丙型肝炎肝组织中HCV RNA和HCV NS5抗原表达及意义

目的研究丙型肝炎肝组织中ＨＣＶＲＮＡ与ＨＣＶＮＳ５抗原分布及其与肝组织损伤的关系．方法应用原位杂交和免疫组化方法检测３４例丙型肝炎病毒（ＨＣＶ）感染者肝组织中ＨＣＶＲＮＡ和ＨＣＶＮＳ５抗原表达状况,采用Ｋｎｏｄｅｌ方法评价肝组织病理损伤程度．结果分别有２１例（６１８％）和２４例（７０６％）患者肝组织中检出ＨＣＶＲＮＡ和ＨＣＶＮＳ５抗原．ＨＣＶＲＮＡ阳性信号主要位于肝细胞浆中,在胆管上皮细胞和炎性浸润细胞中也可检出,其分布与肝组织炎症坏死无固定解剖学关系．ＨＣＶＲＮＡ与肝组织中ＨＣＶＮＳ５抗原表达正相关,但二者在肝组织中的分布存在一定差异．多因素分析显示ＨＣＶＮＳ５抗原的表达与肝组织损伤相关．结论丙型肝炎肝组织中ＨＣＶＲＮＡ和ＨＣＶＮＳ５抗原表达相关,病毒在肝组织中的活跃程度与肝损伤有关     

丙型肝炎病毒感染后肾小球肾炎

目的探讨丙型肝炎病毒（ＨＣＶ）感染与肾小球肾炎病变的关系。方法应用免疫组化技术以丙型肝炎病毒抗体（抗ＨＣＶ）ＮＳ３和抗ＨＣＶＮＳ５单克隆抗体对２１例丙型肝炎患者尸检石蜡包埋肾组织中的丙型肝炎抗原（ＨＣＡｇ）进行检测。结果ＨＣＡｇ的检出率为６１９％（１３／２１），其中膜增殖性肾炎（ＭＰＧＮ）１１例，系膜增殖性肾炎（ＭｓＰＧＮ）１例，１例未见明显病变。ＨＣＡｇ阳性颗粒定位于肾小球系膜区、系膜细胞及肾小管上皮细胞胞浆内。２１例中有１７例血清乙型肝炎病毒（ＨＢＶ）标志呈阳性，同单纯ＨＢＶ感染组相比，本组患者出现肾小球肾炎病变更为多见（χ２＝８５，Ｐ＜００１）。结论ＭＰＧＮ型是ＨＣＶ感染后肾小球肾炎病变的主要类型。     

庚型肝炎病毒在慢性肝炎肝硬化中致病性及病理特征

探讨ＨＢＶ在慢性肝炎肼硬化当中感染率、临床脑病理特征,应用ＥＬＩＳＡ法检测血清抗－ＨＧＶ,用ＨＧＶＮＳ５抗原制备ＭＣＡｂ,检测５４例慢笥肝炎硬化患者肝组织中ＨＧＶ抗原,同时应用ＳＰ法分别对肝组织进行ＨＢｓＡｇ、ＨＢｃＡｇ、ＨＣＶ ＮＳ３Ａｇ肝脏免疫组化检测。结果提示５４例肝病患者肝组织检出２７．７８％ＨＧＶ抗原阳性。其中２４例慢性肝炎轻度中５例;１６例慢性肝炎中度中５２称;１５例肝硬中５例。ＨＧＶ     

66例慢性非甲非乙型肝炎患者的丙型肝炎病毒感染研究

以ＨＣＶ－Ｔ３序列为引物，结合ＲＴ－ＰＣＲ和寡聚核甘酸探针Ｓｏｕｔｈｅｒｎ杂交，检测６６例慢性非甲非乙型肝炎（ＮＡＮＢＨ）患者的血浆ＨＣＶ－ＲＮＡ，阳性４２例（６３．６％）。同样病例以相当于ＨＣＶＣ区基因编码和ＮＳ３区编码的人工合成肽抗原检测抗ＨＣＶ，阳性４９例（７４．２％）。这６６例慢性ＮＡＮＢＨ病例，抗ＨＣＶ和ＨＣＶ－ＲＮＡ双阳性者３８例（５７．６％）；抗ＨＣＶ阴性而ＨＣＶ－ＲＮＡ阳性者４例（６．１％）；抗ＨＣＶ阳性而ＨＣＶ－ＲＮＡ阴性者１１例（１６．７％）。其中诊断为散发型ＮＡＮＢＨ者３５例，检出ＨＣＶ－ＲＮＡ者１７例（４８．６％），为输血后ＮＡＮＢＨ者３１例，检出ＨＣＶ－ＲＮＡ者２５例（８０．７％）。     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒检测及其意义

