合成多肽体外诱导乙肝病毒抗原特异性T杀伤细胞实验研究

目的及方法 为探讨ＨＢＶ抗原特异性细胞毒Ｔ细胞（ＨＢＶ－ＣＴＬ），根据ＨＢｃＡｇ的ＨＬＡ分子结合关键序列合成抗原多肽２段，分别与转铁蛋白（Ｔｆ），抗ＣＤ单克隆抗体（ＣＤ３ｍＡｂ）和ＩＬ－２联合体外诱导ＨＢＶ－ＣＴＬ，应用＾３Ｈ－ＴｄＲ释放法分别测定ＨＢＶ－ＣＴＬ对ＨＢＶＤＮＡ转染的ＨｅｐＧ２．２．１５细胞特异性活性及无ＨＢＶＤＮＡ转染的ＨｅＰＧ２细胞的非特异性杀伤率。结果 ＨＢＶ－ＣＴＬ对ＨＢＶＤ     

重组丙型肝炎病毒基因转染H9细胞模型的建立

目的：建立重组ＨＣＶ基因转染的Ｈ９细胞（人ＣＤ４，Ｔ细胞）模型。方法：以载体ＣＤＺ２（连接有１６９３ｂｐＨＣＶＤＮＡ，包括完整的ＨＣＶ结构区）为模板，通过ＰＣＲ获得高保真的１．７３ｋｂＤＮＡ片段（包括１６９３ｂｐＨＣＶｃＤＮＡ和部分载体ＤＮＡ序列），其特异性被ｓｏｕｔｈｅｒｎｂｌｏｔ证实。将ＨＣＶｃＤＮＡ片段插入载体ｐＣＤ－ＳＲα，经菌落原位杂交以及酶切分斤和ｓｏｕｔｈｅｒｎｂｏＩｔ筛选，获得重组ＨＣＶ（ｐＣＤ－ＨＣＶ），并与ｐＳＶ２－ｇｐｔ载体共转染Ｈ９细胞。结果：从选择性培养基中筛选口阳性克隆细胞，经ＲＴ－ＰＣＲ．ｄｏｔＥＬＩＳＡ和ｗｅｓｔｅｒｎｂｌｏｔ验证表明ｐＣＤ－ＨＣＶ在Ｈ９细胞中可进行复制、转录和表达，表达的约１．３×１０５大小蛋白具有ＨＣＶ抗原性。转染ｐＣＤ－ＨＣＶ的细胞培养至９０天时，仍可检测到ＨＣＶｍＲＮＡ和ＨＣＶ抗原。结论：建立ｐＣＤ－ＨＣＶ转染的Ｈ９细胞模型获得成功。     

国产精制人白细胞α－干扰素体外抗－HBV活性的测定

本研究利用ＨＢＶ体外细胞培养系统（２．２．１５细胞株），观察了国产精制人白细胞α－干扰素的体外抗－ＨＢＶ活性。结果显示：当精制人白细胞α－干扰素达到５Ｘ１０３单位／毫升时，能够明显降低２．２．１５细胞内及细胞培养上清中ＨＢＶＤＮＡ的含量，提示其能够抑制ＨＢＶ复制，具有一定体外抗．ＨＢＶ活性，可以在临床推广使用。但国产精制人白细胞α－干扰素对２．２．１５细胞分泌ＨＢｓＡｇ和ＨＢｅＡｇ未见明显抑制作用，其机理尚待进一步研究。     

海藻硫酸多糖抗乙型肝炎病毒的实验研究

１．材料与方法：海藻硫酸多糖（ＳＰＳ）是自制的一种天然硫酸酯多糖。在ＨＢＶ基因转染的人肝癌细胞系 ２．２．１５细胞中，观察ＳＰＳ在对ＨＢｅＡｇ和ＨＢｓＡｇ的抑制作用，以齐多夫定和灵芝多糖作为对照药物。选用鸭肝炎动物模型，以阿昔洛韦为阳性对照，观察ＳＰＳ对ＤＨＢＶ感染鸭血清ＤＨＢＶ ＤＮＡ水平的抑制作用。 ２．结果：ＳＰＳ在 ２．２．１５细胞培养中对ＨＢｓＡｇ和ＨＢｅＡｇ的分泌有明显抑制作用，随着ＳＰＳ浓度的增高，对ＨＢｅＡｇ和ＨＢｓＡｇ表达的抑制作用增强，最高抑制率分别为７０．１％±４．２％（ＩＣ５…     

