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。     

Human Annexin Ⅴ在不同胎盘中的表达的定量分析

目的观察人膜联蛋白Ⅴ(Human Annexin Ⅴ,HAⅤ)在乙肝病毒感染胎盘、正常中、晚孕胎盘及早孕绒毛中的表达并探讨其意义.方法用SP法检测HBsAg阳性孕妇足月分娩胎盘、HBV血清标志物阴性的正常早孕、中孕和足月胎盘各20例HAⅤ的表达、分布并进行定量分析.结果中孕、足月胎盘和HBV感染的胎盘组织中HAⅤ呈不均一的棕黄色颗粒状染色,泛存在于胎盘组织各类细胞胞浆、胞膜和核膜上.滋养细胞呈强阳性染色,绒毛间质细胞和毛细血管内皮细胞呈弱阳性染色.早孕绒毛HAⅤ棕黄色颗粒主要分布于外层滋养细胞的胞浆、胞膜和核膜上,最内层滋养细胞和间质偶见表达;比较HBV感染组和正常足月组胎盘HAⅤ表达的量,前者的平均灰度比后者明显增高,差异具有显著性(P＜0.01),中孕组与足月组HAⅤ表达的量没有差别(P＞0.05),早孕和中孕组、足月组HAⅤ表达的量的比较,前者显著低于后两者(P＜0.05).结论HAⅤ可能作为受体介导了HBV对胎盘的感染.     

Bcl—2蛋白在正常、妊高征及胎儿宫内发育迟缓胎盘中的表达

目的 通过检测Bcl-2蛋白在正常各期胎盘、妊高征胎盘和胎儿宫内发育迟缓胎盘中的表达，探讨Bcl-2蛋白在胎盘的发育过程中的功能及妊高征和胎儿宫内发育迟缓的病理机制。方法 取正常的8－10周、20－22周、足月胎盘以及妊高征和胎儿宫内发育迟结的胎盘组织固定，石蜡包坦，用ABC法抗Bcl-2免疫组织化学染色，光镜观察。结果 正常早孕绒毛合体细胞滋养怪、细胞滋养层细胞核、绒毛吵轴细胞阳性，细胞滋养层的胞质、绒毛中轴基质阴性。正常中期和足月胎盘滋养层细胞、血管内皮细胞阳性。与正常足月胎盘相比，妊高征、台儿宫内发育迟缓胎盘几科不着色。结论 Bcl-2蛋白在正常各期胎盘中均有表达，而在妊高征和胎儿宫内发育迟缓胎盘几乎不表达，Bcl-2蛋白表达异常与妊高征与胎儿宫内发育迟缓的发病机制有关。     

DC-SIGNR在人胎盘滋养层细胞中的表达及意义

目的:探讨DC-SIGNR在人胎盘绒毛组织中的定位及在体外培养滋养层细胞上的表达.方法:建立稳定的滋养层细胞原代培养体系,采用免疫组化单染及荧光双染的方法检测不同孕期正常胎盘绒毛组织及体外培养滋养层细胞上DC-SIGNR的表达.结果:DC-SIGNR主要表达于胎盘滋养层细胞、Hofbauer细胞及胎盘微血管内皮细胞的胞质及包膜,在原代培养的人绒毛膜滋养层细胞中的DC-SIGNR的表达与在体组织表达一致.结论:DC-SIGNR表达于不同孕期的胎盘组织及体外分离培养滋养层细胞,为研究滋养层细胞上该受体在宫内感染中的作用提供体外实验的细胞学基础.     

我国首次发现胎儿感染乙肝病毒的机制

经过长时间的病例对照研究和复杂的实验观察,我国科学家宣布,他们在世界上首次发现了胎儿在子宫内感染HBV的传播机制。这项名为乙型肝炎病毒宫内传播因素和机制的分子流行病学研究发现:由于某些因素,如先兆早产,引起胎盘微血管的破损,就会使母亲含有高浓度HBV的血液进入胎儿体内。HBV从母亲的脱膜细胞经过滋养层、绒毛间质到胎儿的绒毛毛细血管内皮细胞,有一个逐层转移传递的过程,并且细胞越接近胎儿其感染HBV的机率就越小。以第四军医大学徐德忠教授为首的课题组,为研究这一课题,从1992年起,就开始了病例对照研究和血清与胎盘的多种乙…     

应用原位杂交法检测胎盘组织HLA-G mRNA的表达

应用地高辛标记的特异性寡聚核苷酸探针原位杂交正常妊娠初期胎盘组织切片，检测母胎界面处各类细胞ＨＬＡ ＧｍＲＮＡ的表达与分布。结果显示绒毛内、外的细胞滋养层细胞表达强ＨＬＡ ＧｍＲＮＡ信号；合体滋养层细胞也有ＨＬＡ ＧｍＲ ＮＡ的表达；绒毛内间质细胞、血管内皮细胞以及蜕膜基质细胞不含任何ＨＬＡ Ｇ信号。结果表明地高辛标记的特异性寡聚核苷酸探针原位杂交方法成功显示妊娠期胎盘不同细胞ＨＬＡ ＧｍＲＮＡ转录情况，证明此方法的可行性、可靠性。     

