特异性乙型肝炎病毒X基因反义核酸体外抗病毒作用

为了观察互补于乙型肝炎病毒（ＨＢＶ）Ｘ基因的三段硫代反义核酸（ＡＳＯＮ）体外抗病毒作用，采用ＥＬＩＳＡ和ＰＡＰ－ＥＬＩＳＡ法检测ＡＳＯＮ作用前后２，２，１５细胞上清中乙型肝炎病毒表面抗原、ｅ抗原及Ｘ抗原（ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢｘＡｇ）含量变化及细胞原位杂交检测细胞内ＨＢＶＤＮＡ含量变化。结果表明，三段ＡＳＯＮ均可抑制ＨＢｓＡｇ、ＨＢｅＡｇ和ＨＢｘＡｇ的表达，其抑制率分别为８０．６５％、６２．７６％和７８．０７％；细胞内ＨＢＶＤＮＡ也明显减少。据此认为，ＨＢｘＡｇ表达量下降可能系ＡＳＯＮ序列特异性封闭作用所致，而ＨＢ－ｓＡｇ和ＨＢｅＡｇ表达量以及ＨＢＶＤＮＡ含量降低，可能是通过ＨＢｘＡｇ对ＨＢＶＤＮＡ启动子的反式激活功能降低而实现的。     

乙型肝炎病毒前C／C基因区硫代和脂肪链—硫代反义寡核苷酸的体外抗…

为了研究修饰的反义寡核苷酸（ＡＳＯＮ）的抗乙型肝炎病毒（ＨＢＶ）作用。以２．２．１５细胞为靶细胞，在ＨＢＶ前Ｃ／Ｃ基因区设计合成了１６聚硫代和脂肪链－硫代两种修饰的ＡＳＯＮ，用酶联免疫吸附和斑点杂交技术分别检测作用细胞的乙型肝炎病毒表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ）以及ＨＢＶ ＤＮＡ的分泌情况。结果显示，ＡＳＯＮ在浓度为１０μｍｏｌ／Ｌ时能特异性抑制细胞９２％￣９５％ＨＢｓＡｇ和８４％￣     

成人与小儿肾脏组织中HBV－DNA存在状态的比较

本文分析８例成人与７例小儿肾脏疾病的肾组织中检出的ＨＢＶ－ＤＮＡ。临床诊断：７例小儿均为乙肝病毒相关性肾炎（ＨＢＶ－ＧＮ）；８例成人中３例为ＨＢＶ－ＧＮ，其余为血尿待查、狼疮性肾炎和ＩｇＡ肾病。ＨＢＶ－ＤＮＡ在肾组织的存在状态在成人均为整合型，而小儿有整合型及游离型二种。小儿肾组织中ＨＢｓＡｇ阳性表达率高于成人。作者认为此现象可能与成人肾组织中ＨＢＶ－ＤＮＡ存在状态仅有一种整合型有关。另外发现肾组织中ＨＤｃＡｇ阳性率与ＨＢＶ－ＤＮＡ呈正相关。肾组织中ＨＢｃＡｇ阳性的患者局部有Ｔ细胞的浸润，提示了肾原性抗体存在及其在ＨＢＶ－ＧＮ中激发细胞免疫参与肾脏病变可能性。     

HBV特异性T细胞治疗慢性乙型肝炎的研究

为寻求一种治疗慢性乙型肝炎的新方法,应用ＨＢＶ特异性Ｔ细胞输注治疗慢性乙型肝炎９例,结果显示,在疗程结束后,患者的ＨＢｓＡｇ,ＨＢｅＡｇＨＢＶＤＮＡ的含量均有所下降,其中ＨＢｅＡｇ下降较明显,Ｐ〈０．０１,２例ＨＢｃＡｇ阳性患者,ＨＢｃＡｇ阴转,ＣＤ＾＋３,ＣＤ＾＋４,ＣＤ＾＋４／ＣＤ＾＋８ＮＫ活性上升,ＣＤ＾＋８,ｓＩＬ－２Ｒ较治疗前后所回复,表明特异性Ｔ细胞治疗慢性乙型肝炎有一定疗效,远期疗效     

