原位杂交法检测外周血单个核细胞中HCV RNA

目的比较慢性丙型肝炎患者用干扰素治疗前以及治疗后３个月ＰＢＭＣ中ＨＣＶＲＮＡ。方法应用地高辛素标记ＨＣＶＲＮＡ正链及负链探针，建立原位杂交方法检测外周血单个核细胞（ＰＭＢＣ）中的ＨＣＶＲＮＡ。结果治疗前１９例患者正链ＨＣＶＲＮＡ阳性，８例负链ＨＣＶＲＮＡ阳性，用正链探针杂交在较多的细胞中出现杂交信号，负链探针杂交仅在少数细胞中出现杂交信号，ＨＣＶＲＮＡ在ＰＢＭＣ胞浆中呈均质性分布。用干扰素治疗结束后３个月２０例患者中９例ＨＣＶＲＮＡ转阴性，近期治愈率４５％。结论原位杂交技术的敏感性及特异性较高，且重复性较好，是研究ＨＣＶＲＮＡ在组织中定位分布和病毒复制场所一种切实可行的方法     

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     

周围血白细胞的复制型丙型肝炎病毒RNA的检测及临床意义

用简并引物作套式反转录。聚合酶链反应检测正、负链丙型肝炎病毒（ＨＣＶ）ＲＮＡ。显示３０例急、慢性丙型肝炎患者的血清及血浆中，７例无症状抗－ＨＣＶ阳性者的血清、血浆及周围血白细胞（ＰＢＬ／Ｃ）中，均未检出负链ＨＣＶＲＮＡ。慢性丙型肝炎者ＰＢＩ（中正、负链ＨＣＶＲＮＡ的检出率高于急性丙型肝炎及无症状抗－ＨＣＶ阳性者（Ｐ＜０．０５～０．００１）。１７例经肝组织学检查的患者中，急性肝炎（ＡＨ）者ＰＢＬ／Ｃ的正、负链ＨＣＶＲＮＡ检出率低于慢性活动性肝炎（ＣＡＨ）者（Ｐ＜０．０５）。１例ＡＨ及６例ＣＡＨ患者肝组织内正、负链ＨＣＶＲＮＡ全部阳性。证实丙型肝炎患者的ＰＢＬ／Ｃ确可被ＨＣＶ感染，病程越长，被ＨＣＶ感染的可能性越大；病情活动者，ＰＢＬ／Ｃ中负链ＨＣＶＲＮＡ的检出率越高。提示ＨＣＶ不仅可以感染ＰＢＬＣ，而且可在其中复制；负链ＨＣＶＲＮＡ的出现与病情活动有关。     

不同外周血标本中HCV RNA检出比较

对５４例抗－ＨＣＶ阳性患者，用多聚酶链反应（ＰＣＲ）技术检测其外周血不同检材（血清、血浆、外周血单核细胞）中ＨＣＶ ＲＮＡ，结果检出率分别为５９．３％、５０％、５５．６％，以血清标本检出率最高。外周血单核细胞（ＰＢＭＣ）中亦有较高的检出率，且在部分（６％）血清测不到ＨＣＮ ＲＮＡ时，ＰＢＭＣ中可测及，提示ＰＢＭＣ可贮存ＨＣＶ ＲＮＡ。     

肝癌组织中丙型和乙型肝炎病毒基因状况研究

采用逆转录巢式ＰＣＲ技术检测了５１例ＨＣＣ患者的肝癌及癌周肝组织中ＨＣＶＲＮＡ正负链，同时以巢式ＰＣＲ技术检测了这些组织中的ＨＢＶＤＮＡ。结果，１１．８％（６／５１）检出组织中ＨＣＶＲＮＡ正链：其中５例在癌周肝组织，２例在癌组织中检测出ＨＣＶＲＮＡ负链，由于负链ＲＮＡ为ＨＣＶ的复制中间体，不释放到细胞外，其检出ＨＣＶ感染与ＨＣＣ关系的研究提供了进一步的病因证据，组织中ＨＢＶＤＮＡ检出率高达９２．２     

