慢性肝炎和肝癌病人血清中乙型肝炎病毒DNA的检测

为了了解慢性肝炎和肝癌病人患者体内乙型肝炎病毒（ＨＢＶ）复制与ＨＢＶ血清标志之间的关系，用酶联免疫吸附实验（ＥＬＩＳＡ）、聚合酶链反应（ＰＣＲ）及斑点杂交方法对６１例慢性肝炎和４７例肝癌患者的ＨＢＶ表面抗原（ＨＢｓＡｇ）、相关ｅ抗原（ＨＢｅＡｇ）、表面抗体（抗－ＨＢｓ）、核心抗体（抗－ＨＢｃ）、相关ｅ抗体（抗－ＨＢｅ）进行了检测。结果表明：ＨＢＶＤＮＡ在ＨＢｓＡｇ、ＨＢｅＡｇ、／抗－ＨＢｃ阳性的慢性肝炎和肝癌患者血清中的检出率分别为９０．５０％和５０．００％；在ＨＢｓＡｇ／抗－ＨＢｅ、抗－ＨＢｃ阳性者的检出率分别为４５．４０％和７．１４％；在ＨＢｓＡｇ阳性、ＨＢｅＡｇ阴性／抗－ＨＢｅ阴性者中的检出率分别为６０．００％和４０．００％；ＨＢｓＡｇ阴性、／抗－ＨＢｃ阳性或／抗－ＨＢｅ阳性或／抗－ＨＢｓ阳性者中的检出率分别为２０．００％和２２．２２％；在血清学指标全阴性时，慢性肝炎和肝癌患者血清中ＨＢＶＤＮＡ的检出率均为０。实验提示：无论是肝炎或肝癌，在ＨＢｓＡｇ、ＨＢｅＡｇ同时阳性时，ＨＢＶ复制最为活跃；在单独ＨＢｓＡｇ阳性时，ＨＢＶ有一定程度的复制；ＨＢＶ复制在肝癌细胞中受到一定程度的抑制。     

乙型肝炎病毒前S2抗原／抗体检测方法的改进及其应用

通过改进乙型肝炎病毒表面前Ｓ２抗原（Ｐｒｅ－Ｓ２Ａｇ）、前Ｓ２抗体（Ｐｒｅ－Ｓ２Ａｂ）的检测方法,对１２８份正常人标本Ｐｒｅ－Ｓ２Ａｂ进行测定,确定了Ｐｒｅ－Ｓ２Ａｂ抑制率的阳性界值。用改进后的方法检测不同人群中的Ｐｒｅ－Ｓ２Ａｇ及Ｐｒｅ－Ｓ２Ａｂ,发现在慢性乙型肝炎患者中Ｐｒｅ－Ｓ２Ａｇ比ＨＢｅＡｇ／Ａｂ系统更能反映机体的ＨＢＶＤＮＡ复制水平（Ｐ＜０．０１）,而Ｐｒｅ－Ｓ２Ａｂ的检出则与急性乙型肝炎的预后密切相关,同时,Ｐｒｅ－Ｓ２Ａｂ也可作为评价含有Ｐｒｅ－Ｓ２Ａｇ的乙肝疫苗主动免疫效果的指标。     

乙型肝炎病毒感染者癌基因蛋白的表达及与HBeAg的关系

目的为了阐明癌基因蛋白异常表达与乙型肝炎病毒（ＨＢＶ）复制的关系。方法用链霉菌素－生物素（ＳＬＡＢ）免疫组织化学染色和酶联免疫吸附试验（ＥＬＩＳＡ）方法，检测了６４例慢性ＨＢＶ感染者肝细胞中Ｃ－ｅｒｂＢ－２Ｐ１８５、ｒａｓＰ２１和Ｐ５３等癌基因蛋白和血清乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）。结果Ｃ－ｅｒｂＢ－２Ｐ１８５和ｒａｓＰ２１阳性组中，血清ＨＢｅＡｇ的阳性检出率分别为８９４％和８４６％，而阴性组ＨＢｅＡｇ的检出率分别为２０％和４８％，两组差异显著。结论表明癌基因蛋白Ｃ－ｅｒｂＢ－２Ｐ１８５和ｒａｓＰ２１的异常表达与ＨＢＶ复制密切相关。     