重型病毒性肝炎患者病原学及预后的研究

对９４例重型病毒性肝炎进行了病毒标志的研究，并分析了几种影响重型肝炎预后的因素。结果发现：单纯ＨＢｖ感染４２例（４４．７％）；混合感染共５０例（５３．２％），其中ＨＢＶ与ＨＣＶｌ９例（２０．２％）．ＨＢＶ与ＮＤＶ１３例（１３．８％），ＨＢＶ与ＨＣＶ、ＨＤＶＩ０例（１０．６％）ＨＡＶ与ＫＢＶ３例（３．２％），ＨＡＶ与ＨＢＵ、ＨＣＶ３例（３．２％）ＨＡＶ与ＨＢｖ、ＨＤＶＩ例（１．１％）．ＮＡＶ与ＨＢＶ、ＨＣＶ、ＨＤＶ１例（１．１％）；病毒标志均阴性者２例（２．１％）。ＨＢＶ、ＨＣＶ和ＨＤＶ混合感染者病情重，病死率高。血清总胆红素越高，凝血酶原活性越低，其病死率越高；有并发症者的预后差，而ＡＭＰ升高者的预后较好；重肝的预后可能与年龄、性别无关。     

复方双草退黄冲剂的体外抗乙型肝炎病毒作用

目的：研究中药复方双草退黄冲剂体外抗ＨＢＶ活性。方法：采用ＨＢＶ的体外细胞培养系统（２．２．１５细胞）进行双草退黄冲剂抗ＨＢＶ作用的筛选试验,药物作用后的细胞培养上清中ＨＢｓＡｇ检测采用法,ＭＴＴ法测定细胞存活率,并计算ＩＣ５０、ＴＣ５０和ＴＩ。结果：双草退黄冲剂在对２．２．１５细胞无明显毒性的浓度下,当药物浓度为２０ｍｇ／ｍｌ、１０ｍｇ／ｍｌ、５ｍｇ／ｍｌ和２．５ｍｇ／ｍｌ时,对ＨＢｓＡｇ分泌的     

丙型肝炎病毒体外感染MT-2细胞的研究

目的建立体外丙型肝炎病毒感染的ＭＴ－２细胞模型。方法用ＨＣＶＲＮＡ阳性的丙型肝炎患者血清感染人淋巴细胞株ＭＴ－２以ＨＣＶＲＮＡ阴性血清作对照，用ＲＴ－ＰＣＲ分析ＨＣＶ在体外细胞培养中的复制状态。结果用ＨＣＶ阳性血清感染的ＭＴ－２细胞在培养第７天开始检出ＨＣＶＲＮＡ，并一直持续阳性至第２８天；培养至第１０天可测出ＨＣＶ复制中间体，于第１８天消失。用ＨＣＶ阴性血清感染的ＭＴ－２细胞未检出ＨＣＶＲＮＡ。扩增产物经Ｓｏｕｔｈｅｒｎ转移后用ＨＣＶ探针杂交证实ＨＣＶ特异性序列。结论ＨＣＶ可在ＭＴ—２中短期复制。     

肝舒胶囊在体外细胞培养中抗HBV活性的研究

目的：研究肝舒胶囊在２．２．１５细胞培养中抗乙型肝炎病毒（ＨＢＶ）作用。方法：用２．２．１５细胞体外培养,对肝舒胶囊抗ＨＢＶ活性进行评价。结果：肝舒胶囊５ｇ／Ｌ加药后４ｄ对２．２．１５细胞ＨＢｓＡｇ和ＨＢｅＡｇ的抑制率为５１．１６％和７４．８３％,在加药后第８天抑制作用达高峰,对ＨＢｓＡｇ和ＨＢｅＡｇ的抑制率分别为７５．７２％和８０．０２％。     

基因工程干扰素 α-2b 加CD_3AK治疗慢性乙型肝炎

４５例慢性活动性乙型病毒性肝炎随机分为三组。Ａ组用干扰素α－２ｂ（ＩｎｔｒｏｎＡ，干扰能）加经ＣＤ３单克隆抗体激活的杀伤细胞（ＣＤ３ＡＫ）；Ｂ组单用α－２ｂ；Ｃ组为对照组。结果：Ａ组ＨＢｅＡｇ和ＨＢＶＤＮＡ近期阴转率为７３．３％，远期疗效为５３．３％；Ｂ组分别为４６．７％和３３．３％。两组均明显优于对照组。表明α－２ｂ可有效抑制ＨＢＶ复制，加用ＣＤ３ＡＫ可增强对乙肝病毒的抑制作用。     

