【作者】 陈黎；

【导师】 梁志清；

【作者基本信息】 第三军医大学 ， 妇产科学， 2011， 博士

【摘要】 乙型肝炎是中国的流行病之一,全世界有3.5人口感染乙肝病毒(hepatitis B virus, HBV),其中至少有1/3在中国。中国目前有约3000万慢性乙肝患者和1.2亿无症状的携带者。作为全球免疫疫苗联盟计划的一部分,中国最穷困的省份中至少有1千1百多万人口已经接种到了乙肝疫苗,但是这远远达不到免疫及控制乙型肝炎病毒的传播的程度。尽管大部分的研究人员认同胎儿感染及新生儿早期的垂直传播是HBV传播的主要途径和方式,但是由于具体机制尚不清楚,故在我国HBV感染的高危因素仍然不能确定。目前,人们把HBV的垂直传播主要分为3种不同的方式:胎儿的宫内感染、产时感染及产后感染。对于后两种传播途径而言,HBV疫苗和免疫球蛋白的应用可以有效阻止其传播。因此,更好地了解宫内感染所致的HBV宫内传播对于明确HBV的传播途径是至关重要的。在整个妊娠过程中,胎盘的结构和功能是动态变化。越来越多的证据显示,胎盘对于病毒从母体面到胎儿面的垂直传播中发挥非常重要的作用,如HBV、HCV、HIV及CMV病毒等。先天性的病毒感染可能引起产后感染、胎儿畸形、甚至是胎儿的死亡。虽然经胎盘传播病毒是这种感染的重要方面,但是关于病毒经胎盘的垂直传播及胎盘屏障在病毒从母体到胎儿循环中的传播中所发挥的作用研究还非常少。尽管对于血脑屏障已经有了非常深入的研究,但对于胎盘屏障的建立和鉴定还未见报道,导致宫内感染缺乏一个可靠的体外研究模型,严重影响病毒宫内感染机制的研究,制约了宫内感染的有效治疗。因此,构建一种胎盘屏障体外模型用于病毒宫内感染的研究是当前势在必行解决的问题,从而为病毒宫内感染机制的阐明和治疗方法的探索奠定坚实的基础。同时,已经被证实有3个基本的结构层面:滋养层细胞、间隙细胞(主要是胎盘的单核巨噬细胞,如Hofbauer细胞)及人胎盘微血管内皮细胞参与到宫内感染中。胎盘屏障主要由滋养层细胞、人胎盘微血管内皮细胞及两者的基底层所构成,是营养物质以及某些药物、病毒、激素等从母体进入胎儿的必经之路。本研究运用原代培养的滋养层细胞和人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),采用三种不同的模式来构建胎盘屏障的体外模型:滋养层细胞单独培养,滋养层细胞和内皮细胞非接触性共培养及滋养层细胞和内皮细胞接触性共培养,发现两者的接触性共培养是能够最好的模拟正常胎盘结构和功能的体外胎盘屏障模型;然后我们利用病毒拷贝量高于107/ml HBV患者血清直接感染构建的胎盘屏障模型,进一步研究HBV病毒穿过及感染体外胎盘屏障模型的能力。基于HIV及CMV可以通过受体介导的方式引起宫内垂直感染,同时HBV可以感染外周血单个核细胞(peripheral blood monocyte,PBMC),并在其中复制,我们推测HBV可能通过以下3种途径:1.机械性损伤:炎性反应所致直接或是间接损伤滋养层细胞或是脐静脉内皮细胞,产生细胞裂隙,导致基底膜通透性增高,从而HBV可以突破胎盘屏障第一层(滋养层细胞),然后释放入绒毛间质;2.细胞传递性:感染HBV的PBMC通过与滋养层细胞发生瞬时的微融合而将其内的HBV传递给后者,滋养层细胞再将HBV极性释放入绒毛间质;3.受体介导性:滋养层细胞顶膜侧上某种受体可能介导了游离完整HBV颗粒的进入并在其中复制,然后将部分HBV自其基底侧膜极性释放入绒毛间质。由于受体介导病毒入胞是其最常见的入侵细胞的方式和已经证实HBV在体情况下可以引起胎盘的损伤,所以本研究中,将关注机械性损伤和受体介导性病毒感染途径。HBV受体的探索虽然已经有20余年的历史,先后发现10余种可能的蛋白质,但高亲和力的HBV受体尚未发现。树突状细胞特异的C型外源凝集素(dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin,DC-SIGN)通过甘露糖和海藻糖连接碳水化合物来识别病原微生物从而导致抗病毒的免疫反应。在许多糖化病毒免疫逃逸的过程中,DC-SIGN参与病毒进入并反式感染细胞的过程。同时,亦有研究证实其在HIV的母婴垂直传播中可能发挥重要的作用。除了在未成熟的树突状细胞上有分布和表达外,DC-SIGN也可以在PBMC、胎盘的血管内皮细胞、胎盘巨噬细胞中表达。我们前期的预实验结果也发现,DC-SIGN可以在滋养层细胞上表达。迄今为止,DC-SIGN作为受体的病毒包括HIV、CMV、埃博拉病毒(Ebola virus)、丙型肝炎病毒(hepatitis C virus,HCV),SARS冠状病毒及麻疹等(未见与HBV的相关报道)。所有的结构基础是与病原体的N-高甘露糖型糖链特异性结合,而HBV被膜蛋白的PreS2抗原也存在N-高甘露糖型糖链,HBV有与DC-SIGN结合的结构基础。鉴于DC-SIGN在胎盘上的分布,我们大胆推测DC-SIGN可能和HBV的宫内感染相关。基于以上分析,本课题首先是利用原代培养的滋养层细胞和人脐静脉内皮细胞构建一种胎盘屏障体外模型,并进行系统评价与鉴定;其次,采用HBV病毒感染胎盘屏障体外模型,明确胎盘屏障在HBV宫内感染可能的作用;然后应用胎盘屏障模型进一步研究DC-SIGN在HBV垂直传播中的可能作用,从而为HBV宫内感染机制研究与治疗提供新的实验依据。主要实验结果和结论如下:1、采用胰酶与DNA酶序贯消化法分离人早孕滋养层细胞,通过35%与45%Percoll两个梯度的不连续密度梯度离心对分离细胞加以纯化,使细胞产量达106/g以上。然后用抗HLA-DR的免疫磁珠进一步纯化早孕的绒毛滋养层细胞。应用细胞角蛋白和波形蛋白免疫细胞化学标记,发现细胞纯度达到90%以上,可从数量及纯度上满足后续体外感染要求。利用HUVEC和绒毛滋养层细胞接触共培养构建胎盘屏障体外模型,扫描电镜结果显示滋养层细胞穿过Tranwell小室,与HUVEC接触性共生长;透射电镜和激光共聚焦结果表明屏障构建后,细胞之间存在大量紧密连接;跨膜电阻检测发现构建的胎盘屏障能有效提高跨膜电阻;因此,构建的体外模型从功能和形态符合胎盘屏障的要求,为后续实验奠定基础。2、采用血清直接感染法进行滋养层细胞的HBV感染,模拟自然状态下HBV分别感染胎盘屏障中的滋养层细胞及内皮细胞,然后感染构建的胎盘体外屏障,探讨HBV感染胎盘屏障的可能性。