通过受体介导法将核酶特异导入肝细胞以阻断HBV蛋白表达的初步研究

应用半乳糖末端糖蛋白受体（ＡＳＧＰ－Ｒ）介导的内吞作用，将外源基因导入真核细胞，与脂质体介导的转染和细胞表面转铁蛋白受体（Ｔｆ－Ｒ）介导的内吞作用相比，虽然三种方式均能有效介导外源基因的转移，但ＡＳＧＰ－Ｒ法具有肝细胞特异性，而脂质体法和Ｔｆ－Ｒ法不具此特性。将克隆于真核表达载体的针对乙型肝炎病毒（ＨＢＶ）ｍＲＮＡＰｒｅＣ／Ｃ区的核酶质粒ｐＣＭＶ－Ｒｉｐｃ特异性导入肝细胞并发挥作用，通过酶联免疫吸附法（ＥＬＩＳＡ）检测细胞培养液中的乙型肝炎表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ），评价核酶在细胞水平对ＨＢＶ抗原表达的阻断作用。结果表明当核酶质粒ｐＣＭＶ－Ｒｉｐｃ与ＨＢＶ抗原表达质粒ｐＵＣ－２ＨＢＶ共转染ＨｅｐＧ２细胞时，核酶对ＨＢｓＡｇ和ＨＢｅＡｇ表达的抑制率分别为５５．２９％和６８．７３％。     

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。     

反义寡脱氧核苷酸在鸭体内抑制鸭乙型肝炎病毒复制与 …

目的 研究反义核酸的抗病毒作用。方法 设计合成了针对鸭乙型肝炎病毒（ＤＨＢＶ）前Ｓ（ＰｒｅＳ）基因区第９５１－９６８位核苷酸的硫代反义寡脱氧核苷酸（ＡＳ－ＯＤＮ），以２０μｇ／ｇ体重／日剂量对３只腹腔感染ＤＨＢＶ５．２毒株后，血清ＤＨＢｓＡｇ及ＤＨＢＶ ＤＮＡ阳性鸭连续静脉注射１０天，同时以等体积生理盐水注射另３只感染鸭作为对照。结果 对照鸭注射生理盐水后，血清ＤＨＢｓＡｇ及ＤＨＢＶ ＤＮＡ阳性未     

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，     

黄曲霉毒素高污染区肝细胞癌p（53）蛋白与乙肝病毒感染的相关性研究

应用免疫组化ＳＰ法，检查桂西南黄曲霉毒素（ＡＦＢ１）高污染区１０９例肝细胞癌（ＨＣＣ）组织中突变型ｐ５３蛋白的表达，并与相应病例中乙型肝炎病毒表面抗原（ＨＢｓＡｇ）的表达进行相关性研究。结果发现：ＨＣＣ中ｐ５３蛋白阳性率高达６８．８％（７５／１０９），ｐ５３蛋白表达与病人性别、肿瘤大小及有无乙型肝炎病毒（ＨＢＶ）感染无关（Ｐ＞０．０５）。说明在ＡＦＢ１高污染区，ｐ５３基因突变是ＨＣＣ中非常普遍的现象。在ＨＣＣ病例中，ＨＢｓＡｇ阳性率为８４．２％（８５／１０１），其中同一病例ｐ５３蛋白及ＨＢｓＡｇ均为阳性者占５７．０％（５８／１０１），而ＨＢｓＡｇ阳性、突变型ｐ５３蛋白阴性者为２６．０％（２７／１０１）。表明ＨＢＶ仍然是肝细胞癌发生的主要原因，但ＨＢＶ的致癌机制具有多样性，并非均引起ｐ５３基因的改变。     

针对包装信号起始区的硫代反义核苷酸抗HBV的研究

目的筛选高效特异的抗ＨＢＶ反义核酸药物。方法针对ＨＢＶ包装信号ε起始区设计并合成硫代反义寡聚核苷酸（ｓ－ａｓＯＤＮ）片段，通过ＥＬＩＳＡ检测法、ＭＴＴ法、电子显微镜等观察，研究此ｓ－ａｓＯＤＮ对ＨＢｓＡｇ、ＨＢｅＡｇ和ＨＢｃＡｇ表达，以及对细胞毒性，细胞形态的影响。结果针对ε起始区的ｓ－ａｓＯＤＮ显著抑制ＨＢｓＡｇ、ＨＢｅＡｇ和ＨＢｃＡｇ的表达，其中，对ＨＢｅＡｇ和ＨＢｃＡｇ的抑制率分别为３８．１％和５８．７％高于Ｓ基因起始区（３２．１％和３７．２％，Ｐ＜０．０１），对ＨＢｓＡｇ抑制率为８２％，低于Ｓ基因起始区（９３．４１％），在实验浓度下ｓ－ａｓＯＤＮ对细胞无毒性，对细胞形态无影响。结论ＨＢＶ包装信号区是反义核酸抗ＨＢＶ复制研究的重要的靶序列选择区域。     