鸭乙型肝炎动物模型的建立和DHBV在肝脏的定位研究

本文利用ＤＨＢＶ阳性血清感染１日龄雏鸭，感染后２、４、８周以血清斑点杂交和Ｄｏｔ－ＥＩＡ检测鸭血清中的ＤＨＢＶ－ＤＮＡ和ＤＨＢｓＡｇ，其阳性率分别为５６．３％、６５．６％、７４．２％和５３．１％、７１．９％、７０．９％。进一步采用原位杂交和免疫组化对鸭肝组织内ＤＨＢＶ－ＤＮＡ和ＤＨＢｓＡｇ进行检测，结果表明ＤＨＢＶ－ＤＮＡ主要存在于细胞浆中，而ＤＨＢｓＡｇ主要分布于细胞浆及细胞膜。对肝组织的病理检查及胶原纤维特殊检测发现，ＤＨＢＶ阳性组与阴性组无明显差别。     

A human monoclonal antibody that recognizes viral polypeptides and in vitro translation products of the genome of the hepatitis D virus

Peripheral blood lymphocytes from a patient chronically infected with hepatitis D virus (HDV) were immortalized by Epstein-Barr virus transformation. Two stable monoclonal cell lines, derived from the same parent culture, were established and produced antibodies of the IgG isotype that were specific for the hepatitis delta antigen (HDAg). Both monoclonal antibodies (MAbs) recognized the major HDAg polypeptides of 24 kilodaltons and 27 kilodaltons that were previously detected by polyclonal antibodies to HDAg in both liver and serum from HDV-infected humans, chimpanzees, and woodchucks. This result indicates that the major polypeptides of HDAg share common epitopes. The MAbs also reacted with minor polypeptides of lower molecular weight, which were present in infected liver. In vitro translation products of HDV-specific RNA from infected liver were also detected by the MAbs; these polypeptides were 24 kilodaltons and 27 kilodaltons, respectively, and comigrated with liver- or serum-derived HDAg. In contrast, HDV RNA isolated from virions in serum was not translated into HDAg polypeptides in the in vitro system.     

“Defective” mutations of hepatitis D viruses in chronic hepatitis D patients

AIM: To verify whether "defective" mutations existed in hepatitis D virus (HDV). METHODS: Hepatitis delta antigen (HDAg)-coding sequences were amplified using Pfu DNA polymerases with proof-reading activities from sera of five patients with chronic hepatitis D. Multiple colonies were sequenced for each patient. Pfu analyzed a total of 270 HDV clones. Three representative defective HDV clones were constructed in expression plasmids and transfected into a human hepatoma cell line. Cellular proteins were extracted and analyzed by Western blot. RESULTS: Four of five cases (80%) showed defective HDV genomes in their sera. The percentage of defective genomes was 3.7% (10/270). The majority (90%) of the defective mutations were insertions or deletions that resulted in frameshift and abnormal stop translation of the HDAg. The predicted mutated HDAg ranged from 45 amino acids to >214 amino acids in length. Various domains of HDAg associated with viral replication or packaging were affected in different HDV isolates. Western blot analysis showed defected HDAg in predicted positions. CONCLUSION: "Defective" viruses do exist in chronic HDV infected patients, but represented as minor strains. The clinical significance of the "defected" HDV needs further study to evaluate.     

Hepatitis D virus RNA in serum from patients with hepatitis B

To clarify the correlation of hepatitis D virus (HDV) infection and viral replication in liver diseases, the authors detected HDV RNA and serological HDV markers in serum from 285 patients with hepatitis B and 45 asymptomatic carriers of HBsAg. With dot blot hybridization, serum HDV RNA was detected in 8.8% (29/330) of the patients with HBV infection. The positive rate of HDV RNA in fulminant hepatitis was higher than that in benign hepatitis (15/74 vs 3/47, P less than 0.05). 10 of the 139 patients with chronic active hepatitis and 1 of the 6 cases with cirrhosis were positive for HDV RNA. However, all of the 19 cases with chronic persistent hepatitis and 45 asymptomatic carriers of HBsAg were negative fo, HDV RNA. Serological HDV markers, HDAgr anti-HD and IgM-anti-HD, were determined with ELISA. HDV RNA was detected in all of the serum samples with positive HDAg and/or IgM-anti-HD, in 15 of the 26 cases with positive-anti-HD and in 8 cases without HDV markers. Our results showed that 40 of the 330 patients with HBsAg were infected by HDV. This investigation suggests that HDV is one of the etiological factors for fulminant hepatitis and chronic active hepatitis.     