结果显示HBV DNA在感染后的滋养层细胞及HUVEC的上清液中释放分别在72-96h及48-72h达到高峰。分别应用HBsAg、与CK7及Ⅷ相关抗原、HBcAg与CD105进行免疫组化双标染色,发现HBV可以感染人滋养层细胞和HUVEC及胎盘屏障的体外模型。而且胎盘屏障可以部分阻止HBV穿透母体面至胎儿面,所以在感染后的24h,共培养体系的外室上清液中HBV DNA仅能一过性的达到103/ml。尝试用不同检测法(传统的PCR法、Hirt法及质粒抽提法)检测感染HBV复制的金标准HBV共价闭合环状DNA(covalently closed circular DNA,cccDNA),感染HBV后的胎盘屏障未能检测出HBV cccDNA,所以HBV在胎盘屏障中是一过性的感染还是可以复制,尚没有定论。但至少,扫描电镜中我们在HBV感染后的体外模型中发现了HBV病毒样的颗粒。3、分别采用DC-SIGN抗体及siRNA基因沉默DC-SIGN试验,初步探讨DC-SIGN在HBV宫内传播中的作用,发现DC-SIGN抗体及RNA小片段干扰技术基因沉默DC-SIGN在HBV感染滋养层细胞和HUVEC的过程中有阻断作用,但对于体外构建的胎盘屏障阻断作用未见明显差异。进一步通过受体DC-SIGN重建,利用DC-SIGN质粒转染人胎肝细胞验证DC-SIGN增加了人胎肝细胞HBV易感性,故DC-SIGN是HBV经受体介导性进入胎盘屏障的宫内传播中的可能受体之一。以上结果表明,我们第一次成功构建了人胎盘屏障体外培养模型,也证实HBV可能从滋养层面向内皮细胞面穿过;尽管屏障本身对于病毒的侵入起到一定的阻断作用,HBV DNA在细胞共培养体系中仍然有一过性的增高;同时,高拷贝量的HBV病毒对于HUVEC有机械性损伤,但是共培养中未发现同样的现象;初步探讨通过DC-SIGN这个受体介导HBV入侵滋养层细胞的宫内传播的机制,旨在为HBV宫内感染机制的研究奠定实验基础,为宫内干预提供新的靶标。早产是临床上新生儿致死或是致畸的主要原因之一。尽管在过去20年中,对于早产已展开了深入的实验室和临床研究,其发生率仍然增高超过30%。早产发生主要是产程过早发动,其主要原因有宫内感染、子宫过度牵张和胎盘早剥。正是这些临床病理因素将子宫从静息状态变为收缩,从而导致分娩的发动。3’,5’-环腺苷酸(cyclic adenosine monophosphate, cAMP)作为经典的第二信使,影响了一系列的人体生理和病理反应,包括平滑肌的收缩和炎症反应。事实上,不仅生理状态下产生的化学物质(如松弛素、促肾上腺皮质激素分泌激素及降钙素基因相关肽),就连病理状态下产生的化学物质(如β2肾上腺素激动剂)均是通过cAMP引发子宫舒张。但是,临床上我们也发现除了药物本身的对人体的副作用和快速抗药物反应外,其可能下调人体内?2肾上腺素激动剂受体的表达。所以,需要寻找才一种新的可以增高cAMP水平的其他机制,最近在对于小鼠的研究中发现,磷酸二酯酶4型抑制剂——rolipram,可以降低早产的发生。前列腺素(prostaglandins,PGs)对于早产和足月产产程发动,宫颈成熟和促进子宫肌收缩都发挥非常关键的作用。在临床治疗中,这些特性一方面用于诱导分娩的发生,另一方面前列腺素合成酶抑制剂又应用于抑制早产的发生。目前已知环氧合酶至少有3种不同亚型在子宫平滑肌上表达,而表达最多的环氧合酶2型。环氧合酶-2 (cyclo-oxygenase-2,COX-2)常被转录和转录后水平上被调控,能被生长因子,细胞因子及内毒素刺激增高;在产程发动后,子宫肌和羊膜层中COX-2的表达均上调。我们前面实验中研究证实炎性细胞因子IL-1β可以通过NF-κB增高COX-2的表达,而且IL-1β和牵张也可以通过MAPK上调COX-2的表达。已经有研究表明在不同的组织中cAMP能够降低NF-κB和MAPK活性。然而新近研究发现,PGI2可以通过cAMP/PKA信号转导通路导致一系列收缩相关蛋白的表达,如连接蛋白43、α-平滑肌肌动蛋白、h-钙调蛋白结合蛋白、钙结合蛋白、平滑肌肌凝蛋白重链,这一结果提示在某些特定的信号下,cAMP可能在分娩发动前促进子宫肌的活性。临床上某些cAMP激动剂作为抑制宫缩的药物,可能在某些特殊的环境下也能引起子宫的收缩,所以本研究中我们发现cAMP能够上调COX-2的表达并对其机制进行研究。主要实验结果和结论如下:1、利用不同的cAMP激动剂(8-bromo-cAMP、forskolin、rolipram)在不同浓度作用不同时间刺激原代培养的子宫肌细胞,结果发现8bromo-cAMP提高COX-2表达在6和24h,rolipram和forskolin也用相似的生物学效应。随后的浓度依赖性实验发现,在1、6h时相点,forskolin增高COX-2 mRNA。同时COX-2蛋白合成水平的测试得到相似的趋势结果。ELISA结果也提示COX-2作用产物PGE2、PGI2、PGF2?生成在24和48h是持续增高的。2、采用cAMP下游效应物(PKA、EPAC、AMPK)的激动剂、抑制剂、shRNA技术基因沉默作用于子宫肌细胞,结果显示这些效应即不能被目前所知的cAMP下游效应物(PKA、EPAC和AMPK)激动剂所复制,也不能被其特异的抑制剂所阻截;shRNA技术基因沉默这些效应物,甚至是PDZ-GEF1,2,也不能抑制cAMP对COX-2调节作用。3、进一步利用有丝分裂原激活蛋白激酶(MAPK)信号通路及下游底物验证发现,cAMP通过MAPK信号通路提高COX-2的表达,而PGE2通过其效应物EP-2,再影响MAPK活性,最后促进COX-2的表达。以上结果表明,cAMP能够有效提高子宫肌细胞COX-2的表达,主要通过MAPK信号通路增加COX-2表达,COX-2活性的提高导致PGE2、PGI2、PGF2?生成增高,而PGE2通过其效应物EP-2,再影响MAPK活性,最终反馈调节COX-2的表达。为明确cAMP在子宫肌上的功能机制提供新的实验依据。更多还原

