肝病患者肝组织中Fas抗原表达及其与病毒和凋亡关系的研究

目的 探讨肝组织中Ｆａｓ抗原的表达及其与病毒感染的和细胞凋亡的关系。方法 应用免疫组织化学方法检测了８２例慢性肝病患者肝组织中Ｆａｓ抗原，ＨＢｓＡｇ和ＨＣＶＮＳ５蛋白表达状况，并采用原位末端标记方法对部分患者肝组织中细胞凋亡状况进行研究。结果 ６４例检出Ｆａｓ抗原（７８．１％），检出率慢活肝和肝硬化组较慢迁肝组高，病毒抗原阳性较阴性组高；抗原主要位于肝细胞浆，少数位于胞膜，阳性细胞主要分布在碎屑坏     

慢性病毒性肝炎肝细胞凋亡与Fas抗原和病毒表达的相互关系

为探讨病毒性肝炎肝细胞凋亡及其与病毒和Ｆａｓ抗原表达的关系，应用原位末端标记技术检测一组慢性乙、丙型肝炎患者肝组织中细胞凋亡状况，并以免疫组化方法检测病毒和Ｆａｓ抗原表达。结果３１例患者中，２５例肝组织中检出末端标记阳性细胞，散布于肝小叶和肝窦内，部分炎症坏死灶中和胆管上皮细胞也可检出阳性细胞；组织炎症活动度较大者和病毒抗原阳性者凋亡指数较高；凋亡细胞可为病毒抗原阳性和阴性；Ｆａｓ抗原阳性与阴性组间凋亡程度无显著差异。提示病毒性肝炎患者感染和未感染病毒的肝细胞均可发生凋亡，其机制有待进一步研究。     

病毒性肝炎患者肝组织中bcl-2蛋白的表达

目的探讨抗凋亡基因ｂｃｌ－２蛋白与Ｆａｓ抗原在病毒性肝炎肝组织中表达和分布的关系。方法采用免疫组织化学双标记技术，以ｂｃｌ－２癌蛋白单克隆抗体、抗－ＨＢｓ单克隆抗体及抗－Ｆａｓ多克隆抗体，检测８７例急、慢性肝炎患者肝组织中ｂｃｌ－２、Ｆａｓ及ＨＢｓＡｇ的表达和分布。结果６２例（７１．３％）肝细胞中检出ｂｃｌ－２癌蛋白，阳性物质主要表达于肝细胞胞浆内，少数位于核膜。在急性轻型肝炎，阳性细胞于小叶内弥散分布；在慢性肝炎则多聚集在碎屑样坏死周围。Ｆａｓ抗原的检出率为７５．９％（６６／８７），其阳性细胞的分布类同ｂｃｌ－２阳性肝细胞，但不如后者广泛。ｂｃｌ－２／Ｆａｓ双标记染色显示较多的肝细胞有两种抗原的双表达。结论乙型病毒性肝炎肝组织中ｂｃｌ－２的表达和分布与Ｆａｓ抗原密切相关，ｂｃｌ－２原癌基因可能具有抑制Ｆａｓ介导肝细胞凋亡的作用。     

结合HBV血清学标志和肝内抗原的形态分布探讨与HBV复制、病变程度及预后的关系

通过慢性乙型肝炎和重型乙型肝炎病人肝内ＨＢｓＡｇ和ＨＢｃＡｇ的形态和分布观察，发现血清病毒学标志和肝内病毒抗原特殊形态分布所反映的病毒复制状态是一致的。病毒抗原在肝细胞内的形态和分布与肝炎病变程度密切相关。重型肝炎肝内ＨＢＶ抗原检出低，其预后差，形态分布以ＨＢｓＡｇ膜型、核周分布和ＨＢｃＡｇ浆型为主。核周分布仅见于亚大块坏死灶旁或灶中。     