HBeAg对慢性乙型肝炎孕妇胎盘组织TLR4表达的作用研究

目的探讨HBeAg对慢性乙肝孕妇胎盘组织Toll样受体4（TLR4）表达的作用,及TLR4表达与胎盘持续感染HBV的关系。方法选取22例HBeAg阳性和24例HBeAg阴性慢性乙肝孕妇,及正常孕妇25例。以实时荧光定量RT-PCR（qRT-PCR）和免疫组化法检测胎盘组织中TLR4的表达,并采用实时荧光定量PCR和微粒子酶免疫分析法（MEIA）分别检测其血清HBV DNA和HBeAg水平。结果HBeAg阳性孕妇胎盘组织中TLR4mRNA的表达水平明显低于正常对照组和HBeAg阴性组（P〈0.01）,但与正常对照组相比,其血清HBeAg和HBV DNA水平则显著升高（P〈0.01）;胎盘组织中TLR4 mRNA表达与HBeAg水平呈负相关,而与血清HBV DNA水平无显著相关性。结论HBeAg对胎盘组织TLR4表达起负调节作用,胎盘组织中TLR4的表达与其持续感染HBV有一定关系。     

转化生长因子β1及其受体1在妊娠肝内胆汁淤积症患者胎盘绒毛组织中的表达及其意义

目的探讨妊娠肝内胆汁淤积症(ICP)患者胎盘转化生长因子β1(TGF-β1)及其受体1(TGF-βR1)表达的变化.方法采用S-P免疫组化方法.结果 (1)光镜下发现ICP患者绒毛水肿、绒毛间隙狭窄,绒毛血管密度降低.(2)两组孕妇胎盘绒毛滋养层细胞均有TGF-β1的表达,但研究组胎盘的水平低于对照组(P<0.01).两组孕妇胎盘绒毛滋养层细胞及血管内皮细胞均有TGF-βR1分布, 研究组胎盘的水平低于对照组(P<0.01) .结论 ICP患者胎盘TGF-β1及TGF-βR1的低表达可能导致ICP胎儿早产、胎膜早破及宫内窘迫的发生.     

印迹基因p57~(kip2)蛋白在水泡状胎块诊断和分型中作用

目的 探讨p57kip2蛋白表达在水泡状胎块诊断和分型中的作用和意义.方法 应用免疫组化SP法检测p57kip2蛋白30例在完全性水泡状胎块(complete hydatidiform moles,CHMs)、25例部分性水泡状胎块(partial hydatidiform mole,PHMs)和20例水泡状流产(hydropic abortions,HAs)三组病变中的表达情况,正常成熟胎盘(normal mature placentas,NMPs)为正常对照.结果 p57kip2蛋白在10例NMPs、20例HAs组织中的细胞滋养层细胞、绒毛间质细胞和绒毛外滋养细胞团核表达阳性,合体滋养层细胞不表达;CHMs的绒毛间质细胞和细胞滋养层细胞p57kip2蛋白不表达,阳性表达仅见于绒毛外散在滋养细胞团中;25例CHMs的绒毛间质细胞和细胞滋养层细胞p57kip2均表达阳性.结论 p57kip2蛋白在CHMs和PHMs的表达和分布有明显差异,可作为胎块分型诊断的客观辅助指标,值得在国内妇产科病理诊断常规工作中推广应用.     

人胎盘组织细胞表型特征及其意义

目的:研究足月分娩胎盘组织细胞的表型特征并探讨其意义。方法:采用流式细胞术检测人胎盘组织单个核细胞(MNC)中CD133 细胞及其亚群含量;免疫组织化学法分析胎盘组织CD133、CD34、KDR、vWF和CD144的表达分布。结果:流式细胞仪检测结果表明,在胎盘MNC中,CD133 细胞所占百分率为(1.7±1.1)%,其中CD133 CD34 细胞亚群的比例为(0.5±0.5)%;胎盘CD133 细胞中表达CD34的细胞占(24.1±11.2)%。免疫组织化学结果显示,绒毛间质存在CD133 和CD34 细胞;胎盘绒毛合体滋养层和细胞滋养层细胞也表达CD133;而KDR、vWF和CD144的表达限于胎盘绒毛毛细血管内皮细胞,其表达CD34,但不表达CD133。结论:人足月胎盘组织间质中存在CD34 和CD133 细胞,晚期胎盘绒毛血管无内皮祖细胞存在。     