【Abstract】 Hepatitis BHH is HHendemicHH in HHChinaHH. Of the 350 million individuals worldwide infectedwith the HHhepatitis B virusHH (HBV), one-third resides in China. As of 2006 China hasimmunized 11.1 million children in its poorest provinces as part of several programsinitiated by the Chinese government and as part of the HHGlobal Alliance for Vaccines andImmunizationHH (GAVI). However, the effects of these programs have yet to reach levels ofimmunization that would limit the spread of hepatitis B effectively. The reason for thisincreased HBV infection is unknown, because hepatitis B has no clear transmission routesin many people in China, although it has been widely accepted that HHneonatalHH HHinfectionHH andHHvertical transmissionHH during early childhood are still the most common routes. Also, mostresearchers divided the HHvertical transmissionHH to 3 different approaches: prenatal intrauterinetransmission, intrapartum transmission and postpartum transmission. As for the two of thelatter approaches, the transmission process can be effectively inhibited and blocked byusing of hepatitis B vaccine and hepatitis B immunoglobulin (HBIG), so a betterunderstanding of mechanism of intrauterine infection caused by intrauterine transmissionis critical, according to identify HBV transmission pathway(s).During the whole pregnancy, the placenta is a dynamic organ whose structure andfunction change. There is compelling evidence that the placenta plays an integral role in thevertical transmission of virus, such as HBV, HIV, CMV, and HCV, from mother to the fetus.Congenital viral infection is associated with fetal anomalies, postnatal infection, and fetaldemise. Although transplacental passage of these viruses is a critical aspect of theseinfections, few investigators have characterized placental viral infection and the role ofplacental barrier has in passage of virus from maternal to the fetal circulation. In spite ofintensive research of blood-brain barrier (BBB), a similar effort or interest in characterizingthe placental barrier does not exist yet. The emergence of in vitro models for the humanplacenta barrier will open the door for investigations of virus infection mechanisms andapply that knowledge to inhibiting, even blocking virus to pass through the placenta, thus reducing virus vertical transmission from mother to fetus. Therefore, it is necessary to seek a simple method of construction human placental barrier in vitro.Meanwhile, it has been proved that three different layers: trophoblastic cells, interstitial cells (mainly placental macrophage, i.e. Hofbauer) and human placental microvascular endothelial cells (HPMEC) of placenta are involved in intrauterine transmission from mother to fetus. And placental barrier consisted of trophoblastic cells, HPMEC and their basement