目的 探讨外周血单个核细胞（ＰＢＭＣｓ）在丙型肝炎病毒（ＨＣＶ）的感染中的作用。方法 对２２例慢性丙型肝炎患者２１例抗－ＨＣＶ（＋）血液管析患者及１２例健康献血员的ＰＢＭＣｓ分别进行ＨＣＶＲＮＡ,ＨＣＶ抗原检测及电镜观察。结果 （１）２２生丙型肝炎肝炎患者ＰＢＭＣｓ中有７７．３％（１７／２２）ＨＣＶＲＮＡ阳性,（２）感染ＨＣＶ的ＰＢＭＣｓ中电镜下发现复制的ＨＣＶ颗粒;（３）ＨＣＶ颗粒阳笥者的血清和     

外周血单核细胞中丙型肝炎病毒RNA正负链检测的临床意义

目的通过对外周血单核细胞（ＰＢＭＣ）中ＨＣＶＲＮＡ正负链的检测,来探讨其与丙型肝炎慢性化及干扰素治疗的关系．方法慢性丙型肝炎患者４０例,其中干扰素治疗者１０例,分离血清及ＰＢＭＣ．异硫氰酸胍－酚－氯仿抽提法提取ＨＣＶＲＮＡ,应用逆转录－巢式ＰＣＲ技术检测ＨＣＶＲＮＡ正负链．结果血清正链ＨＣＶＲＮＡ阳性率为６７５％,但负链均为阴性,ＰＢＭＣ中正链ＨＣＶＲＮＡ阳性率为５７５％,负链的阳性率为３５０％．其中３例患者血清中正链ＨＣＶＲＮＡ为阴性,而ＰＢＭＣ中为阳性．１０例干扰素治疗者在治疗结束时血清正链ＨＣＶＲＮＡ６０％转阴,ＰＢＭＣ中负链ＨＣＶＲＮＡ８０％转阴,而正链仅３７５％转阴．结论ＨＣＶ能在ＰＢＭＣ中存在和复制,这可能是导致丙型肝炎易发生慢性化的原因之一．ＰＢＭＣ中ＨＣＶＲＮＡ正负链的检测对于临床判断干扰素的疗效及预后有重要意义     

肝组织内丙型肝炎病毒不同抗原的定位及比较

目的研究ＨＣＶ不同抗原的组织内定位以及与病变和血ＨＣＶＲＮＡ的关系。方法５１份慢性丙型肝炎肝穿刺组织,免疫组化检测ＨＣＶＣＰ１０、ＮＳ３和ＮＳ４,其中３２份还检测ＮＳ５,同时观察组织学病变和血ＨＣＶＲＮＡ（ＲＴＰＣＲ法）。结果ＨＣＡｇ总阳性率＞６０％。不同抗原阳性率不一,以ＣＰ１０和ＮＳ５为高（各＞５０％）。阳性细胞数一般较少,散在分布;灶性集聚时,多与碎屑坏死及炎症反应相伴。几种抗原全阳性者组织病变活动度较高。血ＨＣＶＲＮＡ阳性率８０．３９％,但血与组织检测结果的重合性仅占５０．９８％;血阴性者,８／１０份组织内检出１或多种抗原。结论组织内同时检测多种抗原能提高ＨＣＡｇ检出率。     

丙型肝炎病毒感染与膜性肾病及膜增殖性肾炎的研究

目的：了解我国膜性肾病（ＭＮ），膜增殖性肾炎（ＭＰＧＮ）的丙型肝炎病毒（ＨＣＶ）感染率，ＨＣＶ感染与冷球蛋白血症及肾脏病关系，以期阐明ＨＣＶ在ＭＮ及ＭＰＧＮ中的作用。方法：前瞻性观察４６例健康工作人员以及１９９６年５月至１９９７年１１月期间６７例ＭＮ１２例ＨＰＧＮ患者血清ＨＣＶ抗体ＨＣＶ－ＲＮＡ的阳性率，血清冷球蛋白浓度及冷沉淀物的中的ＨＣＶ抗体及ＨＣＶ－ＲＮＡ阳性率，并结合肾组织ＨＣＶ抗原沉积特     

丙型肝炎患者血清和外周血单个核细胞中HCV RNA检测及意义

目的 了解丙肝患者血清和外周血单个核细胞（ＰＢＭＣ）中ＨＣＶＲＮＡ存在情况及有其临床意义，方法 应用套式ＰＣＲ检测４６例急性丙型肝炎（丙型肝炎以下简称丙肝）和４２例慢性丙型肝炎患者血清和ＰＢＭＣ中ＨＣＶＲＮＡ。结果 慢性丙型肝炎患者ＰＢＭＣ中ＨＣＶＲＮＡ检出率显著高于急性丙型患者（Ｐ〈０．００１），急，慢性丙型肝患者血清和慢性丙肝患者ＰＢＭＣ中ＨＣＶ ＲＮＡ检出率显著高于ＡＬＴ正常的抗－ＨＣＶ阳性     