乙型肝炎e抗原、s抗原对Toll样受体及共刺激分子表达的调控和影响

目的：探讨乙型肝炎e抗原、s抗原（HBeAg、HBsAg）对健康人外周血单个核细胞（PBMC）Toll样受体（TLRs）和共刺激分子表达的影响。方法：分别用HBeAg、HBsAg、空质粒以及卵清蛋白（OVA）作为无关蛋白刺激健康人外周血单个核细胞（PBMC）,采用流式细胞技术检测各刺激组CD14＋细胞TLR2、TLR4、CD86、PD-L1的表达水平。结果：健康人PBMC经HBeAg或HBsAg刺激后,CD14＋细胞表面TLR2、TLR4的表达明显低于未刺激组（P〈0.05）,CD14＋细胞PD-L1的表达则明显增高（P〈0.05）;HBeAg刺激后同时可见CD14＋细胞表面共刺激分子CD86的表达水平明显降低（P〈0.02）。结论 HBeAg和HBsAg通过上调单核细胞表面PD-L1的表达,下调CD14＋单个核细胞表面TLR2、TLR4和共刺激分子CD86的表达,削弱机体固有免疫系统的应答能力,降低启动特异性免疫的活化信号,从而导致免疫清除逃避。因此,HBeAg和HBsAg可能是促进HBV感染慢性化的重要因素。     

A mouse model for HBV immunotolerance and immunotherapy

Lack of an appropriate small animal model remains a major hurdle for studying the immunotolerance and immunopathogenesis induced by hepatitis B virus （HBV） infection. In this study, we report a mouse model with sustained HBV viremia after infection with a recombinant adeno-associated virus （AAV） carrying a replicable HBV genome （AAV/ HBV）. Similar to the clinical HBV carriers, the mice infected with AAV/H BV were sero-negative for antibodies against HBV surface antigen （HBsAg）. Immunization with the conventional HBV vaccine in the presence of aluminum adjuvant failed to elicit an immune response against HBV in these mice. To identify a vaccine that can potentially circumvent this tolerance, the TLR9 agonist CpG was added to HBsAg as an adjuvant. Vaccination of mice with HBsAg/CpG induced not only clearance of viremia, but also strong antibody production and T-cell responses. Furthermore, both the DNA replication and protein expression of HBV were significantly reduced in the livers of AAV/H BV-infected mice. Accordingly, AAV/HBV-infected mice may be used as a robust model for investigating the underlying mechanism（s） of HBV immunotolerance and for developing novel immunotherapies to eradicate HBV infections.     

Perinatal transmission of hepatitis B virus in Thailand

Perinatal transmission of hepatitis B virus (HBV) from asymptomatic HBsAg carrier mothers to their infants was studied in 78 mother-infant pairs by determination of HBsAg, HBeAg and anti-HBe both in the mothers and in their infants at regular intervals for those children up to the time when they reached at least one year of age. Twenty-five out of the 78 (32.1%) infants born to these mothers were HBsAg-positive 2-6 months after birth and they remained so throughout the observation period of at least one year or more. Perinatal HBV transmission occurred only in infants born to HBsAg carrier mothers who were HBeAg-positive (92.6%) but not in those born to HBsAg carrier mothers who had no detectable HBeAg. This study suggests that preventive measures against HBV transmission during the perinatal period should be taken only for infants born to HBsAg carrier mothers who are HBeAg-positive. In addition, the active immune response to HBV was studied in 75 non-HBsAg carrier infants born to HBsAg carrier mothers by determination of anti-HBs at one year of age or older. Forty-three of these infants were treated with HBIG at birth and 32 infants received no treatment. It was found that infants born to HBsAg carrier mothers who were HBeAg-positive had a better active immune response (84.2% positive for anti-HBs) than infants born to HBsAg carrier mothers who had no detectable HBeAg or anti-HBe (14.3% and 20.4% positive for anti-HBs respectively).     

Reversed passive hemagglutination test fails to detect HBsAg in a number of HBeAg positive sera

In endemic areas of hepatitis B virus (HBV) infection, perinatal transmission from asymptomatic HBsAg carrier mothers to infants plays a major role in the transmission of HBV. HBeAg indicates a high level of viral replication and infectivity. Most of the infants born to HBeAg positive mothers become carriers. Prenatal screening of HBsAg would identify infected mothers and thus allow preventive administration of immunoglobulin and immunization to the newborns. Reversed passive hemagglutination assay (RPHA) is commonly used in Thailand for HBsAg screening. However this method has low sensitivity and gives false negative results. Therefore, infants born to HBsAg false negative mothers would not receive proper immunization. This study reveals the rate of false negative results for HBsAg by RPHA in high infectivity sera. Of 985 sera which were HBsAg positive by ELISA, 70 (7.1%) were negative for HBsAg by RPHA. Of these 70 false negative sera, 7 (10%) were HBeAg positive. Our results indicate that RPHA is a less sensitive method for detection of HBsAg than ELISA. RPHA can give false negative results even in sera with high HBV infectivity. Therefore, RPHA should be replaced by EIA for prenatal HBsAg screening or any other screening for HBV infection whenever possible.     