membranes, is the unavoidable access of nutrients and some drugs, viruses and hormones to enter the fetus from mother. We use primary culture cytotrophoblast (CTB), human umbilical vein endothelial cells (HUVEC) and then chose three various ways to construct placental barrier: cytotrophoblast alone, HUVEC and CTB co-culture with or without contact. And we found that contacted HUVEC and CTB co-culture is the best model to mimic in vivo placental barrier construction and function. Then we used HBV copy more than 107 patients serum to infect the in vitro placental barrier, and examined the ability of infectious HBV to cross the placenta in this system.Basing on the research results on human immunodeficiency virus (HIV) and cytomegalovirus (CMV) can via receptor-mediated viral intrauterine infection and HBV is able to infect peripheral blood monocyte (PBMC) and then duplicate inside of the cells, we assumed that HBV probably through the following three approaches invading placental barrier:⒈mechanical injury: inflammation-caused direct or indirect trophoblastic cells and HUVEC injury or more leakage which lead to the increase of permeability of basement membrane so as to allow HBV to get through the first placental barrier (trophoblastic cells) to enter intervillus substance;⒉cell transfer: the microfusion of HBV-infected PBMC and trophoblastic cells cause the transfer of HBV to the latter and further, the trophoblast cells release through HBV polarity into inervillus substance and then infect HUVEC;⒊receptor mediation: some receptors in the superior lateral membrane of trophoblastic cells might have mediated dissociative complete HBV particles into cells to duplicate and then release HBV into intervillus substance.In my project, I will focus on mechanical injury and receptor mediation hypothesis, because the receptor-mediated virus invasion is a very common invasion approach and it has been confirmed that HBV can damage the placenta in vivo. Compelling researches on HBV receptor have been carried out in the past twenty years and nearly 10 candidates of protein have been found. Currently, a high affinity receptor for HBV is still unknown. The dendritic cell specific C-type lectin DC-SIGN is involved in pathogen recognition through mannose and fucose containing carbohydrates leading to the induction of an anti-viral immune response. And DC-SIGN is exploited by many glycosylated viruses which subvert this immune surveillance function as a port of entry and for trans-infection of target cells. Also, it has been confirmed that DC-SIGN might have played an important role in the mother-to-infant transmission of HIV. Besides those distributed in