C—反应蛋白在慢性乙型肝炎中的表达

目的了解慢性乙型肝炎时，Ｃ－反应蛋白（ＣＲＰ）在肝细胞中的表达。方法用本室建立的ＬＢＡ法检测４０例ＨＢｓＡｇ阳性慢性肝炎的肝组织和１５例ＨＢｓＡｇ阴性的正常肝组织的ＣＲＰ原位表达；用免疫双标记染色检测肝细胞中ＣＲＰ、ＨＢｓＡｇ的表达。结果４０例肝炎标本ＣＲＰ均呈阳性，表达位置和形态与ＨＢｓＡｇ相似；免疫双标记染色显示肝细胞中ＣＲＰ与ＨＢｓＡｇ有重叠。此外，Ｔ淋巴细胞也表达ＣＲＰ。结论ＣＲＰ可与ＨＢｓＡｇ形成复合物。ＣＲＰ可能与慢性乙型肝炎的发病机制有关     

慢性乙型肝炎肝细胞内病毒抗原含量测定与肝组织病理变化的关系

作者对２０例慢性乙肝患者进行肝穿活检，并对肝细胞内乙肝病毒抗原含量分组进行定量测定。１组（慢性活动型，ＨＢｓＡｇ在肝细胞胞膜上表达为粗颗粒膜型，ＨＢｃＡｇ为胞膜型）其病毒抗原含量明显高于２组（小叶型肝炎，ＨＢｓＡｇ为细颗粒膜型，ＨＢｃＡｇ为细膜型）。１组与３组（ＨＢｓＡｇ为胞浆型，ＨＢｃＡｇ为胞核型）抗原含量无差异。提示肝损程度与肝细胞膜上表达的ＨＢｃＡｇ含量多少有关。     

乙型肝炎肝组织中Fas抗原表达的研究

目的；探讨乙型肝炎肝组织Ｆａｓ抗原表达民细胞凋亡和肝细胞损伤关系，方法：用免疫组织化学技术，对１０９例乙型肝炎肝组织Ｆａｓ抗原进行了检测。结果：Ｆａｓ抗原总检出率７６．１６％，急性重症肝炎（ＡＦＨ）慢性迁延型肝炎（ＣＰＨ）慢性活动肝炎（ＣＡＨ），活动性肝硬化（ＡＣ）Ｆａｓ抗原检出率分别为９０．９％，４３．４８％，７８．１２％，８５．７１％，急性重症肝炎Ｆａｓ抗原阳性细胞分布于大片坏死区域中残留肝细     

丙型肝炎抗原和乙型肝炎表面抗原在原发性肝细胞癌肝组织中的定位

用免疫组化技术对６４例原发性肝细胞癌（ＨＣＣ）癌组织进行丙型肝炎抗原（ＨＣＡｇ）和乙型肝炎表面抗原（ＨＢｓＡｇ）定位和分布的研究。结果见ＨＣＡｇ呈粗颗粒深着色，弥漫性分布于全核，部分核仁可显色。肝性细胞多数散在分布于癌组织中，少数集中分布。ＨＢｓＡｇ呈棕褐色细颗粒，分布于细胞浆和细胞膜，阳性细胞多数呈聚集性分布，少数呈散在分布。６４例ＨＣＣ癌组织中的ＨＣＡｇ阳性率为１２．５％（８／６４），ＨＢｓＡｇ阳性率为２６．５％（１７／６４）。１５例癌周组织，呈现ＨＣＡｇ或ＨＢｓＡｇ染色阳性者占６．６％，３３％。揭示ＨＣＶ在ＨＣＣ的发生中，有相当一部分以与ＨＢＶ双重感染形式存在。     