丙型病毒性肝炎患者外周血单核细胞中HCV—RNA的存在及其意义

作者用套式ＰＣＲ检测２５例丙型病毒性肝炎（丙肝）患者血清和外周血单核细胞（ＰＢＭＣ）中ＨＣＶ－ＲＮＡ，结果显示血清阳性２１例，ＰＢＭＣ阳性１８例，表现在大部分丙肝患者ＰＢＭＣ中有ＨＣＶ－ＲＮＡ的存在。且急性丙肝患者ＰＢＭＣ中ＨＣＶ－ＲＮＡ阳性率明显低于慢性丙肝患者，提示ＰＢＭＣ中ＨＣＶ－ＲＮＡ的存在可能与感染ＨＣＶ后的慢性化有关。     

丙型肝炎患者胃粘膜细胞中HCV NS3、NS5抗原定位研究

目的了解丙型肝炎病毒（ＨＣＶ）在胃粘膜细胞中的感染状况。方法采用免疫组化Ｓ－Ｐ法以抗－ＨＣＶＮＳ３和抗－ＨＣＶＮＳ５检测了３３例慢性丙型肝炎（ＣＨＣ）患者胃粘膜组织中ＨＣＶＮＳ３及ＨＣＶＮＳ５抗原。结果ＨＣＶＮＳ３、ＨＣＶＮＳ５的检出率分别为３３．３３％（１１／３３）和２７．２７％（９／３３）。阳性物质沉积于胃粘膜上皮腺体细胞胞浆，呈单个或片灶状分布，部分病例淋巴细胞胞浆中也可见到。结论胃粘膜细胞中ＨＣＶ抗原的检出，表明ＨＣＶ可能是一种泛嗜性病毒，其机制及生物学意义须进一步研究。     

HCV replication in PBMC and its influence on interferon therapy

AIM: To study hepatic virus C (HCV) RNA and HCV protein expression in peripheral blood mononuclear cells (PBMCs) of patients with HCV infection, and explore the relationship between the HCV RNA in the PBMCs and response to interferon (IFN) therapy. METHODS: Type-specific primers were designed and RT-nested PCR was used to detect the plus- and minus- strands of HCV RNA in PBMCs of 54 patients with HCV infection; Indirect immunofluorescence assay was applied to identify HCVNS5 protein expression in PBMCs; 6 month-, 3 MU-IFN regiment was administrated to observe the responses to IFN in 35 chronic hepatitis C patients with different HCV RNA status in PBMCs. RESULTS: HCV plus strand RNA was found in 10 of 19 (52.6 %) acute hepatitis C patients and 22 of 35 (62.9 %) chronic hepatitis C patients. HCV minus strand RNA was detected in 14 of 35 (40.0 %) chronic hepatitis C patients, but only one patient (5.3 %) with acute HCV infection was found to be minus HCV RNA positive. Though no HCV NS5 protein expression was found in the examined 10 cases of acute HCV infection, it was positive in 17 of 20 (85.0 %) chronic hepatitis C patients by indirect immunofluorescence assay. There are significant differences of positive rate of the minus-strand and HCVNS5 protein between acute and chronic hepatitis C groups (u=2.07, P<0.05 and u=4.43, P<0.01 respectively). The patients with minus-strand HCV RNA showed a significantly lower 6-month sustained response (SR-6) to IFN compared to those without minus-strand HCVRNA in PBMCs (biologically 14.3 % vs 42.8 %, chi(2)=4.12, P<0.05 and virologically 7.1 % vs 23.9 %, chi(2)=4.24, P<0.05). CONCLUSION: HCV is capable of infecting and replicating in PBMCs, and HCVNS5 protein was expressed in PBMCs. The patients with minus strand HCV RNA in PBMCs showed a significantly lower 6-month sustained response to IFN, suggesting that minus-strand HCV RNA in PBMCs may be one of the factors influencing response to IFN therapy.     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒感染研究

目的 以分子生物学、免疫学有电镜等技术对慢性丙型肝炎患者ＰＢＭＣｓ进行综合检测,以证实其受ＨＣＶ感染,并试图在电镜下发现和证实感染细胞内ＨＣＶ颗粒。方法 对逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和免疫组织化学等技术分别检测患者ＰＢＭＣｓ内的ＨＣＶ ＲＮＡ和ＨＣＶＡｇ,以常规和免疫电镜技术观察研究感染细胞内ＨＣＶ的形态、定位和超微结构。结果 ＨＣＶ ＲＮＡ阳性检出率为７７．２％（１７／２２）,ＨＣＶＡｇ     

