HBV感染与IgA肾病肾小管间质病变的关系

目的探讨ＩｇＡ肾病ＨＢＶ感染与肾小管间质病变的关系。方法利用原位分子杂交（ＨＢＶＤＮＡ）、免疫组化（ＨＢＡｇ、ＣＤ３、ＣＤ８）以及ＨＢＶＤＮＡＨＢＡｇ和ＨＢＡｇＣＤ４３双标记技术，对９１例ＩｇＡ肾病肾穿刺标本进行研究。结果肾组织内ＨＢＡｇ阳性率为６９．２％。ＨＢＶＤＮＡ原位杂交阳性率为４２９％。ＨＢＶＤＮＡ阳性的病例，双重标记染色发现ＨＢＶＤＮＡ阳性的肾小管上皮细胞可表达ＨＢｃＡｇ或／和ＨＢｓＡｇ。ＨＢＶ感染标记（ＨＢＶＤＮＡ、ＨＢｃＡｇ、ＨＢｓＡｇ）阳性组ＣＤ３阳性细胞和ＣＤ８阳性细胞数明显高于阴性组（Ｐ＜００１），并可见数量不等的Ｔ淋巴细胞入侵ＨＢｃＡｇ及ＨＢｓＡｇ阳性肾小管管壁或围绕其周围。结论感染ＨＢＶ的肾组织细胞能够表达ＨＢＡｇ，并诱导ＣＤ３阳性细胞和ＣＤ８阳性细胞浸润，从而加重肾小管、间质损害。ＨＢＶ感染对ＩｇＡ肾病的发生发展可能起着重要作用     

巨细胞病毒宫内感染的诊断及对胎儿的影响

采用地高辛探针斑点杂交技术和聚合酶链反应（ＰＣＲ）技术，对５１例人巨细胞病毒（ＨＣＭＶ）血清学抗体ＩｇＭ阳性孕妇的羊水、脐血和产后两周内的新生儿尿液，进行ＨＣＭＶＤＮＡ检测。同时与酶联免疫吸附法（ＥＬＩＳＡ）检测脐血、羊水中ＨＣＭＶ－ＩｇＭ的结果相比较。结果，地高辛探针斑点杂交检测ＨＣＭＶ－ＤＮＡ的阳性率分别为：羊水２１．５７％，脐血３３．３３％，新生儿尿液２７．４５％。ＰＣＲ检测ＨＣＭＶ－ＤＮＡ的阳性率分别为：羊水２９．４１％，脐血４３．１４％，新生儿尿液３Ｓ，２９％。羊水ＨＣＭＶ－ＩｇＭ阳性率为１１．７６％，脐血ＨＣＭＶ－ＩｇＭ阳性率为２３．５３％。脐血ＨＣＭＶ－ＤＮＡ阳性的新生儿平均出生体重明显低于脐血ＨＣＭＶ－ＤＮＡ阴性的新生儿，而脐血ＨＤＷ－ＤＮＡ阳性的新生儿肝功能异常、血小板减少以及低Ａｐｇａｒ评分的发生率明显高于脐血ＨＣＭＶ－ＤＮＡ阴性的新生儿。结果表明：采用ＥＲ技术检测羊水、脐血或产后两周内新生儿尿液ＨＣＭＶＤＮＡ有助于ＨＣＭＶ官内感染的早期诊断，便于临床早期干预，ＨＣＭＶ官内感染严重影响胎儿的生长发育，可造成胎儿肝功能异常、血小板减少和新生儿窒息的发生。     

