HCV的研究进展

近年来，由于血液和血制品的广泛应用，ＨＣＶ的感染越来越引起人们的重视，仅美国每年大约就有５万人左右被ＨＣＶ感染。以下将从ＨＣＶ的病原学、自然史、流行病学、临床特征及实验室检测几方面作一综述。１病原学ＨＣＶ是一种球型外壳病毒，直径约５０ｎｍ。ＨＣＶ基因...     

HCV亚型的临床研究进展

许多学者从研究ＨＣＶ基因型中发现：ＨＣＶ基因型与致病性、地区聚集性及对干扰素的应答可能有关。     

1年以上血透患者HCV感染状况的调查

目的 研究1年以上的长期血液透析患者丙型肝炎（HCV）感染状况。方法 用ELISA法和RT－PCR法检测137例长期血透患者血清中的抗－HCV和HCVRNA,并且同时检测谷丙转氨酶（ALT）和谷草转氨酶（AST）,计算其变动率。结果 透析时间超过1年以上的137例患者中仅抗－HCV阳性8例,仅HCV－RNA阳性13例,抗－HCV与HCVRNA同时阳性者24例,感染率34．3％。且透析时间小于2年的,HCV感染率为15％,透析时间大于2年以上的其感染率增至37．6％。结论 血透患者中HCV的感染应引起重视,透析的年限越长,被HCV感染的机率就越大。酶学指证的变动率不能作为长期透析患者HCV感染的敏感指标。     

从随机9肽库中筛选HCV抗原表位

目的 :用患者血清中抗HCV抗体从随机 9肽库中筛选HCV抗原表位。方法 :将病人血清用硫酸铵粗提后 ,用ProteinA亲和层析柱纯化和制备抗HCV多克隆抗体 ;以此为筛选配基 ,对噬菌体表面展示的随机 9肽库进行亲和筛选。结果 :三轮筛选的投入产出比逐轮升高至 5 0× 10 - 3 、假阳性率逐轮降低至 0 2 % ,提示具有良好的富集效果。从第三轮挑选出的 15个克隆进行结合试验 ,发现 7个克隆只与HCV抗体有较强的结合力而不与正常人血清反应 ;测序表明 6个克隆的外源肽含有核心序列SPVAXVLXT。用阳性噬菌体克隆检测 2 0例病人血清 ,有程度不等的阳性反应。结论 :用多克隆抗体从噬菌体随机肽库中筛选得到了有一定功能的模拟表位。     

慢性丙型肝炎患者HCV基因型调查

目的 研究丙型肝炎病毒（ＨＣＶ）基因型的流行病学特点。方法 采用ＩＮＮＯ－ＬＩＰＡ反向线形探针杂交法，对来自我国南方（上海，武汉，徐州）、北方（北京、天津）和东北（大连、沈阳）７市的１０７例慢性丙型肝炎患者进行了ＨＣＶ基因型调查。结果（１）地域性，ＨＣＶ１ｂ是主要的基因型，其检出率达８３．１７％，其中东北２市（７２．２２％）低于北方２市（８７．６５％，Ｐ〈０．０１）；２型的检出率较低，仅占６．８６     

用抗HCV多抗从随机15肽库中筛选抗原表位

目的:分析丙肝患者血清中抗HCV抗体的抗原表位。方法: 制备Protein A亲和层析柱,从丙肝患者血清中纯化、制备抗HCV多克隆抗体;并以此为筛选配基,对噬菌体表面展示的随机15肽库进行亲和筛选。结果:三轮筛选的投入产出比逐轮升高至3.3×10-3,假阳性率逐轮降低至0.2％,提示具有良好的富集效果。对从第3轮挑选出的16个克隆进行结合试验,发现9个克隆只与HCV抗体有较强的结合力而不与正常人血清起反应。测序表明,8个克隆的外源肽含有核心序列WPWS。用阳性噬菌体克隆检测20例丙肝患者血清,有程度不等的阳性反应。结论:用多克隆抗体从噬菌体随机肽库中,筛选到有一定功能的模拟表位。     