premature dendritic cells (DC), DC-SIGN are also expressed in PBMC, placental vascular endothelial cells, placental macrophage and our group’s preliminary data also showed that it is can be immunostained in trophoblastic cells. So far, reports have been found that DC-SIGN can serve as the receptor of HIV, CMV, Ebola virus, hepatitis C virus (HCV), SARS coronavirus, measles and etc ( no HBV-related reports have been found). The glycosylation pattern on HBV surface antigens (HBsAg) together with the tissue distribution of HBV would allow interaction between HBV and DC-SIGN. And because the N- high-mannose glycans also exists in the PreS2 antigen of HBV envelope protein, HBV has the structure base of combining with DC-SIGN. The HBV glycosylation pattern together with the cellular localization prompted us to investigate the possible role DC-SIGN in binding HBV. Also, according to the distribution of DC-SIGN in placenta, we hypothesize the possibility of DC-SIGN could be related with HBV intrauterine infection. Therefore, our project are mainly focus on the placenta barrier construction and HBV intrauterine infection model in vitro, then to further investigate the potential role DC-SIGN on HBV vertical transmission. Main results are as follows:1. Trypsin and DNAse sequential digestion method is used to separate human pregnant chorionic trophoblastic cells. By gradients of 35% and 45% Percoll noncontinuous density centrifugation method to purify separated cells to make the cell production exceed 106/ml. Then, Anti-HLA-DR Dynabeads were used for the purification and enrichment in the primary culture of human early pregnant chorionic trophoblastic cells. By using cell keratin and vimentin immunocytochemistry(ICC) staining, the positive staining rate of keratin exceeds 90% and that of vimentin is lower than 10%. The cell purity of over 90% can satisfy the purity requirements of the following experiments. Collagenase I is used for isolation of human umbilical vein endothelial cell. Then, both of the cells are contacted co-cultured to construct human placental barrier in vitro. From the functional and structural aspects, the barrier model has been identified and could be used for the following experiment.2. The direct serum infection method is used to simulate the natural HBV infection of trophoblastic cells or HUVEC separately, and then the possibility of HBV infection of human placental barrier is discussed. We find out that the HBV DNA in culture supernatant reaches the peak in 72-96 or 48-72 hours when the primarily cultured HBV infected trophoblastic cells or HUVEC is released. Furthermore, we use immunoenzyme double Staining method to determine and