硫代磷酸反义寡核苷酸对乙型肝炎病毒抗原表达的体外抑制作用

目的 研究硫代磷酸反义寡核苷酸（Ｓ－ＡＳＯＮ）的抗乙型肝炎病毒（ＨＢＶ）作用。方法 作者以２．２．１５细胞作为研究对象，在ＨＢＶ基因Ｓ区和Ｃ区的翻译起始位点设计合成了２段１６聚Ｓ－ＡＳＯＮｓ，采用酶联免疫吸附试验检测了培养细胞上清中表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ）的分泌情况。结果 当Ｓ－ＡＳＯＮ浓度为每天２μｍｏｌ／Ｌ时，对ＨＢｓＡｇ和ＨＢｅＡｇ的抑制率可分别达到８８％和７５％，而无关     

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     

DNA疫苗诱导小鼠细胞免疫及抗肿瘤免疫研究

目的 观察ＨＢＶＤＮＡ疫苗诱导ＢＡＬＢ／Ｃ小鼠（Ｈ－２＾ｄ）的特异性细胞免疫应答及其对稳定表达ＨＢｓＡｇ的小鼠肥大细胞瘤Ｐ８１５细胞（这５－ＨＢＶ－Ｓ）（Ｈ－２＾ｄ）成瘤性的影响。方法 肌肉注射ＤＮＡ疫苗,背部皮下接种Ｐ８１５－ＨＢＶ－Ｓ细胞,观察成瘤情况,４ｈ＾５１Ｃｒ释放法不细胞细胞毒Ｔ淋细胞（ＣＴＬ）活性。结果 ＤＮＡ疫苗可以降低成瘤率,抑制成长小鼠存活期和提高小鼠存活率。ＣＴＬ细胞杀伤活性     

反义乙肝病毒S基因真核载体构建及体外抗乙肝病毒作用

目的：探讨反义ＲＮＡ抗乙肝病毒（ＨＢＶ）作用。方法：构建了正，反义ＨＢＶＳ基因重组ＥＢ病毒载体ｐＭＥＰ４ｓ，ｐＭＥＰ４Ｓａｓ，ＤＮＡ－磷酸钙共沉淀法将重组载体ＤＮＡ转染２．２．１５细胞，潮霉素筛选１个月得到抗性细胞克隆，ＥＬＩＳＡ，斑点杂交法分别检测抗性细胞上清ＨＢｓＡｇ，ＨＢｅＡｇＨＢＶＤＮＡ水平，结果：转染后１，２月，反义载体ＨＢｓＡｇ，ＨＢｅＡｇ的抑制率分别达７５％，５１．６％，７０％，４６     

聚合酶链反应检测HBV－DNA的临床意义

本文采用聚合酶链反应（ＰＣＲ）和ＥＬＩＳＡ法对２７３７例乙肝患者血清中ＨＢＶ－ＤＮＡ和乙肝病毒标志物进行检测，结果发现各组ＨＢＶ－ＤＮＡ的检出率：①ＨＢｓＡｇ（＋）、ＨＢｅＡｇ（＋）和抗ＨＢｃ（＋）组为９９．２７％；②ＨＢｓＡｇ（＋）、抗ＨＢｅ（＋）和抗ＨＢｃ（＋）组为４４．７７％；③ＨＢｓＡｇ（＋）和抗ＨＢｃ（＋）组为４２．８６％；④ＨＢＶ标志物均阴性为１５．９２％。结果表明ＨＢＶ－ＤＮＡＰＣＲ检出先于其它乙肝病毒标志物，可早期发现乙肝。ＰＣＲ法直接检测ＨＢＶ－ＤＮＡ更有利于临床对乙肝的诊断和治疗。     