慢性丙型肝炎患者HCV血清学分型研究

目的探讨HCV基因分型与血清学分型的关系。方法对来自14家医院的104例已知HCV基因型的慢性丙型肝炎患者血清，经ELISA方法，用Murex HCV Serotyping 1-6 Assay血清分型试剂进行HCV的血清学分型。结果104例血清中的86（82，69％）例可分出血清型，检出血清型病毒株91株，病毒株检出率为78．4％。血清型与基因型的总符合率为62，1％，血清型1型、2型和3型的符合率分别为69，4％、51，2％和70．0％，以2b基因型的符合率和漏检率最低（54．5％）。结论HCV血清型特异性受病毒基因型的影响，与基因型存在一定的差异。     

线探针核酸杂交分析贵州地区HCV感染株基因型

目的　分析贵州地区丙型肝炎病毒（ＨＣＶ）感染株的基因型。方法　用第二代线探针核酸杂交方法,对９８ 例ＨＣＶ感染者血清进行基因分型。结果　１２ 例献血员血清,均为ＨＣＶ１ｂ 型;１５ 例血液病患者中,１４ 例（９３－３％ ）为ＨＣＶ１ｂ ,１ 例（６－７％ ）为ＨＣＶ１ｂ ＋ ２ 型。慢性丙肝患者３７ 例中,ＨＣＶ１ｂ 型３４例（９１－９％ ） ,混合感染基因型３ 例（ＨＣＶ１ａ ＋ １ｂ 、ＨＣＶ１ｂ ＋ ２ 、ＨＣＶ１ａ ．１ｂ ＋ ２ａ２ｃ 各１ 例）。静脉药瘾者３４ 例,其中ＨＣＶ１ｂ 感染１３ 例（３８－２％ ） ,ＨＣＶ６ａ１０ 例（２９－４％ ） ,ＨＣＶ３ｂ４ 例（１１－８％ ） ,ＨＣＶ３ａ１ 例（２－９ ％） ,混合感染６ 例（１７－６ ％） ,大多为ＨＣＶ２ 复合其他基因型。结论　贵州地区ＨＣＶ感染者中,以ＨＣＶ１ｂ 基因型为主,其次为ＨＣＶ６ａ ,尚存在ＨＣＶ３ａ 和ＨＣＶ３ｂ 。其中ＨＣＶ３ａ、３ｂ 、６ａ ,在本地区系首次报告,但主要存在于静脉药瘾者中。虽然有混合感染基因型的存在,但其基因基础需测序分析     

用噬菌体展示随机12肽库筛选HCV B细胞抗原表位

目的用患者血清中抗丙型肝炎病毒(HCV)抗体从噬菌体展示随机12肽库筛选HCV抗原表位。方法用硫酸铵粗提HCV患者血清中Ig后用DEAE—Sephadex A50层析纯化并作为筛选的配基;对噬菌体展示随机12肽库采用配基亲和富集、阴性血清吸收的方法进行生物淘洗;ELISA鉴定筛选克隆的结合特性;测定阳性克隆所携带DNA序列并进行计算机辅助分析。结果三轮生物淘洗后特异性克隆得到了富集。用第3轮淘洗出的26个克隆进行结合实验,其中有3个克隆与HCV患者血清结合力较高而与正常人血清结合能力较弱;序列分析发现2个序列与HCV蛋白第1082～1093和1954～1965位(1082YHGAGTRTIASP 1093和1954TQLLRRLHQWIS 1965)氨基酸有较高的同源性;计算机辅助分析提示这两个位点具有形成表位的性质。结论获得了两个HCV抗原表位,在HCV感染的检测中具有潜在的应用价值。     

Cyclosporine A for the treatment of autoimmune disorders in HCV infected patients