transmission electron microscope to identify that HBV can infect both trophoblastic cells/HUVEC and human placental barrier in vitro. Also, this barrier can block part of HBV transmission from mother side to fetal side, so HBV DNA in co-culture system outside supernatant only transiently can reach 103/ml after 24h infection. Try three different methods (traditional PCR method, Hirt method and plasmid extraction method) to detect the gold standard of infected HBV duplication---HBV covalently closed circular DNA (cccDNA). Because HBV cccDNA hasn’t been detected in infected HBV human placental barrier in vitro, it is hard to conclude that HBV infection of trophoblastic cells is a transient infection or it can be duplicated within placental barrier. At least, the virus-like particle had been found in our in vitro model.3. Antibody DC-SIGN and siRNA DC-SIGN is carried out to preliminarily discuss the action of DC-SIGN in HBV intrauterine transmission. We find out that antibody DC-SIGN and siRNA DC-SIGN can significantly inhibit HBV infection in trophoblastic cells and HUVEC, but the blocking function of them are not prominent in placental barrier in vitro. Further reconstruct the receptor and use DC-SIGN plasmid transfection fetal hepatocyte to validate that DC-SIGN might have played an important role in HBV infection and it could be regarded as one of the potential receptors of HBV which is involved intrauterine transmission.In conclusion, our experiment successfully set up the primary culture system of human placental barrier in vitro and proves that HBV has the possibility of infecting through trophoblastic cells to HUVEC. Furthermore,although placental barrier can block HBV transmission from mother side to fetal side, HBV DNA still can be temporary induced in placental barrier in vitro. Also, high copies HBV can mechanical injury HUVEC, but in co-culture system this phenomena can be avoided. Preliminary research on the intrauterine transmission mechanism of trophoblastic cells and HUVEC invaded by DC-SIGN receptor-mediated HBV aims to lay down the foundation for further research on HBV intrauterine infection mechanism and to provide new guide for intrauterine intervention. Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI2, acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE2 and PGI2 in culture supernatant) in primary cultures of uterine smooth muscle cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF1-2, without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE2 acts through EP-2 to activate MAPK and increase COX2. These data provide further evidence in support of a dual role for cAMP in determining myometrial function.更多还原