逆转录－套式－聚合酶链反应检测HCV RNA正链和负链及与肝细胞癌的关系

为了解ＨＣＶ与肝癌的关系，采用ＨＣＶ５－ＮＣＲ正相引物和反相引物逆转录－套式－聚合酶链反应（ＲＴ－Ｎｅｓｔｅｄ－ＰＣＲ）检测了２２例肝癌手术患者血清、癌组织和癌旁组织ＨＣＶＲＮＡ正链和负链；用ＨＢＶＣ保守区引物ＰＣＲ法检测了ＨＢＶＤＮＡ。结果发现：５例抗－ＨＣＶ阳性的肝癌患者中，３例血清、癌组织和癌旁组织ＨＣＶＲＮＡ正链扩增试验均阳性；１例三者均阴性；１例血清和癌旁组织阳性。３例正链扩增试验阳性的癌组织和癌旁组织均检出了ＨＣＶＲＮＡ负链，但血清中未检出负链。在２２例ＨＣＣ中１３例血清、８例癌组织和１５例癌旁组织中检出ＨＢＶＤＮＡ。５例有ＨＣＶＲＮＡ和／或抗－ＨＣＶ阳性的肝癌患者血清、和／或癌组织、和／或癌旁组织中均伴随ＨＢＶＤＮＡ和／或ＨＢ－ＶＭ阳性。提示ＨＣＶ可在癌旁肝细胞或肝癌细胞中感染并复制；ＨＢＶ感染与肝癌发生的关系比ＨＣＶ感染更密切。     

肝细胞癌组织中HCV复制中间体的研究

本文报道在逆转录反应时仅加入与负链（复制中间体）ＨＣＶＲＮＡ互补的有义外引物，待将逆转录酶变性灭活后，再加入反义外引物，然后进行两轮聚合酶链反应（ＰＣＲ）扩增。用这种逆转录反应和套式ＰＣＲ方法，检测了ＨＣＣ组织和对照血清中负链ＨＣＶＲＮＡ的存在状况。结果负链ＨＣＶＲＮＡ在１０例（其中３例为已测血清总ＨＣＶＲＮＡ阳性，余７例为随机选择）ＨＣＣ组织中检出５例，而在５例（其中３例与组织配对）总ＨＣＶＲＮＡ的ＨＣＣ阳性病人血清则均阴性。表明ＨＣＣ组织中确有ＨＣＶ复制，从而进一步提示ＨＣＶ感染与我国ＨＣＣ的发生密切相关。     

地高辛标记原位杂交技术检测骨髓单个细胞中HCV RNA

为探讨丙型肝炎病毒（ＨＣＶ）肝外感染、复制场所与肝损害的关系，应用地高辛标记ＨＣＶ ＲＮＡ探针原位杂交技术，对２０例慢性丙型肝炎患者骨髓单个核细胞中的ＨＣＶ ＲＮＡ（包括正边ＲＮＡ和负链ＲＮＡ）进行了检测。结果有１８例骨髓单个核细胞用正链ＨＣＶ ＲＮＡ探针杂交在较多细胞中出现杂交信号（９０％），而所有患者骨髓竟单个核细胞用负链ＨＣＶ ＲＮＡ探针杂交无杂交信号出现。结果提示：慢性丙肝患者骨髓中存在的     

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。     

干扰素联合病毒唑对肝炎患者正,负链丙型肝炎病毒RNA的影响

为评价干扰素联合病毒唑的抗病毒效果，用套式反转录－聚合酶链反应技术观察其对慢性丙型肝炎患者正、负链丙型肝炎病毒ＲＮＡ（ＨＣＶＲＮＡ）的作用。结果显示，在对联合抗病毒治疗有反应的患者中，多数患者ＡＬＴ降至正常，血清正链阳性率由９２．３１％降至３８．４６％（Ｐ＜０．００５），周围血单核细胞（ＰＢＭＣ）中负链阳性率由７６．９２％降至３８．４６％（Ｐ＜０．０５），但对ＰＢＭＣ中正链ＨＣＶＲＮＡ影响不大。治疗停止后，半数以上患者病情复发，病情复发者均为ＰＢＭＣ中正链及负链ＨＣＶＲＮＡ持续阳性者。提示单纯检测血清正链ＨＣＶＲＮＡ作为疗效考核及病情转归的判断指标有一定的局限性。同时检测血清及ＰＢＭＣ中正、负链ＨＣＶＲＮＡ，具有重要的临床意义。本研究干扰素联合病毒唑的抗病毒效果与单用干扰素接近，似说明病毒唑并未增加干扰素的疗效     

Detection of replicative form of hepatitis C virus RNA in peripheral blood mononuclear cells.