Due to the relatively high prevalence of both HCV infection and autoimmune disorders (AD), it is not rare to encounter patients with AD also carrying HCV. Considering that the use in HCV infected individuals of corticosteroids or immunosuppressant drugs, that are indeed needed to treat AD, is considered a risk for worsening the clinical outcome of HCV infection, rheumatologist have often refrained from using these drugs in AD when HCV-RNA is also present. Cyclosporine (CsA) is an immunosuppressive agent used to treat a wide range of autoimmune disorders but there is in literature a large body of evidence suggesting that CsA also exerts an inhibitory effect on HCV replication at standard therapeutic dose. The anti-HCV effect of CsA has been demonstrated both in vitro and in vivo. Therefore, these evidences have opened new ways to improve the therapy and the prognosis in patients with HCV-related liver diseases including transplanted ones. Recent reports, although limited in number, also suggest the safety of CsA, in the treatment of patients with AD and concomitant HCV infection. Good results have also been obtained in the treatment in rheumatoid arthritis patients even in association with anti-TNF agents.     

Potential ultrastructure predicting factors for hepatocellular carcinoma in HCV infected patients

Hepatitis C virus represents one of the rising causes of hepatocellular carcinoma (HCC). Although the early diagnosis of HCC is vital for successful curative treatment, the majority of lesions are diagnosed in an irredeemable phase. This work deals with a comparative ultrastructural study of experimentally gradually induced HCC, surgically resected HCC, and potential premalignant lesions from HCV-infected patients, with the prospect to detect cellular criteria denoting premalignant transformation. Among the main detected pathological changes which are postulated to precede frank HCC: failure of normal hepatocyte regeneration with star shape clonal fragmentation, frequent elucidation of hepatic progenitor cells and Hering canals, hepatocytes of different electron density loaded with small sized rounded monotonous mitochondria, increase junctional complexes bordering bile canaliculi and in between hepatocyte membranes, abundant cellular proteinaceous material with hypertrophied or vesiculated rough endoplasmic reticulum (RER), sequestrated nucleus with proteinaceous granular material or hypertrophied RER, formation of lipolysosomes, large autophagosomes, and micro-vesicular fat deposition.

In conclusion, the present work has visualized new hepatocytic division or regenerative process that mimic splitting or clonal fragmentation that occurs in primitive creature. Also, new observations that may be of value or assist in predicting HCC and identifying the appropriate patient for surveillance have been reported. Moreover, it has pointed to the possible malignant potentiality of liver stem/progenitor cells.

For reliability, the results can be subjected to cohort longitudinal study.     

不同感染途径慢性丙型肝炎患者HCV基因型分布的差异

目的 探讨丙型肝炎病毒(HCV)基因型在中国部分城市输血与非输血途径感染者之间的分布。方法 对来自中国南北地区9个城市的慢性HCV肝炎患者的血清，用5′非编码区酶切分型方法进行HCV基因分型，分析HCV基因型在不同地区和感染途径之间的分布差异。结果 在219例慢性HCV肝炎血清中，有214例(97．7％)检测出HCV基因型，其中197例为单基因型HCV感染，主要的HCV流行株为1b(76．64％)和2a(18．22％)，并有5．14％的患者感染基因3b型，且首次在中国检测出4a型。HCV在中国南北地区城市的分布差异无显著意义，但输血感染者和非输血感染者之间的HCV基因型分布差异有显著意义，输血感染者中，93．88％为单基因型HCV感染，1b占76．87％，高于非输血途径感染患者中单基因型HCV感染百分率(86．57％)和1b的感染百分率(58．21％)，非输血感染者中的混合HCV基因型比例(13．43％)高于输血感染者(6．12％)。结论 中国南北部分地区的HCV基因型分布差异无显著意义，但经输血感染和非输血感染的慢性丙型肝炎患者之间的HCV基因型分布差异有显著意义。     

