋养层细胞功能的研究。本研究拟分别构建1.3倍HBV基因组及HBeAg的真核表达载体,转染Bewo细胞,建立HBV和HBeAg表达的细胞模型,并以此研究HBeAg对人胎盘滋养层细胞TLR3／4的表达及其生物效应的影响,探讨胎盘HBV持续感染并垂直传播给胎儿的机制,为防治HBV宫内感染提供新的理论和实验依据。方法1. HBeAg阳性慢性乙型肝炎孕妇胎盘组织TLR3、TLR4的表达及意义：选取22例HBeAg阳性和24例HBeAg阴性CHB孕妇,及正常孕妇25例。建立实时荧光定量RT-PCR检测胎盘组织中TLR3、TLR4mRNA水平,同时以免疫组化法检测其在胎盘组织中的表达,并采用荧光定量PCR(FQ-PCR)和微粒子酶免疫分析法(MEIA)分别定量检测其血清HBVDNA和HBeAg水平。2.TLR3、TLR4介导的天然免疫对Bewo细胞中HBV复制的作用：以质粒pMD18T-HBV上的HBVDNA序列为模板,构建1.3倍HBV全基因序列基因,将其插入到真核表达载体pcDNA3.1(+),用酶切、PCR及测序鉴定,并把该载体转染Bewo细胞,以Western免疫印迹、MEIA和FQ-PCR分别检测胞内和上清中HBsAg、HBeAg的表达及HBV DNA水平。以RT-PCR和ELISA分别检测细胞TRIF、MyD88 mRNA表达及其上清IFN-β、TNF-α水平。3. HBeAg对Bewo细胞TLR3、TLR4表达及其生物效应的影响：用PCR方法从质粒pMD18T-HBV中扩增HBeAg基因,克隆到pcDNA3.1(+),构建真核表达载体pcDNA3.1(+)-HBe,通过酶切、PCR及测序鉴定,并把该载体转染Bewo细胞,用Western免疫印迹和MEIA检测胞内和上清中HBeAg的表达。以FQ-PCR和ELISA分别检测细胞TLR3、TLR4 mRNA表达及细胞上清IFN-β、TNF-α水平,并采用免疫荧光染色法检测细胞NF-κB p65核转运。结果1. HBeAg阳性CHB孕妇胎盘组织中TLR3、TLR4 mRNA表达水平显著低于正常对照组和HBeAg阴性组,两组相比差异有统计学意义(P=0.000)；但HBeAg阳性CHB孕妇血清HBeAg及HBV DNA水平显著高于正常对照组,两者比较差异有统计学意义(P=0.000)。且免疫组化结果显示,TLR3主要表达于胎盘合体滋养层细胞和绒毛滋养层细胞的胞膜和胞浆,而TLR4主要表达于该细胞的胞膜。HBeAg阳性CHB孕妇胎盘组织中TLR3和TLR4表达的平均光密度值明显低于正常对照组和HBeAg阴性组,两者比较差异有统计学意义(P=0.000)。胎盘组织中TLR3、TLR4 mRNA表达与HBeAg水平呈负相关(r=-0.495,P<0.05,r=-0.450,P<0.05),而与血清HBV DNA水平无显著相关性。2.通过酶切、PCR及测序鉴定,成功构建了1.3倍HBV全基因表达载体,该载体可以在Bewo细胞中表达和分泌HBsAg与HBeAg,并可检测到高水平的HBV DNA。对Bewo细胞转染2μg或8μg HBV重组质粒,与对照组比较,polyI:C和LPS可显著抑制HBV复制(P<0.01),但转染8μg HBV重组质粒时,polyI:C和LPS对HBV复制的抑制率显著下降(P<0.01)。抗TLR3、抗TLR4可显著逆转polyI：C和／或LPS对HBV复制的抑制作用(P<0.01),抗IFN-β也可逆转polyI：C对HBV复制的抑制作用(P<0.01),但不能逆转LPS的作用(P>0.05)。与对照组相比,polyI:C可显著诱导Bewo细胞产生IFN-β和TNF-α(P<0.05或0.01),LPS可诱导细胞产生TNF-α,差异有显著性意义(P<0.05或0.01),但不能诱导IFN-β表达,二者均呈剂量和时间依赖性。NF-κB拮抗剂PDTC可抑制polyI:C和LPS诱导细胞产生TNF-α,显著低于对照组(P<0.01),但对IFN-β无显著作用。以polyI:C或LPS处理HBV重组质粒转染的Bewo细胞,可分别诱导TRIF mRNA或MyD88 mRNA表达,显著高于对照组(P<0.01)。3.通过酶切、PCR及测序鉴定,成功构建HBeAg表达载体,该载体可以在Bewo细胞系中表达HBeAg,并可分泌HBeAg。与对照组相比,polyI：C和LPS可显著诱导Bewo细胞TLR3和TLR4 mRNA表达(P<0.05或0.001),呈剂量和时间依赖性；IFN-β可诱导细胞TLR3 mRNA表达,显著高于对照组(P<0.01),但对TLR4 mRNA的表达无明显作用。对Bewo细胞转染2μg或8μg HBeAg重组质粒,再以polyI:C和LPS处理,转染8μg HBeAg重组质粒组其TLR3或TLR4的表达及IFN-β、TNF-α水平显著低于对照组(P<0.01)。外源性HBeAg可显著下调polyI：C诱导Bewo细胞TLR3 mRNA表达及IFN-β、TNF-α产生(P<0.05或0.01),也可抑制LPS诱导TLR4 mRNA表达及TNF-α产生(P<0.05或0.01),呈剂量依赖性。Bewo细胞在polyI:C或LPS作用下,NF-κB p65入核的数量显著高于对照组(P<0.01),但内源性和外源性HBeAg均可显著抑制polyI:C或LPS激活NF-κB(P<0.01)。结论1. HBeAg阳性CHB孕妇血清]HBeAg持续高表达,且其宫内感染率显著高于正常及HBeAg阴性孕妇,但胎盘组织TLR3、TLR4的mRNA、蛋白水平表达均显著低于其余两组；胎盘组织中TLR3、TLR4 mRNA表达与HBeAg水平呈负相关。此提示,HBeAg阳性CHB孕妇宫内感染HBV与TLR3、TLR4低表达关系密切,其对胎盘TLR3、TLR4表达可能起负调节作用。2.成功构建了1.3倍HBV全基因真核表达载体pcDNA3.1(+)-HBV1.3,为研究HBV宫内感染的机制奠定了基础。通过诱导Bewo细胞产生IFN-β和TNF-α,TLR3、TLR4介导的天然免疫应答可抑制HBV复制,但随着HBV感染量的增大,其对HBV复制的抑制作用显著下降。TLR3介导抑制Bewo细胞中HBV复制主要呈TRIF依赖性,而TLR4主要依赖于调节蛋白MyD88。此提示,激活TLR3、TLR4可能成为一种新颖和有效防治HBV宫内感染的方法。3.成功构建了HBeAg真核表达载体pcDNA3.1(+)-HBe,为研究内源性HBeAg对Bewo细胞TLRs表达的作用奠定了基础。polyI：C和LPS可显著诱导Bewo细胞TLR3、TLR4 mRNA表达,且可通过IFN-β自分泌反馈诱导TLR3表达。HBeAg通过下调细胞TLR3、TLR4 mRNA的表达,抑制NF-κB激活和细胞因子产生,从而干扰Bewo细胞的免疫功能。此提示,HBeAg下调胎盘滋养层细胞TLR3和TLR4表达效应可能是引起HBV宫内感染的一个重要的原因。主要创新点1.成功构建了1.3倍HBV全基因真核表达载体pcDNA3.1(+)-HBV1.3,建立了新型的滋养层细胞HBV表达模型,为研究HBV宫内感染奠定了基础。2.成功构建了HBeAg真核表达载体pcDNA3.1(+)-HBe,建立了滋养层细胞]HBeAg表达模型,为研究胞内HBeAg对滋养层细胞TLRs表达及其生物效应的影响提供了一个良好的平台。3. HBeAg可下调胎盘滋养层细胞TLR3、TLR4表达及其生物效应,可能是胎盘持续感染HBV,并引起宫内感染的一种新的机理,对防治HBV宫内感染提供了新的理论和实验依据。更多还原