HBcAg DNA疫苗诱导小鼠（H—2^b）特异性细胞和体液免疫应答

目的 观察ＨＢｃＡｇ ＤＮＡ疫苗（ｐＪＷ４３０３／ＨＢｃ）免疫Ｃ５７ＢＬ／６小鼠（Ｈ－２＾ｂ）的特异性细胞和体液免疫应答。方法 基因枪和肌肉注射两种方法接种ＤＮＡ疫苗;ＥＬＩＳＡ法检测小鼠血清抗－ＨＢｃ（ＩｇＧ）及ＩｇＧ亚类（ＩｇＧ１,ＩｇＧ２ａ）;＾５１铬释放法检测小鼠脾细胞ＨＢｃＡｇ特异性ＣＴＬ活性。结果 该ＤＮＡ疫苗体外可表达ＨＢｃＡｇ;小鼠经基因枪或肌肉注射接种该疫苗后血清抗－ＨＢｃ滴度分     

Hepatitis B virus antigens in liver tissue in hepatocellular carcinoma and advanced chronic liver disease,relationship to liver cell dysplasia

ABSTRACT— Hepatitis B surface (HBs) and core (HBc) antigens (Ag) were studied in liver tissue in HBsAg seropositive patients with chronic liver disease complicated (n=32) and not complicated (n=36) by hepatocellular carcinoma. Both groups were matched by age, sex and underlying disease. There was no qualitative and quantitative difference in tissue HBsAg between the two groups. However, HBcAg was significantly less in quantity in hepatocytes of patients with hepatocellular carcinoma compared to chronic liver disease without cancer. Serum hepatitis B e antigen tested by radioimmunoassay was also less frequently positive in patients with hepatocellular carcinoma. These findings seem to suggest that hepatitis B virus replication becomes less active in the process of hepatocarcinogenesis. The relationship between intrahepatic hepatitis B antigens and liver cell dysplasia was also studied. In hepatocellular carcinoma, tissue hepatitis B antigens often coexisted in the same liver having liver cell dysplasia, but no such association was observed in chronic liver disease without cancer. However, no indication was obtained that the dysplastic cells harbor HBsAg more frequently than non-dysplastic cells.     

Hepatitis B virus antigens in liver tissue in hepatocellular carcinoma and advanced chronic liver disease-relationship to liver cell dysplasia

Hepatitis B surface (HBs) and core (HBc) antigens (Ag) were studied in liver tissue in HBsAg seropositive patients with chronic liver disease complicated (n = 32) and not complicated (n = 36) by hepatocellular carcinoma. Both groups were matched by age, sex and underlying disease. There was no qualitative and quantitative difference in tissue HBsAg between the two groups. However, HBcAg was significantly less in quantity in hepatocytes of patients with hepatocellular carcinoma compared to chronic liver disease without cancer. Serum hepatitis B e antigen tested by radioimmunoassay was also less frequently positive in patients with hepatocellular carcinoma. These findings seem to suggest that hepatitis B virus replication becomes less active in the process of hepatocarcinogenesis. The relationship between intrahepatic hepatitis B antigens and liver cell dysplasia was also studied. In hepatocellular carcinoma, tissue hepatitis B antigens often coexisted in the same liver having liver cell dysplasia, but no such association was observed in chronic liver disease without cancer. However, no indication was obtained that the dysplastic cells harbor HBsAg more frequently than non-dysplastic cells.     

Biologic significance of the detection of HBsAg and HBcAg in liver and tumor from 204 HBsAg-positive patients with primary hepatocellular carcinoma