HCV患者外周血单个核细胞蛋白质表达质谱分析

目的:应用比较蛋白质组学的方法分析丙型肝炎患者外周血单个核细胞蛋白质表达模式的变化及寻找丙型肝炎相关生物标记分子,进一步识别鉴定其差异表达蛋白质,分析其对丙型肝炎慢性化机制的意义.方法:应用固相化pH梯度双向凝胶电泳(2-DE)分离健康者(10)及HCV患者(28)PBMC的总蛋白质,凝胶银染显色后,PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)获得差异蛋白点的肽质指纹图谱,通过SWISS-PROT数据库鉴定蛋白质.结果:得到两张2-DE图谱,HCV患者及健康者PBMC凝胶的蛋白质点数分别为625及614;初步筛选出HCV患者与健康者存在明显差异的12个蛋白点,经质谱分析,初步鉴定了10种蛋白质.这些差异蛋白质包括病毒蛋白、蛋白质合成与分解、三大代谢相关酶类、细胞结构相关蛋白质以及信号转导相关蛋白质.结论:应用2-DE及MALDI-TOF-MS方法建立了HCV患者PBMC双向凝胶电泳图谱,分离并初步鉴定了10种与HCV感染相关的差异表达的蛋白质,为研究HCV慢性化相关机制提供新的线索.     

Differences in HCV antibody patterns in haemodialysis patients infected with the same virus isolate

Eight cases of de novo hepatitis C virus (HCV) infection in a haemodialysis unit in Rio de Janeiro, Brazil, were retrospectively studied. HCV viraemia was demonstrated by RT nested PCR in seven of the seroconverters. Genotyping showed that six patients were infected with a genotype 1b strain and one with a genotype 1a strain. A phylogenetic analysis of nucleotide sequences of the HCV core region revealed that five of the six 1b isolates form a separate cluster when compared with other 38 HCV 1b core sequences randomly chosen from the GenBank. The revealed sequence similarities indicated the nosocomial spread of a single HCV strain within the unit. To investigate whether the patients infected with the same viral isolate display similar patterns of antibody response to individual proteins, serial serum samples were examined. A line immunoassay for qualitative and semi-quantitative determination of specific antibodies against recombinant and synthetic HCV antigens was employed. Despite infection with the same virus strain, the patients sera demonstrated different patterns of reactivity against individual structural and nonstructural HCV proteins immediately after seroconversion. For each patient, however, antibody responses remained mostly stable throughout the follow-up of 8 to 24 months.     

丙型肝炎病毒感染患者PBMC细胞因子的分泌水平

目的 :检测丙型肝炎病毒 (HCV)感染患者外周血单个核细胞(PBMC)体外培养后 ,培养上清中细胞因子分泌的水平 ,以反映HCV患者体内的免疫状况。方法 :患者PBMC培养上清中的细胞因子 (IL 2、IL 4、IL 10、IL 12、TNF γ、TNF α)采用ELISA进行检测。结果 :(1)HCV感染患者的PBMC培养 72h后 ,细胞因子检测结果表明 :与正常对照组相比较 ,HCV患者PBMC培养上清中TNF γ、IL 10和TNF α的水平明显升高 ,而没有检测到IL 2、IL 4、和IL 12的分泌产物。 (2 )轻、中度慢性肝炎 ,以及代偿期、失代偿期肝硬化患者间细胞因子的水平未见明显差异。结论 :(1)HCV感染患者体内细胞因子的分泌倾向于Th2型细胞因子占优势。 (2 )HCV感染患者PBMC分泌IL 2水平的降低可能是HCV免疫逃逸的原因之一     

MHC class II genes in HCV viral clearance of hepatitis C infected Hispanic patients

The frequency of class II human leukocyte antigen (HLA) alleles in 112 infected patients of Hispanic ancestry with serology positive for hepatitis C virus (HCV) was investigated. Our studies failed to demonstrate significant association between class II HLA alleles and the outcome of HCV infection: chronic viremia versus spontaneous viral clearance. Our results suggest that the genes responsible for the outcome are unknown, so far, and those HLA associations reported in several ethnic groups may represent genetic markers in nonrandom association with the responsible genes involved in determining viral clearance or chronic viremia following HCV infection.