HBV感染宿主细胞的早期过程

自从HBV基因组被确定后,就阐明了HBV编码的蛋白。在研究HBV受体、吸附、穿入靶细胞等方面有很大的进展,但HBV感染的分子机制目前仍然不清楚。本文就HBV细胞受体及其感染宿主细胞早期过程的最新进展作一简要综述。     

HBV感染宿主细胞的早期过程

自从HBV基因组被确定后,就阐明了HBV编码的蛋白。在研究HBV受体、吸附、穿入靶细胞等方面有很大的进展,但HBV感染的分子机制目前仍然不清楚。本文就HBV细胞受体及其感染宿主细胞早期过程的最新进展作一简要综述。     

乙型肝炎病毒Ⅱ型感染的初步探讨

1978年先后在西非塞内加尔发现HBsAg阳性,而抗-HBc阴性的乙型肝炎患者,人体内单项抗-HBs阳性也不一定能保护机体免受HBV感染,从而表明了乙型肝炎疫苗免疫后所产生的高滴度抗-HBs不能防止一种新的乙型肝炎病毒感染。1987年将这种病毒命名为乙型肝炎病毒Ⅱ型(HBV_2)。我院于1992年～1996年对1800例乙型肝炎病人进行HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc检测,现将结果报告如下。     

影响HBV宫内感染的相关临床因素的探讨

目的探讨乙型肝炎病毒（HBV）宫内感染的高危因素或保护因素。方法对2006年6月至2008年2月在中山大学附属第三医院产科分娩的417例血清HBsAg阳性孕妇的新生儿进行回顾性分析。结果HBV宫内感染组33例,非感染组384例,感染组的母亲HBeAg阳性率、HBV DNA阳性率和非感染组的具有显著性差异（P〈0.05）,母亲孕晚期注射HBIG在2组中均无显著性差异（P〉0.05）;在母亲各HBV DNA水平级上分析,新生儿HBV宫内感染和母亲使用HBIG均无明显相关（P〉0.05）;结论1.HBV DNA阳性是HBV宫内感染的高危因素。2.HBsAg阳性孕妇晚孕期间肌注HBIG对HBV宫内感染未见明显的保护作用。     

抗-HBc单项阳性患者中的隐匿性HBV感染

目的 研究抗-HBc单项强阳性患者中的隐匿性HBV感染发生率并分析发生原因.方法 收集183例血清学检测抗-HBc单项强阳性（A≤0.1）患者血清标本,采用实时定量PCR进行HBV DNA含量检测.对于DNA定量阳性的标本,PCR扩增HBV pre-S/S区基因,并进行克隆测序.结果 183例血清标本中3例HBV DNA定量结果大于103拷贝/ml,占1.6%.这3例标本中有2例得到pre-S/S区测序结果,2例标本均存在S基因＂a＂决定簇内Q129R/P点突变,且突变株与野生型共存.结论 抗-HBc单项阳性患者中存在隐匿性HBV感染,HBsAg血清免疫学方法的漏检与HBV S基因突变有关,同时与循环中HBsAg量低于检测限也有一定关系.HBV隐匿感染不仅可以造成临床诊断失误,更为严重的是献血员HBV隐匿感染造成血液的污染.     

HBV感染的CD68细胞在隐匿性HBV感染产妇胎盘中的分布及与宫内感染的关系

目的探讨HBV感染的外周血单个核细胞(peripheral blood mononuclear cells,PBMC)在乙肝宫内传播中的作用及机理。方法12例血清HBV DNA(-)、PBMC HBV DNA(+)产妇分娩的新生儿血清HBV DNA(+)和/或PBMC HBV DNA(+)的胎盘作为实验组,10例乙肝标志物均为阴性产妇的胎盘作为对照。采用SP法在连续切片上检测HBsAg和HBcAg在胎盘CD68细胞及各类细胞中的表达。结果8例新生儿血清HBV DNA(-)、PBMCsHBV DNA(+)胎盘绒毛间质5例CD68细胞HBsAg阳性,6例CD68细胞HBcAg阳性;毛细血管内5例CD68细胞HBsAg阳性,8例CD68细胞HBcAg阳性;滋养层细胞和血管内皮细胞均未见HBsAg、HBcAg阳性信号;2例新生儿血清HBV DNA(+)、PBMCs HBV DNA(-)的胎盘滋养层细胞、绒毛间质、毛细血管内皮细胞均有HBsAg、HBcAg的表达,而绒毛毛细血管内CD68细胞未见表达。2例新生儿血清和PBMC HBV DNA均阳性的的胎盘滋养层细胞、绒毛间质、CD68细胞和毛细血管内CD68细胞均有HBsAg、HBcAg的表达,毛细血管内皮细胞无表达。10例乙肝标志物全阴性产妇胎盘中均无阳性信号。结论HBV感染的外周血单个核细胞可作为宫内传播的载体。     