肝硬变内HBV DNA及其五种抗原的表达及意义

取２２５例人肝硬变活检组织石蜡切片，检测了ＨＢＶＤＮＡ及其５种抗原。分别用免疫组化ＡＢＣ法检测ＨＢｘＡｇ、ｐｒｅ－Ｓ＿１和ｐｒｅ－Ｓ＿２抗原；用ＰＡＰ法检测ＨＢｓＡｇ和ＨＢｃＡｇ；用原位杂交方法检测ＨＢＶＤＮＡ；用免疫组化、原位杂交双标记方法检测ＨＢＶＤＮＡ和ＨＢｓＡｇ、ＨＢｘＡｇ或ＨＢｃＡｇ。结果显示，阳性检出率ＨＢｓＡｇ为７０．０％（１２８／１８３例），ｐｒｅ－Ｓ＿１抗原为６４．４％（８５／１３２例）、ｐｒｅ－Ｓ＿２抗原为６１．４％（８１／１３２例），ＨＢｘＡｇ为７５．３％（１１３／１５０例），ＨＢｃＡｇ为２２．４％（３９／１７４例），ＨＢＶＤＮＡ为６２．４％（５８／９３例）。双标阳性检出率ＨＢＶＤＮＡ和ＨＢｓＡｇ为３７．３％（１９／５１例），ＨＢＶＤＮＡ和ＨＢｘ－Ａｇ为８６．３％（４４／５１例），ＨＢＶＤＮＡ和ＨＢｃＡｇ为３９．２％（２０／５１例）。ＨＢＶＤＮＡ和ＨＢＶ５种抗原阳性病例中８０％以上均伴有肝细胞不典型增生。这一结果表明，在我国肝硬变的发生发展与ＨＢＶ慢性感染有密切的关系。     

肝癌及癌旁组织HBVDNA原位杂交HBsAg免疫组织化学双标记研究

用原位分子杂交和免疫组化双标记技术，检测４０例肝癌（ＨＣＣ）及癌旁组织中ＨＢＶＤＮＡ和ＨＢｓＡｇ，癌及癌旁中ＨＢＶＤＮＡ阳性率为６５％，ＨＢｓＡｇ阳性率为８２．５％，表明ＨＢＶ感染与ＨＣＣ发生密切相关。二者在ＨＣＣ中表达较癌旁组织中少而弱，可能系ＨＣＣ中ＨＢＶＤＮＡ整合后复制减弱之故。发现的碎点状ＨＢｓＡｇ小包涵体可能为ＨＣＣ中ＨＢｓＡｇ的特有表现形式。小细胞ＬＣＤ中ＨＢ－ｓＡｇ表达显著高于癌旁其他类型病变，支持其更接近癌前病变的观点。     

用ELISA微板法检测乙型肝炎病毒核心抗原

以双抗体包被的抗体夹心法，用微板ＥＬＩＳＡ检测血清乙型肝炎病毒核心抗原（ＨＢＣＡｇ），确定双包被工作浓度ＭＣ－抗－ＨＢｃ（效价１０００）为０．０４μｌ／孔，ＭＣ－抗－ＨＢｓ（１ｍｇ／ｍｌ）为３～４μｌ／孔；最佳裂解剂及其工作浓度为７％ＮＰ－４０巯基乙醇溶液。分别用不同的酶标记抗体检测，均证明双包被具有特异性。加入抗－ＨＢｃ进行阻断试验，其阻断率为７９．３％。对８４４例ＨＢｓＡｇ阴性的血清及１１４例ＨＢＶ－ＤＮＡ探针阴性血清用本法进行ＨＢｃＡｇ检测，均为阴性。在临床应用上，本法的阳性率明显高于试管法的，与ＨＢＶ－ＤＮＡ探针的阳性符合率为９１．４％，并且特异性与ＨＢＶ－ＤＮＡ探针的一致。     