Hepatitis B virus surface and core antigens (HBsAg, HBcAg) were examined in the resected primary hepatocellular carcinoma from 204 patients who had HBsAg in serum. Ninety patients had small (less than 5 cm) and 114 had large hepatocellular carcinoma (greater than 5 cm). HBsAg was detected in hepatocellular carcinoma in 65 cases (32%) and HBcAg in 30 cases (14.7%); hepatitis B virus antigens were more frequently detected in small (HBsAg in 42.2% and HBcAg in 20%) than in large hepatocellular carcinoma (HBsAg 23.7% and HBcAg 10.5%). These results suggest that replicative forms of hepatitis B virus DNA may exist in hepatocellular carcinoma more frequently than previously believed and that the malignant hepatocytes can support hepatitis B virus replication. A lymphocytic infiltration in hepatocellular carcinoma was more often observed in hepatocellular carcinoma expressing HBsAg (71%) or HBcAg (63%) than in hepatocellular carcinoma with no detectable HBsAg (26%) or HBcAg (37%), p less than 0.01. The reaction was mild in the majority (85%) of the cases. These findings suggest that hepatitis B virus antigen expression in hepatocellular carcinoma can provoke a local immune response. The most striking finding was that patients with hepatitis B virus antigens in small hepatocellular carcinoma had a 5-year survival rate (13%) lower than that (50%) of the antigen-negative patients (p less than 0.05). In contrast, patients with a marked local immune response in hepatocellular carcinoma, regardless of the viral antigen status, had significantly better 5-year survival rates (43%) than those with no or a mild lymphocytic reaction (18%). These findings indicate that a marked immune response in hepatocellular carcinoma is a favorable prognostic sign.(ABSTRACT TRUNCATED AT 250 WORDS)     

A lack of direct role of hepatitis B virus in the activation of ras and c-myc oncogenes in human hepatocellular carcinogenesis

This study was performed to determine the relationship of the activation of ras and c-myc oncogenes in human hepatocellular carcinoma to the hepatitis B virus gene expression or the presence of hepatitis B virus DNA/RNA at the cellular level. This was done using immunocytochemical analysis with two different antibodies on serial sections. In addition, immunocytochemical assay for the detection of ras p21 or c-myc protein was performed in combination with in situ hybridization for hepatitis B virus DNA/RNA using 35S-labeled hepatitis B virus DNA as a probe. Investigation of a total of 14 paired human hepatocellular carcinoma and adjacent nontumorous hepatic tissues revealed enhanced expression of ras p21 in one human hepatocellular carcinoma whereas c-myc protein was found in one paired human hepatocellular carcinoma and nontumorous tissue of the same patient. Only a small proportion of human hepatocellular carcinoma cells or hepatocytes among a large number of cells on a given section showed enhanced expression, and the distribution of the oncogene product-expressing cells was focal. However, the cells overexpressing these oncogenes did not show hepatitis B surface antigen in the serial sections. Furthermore, the combined immunocytochemical and in situ hybridization assays revealed that human hepatocellular carcinoma cells overexpressing ras p21 did not show hepatitis B virus DNA/RNA, whereas some human hepatocellular carcinoma cells and nontumorous hepatocytes located away from the foci of oncogene-expressing cells gave positive signals. These findings suggest that continued expression of HBsAg or the presence of hepatitis B virus DNA/RNA in a given human hepatocellular carcinoma cell id not necessary for enhanced expression of ras or c-myc proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of receptors for polymerized albumin in HBsAg-containing hepatocytes and hepatoma cell line

Recent evidence suggests that hepatitis B virions (HBV) and HBsAg particles contain receptors for polymerized human serum albumin (pHSA). We studied, by immunohistochemical techniques, the relationship between HBsAg and pHSA receptors in liver tissue from eight patients with chronic HBV infection and in a human hepatocellular carcinoma cell line (PLC/PRF/5) producing HBsAg. Both parallel sections and double fluorescent antibody staining of liver tissue demonstrated that only HBsAg-containing hepatocytes expressed pHSA receptors. The receptors could not be demonstrated in eight HBsAg negative livers. Sequential studies of PLC/PRF/5 cells revealed that pHSA and HBsAg emerged simultaneously in the cytoplasm, on the cell surface, and in the supernatant culture media. These findings indicate that pHSA receptors are closely associated with HBsAg during its synthesis and secretion by hepatocytes and suggest that the receptors are HBV-specific.     

Expression of hepatitis B surface antigen subtypes in liver of patients with hepatocellular carcinoma; comparison of subtypes in serum and liver.