广州地区HBV/HCV重叠感染患者流行及临床特征研究

目的 探讨广州地区乙型肝炎病毒/丙型肝炎病毒（HBV/HCV）重叠感染患者的流行及临床特征,为提高其诊治提供依据.方法 回顾2005年4月至2011年10月我院收治慢性HBV感染患者临床资料,分析HBV/HCV重叠感染患者流行特征,按照HBeAg状态以及HBV DNA、HCV RNA水平分组分析HBV/HCV重叠感染患者临床特征.结果 HBV/HCV重叠感染率为1.9％（128/6604）,主要见于伴静脉吸毒史的男性中年患者.HBeAg阳性与阴性重叠感染患者之间的生化指标和终末期肝病发生率均无差异.HBeAg阳性患者HBV DNA阳性率高于阴性患者（P ＜0.001）,而HCV RNA水平阳性率低于阴性患者（P =0.007）.HBV DNA阳性患者终末期肝病发生率较阴性组升高（58.1％比37.9％,P=0.027）,而HCV RNA阳性与阴性患者之间发生率相似（43.6％比49.2％,P=0.540）.结论 广州地区HBV/HCV重叠感染率较低,HBV DNA水平可能与患者的疾病进展相关.     

HBV感染孕妇胎盘绒毛Hofbauer细胞并与HBV复制水平有关

目的探讨胎盘Hofbauer细胞在乙型肝炎病毒(HBV)垂直传播中的作用。方法以酶消化法、机械法、FicollHypaque分离法等多种方法分离纯化HBV感染孕妇流产绒毛Hofbauer细胞,CD163免疫组织化学染色鉴定Hofbauer细胞,PCR法检测绒毛Hofbauer细胞内HBV-DNA,实时定量PCR(qRT-PCR)检测Hofbauer细胞内CD16(FcγRⅢ)mRNA表达,免疫组织化学染色检测Hofbauer细胞CD16蛋白表达。结果 HBV感染孕妇流产绒毛Hofbauer细胞内HBV-DNA阳性率为31.67%(19/60),其中乙型肝炎e抗原阳性(HBe Ag+)和HBe Ag-孕妇绒毛Hofbauer细胞内HBV-DNA阳性率分别为46.4%(13/28)和18.75%(6/32)。qRT-PCR检测发现在HBV-DNA+的Hofbauer细胞内CD16 mRNA表达增加,其表达水平与HBV-DNA载量明显相关。免疫组织化学染色显示HBV-DNA+患者Hofbauer细胞胞膜和胞质CD16着色较HBV-DNA-者明显增强。结论 Hofbauer细胞作为胎盘巨噬细胞可被HBV感染,其感染率与体内病毒复制水平密切相关。     

抗-HBc单项阳性患者中的隐匿性HBV感染

Objective This study was designed to explore the incidence rate of occuIt HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occuIt infection.Methods Sera of 183 patients carrying anti-HBc alone(A≤0.1) were collected and real-time PCR was used to select samples with HBV DNA positive.HBV pre-S/S amplification products were obtained by PCR,and clonal sequencing were then used for these samples with HBV DNA positive.Results DNA quantitative results of three samples were greater than 103 copies/ml in 183 samples,with a fraction of 1.6%.Pre-S/S sequencing results of two samples from these three samples were obtained.Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.Conclusions Occult HBV infection existed in samples with anti-HBc alone.Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of cireulating antigen which are below the assay limit of detection.Occult HBV infection not only can lcad to a false clinical diagnosis,but also can result in hematological pollution due to such occult infection of blood donors.     