用聚合酶链反应检测人肝细胞癌乙型肝炎病毒DNA和丙型肝炎病毒RNA

为研究乙型肝炎病毒ＤＮＡ（ＨＢＶＤＮＡ）和丙型肝炎病毒ＲＮＡ（ＨＣＶＤＮＡ）与肝细胞癌的关系，用聚合酶链反应（ＰＣＲ）和巢式ＰＣＲ（ｎｅｓｔｅｄ－ＰＣＲ）分别检测４２例肝肿瘤组织中ＨＢＶＤＮＡ和ＨＣＶＲＮＡ。结果：１例胆管细胞癌组织ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性，１例胆管囊腺瘤ＨＢＶＤＮＡ阳性。４０例肝细胞癌组织中，单纯ＨＢＶＤＮＡ阳性１９例，单纯ＨＣＶＲＮＡ阳性３例，二者均阳性１０例。ＨＢＶＤＮＡ阳性率７２．５％，ＨＣＶＲＮＡ阳性率３２．５％。ＨＢＶＤＮＡ和ＨＣＶＲＮＡ感染与肝癌组织学分型无关；且肝细胞癌中ＨＣＶ感染与ＨＢＶ未见相关。结果提示，我国ＨＢＶ感染仍是引起肝细胞癌的主要原因。但由于肝细胞癌患者中ＨＣＶ的感染率也较高，且有上升趋势，因此ＨＣＶ可能也是肝细胞癌发生的重要原因之一。     

应用聚合酶链反应技术检测肝组织中的HBV－DNA

采用聚合酶链反应（ＰＣＲ）技术，对４２例肝活切组织石蜡切片中乙型肝炎病毒（ＨＢＶ）ＤＮＡ进行检测，并与乙肝表面抗原（ＨＢｓＡｇ）的免疫组织化学及血清学检测进行比较，ＨＢＶ－ＰＣＲ阳性率为７３．８％，高于组织及血清ＨＢｓＡｇ阳性率（分别为５９．５％和５０．０％）。３例病理形态呈肝炎改变，而血清ＨＢｓＡｇ（─）的肝组织中有２例检出ＨＢＶ－ＤＮＡ，提示ＰＣＲ的高度敏感性和准确性。８３．３％的门脉性肝硬变和８７．５％的肝细胞癌组织中ＨＢＶ－ＰＣＲ呈阳性，进一步证实了上述两病与ＨＢＶ的关系密切。我们还发现肝细胞淤胆患者ＨＢＶ感染率较高，ＨＢＶ－ＤＮＡ及组织ＨＢｓＡｇ阳性比例各为６／９和４／８。     

乙/丙型肝炎病毒双重感染患者前C区终止变异低频率

目的了解乙型肝炎病毒（ＨＢＶ）与丙型肝炎病毒（ＨＣＶ）双重感染患者前Ｃ区基因变异，及其可能的临床意义。方法用聚合酶链反应（ＰＣＲ）与限制片段长度多态性（ＲＦＬＰ）来分析２５例ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性（Ａ组）和３１例ＨＢｓＡｇ和ＨＢＶＤＮＡ阳性但抗－ＨＣＶ和ＨＣＶＲＮＡ均阴性（Ｂ组）的慢性肝病患者前Ｃ区密码２８终止变异（终２８）。结果ＨＢＶ和ＨＣＶ双重感染患者（Ａ组）血清ＨＢＶＤＮＡ第１次ＰＣＲ阳性率（１６％）明显低于单独ＨＢＶ感染组（６５％）（Ｐ＜０．００１）；前Ｃ终２８检出率（２８％）亦明显低于单独ＨＢＶ感染（６８％）（Ｐ＜０．００１）。结论提示双重感染患者ＨＢＶ前Ｃ终止变异低频率可能与ＨＢＶ低水平复制有关     

孕产妇和新生儿血清乙肝免疫标志物与HBV—DNA检测结果的比较…

利用ＨＢＶ－ＤＮＡ出现先于血清其它血清学指标理论依据。采用聚合酶链式反应（ＰＣＲ）对乙肝免疫标志物至少一项改变的２００例孕产妇、新生儿血清进行ＰＣＲ扩增，结果有１０８份标本ＨＢＶ ＤＮＡ阳性、总阳性率为５４％。其中ＨＢｅＡｇ阳性率８３．３％（７０／８４），ＨＢｓＡｇ阳性率４８％（２４／５０），ＨＢｅＡｂ阳性率１０．５％（２／１９），ＨＢｓＡｂ９．８％（２／２２），可见ＰＣＲ早期诊断，判断其传染性，     