乙型肝炎病毒基因注入大鼠肝脏后的短暂抗原表达

目的建立ＨＢＶ感染的急性乙型肝炎大鼠模型。方法将经磷酸钙沉淀的含３．２ｋｂ全序列ＨＢＶＤＮＡ的ＨＢＶ－ＰＣＮＣＸ质粒直接导入大鼠肝脏。结果１０只实验动物中，８只大鼠血清ＨＢｓＡｇ和ＨＢｅＡｇ阳性，肝细胞中ＨＢＶｍＲＮＡ及ＨＢｓＡｇ得到表达，肝脏出现局灶性炎性细胞浸润，肝细胞坏死、浊肿等典型的病毒性肝炎病理变化。结论将磷酸钙沉淀后的ＨＢＶＤＮＡ导入大鼠肝脏细胞，可产生病毒抗原血症和典型的急性病毒性肝炎病理变化     

肝细胞癌患者肝组织中丙型肝炎病毒抗原的定位研究

用过氧化物酶与抗过氧化物酶（ＰＡＰ）法检测了７３例肝细胞癌（ＨＣＣ）患者肝组织中的丙型肝炎病毒抗原（ＨＣＶＡｇ）和ＨＢｓＡｇ及ＨＢｃＡｇ。结果显示：ＨＣＶＡｇ，ＨＢｓＡｇ和ＨＢｃＡｇ的检出率分别为２８．８％，７５．３％和２６．０％，ＨＣＶＡｇ阳性的肝细胞多呈散在分布，ＨＣＶ染色颗粒既可见于细胞浆也可见于细胞核，ＨＣＶＡｇ的检出与ＨＢＶ的感染态多显著相关，提示，ＨＣＶ可在ＨＣＣ患者的肝细胞中表达，Ｈ     

Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection

The expression of hepatitis B virus antigens was studied by double staining liver tissue with appropriate antisera and correlated with serum hepatitis B viral DNA and histology in 28 patients with disease related to chronic hepatitis B virus infection. The cellular localization of hepatitis B core and hepatitis B e antigens generally coincided, but there were important differences at a subcellular level. Thus, hepatitis B e antigen was detected in nuclei and/or cytoplasm but strong cytoplasmic hepatitis B e antigen was associated with a high serum hepatitis B viral DNA (P = 0.0017) but not with active liver disease. Hepatitis B core antigen could also be detected in nuclei and/or cytoplasm, but strong cytoplasmic hepatitis B core antigen expression, exceeding that of hepatitis B e antigen, was associated with active liver disease (P = 0.041) and not with serum hepatitis B virus DNA. The proportion of hepatocytes expressing hepatitis B surface antigen correlated inversely with the serum titer (P = 0.0017), whereas hepatitis B surface and nucleocapsid antigens were usually expressed independently. The data support the hypothesis that cytoplasmic hepatitis B core antigen and not hepatitis B e antigen is the target for immune system-mediated cytolysis of hepatocytes. Cytoplasmic hepatitis B e antigen is not associated with liver damage but is instead associated with high levels of hepatitis B virus replication.     

Latent hepatitis B virus infection with full-length viral genome in a patient serologically immune to hepatitis B virus infection

The presence, state, physical structure and cellular localization of hepatitis B virus (HBV) DNA were investigated in a patient with hepatitis B surface antigen (HBsAg)-negative chronic liver disease. HBV serology was positive for antibodies to hepatitis B core antigen (anti-HBc), to hepatitis B e antigen (anti-HBe) and to HBsAg (anti-HBs); no HBV DNA was detectable in serum. Southern blot analyses of DNA extracted from the liver demonstrated free monomeric HBV DNA as two distinct species: a predominant species of fully double-stranded relaxed circular molecules and a minor species of linear molecules of 3.2 kilobase pairs (kbp) length. Restriction enzyme analyses identified the HBV genome as HBsAg subtype adw2. Cell fractionation studies further revealed that the free viral DNA species were localized exclusively in liver cell nuclei. These findings in a patient serologically immune to HBV infection demonstrate that in hepatocytes HBV can establish a latent infection, characterized by the extrachromosomal presence of a full-length viral genome without production of infectious virus or synthesis of viral antigens.     

Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance

Summary. Seroconversion from hepatitis B surface antigen (HBsAg) to antibodies against HBsAg (anti‐HBs) usually indicates resolution of hepatitis B virus (HBV) infection. Here, two HBV‐infected patients with seroconversion to anti‐HBs were found to be persistently positive for HBeAg and HBV DNA. Immunohistology of liver biopsies confirmed the expression of HBV proteins in the liver of one patient. The neutralizing ability of anti‐HBs in patient sera was demonstrated by blocking HBV infection of primary tupaia hepatocytes. Analysis of the HBsAg‐encoding region of HBV isolates from patients indicated the coexistence of heterogeneous HBV genomes in patients. The majority of recombinant variant HBsAg was reactive in HBsAg assays and was able to bind to anti‐HBs. Circulating immune complexes (CIC) of HBsAg in patient sera could be detected by polyethylene glycol precipitation and trypsin digestion. Thus, neutralizing anti‐HBs may appear in chronic HBV carriers for long periods but does not necessarily lead to complete viral clearance.     

Liver histology and immunohistochemical findings in asymptomatic Indians with incidental detection of hepatitis B virus infection.

BACKGROUND AND OBJECTIVES: The relationship between hepatocyte expression of hepatitis B virus (HBV) antigens, liver histology and viral replication in asymptomatic subjects with incidental detection of hepatitis B surface antigen (HBsAg) remains unclear. We evaluated the histological activity index (HAI) and hepatocyte expression of viral antigens with replicative status in asymptomatic chronic HBV infection. METHODS: Asymptomatic subjects with incidental detection of HBsAg and ALT levels less than twice the upper limit of normal were grouped as follows: Group A - negative for HBeAg and HBV DNA (no HBV replication); B - HBeAg negative, HBV DNA positive (low HBV replication or pre-core mutant); C - positive HBeAg and HBV DNA (high viral replication). Liver biopsies were assessed for HAI (Ishak's scoring system). These were also subjected to immunohistochemistry for expression of HBsAg and hepatitis B core antigen (HBcAg); distribution, staining pattern and quantitative measurement of antigen expression were assessed. RESULTS: Median HAI was similar in the three groups (1.0, 2.0 and 2.0 in groups A, B and C, respectively). All subjects in Group C showed discrete cytoplasmic expression of HBsAg, whereas the other two groups showed heterogeneity in distribution and pattern of HBsAg staining. Quantitative measurement of cytoplasmic HBsAg revealed similar results in the three groups. Core antigen (nuclear) was detected in 4 of 5 subjects in Group C and none of those in Groups A and B. Ground-glass hepatocytes were seen in 20 and orcein-positive cells in 26 cases. HBsAg was detected by immunohistochemistry in 37 biopsies. CONCLUSIONS: Among asymptomatic subjects with chronic HBV infection, those with high rate of viral replication had discrete cytoplasmic HBsAg expression and nuclear expression of core antigen; these findings were uncommon in subjects with low or no viral replication.     

High-level expression of hepatitis B viral antigens in fibrosing cholestatic hepatitis.

The expression of hepatitis B viral antigens was quantified in liver tissue from four transplant recipients with fibrosing cholestatic hepatitis (FCH) and compared with five other transplant recipients who did not develop this syndrome and 30 patients with chronic hepatitis B virus (HBV) infection. As measured by radioimmunoassays, the liver tissue from patients with FCH had significantly greater amounts of both hepatitis B surface antigen (HBsAg) and nucleocapsid antigens than to transplant patients without this syndrome (P less than 0.01) or patients with chronic HBV infection (P less than 0.001). Intrahepatic expression of pre-S1/pre-S2 in FCH was also extensive with a distribution parallel to that of HBsAg. High-level expression of intrahepatic HBsAg and hepatitis B core antigen in the explanted liver was associated with subsequent development of FCH in the liver graft, suggesting that viral/host factors may also be important. This pattern of intrahepatic hepatitis viral antigen expression, by analogy with Chisari's transgenic mice model and Roingeard's HBV-transfected HepG2 cell model, may be the cause of direct hepatocytopathic injury in this condition.     

Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis

The intrahepatic distribution of hepatitis B core antigen (HBcAg) and surface antigen (HBsAg) was studied in 32 patients with chronic type B hepatitis, and the results were correlated with the status of hepatitis B e antigen/antibody (HBeAg/anti-HBe) and with the histologic activity of the patients. In HBeAg-positive patients with minor hepatitis activity, HBcAg was distributed mainly in the nuclei, whereas HBsAg was diffusely located on the plasma membrane as well as focally in the cytoplasm. In HBeAg-positive patients with chronic active liver disease, although the distribution pattern of HBsAg in liver remained unchanged, the expression of nuclear HBcAg decreased significantly with concomitant increase in cytoplasmic/membranous HBcAg expression. In HBsAg carriers who were anti-HBe positive, HBcAg was undetectable anywhere, whereas HBsAg could be seen only in the cytoplasm. These results suggest that membranous expression of HBsAg relates closely to active viral replication but is probably not responsible for the observed histologic activities. In contrast, cytoplasmic/membranous expression of HBcAg correlates with high degree of liver inflammatory activity. It is therefore suggested that hepatocytes with cytoplasmic/membranous HBcAg expression might be the target cells for immune hepatocytolysis.     

HBcAg and HBsAg expression in ductular cells in chronic hepatitis B

The possible involvement of bile duct epithelium (BDE) in chronic hepatitis B was examined by immunohistochemical investigation of HBcAg and HBsAg expression in biliary cells in 47 liver biopsies with both viral antigens detectable in hepatocytes. HBsAg- and HBsAg-positive cells were identified in nine and five cases, respectively, in atypical and occasionally in typical ductules in cases of acute excacerbation, chronic active hepatitis and active cirrhosis. Atypical ductules were usually located in areas of periportal fibrosis and in cirrhotic septa. Liver cell plates expressing viral markers and undergoing ductular transformation (positive reaction of hepatocytes to BDE-specific, wide-spectrum keratin) were also observed in acinar zone 1, at the periphery and within parenchymal nodules in a number of cases. The presence of both viral antigens in atypical ductules in cases of advanced chronic liver disease most probably expresses the persistence of the virus in cells deriving from biliary metaplasia of infected hepatocytes. However, the detection of the virus in a few typical ductules is indicative of a direct viral infection. According to these findings, ductular cells seem to serve as a suitable host for HBV, their genotype permitting viral replication and antigen production.     

肿瘤坏死因子受体和Fas在乙型肝炎肝细胞凋亡中的意义

目的研究肿瘤坏死因子1型受体(TNFR1)和Fas在乙型肝炎肝细胞凋亡中的意义.方法用免疫组织化学方法和原位末端标记技术(TUNEL试验)检测70例各型乙型肝炎患者肝组织TNFR1、Fas的表达和肝细胞凋亡情况.结果不同类型乙型肝炎肝细胞均有TNFR1和Fas表达,其表达程度与组织学类型相关(P＜0.05).TNFR1在细胞浆和细胞膜的分布相似,而Fas主要在细胞浆分布,TNFR1与Fas在肝细胞上的表达无明显关系(P＞0.05).不同类型肝炎肝细胞凋亡与病变程度密切相关,Fas的表达与肝细胞凋亡也有密切关系(P＜0.005),但TNFR1的表达强弱与肝细胞凋亡程度不呈正比(P＞0.05).在46例中度以上肝细胞凋亡患者中,同时伴有单纯TNFR1中度以上阳性者有4例(8.7%),同时伴有单纯Fas抗原中度以上阳性者有12例(26.1%),而同时伴有TNFR1和Fas抗原均为中度以上阳性者有28例(60.9%),另有2例患者TNFR1和Fas在肝细胞均无明显表达(4.35%).结论在乙型肝炎肝细胞凋亡中,Fas似较TNFR1的作用更重要;TNFR1和Fas在肝细胞的同时表达可能对肝细胞凋亡有相加作用.