抗-HBc单项阳性患者中的隐匿性HBV感染

Objective This study was designed to explore the incidence rate of occuIt HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occuIt infection.Methods Sera of 183 patients carrying anti-HBc alone(A≤0.1) were collected and real-time PCR was used to select samples with HBV DNA positive.HBV pre-S/S amplification products were obtained by PCR,and clonal sequencing were then used for these samples with HBV DNA positive.Results DNA quantitative results of three samples were greater than 103 copies/ml in 183 samples,with a fraction of 1.6%.Pre-S/S sequencing results of two samples from these three samples were obtained.Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.Conclusions Occult HBV infection existed in samples with anti-HBc alone.Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of cireulating antigen which are below the assay limit of detection.Occult HBV infection not only can lcad to a false clinical diagnosis,but also can result in hematological pollution due to such occult infection of blood donors.     

Herpes simplex virus infection of cultured human term trophoblast.

Mononuclear trophoblast cells were isolated from term placentas of uncomplicated pregnancies, purified to homogeneity by negative immunomagnetic separation using monoclonal antibodies to the major histocompatibility complex, and challenged with herpes simplex virus (HSV). The cultures were highly susceptible to virus-induced cytopathic effect as evidenced by cytopathic alteration and inhibition of human chorionic gonadotropin (hCG) secretion. Both HSV I and II underwent a full replicative cycle in the trophoblast, although the production of progeny virus was 10-100 times less than that obtained with placental fibroblasts or choriocarcinoma cells. The permissiveness was independent of in vitro syncytial differentiation of the trophoblast. The results suggest that the trophoblast layer may be involved in intrauterine HSV infection.     

Heparin prevents programmed cell death in human trophoblast

Heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, especially antiphospholipid antibody syndrome. Recent studies have suggested that heparin may exert direct effects on placental trophoblast, independently of its anticoagulant activity. We now demonstrate that heparin abrogates apoptosis of primary first trimester villous trophoblast in response to treatment with the pro-inflammatory cytokines interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. This multifunctional glycosaminoglycan also inhibited apoptosis induced by other agents, including staurosporin, broad-spectrum kinase inhibitor and thrombin. Furthermore, heparin attenuated caspase-3 activity, a hallmark of apoptosis, in human first trimester villous and extravillous trophoblast cell lines treated with peptidoglycan, a Toll-like receptor-2 agonist isolated from Staphylococcus aureus. The ability of heparin to antagonize cell death induced by such diverse apoptotic signals suggested that it acts as a survival factor for human trophoblast. We demonstrate that heparin, like epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), elicits phosphorylation of the EGF receptor and activation of the phosphatidyl inositol 3-kinase (PI3K)-, the extracellular signal-related kinase 1/2 (ERK1/2)- and the c-Jun NH2 terminal kinase (JNK)-signal transduction pathways in primary villous trophoblast. In summary, we have demonstrated that heparin activates multiple anti-apoptotic pathways in human trophoblast. Our results suggest that heparin may be useful in the management of at-risk patients, even in the absence of an identifiable thrombophilic disorder.     

人早孕绒毛细胞滋养层细胞培养方法的改良

目的 探索一种简便高纯度的早孕滋养细胞的体外培养方法。方法 采用完整早孕绒毛通过胰蛋白酶联合胶原酶消化法分离,Percoll梯度分离法提纯体外培养,应用光镜HE染色和免疫细胞化学法检测细胞纯度。 结果 CK-7表达阳性细胞数可以达到90%以上。结论 完整绒毛消化和Percoll纯化联合可在短期内获得高纯度、高产量的滋养细胞以供后期试验,方法更加简便。     

Intermediate filaments of human trophoblast and choriocarcinoma cell lines

Summary Human term placenta and two human choriocarcinoma cell lines were studied immunohistochemically and by immunoblotting with monoclonal antibodies to keratin polypeptides and vimentin. Four keratin polypeptides (40, 45, 52, 54 K) were detected in both normal and malignant trophoblastic cells. The presence of the 54 K keratin polypeptide distinguishes the benign and malignant trophoblastic cells from human embryonal carcinoma cells and a yolk sac carcinoma cell line.     