基因重组HBeAg一步法检测抗HBe的实验研究

本文应用基因重组ＨＢｅＡｇ与大鼠抗ＨＢｅ单克隆抗体，建立了ＥＬＩＳＡ一步快速法检测病人血清中抗ＨＢｅ，并与常规ＥＬＩＳＡ法做了比较，结果批内，批间ＣＶ分别为１１．２９％与１３．２％，两法敏感一致，准确性两法符合率９８．６％，临床检测２８０例标本，急慢性乙肝阳性７８％（３９／５０），可疑肝病２６％（４２／１６０），正常人群１２．９％（９／７０），此法简便，快速，有较高的敏感度与特异性，是临床值得推广     

Evaluation of the pre-S (pre-S(1)Ag/pre-S(2)Ab) system in hepatitis B virus infection.

The diagnostic and prognostic value of pre-S(1)Ag and pre-S(2)Ab was investigated in 69 HBsAg surface antigen positive patients--14 with acute hepatitis B, 30 with chronic liver disease (six chronic persistent hepatitis, 14 chronic active hepatitis, 10 with cirrhosis) and in 25 asymptomatic carriers. Pre-S(1)Ag was found in all patients with chronic hepatitis B virus (HBV) infection regardless of viral replication. In contrast, pre-S(2)Ab was not detected in any patients. Acute hepatitis was studied sequentially with periodic controls at 20 day intervals. Pre-S(1)Ag cleared before HBsAg in six of 14 (43%) patients who progressed favourably, and the two antigens cleared simultaneously in eight of 14 (57%) cases. Patients with early clearance of pre-S(1)Ag progressed favourably, thus indicating the prognostic value of this test, which, however, is still of limited practical application given the small temporal difference between the moment of clearance of the two antigens. The first markers to clear, however, were HBeAg and DNA-HBV, which showed significant differences with respect to the clearance of HBsAg. Moreover, pre-S(2)Ab appeared before HBsAb in 57.1% of our patients and was found in some patients before pre-S(1)Ag and HBsAg had cleared (42.8%), thus allowing complete viral clearance and acute HBV infection to be predicted earlier.     

Detection of hepatitis B virus pre-S1 and pre-S2 determinants in paraffin wax embedded liver tissue: importance of reagents used.

The presence of pre-S polypeptides in paraffin wax embedded liver sections from the biopsy specimens of 15 hepatitis B surface antigen (HBsAg) seropositive patients (five with chronic persistent hepatitis (CPH), four with chronic active hepatitis (CAH), four with cirrhosis and two "healthy" HBsAg carriers) was investigated using monoclonal antibodies directed to distinct epitopes on pre-S1 (18/7 and TO 606) and pre-S2 (5535 and Q 19/10). Pre-S1 was found in 13 cases when MA 18/7 was used but in only one specimen when TO 606 was used. Pre-S2 was detected in all the biopsy specimens with 5535 and in eight samples with Q 19/10. Mild enzymatic digestion annulled the staining of all monoclonal antibodies but Q 19/10. No association was observed between pre-S polypeptide expression and hepatitis B virus (HBV) replication or disease severity. Pre-S polypeptides can be detected readily in paraffin wax embedded material but the results obtained largely depend on the monoclonal antibodies used.     

血清乙型肝炎病毒前S1抗原检测及其与病毒复制的关系

用抗Ｓ和抗前Ｓ１单抗的双抗体夹心ＥＬＩＳＡ法检测１５０例慢性乙型肝炎患者、乙型肝炎病毒表面抗原（ＨＢｓＡｇ）携带者和健康人血清中的ＨＢＶ前Ｓ１抗原，其结果和ＨＢＶＤＮＡ聚合酶链反应（ＰＣＲ）、乙型肝炎血清标志的检测结果进行比较。结果表明：前Ｓ１抗原在乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）阳性组中的检出率和相对滴度显著高于ＨＢｅＡｇ阴性组（Ｐ＜０．０１）；在ＨＢｅＡｇ阴性组中，抗－ＨＢｅ阴性人群前Ｓ１抗原的检出率和相对滴度也显著高于抗－ＨＢｅ阳性人群（Ｐ＜０．０１）。前Ｓ１抗原和ＨＢＶＤＮＡ检测结果的符合率达８０％，两者检出率的相关系数ｒ＝０．９８２６（Ｐ＜０．０１）。结论：血清前Ｓ１抗原和乙型肝炎病毒的存在关系密切。     

Pre-S1 and Pre-S2 gene-encoded proteins in liver and serum in chronic hepatitis delta infection