初步探讨丙型肝炎病毒体外感染的致病机理

目的:初步探讨原代培养肝细胞对HCV的易感性,并在此基础上观察HCV感染后肝细胞形态学的变化。方法:用HCV RNA阳性血清与培养肝细胞共同孵育后,收集不同时相点标本,以逆转录聚合酶链反应(RT-PCR)、分别细胞内或上清中正负链RNA,免疫组化检测细胞内HCV NS3、NS5抗原的表达以及原位杂交检测细胞内HCV负链RNA,同时在电镜下观察细胞形态学变化。结果:接种感染血清后第3～5d,即可在细胞内或培养上清中检出HCV正负链RNA;感染肝细胞内可见HCV NS3、NS5抗原表达,阳性物质位于胞浆中;原位杂交法证实细胞内存在负链RNA,也位于胞浆中;光镜下未发现感染细胞形态学变化,电镜下发现胞浆内含有大量的脂肪空泡、多泡体,并可见疑似病毒样结构。结论:HCV不但能感染树鼩肝细胞,而且在体外能直接致细胞病变。     

T-cell antigenic determinants within hepatitis C virus nonstructural protein 3 and cytokine production profiles in hepatitis C

summary . The aim of this study was to further investigate the role of T-helper cells in hepatitis C virus (HCV) infection, focusing on the T-cell antigenic determinants and cytokine profiles of nonstructural 3 (NS3) protein-stimulated peripheral blood mononuclear cells (PBMCs) of HCV patients. A total of 12 recombinant proteins of theNS3 region were purified and used to test T-cell proliferative response and antigenic determinants of HCV-seropositive patients. In addition, cytokines produced by antigen stimulated PBMCs were measured. Our data showed that PBMCs from 55.7% (34/61) of HCV patients proliferated to at least one antigen, but PBMCs of HCV seronegative patients did not. In addition, PBMCs from about 82.0% (32/39) HCV-seropositive patients produced significant amounts of cytokines (10 pg/mL). Interestingly, PBMCs from 66% of patients produced TH₂-related cytokines such as interleukin (IL)-4 and IL-5. In mappingexperiments, the data showed multiple T-cell antigenic determinants. Our data demonstrated that NS3 antigen-stimulated PBMCs of HCV patients recognized multiple T-cell antigenic determinants and produced significant amounts of TH₀ or TH₂-related cytokines, which might play a critical role in the chronicity of HCV infection.     

实时荧光定量RT-PCR检测血清和单个核细胞中HCV RNA的意义

目的探讨实时荧光定量聚合酶链反应(FQ-RT-PCR)检测血清和外周血单个核细胞(PBMCs)中HCV RNA含量及其临床价值。方法采集可疑丙型病毒性肝炎病人血,用FQ-RT-PCR分别检测血清和PBMCs中HCV RNA含量。血清或PBMCS HCV RNA阳性者都视为HCV RNA阳性。结果检测可疑患者180例,HCV RNA阳性106例。其中血清HCV RNA阳性84例,占79.25%;PBMCs HCV RNA阳性63例,占59.43%。HCV RNA阳性比率血清高于PBMCs(χ2=9.78,P<0.01)。单独血清HCV RNA阳性43例,占40.57%;单独PBMCs HCV RNA阳性22例,占20.75%;血清和PBMCs中HCV RNA同时阳性41例,占38.68%。血清和PBMCs HCV RNA含量差异无显著性。结论同时检测血清和PBMCs HCV RNA可提高HCV感染诊断的阳性率,检测PBMCs HCV RNA对抗病毒治疗的疗效评价及治疗时间有重要意义。     

原位反转录PCR检测慢性丙型肝炎患者外周血单个核细胞中HCV

目的研究慢性丙型肝炎患者外周血单个核细胞（ｐｅｒｉｐｈｅｒａｌｂｌｏｏｄｍｏｎｏｎｕｃｌｅａｒｃｅｌ，ＰＢＭＣ）中丙型肝炎病毒的定位分布及复制。方法直接原位反转录－ＰＣＲ（标记引物）对２０例慢性丙型肝炎患者的ＰＢＭＣ内丙型肝炎病毒（ＨＣＶ）的分布、定位及存在形式进行研究。结果１６例患者外周血单个核细胞的胞浆内发现ＨＣＶＲＮＡ阳性信号，并有一例患者的胞浆内发现ＨＣＶＲＮＡ负链信号。结论ＨＣＶ可感染ＰＢＭＣ并在其中复制。原位ＲＴ－ＰＣＲ为进一步研究ＨＣＶ进入人体内的分布、潜伏、复制状况以及丙型肝炎治疗、药物研究奠定了基础。     

Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology.

A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or delta, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver cell membrane.