To clarify the discrepancy in hepatitis B surface antigen (HBsAg) subtypes present in the serum and liver, as well as among hepatocytes, liver specimens which were resected from 37 HBsAg-positive patients with hepatocellular carcinoma (HCC) were examined. We evaluated HBsAg and the subtypic determinants of HBsAg and hepatitis B core antigen (HBcAg) using the peroxidase-antiperoxidase (PAP) staining method. Hepatitis B antigens were more frequently detected in small tumors (HBsAg in 67%. HBcAg in 40%) than in large ones (HBsAg in 36%, HBcAg in 14%). The prevalence of each subtypic determinant in the HBsAg positive non-tumorous vs. tumorous areas was 100% vs. 67% in a, 100% vs. 57% in d, 100% vs. not tested in y, 100% vs. 53% in r and 25% vs. 0% in w (a, d, y, r and w represent subtypic determinants). There was virtually no difference in a set of subtypic determinants between the serum and liver. However, there were some variations in a set of subtypic determinants among the hepatocytes. On the other hand, liver tissue of compound subtype adyr in serum contained both cells with a,d,r and with a,y,r as well as a few cells with a,d,y,r. These findings suggest that HBV genomes in hepatocytes of type B chronic liver disease may differ genetically among cells even in the same liver tissue.     

Immunohistochemical analysis of retinoblastoma gene product (pRB) expression in malignant and non-malignant liver diseases

ABSTRACT: The retinoblastoma gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To analyze the expression of retinoblastoma gene product in the process of liver regeneration and the initiation of hepatocellular carcinoma, we studied immunohistochemically the expression of retinoblastoma gene product and DNA polymerase alpha (DPA) in 33 patients with various liver diseases. Only a few hepatocytes positive for retinoblastoma gene product were found in undamaged, nonregenerating liver tissues, whereas many hepatocytes positive for retinoblastoma gene product were detected in specimens of regenerating liver obtained from patients with acute or chronic liver diseases. Similarities were found between distribution patterns of hepatocytes positive for retinoblastoma gene product and those of hepatocytes positive for DPA, and a highly significant positive correlation was found between the number of hepatocyte nuclei stained for retinoblastoma gene product per 1000 nuclei examined (R-LI) and the number of hepatocyte nuclei stained for DPA per 1000 nuclei examined (D-LI) in tissues obtained from patients with nonmalignant liver disease. Hepatocellular carcinoma cells positive for DPA were detected in the 14 hepatocellular carcinoma specimens tested. In ten of these specimens, hepatocellular carcinoma cells positive for retinoblastoma gene product were found but not in the other four. For all hepatocellular carcinoma specimens, R-LI was proportional to D-LI. Thus in both nonmalignant and malignant liver, retinoblastoma gene product increased in proportion to proliferation of hepatocytes or hepatocellular carcinoma cells.     

Expression of hepatitis B surface antigen subtypes in liver of patients with hepatocellular carcinoma; comparison of subtypes in serum and liver

ABSTRACT— To clarify the discrepancy in hepatitis B surface antigen (HBsAg) subtypes present in the serum and liver, as well as among hepatocytes, liver specimens which were resected from 37 HBsAg-positive patients with hepatocellular carcinoma (HCC) were examined. We evaluated HBsAg and the subtypic determinants of HBsAg and hepatitis B core antigen (HBcAg) using the peroxidase-antiperoxidase (PAP) staining method. Hepatitis B antigens were more frequently detected in small tumors (HBsAg in 67%, HBcAg in 40%) than in large ones (HBsAg in 36%, HBcAg in 14%). The prevalence of each subtypic determinant in the HBsAg positive non-tumorous vs. tumorous areas was 100% vs. 67% in a, 100% vs. 57% in d, 100% vs. not tested in y, 100% vs. 53% in r and 25% vs. 0% in w (a, d, y, r and w represent subtypic determinants). There was virtually no difference in a set of subtypic determinants between the serum and liver. However, there were some variations in a set of subtypic determinants among the hepatocytes. On the other hand, liver tissue of compound subtype adyr in serum contained both cells with a,d,r and with a,y,r as well as a few cells with a,d,y,r. These findings suggest that HBV genomes in hepatocytes of type B chronic liver disease may differ genetically among cells even in the same liver tissue.     