“中间型”滋养细胞的超微结构观察

为系统的阐明正常及肿瘤的“中间型”滋养细胞微细形态，应用透射电镜观察了正常胎盘绒毛标本６７例、胎盘床１０例、未化疗的葡萄胎、恶葡各１０例、绒癌５例，结果显示胎盘绒毛过渡型（中间型）滋养细胞是细胞滋养细胞分化、融合形态，演变的合体滋养细胞是其成熟和老化的形态，它具备细胞滋养细胞核与合体滋养细胞质的特点，胎盘床浸润的过渡型保持与绒毛过渡型类似的形态，滋养细胞肿瘤的过渡型形态，为既有正常的形态又具有细胞增生、异型和肿瘤超微结构特征，后者突出表现为多量的高电子致密颗粒与充塞绒毛的多形状的囊腔。显示胎盘与滋养细胞肿瘤中的“中间型”滋养细胞形态相似，肿瘤时细胞形态不同程度的异形。     

Occult HBV infection

The long-lasting persistence of hepatitis B virus (HBV) genomes in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for the HBV surface antigen (HBsAg) is termed occult HBV infection (OBI). Although in a minority of cases the lack of HBsAg detection is due to infection with variant viruses unrecognized by available assays (S-escape mutants), the typical OBI is related to replication-competent HBVs strongly suppressed in their replication activity. The causes of HBV suppression are not yet well clarified, although the host’s immune surveillance and epigenetic mechanisms are likely involved. OBI is a worldwide diffused entity, but the available data of prevalence in various categories of individuals are often contrasting because of the different sensitivity and specificity of the methods used for its detection in many studies. OBI may have an impact in several different clinical contexts. In fact, it can be transmitted (i.e., through blood transfusion and liver transplantation) causing classic forms of hepatitis B in newly infected individuals. The development of an immunosuppressive status (mainly by immunotherapy or chemotherapy) may induce OBI reactivation and development of acute and often severe hepatitis. Finally, evidence suggests that OBI can favor the progression of liver fibrosis, in particular in HCV-infected patients. The possible contribution of OBI to the establishment of cirrhosis also implies its possible indirect role in the development of hepatocellular carcinoma. On the other hand, OBI may maintain most of the direct transforming properties of the overt HBV infection, such as the capacity to integrate in the host’s genome and to synthesize pro-oncogenic proteins.     

胰岛素对滋养层细胞凋亡的调节作用及机制

目的 :探讨胰岛素对培养的滋养细胞凋亡的影响及可能的机制。方法 :将培养的妊娠早期滋养层细胞 ,分为正常对照组(细胞 +培养液 )、H2 O2 组 (细胞 +培养液 +H2 O2 )和胰岛素 +H2 O2 组 (细胞 +H2 O2 +胰岛素 )。采用透射电镜观察及流式细胞术 ,观察H2 O2 诱导的细胞凋亡及胰岛素对H2 O2 诱导的细胞凋亡的抑制作用。并检测胰岛素对滋养细胞caspase 3的活性及Bcl 2蛋白表达的影响。结果 :H2 O2 可诱导培养的滋养细胞凋亡 ,透射电镜下可见特征性的细胞核改变。胰岛素可显著抑制H2 O2 诱导的细胞凋亡 ,流式细胞仪检测其凋亡率较H2 O2 组显著下降 (P <0 .0 1)。H2 O2 组滋养细胞中caspase 3的活性较对照组显著增高 (P <0 .0 1) ,而Bcl 2蛋白的表达则较对照组显著下降 (P <0 .0 1)。结论 :胰岛素可明显抑制H2 O2诱导的滋养细胞凋亡 ,其机制可能与降低caspase 3的活性和促进Bcl 2蛋白的表达有关     

Intrauterine growth restriction, human placental development and trophoblast cell death

Intrauterine growth restriction (IUGR) is a failure to achieve the growth potential of a fetus that is promised by the genetic constitution and environmental influences endogenous to the pregnancy. Optimal placental development and the ability of the placenta to compensate for stimulus-induced injury are central in promotion of normal fetal growth. In this review, we will overview placental development with a focus on how villous structure relates to function. We will also describe the differentiation and turnover of villous trophoblast while highlighting selected features of microscopic placental injury. Histopathological studies of the placenta in IUGR indicate that abnormalities of the maternal spiral arterioles, dysregulated villous vasculogenesis, and abundant fibrin deposition are characteristic of the injuries associated with this condition. We identify selected insults, including oxidative stress and complement activation, and key pathways that regulate apoptosis in villous trophoblast, including increased p53 activity, altered translation of AKT and mTOR proteins, and the stress response of the endoplasmic reticulum. We surmise that trophoblast dysregulation at a subcellular level and loss of functional mass of villous trophoblast via cell death pathways are key contributors to the suboptimal placental performance that yields IUGR. We predict that a better understanding of placental dysfunction in IUGR will lead to targeted therapeutic options for this important clinical condition.