【Abstract】 Backgroud & ObjectiveHepatitis B virus (HBV), a hepatotropic DNA virus, severely threats human health. HBV may vertically transmit into fetus from placenta in pregnant women infected by HBV, and this is one of important routes of HBV transmission. HBV e antigen (HBeAg) is an accessory protein not required for viral replication and assembly. HBV may establish persistent infection in pregnant women HBeAg-positive for chronic hepatitis B(CHB), which probably results in HBV intrauterine infection. It is shown that HBeAg inhibits TLR2 expression and TLR2-mediated innate immunity response in hepatocyte, peripheral blood monocyte and kupffer cells (KCs). Similarly, the expression of TLR3 in dendritic cells (DCs) of peripheral blood is attenuated. This suggests that HBeAg may play roles in immune-regulation which in turn to promote viral persistent infection.Toll-like receptors (TLRs), identified as pattern recognition receptors in recent years, are capable of recognizing and responding to the pathogen-associated molecular patterns(PAMPs), and thus play important roles in defending microorganisms.The innate immunity system, in which the TLRs are key components, represents the first defense line against invading pathogens in organism.TLRs are widely expressed in mammalian tissues and cells. Up to now,10 TLRs have been characterized in human being, and all of them express in human placenta tissues whereas TLR3 and TLR4 mainly express in human placenta trophoblast cells. There are 2 signal pathways related to TLRs based upon its dependence or independence on MyD88. TLR3 belongs to the MyD88-independent but TRIF-dependent pathway, whereas TLR4 pathways are both MyD88-and TRIF-dependent. Inhibition of HBV replication are mediated by TLR3 and TLR4 in hepatocytes and peripheral blood dendritic cells TLR3 recognize virus double strands RNA leading to immune response. Stimulated with TLR3 ligands polyI:C, trophoblast cells will produce the typeⅠinterferon(IFN-α/β), and IFN-P is the key cytokine from the TLR3-induced cells. However, trophoblast cells treated with TLR4 ligands LPS will result in up-regulation of proinflammatory cytokines(TNF-a) and chemotatic factor (IL-8), indicating that the placenta trophoblast cells play a key role in recognizing PAMPs to trigger anti-virus responses.As a physical and immunologic barrier, placenta is important for defending the diverse array of microorganisms invaded. Trophoblast cells, directly connecting with mother blood, are the first cell layer in placental barrier and are able to recognized and respond to pathogenic microorganisms. Therefore, trophoblast cells may be key players in the prevention of intrauterine infection by HBV.However, the mechanisms by which the pregnant women HBeAg-positive for chronic hepatitis B(CHB) have persistent infection of HBV are poorly understood. Bewo cells, derived from a human choriocarcinoma, are widely used to study the functions of trophoblast cells.In this study, we have constructed eukaryotic expression plasmids of HBeAg and 1.3 fold of HBV genome, separately, and have established the Bewo cells overexpressing HBV or HBeAg. Using these cells, we have investigated regulation of TLR3 and TLR4 expression and biological effect by HBeAg in human placental trophoblast cells, and have further explored the potential mechanisms for persistent infection or vertical transmission of HBV. It will be helpful to prevent and/or cure HBV intrauterine infection.Methods1. TLR3 and TLR4 expression in placenta of pregnant women with HBeAg- positive chronic hepatitis B and its significance46 Pregnant women with chronic hepatitis B (CHB), including 22 HBeAg-positive and 24 HBeAg-negative,and 25 normal pregnant women were chosen from November 2008 to May 2009. The mRNA and protein levels of