Expression of transforming growth factor-alpha and hepatitis B surface antigen in human hepatocellular carcinoma tissues and its significance

AIM:To evaluate the expression of transforming growthfactor-alpha (TGF-α) and hepatitis B surface antigen (HBsAg) in human hepatocellular carcinoma (HCC) tissues and its significance.METHODS:Seventy specimens of HCC tissues were detected by immunohistochemical method. Five specimens of normal human liver tissues were used as control.RESULTS: The TGF-o~ positive expression rates in HCC and its surrounding tissues were 74.3%(52/70) and 88.1%(52/59), respectively. TGF-α positive granules were mainly in the cytoplasm and fewer existed on the karyotheca. The TGF-α positive expressing rate in well differentiated HCC was significantly higher than that in moderately and poorly differentiated HCC (P<0.05).The TGF-α positive expression also was observed in intrahepatic bile ducts (part of those were hyperplastic ducts).The HBsAg positive expression rates in HCC and its surrounding tissues were 21.4%(15/70) and 79.7%(47/59), respectively.HBsAg positive granules were in the cytoplasm, inclusion and on the karyotheca.There was a prominent positive correlation between TGF-α and HBsAg expression in HCC surrounding tissues (P<0.05,γ=0.34). TGF-α was usually existed with HBsAg in regenerated and/or dysplastic liver cells.In the five normal liver tissues, TGF-α and HBsAg were not detectable in hepatocytes and bile ducts.CONCLUSION:Hepatitis B virus infection is dosely related with hepatocarcinogenesis.The overexpression of TGF-α in the liver seems to be associated with the regeneration of hepatocytes injured by HBsAg.The continued expression of TGF-α might lead to dysplasia of liver cells and development of HCC. Furthermore, TGF-α might play a role in morphogenesis and regeneration of intrahepatic bile ducts.     

Specificities of serum alpha-fetoprotein in HBsAg+ and HBsAg- patients in the diagnosis of hepatocellular carcinoma.

Serum alpha-fetoprotein level is often elevated in patients with chronic liver disease and patients with hepatocellular carcinoma. One of the most difficult problems frequently encountered in practice is differentiating hepatocellular carcinoma from chronic liver disease. This study investigated the specificity and predictive value positive of serum alpha-fetoprotein at various levels in the diagnosis of hepatocellular carcinoma, using 54 patients with histologically proven hepatocellular carcinoma and 200 patients with chronic liver disease (40 patients with chronic active hepatitis and 160 patients with cirrhosis) as nontumor controls. Among 254 patients, 170 (66.9%) were HBsAg+. A wide range of overlap (from 0 to 6,400 ng/ml) in the distribution of serum alpha-fetoprotein levels between hepatocellular carcinoma and chronic liver disease patients was observed mainly among HBsAg+ patients. In contrast, the overlapping range of serum alpha-fetoprotein levels between HBsAg- patients with hepatocellular carcinoma and chronic liver disease was remarkably narrow (from 0 to 200 ng/ml). Therefore the specificity and predictive value positive of alpha-fetoprotein at a given level were significantly lower in HBsAg+ than in HBsAg- patients, especially when alpha-fetoprotein was between 25 and 200 ng/ml. The specificities of alpha-fetoprotein at 200 ng/ml and 400 ng/ml in HBsAg+ patients were 79.8% and 91.5%, respectively, whereas these specificities were both 100% in HBsAg- patients. The predictive values positive at 200 ng/ml and 400 ng/ml in HBsAg+ patients were 53.6% and 72.5%, respectively, in contrast to 100% at both levels in HBsAg- patients. The serum alpha-fetoprotein level, which showed a predictive value positive of 95% in HBsAg+ hepatocellular carcinoma patients, was 3,200 ng/ml, whereas that in HBsAg- hepatocellular carcinoma patients, was 200 ng/ml. We conclude that serum HBsAg status should be considered when serum alpha-fetoprotein is measured as an independent test to diagnose hepatocellular carcinoma, and suggest that regular serum alpha-fetoprotein determination may be more useful in HBsAg- patients with chronic liver disease for the early diagnosis of hepatocellular carcinoma than in HBsAg+ patients.