我国九个地区丁型肝炎病毒感染状况及其与乙型肝炎病毒复制指标关系的研究

对１５４５例各类乙型肝炎病毒表面抗原（ＨＢｓＡｇ）阳性肝病和无症状ＨＢｓＡｇ携带者的血清进行了乙型肝炎病毒（ＨＢＶ）与丁型肝炎病毒（ＨＤＶ）感染标记物的测定。结果表明，ＨＤＶ感染率为１３．０１％，其中ＨＤＡｇ和抗－ＨＤ阳性率分别为２．９１％和１０．０９％。而且在全国九个地区均有ＨＤＶ感染者存在，说明其分布是较为广泛的。同时还表现出，男性高于女性，慢性肝炎、重型肝炎及原发性肝癌高于急性肝炎和无症状ＨＢｓＡｇ携带者。提示ＨＢＶ与ＨＤＶ合并感染或重叠感染可能导致病情加重和感染的慢性化。本项研究结果还揭示，在ＨＢＶ与ＨＤＶ合并或重叠感染时，可能对ＨＢＶ的复制指标（ＨＢｅＡｇ·ＨＢＶＤＮＡ）有一定的抑制现象。     

血清HBsAg阳性IgA肾病肾组织HBV抗原蛋白及血清和肾组织HBV-DNA检测分析

目的探讨乙型肝炎病毒（HBV）感染与IgA肾病发病的关系。方法32例肾活检冰冻切片组织HBsAg和HBcAg蛋白和42例HBsAg阳性的肾活检石蜡切片组织及其部分血清HBV-DNA的检测。结果HBsAg和HBcAg在IgA肾病肾活检组织的总阳性率为59．1％，在非IgA肾病中的总阳性率为63．6％，二者差异无统计学意义。42例肾活检组织中，仅发现有5例（11．9％）在肾活检组织中有HBV-DNA的存在。且5例均为大三阳患者，其病理诊断为系膜增生性肾小球肾炎2例，轻微肾小球病变1例，基底膜病变1例，IgA肾病仅1例。血清HBsAg阳性的患者，同时进行了42例肾活检组织的血清HBV-DNA检测，其中大三阳患者为12例，其血清HBV-DNA均为阳性，而这12例血清阳性的肾活检组织中仅有5例HBV-DNA为阳性，其余30例血清及肾活检组织中HBV-DNA为阴性。结论HBsAg和HBcAg蛋白在IgA肾病肾活检组织和非IgA肾病肾活检组织表达差异无统计学意义，表明HBV感染与IgA肾病并无直接关系。     

IgA肾病肾组织内乙型肝炎病毒感染的发病机制研究

目的：探讨乙型肝炎病毒感染致IgA肾病肾损伤的发病机制。方法： 随机选取48例IgA肾病肾穿刺组织，参照Meadow病变分级标准分为Ⅰ-Ⅴ级5个实验组，应用Envision免疫组织化学方法检测各级肾组织内HBsAg和HBcAg；同时用直接IS-PCR技术检测其中18例IgA肾病肾组织内HBV DNA。结果： 48例IgA肾病肾组织内HBcAg和HBsAg总的阳性检出率分别为75.00%(36/48)和43.75%(21/48)；18例IgA肾病肾组织内HBV DNA阳性检出率为61.11%(11/18)；3者均表现为肾小管阳性检出率高于肾小球(P＜0.05)，但各级之间，HBcAg、HBsAg和HBV DNA检出率均无显著差异(P＞0.05)。结论： HBV参与了IgA肾病的发生，其导致肾组织损伤的机制可能主要是由细胞免疫或一系列细胞因子介导，并非病毒直接所致；肾小管上皮细胞可能是HBV感染的靶对象。     