反义寡核苷酸对H1δ9细胞中丁型肝炎病毒基因复制与表达的抑制作用

目的在Ｈ１δ９细胞中观察反义寡核苷酸（ＡＳＯＤＮ）及其硫代修饰物（Ｓ－ＡＳＯＤＮ）对丁型肝炎病毒（ＨＤＶ）基因复制与表达的抑制作用。方法 在分子水平证实互补于ＨＤＶ基因链球核酶自裂位点和ｓｔｅｍⅠ区的ＡＳＯＤＮ可抑制核酶自裂的基础上,进一步合成针对该区６８４－６９８位核苷酸的１５聚ＡＳＯＤＮ及Ｓ－ＡＳＯＤＮ,在培养的Ｈ１δ９细胞中加入不同寡核苷酸,分别采用ＥＬＩＳＡ和斑点杂交法检测上清中ＨＤＡｇ及     

Hepatitis D virus: an update

Hepatitis D virus (HDV) infection involves a distinct subgroup of individuals simultaneously infected with the hepatitis B virus (HBV) and characterized by an often severe chronic liver disease. HDV is a defective RNA agent needing the presence of HBV for its life cycle. HDV is present worldwide, but the distribution pattern is not uniform. Different strains are classified into eight genotypes represented in specific regions and associated with peculiar disease outcome. Two major specific patterns of infection can occur, i.e. co‐infection with HDV and HBV or HDV superinfection of a chronic HBV carrier. Co‐infection often leads to eradication of both agents, whereas superinfection mostly evolves to HDV chronicity. HDV‐associated chronic liver disease (chronic hepatitis D) is characterized by necro‐inflammation and relentless deposition of fibrosis, which may, over decades, result in the development of cirrhosis. HDV has a single‐stranded, circular RNA genome. The virion is composed of an envelope, provided by the helper HBV and surrounding the RNA genome and the HDV antigen (HDAg). Replication occurs in the hepatocyte nucleus using cellular polymerases and via a rolling circle process, during which the RNA genome is copied into a full‐length, complementary RNA. HDV infection can be diagnosed by the presence of antibodies directed against HDAg (anti‐HD) and HDV RNA in serum. Treatment involves the administration of pegylated interferon‐α and is effective in only about 20% of patients. Liver transplantation is indicated in case of liver failure.     

HIF-1α质粒转染NIH3T3细胞的体外实验

目的探讨低氧诱导因子1(HIF-1)在NIH3T3细胞的体外表达.方法首先构建真核表达质粒,以脂质体为介导,体外转染细胞,经RT-PCR,Western blot,ELISA检测基因在mRNA、蛋白质等方面的表达.结果半定量RT-PCR证实在NIH3T3细胞表达外源基因,Western blot检测到外源HIF-1蛋白质表达,ELISA试验证明表达的基因产物具有生物活性.结论PcDNA3-HIF1真核表达质粒能够有效地在体外表达目的基因,为进一步的动物实验奠定了基础.     

Varied assembly and RNA editing efficiencies between genotypes I and II hepatitis D virus and their implications