TLR3 or TLR4 in placentas were detected by real-time quantitative RT-PCR(qRT-PCR) and immunohistochemitry (IHC), respectively. The levels of HBV DNA and HBeAg were assayed by real-time fluorescence quantitative PCR(FQ-PCR) and microparticle enzyme immunoassay (MEIA),respectively.2. The effect of TLR3-and TLR4-mediated innate immune on HBV replicationThe 1.3-fold HBV genome DNA was constructed from a recombinant plasmid called pMD18T-HBV and was sub-cloned into the eukaryotic expression plasmids pCDNA3.1(+).The recombinant vector was confirmed by restriction enzyme digestion,PCR and sequencing, then was transfected into Bewo cells.The extracellular and intracellular HBsAg,HBeAg expression and HBV DNA level was detected by Western blotting,MEIA and FQ-PCR, respectively. The mRNA of TRIF and MyD88 in Bewo cells were determined by RT-PCR, whereas the levels of IFN-P and TNF-a in supernatant were detected by ELISA.3. HBeAg affects TLR3 and TLR4 expression and biological effect in Bewo cellHBeAg gene was amplified from the pMD18T-HBV vector by PCR and then was cloned into the pcDNA3.1(+),the recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing, and then was transfected into Bewo cells.The extracellular and intracellular HBeAg expression was detected by Western blotting and MEIA, and the same methods as described above were applied for mRNA levels of TLR3 and TLR4 in Bewo cells and IFN-βand TNF-αlevel in supernatant. NF-κB p65 protein nuclear translocation was detected by immunofluorescence (IF).Results1. The levels of TLR3 and TLR4 were significantly lower in the CHB pregnant women HBeAg-positive than those HBeAg-negative or normal pregnant women (P=0.000). But the serum level of HBeAg and HBV DNA were significantly higher in the CHB pregnant women HBeAg-positive than those HBeAg-negative or normal pregnant women (P=0.000). Moreover, strong staining of TLR3 was observed by IHC in cytomembrane and endochylema of syncytiotrophobalst (SCTs) and villous cytotrophoblast. In addition,TLR4 mainly localized to cytomembrane of these extravillous trophoblasts. The average levels of TLR3 and TLR4 were significantly lower in the CHB pregnant women HBeAg-positive than those HBeAg-negative or normal pregnant women (P=0.000). There was a reverse correlation between both of TLR3 and TLR4 mRNA in placentas and the levels of HBeAg in serum (r=-0.495,P <0.05,r=-0.450,P<0.05), while there was no relationship between TLR3 and HBV DNA.2. The 1.3-fold HBV genome DNA inserted into pCDNA3.1 was confirmed by the restriction enzyme digestion,PCR and sequencing, and Bewo cells transfected with this vector are able to express HBsAg and HBeAg proteins in cells or supernatant, and high level of HBV DNA were detected in supernatant.2μg or 8μg HBV recombinant vector was transfected into Bewo cells,compared with control,polyI:C and LPS could significantly suppressed HBV replication (P<0.01), but the inhibitory rate of polyI:C and LPS were strikingly lower after transfecting 8μg HBV recombinant vector into Bewo cells (P<0.01).The suppressive effect of polyI:C and LPS on HBV replication was reversed by neutralizing antibodies against human TLR3 and TLR4 and IFN-β, but anti-IFN-βantibody did not inhibit the antiviral effect of LPS (P>0.05). Compared with control, polyI:C could significantly induce the production of IFN-P and TNF-αin Bewo cells (P<0.05 or 0.01),whereas LPS only obviously up-regulated TNF-αexpression, but not IFN-P (P<0.05 or 0.01), in time-and dose-dependent manners.Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, strongly inhibited polyI:C-and LPS-indued TNF-a production, not IFN-P in Bewo cells(P<0.01).The mRNA levels of TRIF or MyD88 were significantly induced by polyI:C and LPS in the Bewo cells transfected with this recombinant vector (P<0.01). 3. The HBeAg gene inserted into the pcDNA3.1(+) was confirmed by restriction enzyme digestion,PCR and sequencing. Bewo cells transfected with this vector are able to express HBeAg protein in cells or supernatant. Compared with control,polyI:C and LPS could significantly induce TLR3 and TLR4 mRNA expression in Bewo cells (P<0.05 or 0.001),in time-and dose-dependent manners. IFN-βsignificantly induced TLR3, not TLR4 mRNA in Bewo cells(P<0.01). However,2μg or 8μg HBeAg recombinant vector was transfected into Bewo cells, then the cells were treated with polyI:C and LPS,TLR3 mRNA level and IFN-βand TNF-αproduction in transfecting 8μg HBeAg recombinant vector group were obviously lower than those in control (P <0.01). Exogenous HBeAg remarkably suppressed TLR3 mRNA level and both of IFN-βand TNF-αproduction induced by polyI:C in Bewo cells (P<0.05 or 0.01),and TLR4 mRNA and TNF-a production induced by PLS were also inhibited by exogenous HBeAg (P<0.05 or 0.01) in a dose-dependent manner. PolyI:C or LPS induced NF-κB p65 translocation into nuclei (NF-κB activation) in Bewo cells measured by IF (P<0.01),which was abolished by endogenous or exogenous HBeAg (P<0.01).Conclusions1. There was a persistently high level of HBeAg in serum of HBeAg-positive CHB pregnant women,furthermore, its rate of intrauterine infection rised significantly than normal and HBeAg-negative CHB pregnan women,but the mRNA level and protein expression of TLR3 and TLR4 were lower in HBeAg-positive CHB pregnant women than that in other 2 groups. There was a reverse correlation between both of TLR3 and TLR4 mRNA in placentas and the levels of HBeAg in serum.This may imply that the low expression of TLR3 and TLR4 have close relationships with HBV intrauterine infection of pregnant women,and it plays a negative role in the expression of TLR3 and TLR4 in placentas.2. The 1.3-fold HBV genome DNA inserted into pCDNA3.1 was constructed successfully, setting up the foundation for future research on intrauterine HBV infection. TLR3-and TLR4-mediated innate defence responses could suppress HBV replication in Bewo cells via its ligands-induced production of IFN-P and TNF-a. But with the increase of HBV infectious dose, their suppressive effect on HBV replication was strikingly decreased.TLR3-mediated suppression HBV replication was TRIF-dependent,while TLR4 mainly depended on regulatory protein MyD88. This indicated that TLR3 and TLR4 activation could represent a powerful and novel therapeutic approaches against HBV intrauterine infection.3. The HBeAg gene inserted into the pcDNA3.1(+) was constructed successfully, allowing for future research on the effect of intracellular HBeAg on the expression of Toll-like receptors in Bewo cells.polyI:C and LPS could significantly induce TLR3 and TLR4 mRNA expression in Bewo cells. TLR3 expression was induced through IFN-βautocrine feedback. HBeAg could impair the immune function by down-regulating TLR3 and TLR4 mRNA level, abrogating NF-κB activation, and decreasing the production of cytokines in Bewo cells. This may imply that HBeAg diminished TLR3 and TLR4 mRNA expression and biological effect in placenta trophoblasts to lead to HBV intrauterine infection.更多还原