Frozen cryostat sections and sera from 30 patients with chronic delta infection were examined for pre-S1 and pre-S2 gene-encoded proteins, and the results were compared to markers in liver and serum HBV and HDV replication. Pre-S1 and pre-S2 were detected by indirect immunofluorescence (IF) in the liver in all 26 patients with histochemically demonstrable HBsAg. Pre-S peptides were found by double IF to have a predominantly cytoplasmic expression and to be located in the same hepatocytes expressing HBsAg. Liver cells expressing hepatitis delta antigen (HDAg) were frequently negative or very weakly positive for HBsAg and pre-S peptides, but occasional HDAg positive hepatocytes were also strongly positive for HBsAg and for pre-S peptides, particularly pre-S2. Circulating pre-S1 was detected in 24 patients (80%) and pre-S2 in 27 (90%). Detection of pre-S peptides in liver and serum was independent of HBV and HDV replication and of the HBV-DNA integration state. There was no correlation between the amount of circulating pre-S peptides and serum HBV-DNA and HDV-RNA. These results indicate that in chronic HDV infection, formation and secretion of pre-S peptides and of HBsAg occur independently of HBV and HDV replication and secretion. They further indicate that in the acquisition by replicating HDV of an HBV-derived envelope in the liver, both HBsAg and pre-S peptides are concomitantly available but circulating HDV-RNA is not invariably associated with the presence of these peptides in serum.     

Detection of hepatitis B viral DNA by polymerase chain reaction in patients with hepatitis B surface antigen

Hepatitis B virus (HBV) DNA was assayed using the polymerase chain reaction in serum samples of 116 hepatitis B surface antigen (HBsAg) carriers, including 30 positive for hepatitis B e antigen (HBeAg) and 86 negative for HBeAg. In the HBeAg-positive group, all were positive for HBV DNA. In the HBeAg-negative group, 80.2% were positive for HBV DNA (80.0% in the healthy carrier group, 90.0% in the chronic active liver disease group, and 69.2% in patients with cirrhosis). This study indicated that every HBeAg-positive carrier as well as the majority of HBeAg-negative carriers were infectious and, in the latter group, that viral replication is most active in patients with chronic active liver disease.     

慢乙肝患者HBV-DNA和Pre-S1Ag及HBVM与肝脏功能的检测意义

目的:了解乙型肝炎病毒HBV-DNA、Pre-S1Ag、乙肝标志物(HBVM)和肝脏功能之间的关系及临床意义.方法:采用荧光定量聚合酶链式反应(FQ-PCR)和ELISA分别检测169例乙肝病人血清HBV-DNA含量和乙肝标志物及Pre-S1Ag与肝功能,并对结果进行对比分析.结果:各种不同类型乙肝HBsAg的阳性率均高于91.1%,HBeAg、Pre-S1Ag的阳性率随HBV-DNA拷贝数的升高而升高,但肝功能和HBV-DNA拷贝数之间不存在相关关系.结论:同时检测血清乙肝标志物、Pre-S1Ag、HBV-DNA和肝脏功能对临床HBV感染、复制及传染性的判断以及肝功能损伤程度均有重要意义.     

乙肝不同血清模式及乙肝DNA定量与前S1抗原联合检测的临床意义

目的 探讨乙型肝炎患者不同的血清学模式、乙肝病毒DNA(HBV-DNA)与乙肝前S1抗原(Pre-S1 Ag)联合检测的临床意义.方法 采用化学免疫发光法(CLIA)定量筛选339例乙肝血清标志物阳性血清,采用荧光定量聚合酶链反应法(FQ-PCR)检测HBV-DNA,采用酶联免疫吸附法(ELISA)检测Pre-S1 Ag.结果 乙肝不同血清模式下,HBV-DNA与Pre-S1 Ag检测结果比较差异无统计学意义(P＞0.05).HBsAg、HBeAg、抗-HBc阳性组HBV-DNA检出率93.1％,Pre-S1Ag检出率86.1％.HBsAg、HBeAb、抗-HBc阳性组HBV-DNA检出率45.9％,Pre-S1 Ag检出率69.2％.HBsAg、抗-HBc阳性组HBV-DNA检出率61.0％,Pre-S1 Ag检出率72.9％.HBsAg、HBeAg阳性组HBV-DNA及Pre-S1 Ag检出率均为100％.以HBeAg阳性为对照HBV-DNA及Pre-S1 Ag检出率分别为87.3％和93.7％.HBV-DNA与Pre-S1 Ag检测结果比较差异有统计学意义(P＞0.05).结论 乙肝五项、HBV-DNA、Pre-S1Ag联合检测能够对乙肝病毒的感染、复制程度做出准确的判断,为临床治疗方案的选择和疗效的观察提供可靠的依据.     