Intrauterine transmission of hepatitis B virus is closely related to placental leakage

Hepatitis B virus (HBV), transmitted by hepatitis B e antigen (HBeAg)-positive mothers by intrauterine infection, infecting newborns, is closely related to signs and symptoms associated with miscarriage. However, no correlation was observed between intrauterine infection of infants and the presence of antibodies of immunoglobulin M (IgM) class antibodies against hepatitis B core antigen (anti-HBc) in maternal blood, nor was HBeAg found in maternal or cord sera. These results indicate that contamination by the mother's blood, through placental leakage, plays an important role in HBV infection in utero. Without placental leakage, maternal blood could not pass through the placenta and enter fetal circulation, and so intrauterine infection would not occur, even if very high titers of hepatitis B surface antigen (HBsAg) and HBeAg were present in maternal blood.     

HBsAg seropositivity among multi-transfused thalassemic children

Thalassemia major patients are transfusion dependent; they are at high risk of post transfusion viral infections including Hepatitis B virus (HBV). The present study was undertaken to find out the proportion of HBV infection among multiple transfused patients. This cross-sectional study was conducted among thalassemic children of either sex between 2 to 13 years of age, who attended the tertiary care hospital (G. G. Hospital, Jamnagar). Subjects were divided according to number of transfusions and the immunization status. HBsAg was detected by ELISA. Of 90 patients 6 (6.6%) were positive for HBsAg. Of 29 patients who had received above hundred transfusions 3 (10.34%) were sero-positive for HBV. Un-immunized patients were at double risk for acquiring HBV infection. This study suggests that the screening of blood of donors for HBV should be strictly followed and implementation of immunization against HBV is a must, especially in a high-risk group like thallasemic patients.     

Unusual hepatitis B surface antigen variation in a child immunised against hepatitis B

Perinatal transmission of and infection with hepatitis B (HBV) in early childhood are observed in a small proportion of the offspring of hepatitis B surface antigen (HBsAg)-positive mothers who are vaccinated against HBV immediately after giving birth. The children may be infected by wild-type HBV or by variants with amino acid substitutions in the "a" determinant of HBsAg, particularly at position 145 and, rarely, at positions 120, 126, 129, 131, 141, and 144. Four hundred and forty-six newborn infants of HBsAg-positive mothers in the northeastern part of the Czech Republic received combined active and passive immunisation against HBV. Only one child became an HBsAg carrier. This followed a mild, acute HBV illness in the beginning of the second year of his life. HBV DNA encoding the "a" determinant and surrounding region of HBsAg was sequenced after amplification from the plasma of the child and his mother. The child was infected with variants of HBsAg with substitutions at residues 137 and 139. The virus of the mother had changes at residues 120 and 121. HBV from both child and mother had an unusual substitution at residue 118 and seemed to be of the ayw subdeterminant.     

Prophylactic vaccination against hepatitis B: achievements,challenges and perspectives

Large-scale vaccination against hepatitis B virus (HBV) infection started in 1984 with first-generation vaccines made from plasma of chronic carriers containing HBV surface antigen (HBsAg). Thereafter, it was replaced in most countries by second-generation vaccines manufactured in yeast cells transformed with gene S encoding HBsAg. Both generations of vaccines have been applied for universal neonate and early childhood vaccination worldwide and have led to a 70–90 % decrease in chronic HBV carrier rates. However, 10–30 % of newborns from HBsAg/HBeAg-positive mothers cannot be protected by passive/active vaccination alone and become chronic HBV carriers themselves. Asymptomatic occult HBV infections are frequent even in those who have protective levels of anti-HBs. Suboptimal protection may be due to heterologous HBsAg subtypes that are present in 99 % of HBV carriers worldwide. Second-generation vaccines contain partially misfolded HBsAg and lack preS1 antigen that carries the major HBV attachment site and neutralizing epitopes. Third-generation vaccines produced in mammalian cells contain correctly folded HBsAg and neutralizing epitopes of the preS antigens, induce more rapid protection, overcome nonresponse to second-generation vaccines and, most importantly, may provide better protection for newborns of HBV-positive mothers. PreS/S vaccines expressed in mammalian cells are more expensive to manufacture, but introduction of more potent HBV vaccines should be considered in regions with a high rate of vertical transmission pending assessment of health economics and healthcare priorities. With optimal vaccines and vaccination coverage, eradication of HBV would be possible.