乙型肝炎病毒感染在肾小球肾炎发病中的作用

为探讨ＨＢＶ感染于肾小球肾炎发病机制中的作用，收集５０例血清ＨＢＶ感染标志阳性或／和肾组织免疫组化证实ＨＢＡｇ阳性肾炎患者的肾穿刺组织，应用Ｓｏｕｔｈｅｒｎ印迹杂交和原位分子杂交观察ＨＢＶＤＮＡ的存在状态和定位。Ｓｏｕｔｈｅｒｎ印迹杂交阳性率为７３．９％，其中８２％属整合型；原位杂交显示肾小管和肾小球ＨＢＶＤＮＡ的阳性率分别为７２％和５６．５％。其结果提示肾组织本身感染ＨＢＶ，因而考虑沉积于肾小球上的ＨＢＡｇ除源于血循环（肝原）外，尚有原位合成的可能；认为ＨＢＶ相关性肾炎的发病除ＨＢＶ抗原、抗体在肾小球沉积导致的体液免疫损伤机制外，应考虑因肾组织感染ＨＢＶ而导致的细胞免疫机制参与作用。     

In situ hybridization study of HBV DNA in chronic active hepatitis

Liver biopsies from 50 patients with chronic active hepatitis B were analysed immunohistochemically for HBcAg and HBsAg, and with in situ hybridization for HBV DNA. Double labelling technique for detecting HBV DNA and viral antigens simultaneously was also performed in some of these cases. The results showed that localization of HBV DNA in hepatocytes could be classified into whole cytoplasmic, submembranous, nucleic and intermembranous types. The last type suggests that HBV DNA might be transmitted directly to the adjacent hepatocytes through the cell membrane. By double labelling technique, it was disclosed that most hepatocytes with high level of HBV replication did not contain HBcAg or HBsAg. Conversely and most liver cells strongly positive for HBAg have low or negligible level of viral replication. Additionally, in a few cases, HBV DNA was found in the cytoplasm of bile ductule epithelia and sinusoidal endothelia.     

Hepatitis B virus infection and replication in primary cultured human granulosa cells

To investigate the infection and replication of hepatitis B virus (HBV) in primary cultured human granulosa cells. Human granulosa cells were cultured with HBV-positive serum. Media were collected and assayed for HBsAg and HBeAg by ELISA, and HBV DNA by quantitative PCR. HBsAg and HBcAg were detected by immunocytochemistry in cultured cells. HBV DNA and RNA were extracted and amplified by nested PCR. Intracellular HBV DNA was localized by in situ hybridization. By co-cultivation of human GCs with HBV-positive serum, a system was established to study HBV infection and replication in GCs. HBsAg in medium could be detected from 4 to 96 h, and HBV DNA could be detected from 12 to 96 h after exposure. HBsAg and HBcAg showed positive signals by immunocytochemistry. A 206-bp fragment was amplified by nested PCR to detect HBV DNA and RNA in granulosa cells. HBV DNA was detected in GC nuclei by in situ hybridization. HBV can infect and replicate in human primary granulosa cells. This culture system could enable us to study infection of ova by HBV.     

HBV体外感染卵巢颗粒细胞

目的建立HBV体外感染颗粒细胞模型,研究HBV在颗粒细胞中的复制情况,为深入研究HBV经卵细胞母婴垂直传播提供研究平台。方法原代颗粒细胞体外培养后用HBV阳性血清感染。收集培养上清,在不同时点检测HBsAg、HBeAg定量,实时定量PCR检测HBVDNA。免疫组化检测培养细胞中的HBsAg和HBcAg。巢式PCR检测细胞中的HBVDNA及HBV-mRNA。原位杂交检测细胞内的HBVDNA。结果成功建立了HBV体外感染颗粒细胞模型,在培养上清中可以持续96h检测到HBsAg和HBV DNA,在细胞内检测到HBsAg和HBcAg的阳性信号,PCR扩增显示细胞内有HBVD-NA及HBV-mRNA的存在,原位杂交证实细胞内HBVDNA阳性。结论 HBV能够在体外感染颗粒细胞,并在其内复制,该结果为深入研究HBV经卵细胞传播机制提供了很好的研究平台。     