Differential effect of chronic hepatitis D virus infection on intrahepatic expression of hepatitis B viral antigen.

AIMS: To determine how chronic hepatitis D virus (HDV) infection affects intrahepatic hepatitis B virus (HBV) antigen expression. METHODS: Ninety eight liver biopsy specimens from 68 patients seropositive for total antibody to HDV were studied by immunohistochemistry, and the amount of HBV antigens was also quantified by radioimmunoassay in 12 patients and compared with 30 patients with chronic HBV infection. RESULTS: Forty nine of the 68 patients were positive for intrahepatic HDV antigen and only five were positive for HBV core antigen (HBcAg). HBV surface antigen (HBsAg) was present in 55 (80.9%) patients and was always cytoplasmic in distribution. Hepatic pre-S1 and pre-S2 expressions paralleled that of HBsAg, and were detected in 53 (77.9%) and 54 (79.4%) patients, respectively. There was no relation between the intrahepatic expression of HDV antigen and HBsAg/pre-S1/pre-S2. Follow up biopsy specimens in 25 patients showed either static or deteriorating histology while intrahepatic HDV antigen remained the same or fell. The patients with intrahepatic expression of HBcAg had either absent or noticeably decreased expression of HBcAg in their follow up biopsy specimens (median two years). In contrast, HBsAg/pre-S1/pre-S2 were the same or increased (p less than 0.001). Quantification of intrahepatic HBsAg in patients with chronic HDV infection (0.61 pg/hepatocyte, range: 0.05-1.08, n = 12) showed no difference with patients with chronic HBV infection alone (0.64 pg/hepatocyte, range: 0.02-1.02, n = 30, p = NS). CONCLUSION: These data indicate that chronic HDV infection suppresses intrahepatic expression of HBcAg but not HbsAg and pre-S antigens, suggesting a differential effect of chronic HDV infection on HBV gene expression.     

Immune response to hepatitis B virus surface antigen peptides during HBV infection.

Antibody responses of patients with acute (n = 73), fulminant (n = 30) and chronic (n = 51) hepatitis B virus (HBV) infection as well as recovered individuals (n = 7) were studied against three synthetic peptides, Pre-S1 amino acids (aa. 12-32), Pre-S2 amino acids (aa. 120-145), and S amino acids (aa. 124-147) of the envelope region (HBsAg). T cell blastogenic response was investigated in a proportion of the patients (27 acute, nine fulminant, 13 chronic hepatitis and seven recovered individuals) along with seven HBV vaccinated and three normal individuals. The presence of T cell response against S peptide was observed in all the cases (9/9, 100%) during early acute hepatitis. This was suppressed during late stages (8/18, 44%) followed by partial reversal during recovery (5/7, 71%). T cell response and antibodies to Pre-S1 and Pre-S2 peptides were present only in one-third of the patients throughout these periods. The T cell blastogenic response as well as antibody reactivity against these peptides were absent and minimal in chronic hepatitis. Immune response against envelope protein appears to play a major role in acute hepatic injury due to HBV infection and help in virus clearance.     

Hepatitis D virus antibody in HBsAg-positive and HBsAg-negative substance abusers with chronic liver disease

The hepatitis D virus (HDV; previously called the "delta agent") is a defective organism which can replicate only in the presence of the hepatitis B virus (HBV). We tested the serum of 95 substance abusers, all of whom had sufficient evidence of chronic liver disease to warrant a liver biopsy, for hepatitis D virus antibody (anti-HDV). Anti-HDV was detected in five of eight hepatitis B surface antigen (HBsAg)-positive patients and 12 of 87 (14%) HBsAg-negative patients. Antibody to the hepatitis B core antigen (anti-HBc) was the sole hepatitis B marker in eight of the 12 (67%) anti-HDV-positive, HBsAg-negative patients but in only 14 of 75 (19%) anti-HDV-negative, HBsAg-negative patients (P less than 0.005). None of the anti-HDV-positive, HBsAg-negative patients had detectable IgM anti-HBc in the serum or hepatitis D antigen in liver tissue, and they had similar clinical features and liver biopsy diagnoses to HBsAg-negative patients without anti-HDV. We conclude that anti-HDV in HBsAg-negative substance abusers reflects infection with HDV and HBV in the distant past and does not indicate more severe liver disease than that seen in HBsAg-negative patients without anti-HDV.