The mechanisms that link genotypes of hepatitis D virus (HDV) with clinical outcomes have not yet been elucidated. Genotypic variations are unevenly distributed along the sequences of hepatitis delta antigens (HDAgs). Of these variations, the packaging signal at the C-terminus has a divergence of 74% between genotypes I and II. In this report, we address the issue of whether these high variations between genotypes affect assembly efficiency of HDV particles and editing efficiency of RNA. Viral package systems of transfection with expression plasmids of hepatitis B surface antigen and HDAgs or whole genomes of HDV consistently indicate that the package efficiency of genotype I HDV is higher than that of genotype II. Segment swapping of large-form HDAg indicates that the C-terminal 19-residue region plays a key role for the varied assembly efficiencies. Also, the editing efficiency of genotype I HDV is higher than that of genotype II. The nucleotide and structural changes surrounding the editing site may explain why genotype II HDV has a low RNA editing efficiency. The findings of in vitro assembly systems were further supported by the observations that patients infected with genotype II had significantly lower alanine transaminase (ALT) levels, more favorable outcomes (P <.05), and a trend to have lower serum HDV RNA levels as compared with those infected with genotype I HDV (P =.094). In conclusion, genotype II HDV secretes fewer viral particles than genotype I HDV does, which in turn may reduce the extent of infection of hepatocytes and result in less severe hepatic inflammation.     

Serum hepatitis delta virus RNA in patients with delta hepatitis and in liver graft recipients

We have investigated the usefulness of serum hepatitis delta virus (HDV) RNA detection using a slot hybridization analysis of serum samples from ten patients with acute hepatitis and delta markers (group I), from 28 patients with chronic delta hepatitis (group II) and from seven liver graft recipients with hepatitis B virus (HBV) and HDV related cirrhosis or fulminant hepatitis (group III). The slot-blots were hybridized with both HDV-complementary DNA and single-stranded RNA probes. With the single-stranded RNA probe, HDV RNA was detected in the first serum sample available in 9/10 of the patients with acute hepatitis (group I). In addition, HDV RNA was detected in 8/9 and 7/8 of the samples obtained within and after 1 month of the onset of hepatitis. Five of the ten patients scored positive for HDV RNA and negative for hepatitis delta antigen (HDAg) while one was negative for HDV RNA and positive for HDAg. The same RNA probe enabled the detection of serum HDV RNA in 21/28 chronic hepatitis patients (liver HDAg and/or IgM anti-HD positive) (group II). Among the liver graft recipients (group III), 7/7 had a recurrent delta infection. Serum HDAg, liver HDAg and anti-HD IgM were identified in 3/7, 6/7 and 5/7 of the patients, respectively. HDV RNA was detected in the seven patients with either persistent (4/7) or transient (3/7) positivity. In addition, HBsAg and HBV RNA were persistently shown in 4/7 patients with continuous HDV replication. In the remaining three patients, HDV RNA was detectable despite the absence of HBsAg.(ABSTRACT TRUNCATED AT 250 WORDS)     

丁型肝炎病人肝组织HDAg和 Fas表达及其相互关系

目的 研究丁型肝炎病人肝组织Fas和HDAg表达及其相互关系,了解Fas在HDV致病机制中的作用。方法 应用免疫组化单、双标记技术,检测48例丁型肝炎病人肝组织Fas、HDAg表达,以54例乙型肝炎病人肝组织Fas表达作对照。结果 Fas以肝细胞浆表达为主,HDAg以肝细胞核和胞浆表达为主,二抗原表达及分布呈一致性。各型肝炎中Fas和HDAg表达有统计学差别,两种抗原的分布和表达强度与肝细胞炎症活动和病理损害程度相关。结论 Fax和HDAg表达及分布密切相关,与肝组织炎症活动和病理损害相关,此提示HDV感染可诱导肝细胞表达Fas,Fas及其所介导的肝细胞凋亡在HDV致病机制中可能有重要作用。     

聚合酶链反应检测慢性丁型肝炎血清HDV RNA的实验研究和临床观察

本文建立一种高度敏感和特异性的检测丁型肝炎病毒(HDV)RNA的方法,提高了丁型肝炎的诊断水平。以HDV RNA保守区ORF5′末端第929～1640位核苷酸为靶基因设计一对引物,采用逆转录-聚合酶链反应(RT-PCR)检测了35例慢性丁型肝炎患者血清HDV RNA,并同时检测HBVM。35例中共检出HDV RNA421例(60％),10例HDAg阳性者HDV RNA全部阳性,25例抗-HD阳性者中有11例HDV RNA阳性(44％)。表明采用RT-PCR可以准确、快速、敏感地检测出HDV RNA,具有很强的特异性。35例中HBV DNA的检出率为34.2％,而HDV RNA的检出率为60％,HBV DNA和HDV RNA同时阳性者共9例占25.7％,提示慢性乙型肝炎病人合并丁型肝炎时,HBV的复制受到不同程度的抑制而HDV的复制较为活跃。