Hepatitis B virus (HBV) antigen-pulsed monocyte-derived dendritic cells from HBV-associated hepatocellular carcinoma patients significantly enhance specific T cell responses in vitro

To investigate whether hepatitis B virus (HBV) antigen-pulsed monocyte-derived dendritic cells (MoDC) could mount a T cell response in hepatocellular carcinoma (HCC) patients associated with chronic HBV infection, peripheral blood mononuclear cells (PBMCs) from 36 HBV-associated HCC patients were induced into MoDC and pulsed with hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg), alone and in combination. Co-stimulatory molecules CD80, CD86 and CD40, as well as human leucocyte antigens D-related (HLA-DR) were found to express at the highest level on MoDC pulsed with HBcAg or HBsAg + HBcAg, at a median level on MoDC pulsed with HBcAg or HBsAg alone, and at the lowest level on non-antigen-pulsed MoDC. Interleukin (IL)-10 and IL-12 cytokines were released by antigen-pulsed MoDC at increased levels in the order: no-antigen < HBsAg < HBcAg < HBcAg + HBsAg. MoDC pulsed with HBcAg or HBsAg + HBcAg also had the strongest ability to stimulate autologous T cell proliferation and intracellular interferon (IFN)-gamma production. HBcAg- or HBsAg + HBcAg-pulsed MoDC could also induce HBV core peptide-specific CD8(+) T cell proliferation determined by tetramer staining. In addition, the antigen-pulsed MoDC were found to have a stronger capacity to produce IL-12 and induce T cell response in vitro for patients with higher alanine transaminase (ALT) levels than those with lower ALT levels, indicating that antigen pulse could substantially reverse the impaired function of MoDC in primary HCC patients with active chronic hepatitis B. In conclusion, HBV antigen-pulsed MoDC from HCC patients with chronic hepatitis B could induce HBV-specific T cell response in vitro.     

Detection of HBV DNA in hepatocellular carcinoma by in situ hybridization using a biotin-labelled probe

Eight cases of hepatocellular carcinoma were hybridized in situ with a biotin-labelled HBV DNA probe on formalin fixed paraffin embedded sections. HBV DNA was detected in 218 cases both in cancer and pericancerous tissue of the liver, and both carcinomas were well differentiated. In three cases, HBV DNA was only present in pericancerous tissue and no HBV DNA could be identified in the remaining three cases. The positive rate of HBV DNA was 25% in tumor and 62.5% in the pericancerous area of liver. HBcAg was negative in all the eight tumors, nevertheless, HBsAg was present in one case. Both HBsAg and HBcAg were positive in the peri-cancerous tissue of liver in 6 out of 8 cases. In the remaining two, one was only HBsAg positive while the other was HBcAg positive. HBV DNA was identified mainly in the cytoplasm of tumor cells. In certain cells it was seen in the perinuclear cytoplasm or beneath the nuclear envelope. Only in a few cells, HBV DNA was distributed in the nuclei diffusely. Since HBV DNA was present both in the cytoplasm and nuclei, suggesting that HBV DNA was present in either integrated or free forms.     

HBV DNA及HBV抗原在血清HBV标志物阴性肝炎肝组织中表达的研究

目的 研究HBV DNA及HBV抗原在血清HBV标志阴性的肝炎肝组织中的表达。方法 对45例HBV血清标志阴性阴性肝炎患者，进行肝组织HBV DNA的原位杂交及免疫组织化学染色检测。结果 原位杂交表明，HBV DNA阳性者7例，（阳性率15．56％），阳性信号主要存在于肝细胞的胞核中，少数位于胞浆内；免疫组化染色表明，HBsAg及HBcAg均呈阴性。结论 血清HBV标志阴性的肝炎肝组织中可检出HBV DNA，有利于提高对HBV感染的诊断。