Paired samples of plasma and peripheral blood mononuclear cells (PBMC) from 7 patients with posttransfusion hepatitis C were collected and studied for the presence of replicative forms of hepatitis C virus (HCV). RNA was extracted and tested for both positive and negative strands of HCV RNA by polymerase chain reaction. Positive-strand RNA of HCV was found in both plasma and PBMC of all 7 patients. However, negative-strand RNA was found in PBMC of 3 patients while none was found in the plasma. Levels of negative-strand RNA were about 10-100 times less than those of positive-strand RNA by semiquantification with serial dilution of viral cDNA. The results suggest that active infection and replication of HCV in PBMC is present in patients with posttransfusion hepatitis C and implicate the extrahepatic infection of HCV.     

Hepatitis C virus RNA in peripheral blood mononuclear cells: Comparing acute and chronic hepatitis C virus infection

Hepatitis C virus (HCV) can infect peripheral blood mononuclear cells (PBMC) of patients with chronic HCV infection. No data are available on PBMC testing for HCV RNA in acute hepatitis C. This study investigated the presence of HCV RNA in PBMC of patients with acute posttransfusion hepatitis C, compared with those with chronic HCV infection. Nested polymerase chain reaction (PCR) was applied to detect HCV RNA in 111 and 48 paired samples of serum and PBMC of 11 patients with acute posttransfusion hepatitis C and 48 patients with chronic HCV infection, respectively. In patients with acute posttransfusion hepatitis C, HCV RNA was detected in 17 of 29 (59%) and 67 of 82 (82%) serum samples collected during the incubation period and acute phase, respectively. Meanwhile, of the 48 patients with chronic HCV infection, 41 had serum HCV RNA (85%). HCV RNA was not detected in PBMC samples from incubation period or from acute-phase hepatitis, although it was detected in 12 of the 48 PBMC samples of chronically infected patients (25%) P < .005). Of the 12 PBMC specimens positive for positive-stranded HCV RNA, 6 were also positive for negative-stranded HCV RNA. Among patients with chronic HCV infection, HCV infection of PBMC was not related to age, sex, blood transfusion, serum alanine transaminase (ALT) levels, or serum virus titers. In conclusion, HCV infection of PBMC rarely exists in patients with acute hepatitis C. As HCV infection persists, the incidence of HCV infection of PBMC becomes higher. (Hepatology 1996 May;23(5):977-81)     

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     

慢性丙型肝炎患者外周血单个核细胞中负链HCV RNA的检测及意义

19例临床诊断慢性丙型肝炎患者,检测其外周血单个核细胞中丙型肝炎病毒(HCV)RNA的存在及复制状态。所选检测对象血清标本抗-HCV及HCV RNA均为阳性。采用逆转录-套式PCR法检测其外周血单个核细胞中HCV正、负链RNA,结果19例患者外周血单个核细胞中14例HCV正链RNA阳性;6例HCV负链RNA阳性。实验结果证实部分慢性丙型肝炎患者外周血单个核细胞中存在HCV复制,表明肝细胞并非为HCV感染与复制的唯一场所。HCV感染慢性化与其外周血单个核细胞中存在HCV复制可能有一定关系。     

Hepatitis C virus is detected in a monocyte/macrophage subpopulation of peripheral blood mononuclear cells of infected patients.

Hepatitis C virus (HCV) is the primary agent of posttransfusion non-A, non-B hepatitis. HCV RNA was detected in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction in 17 of 24 HCV-infected patients with chronic hepatitis with or without cirrhosis. One of 5 patients whose PBMC contained HCV RNA also had negative-stranded HCV RNA in the PBMC. In 3 of 11 patients whose PBMC contained HCV RNA, flow cytometry with a murine monoclonal antibody to HCV core epitope revealed cytoplasmic staining of peripheral blood monocytes. The monocyte surface and the peripheral blood lymphocytes did not stain for HCV core epitopes. No correlation could be made between the presence of HCV RNA or antigen in PBMC and any serologic markers of HCV infection. These results indicate that monocyte uptake of HCV by either phagocytosis or infection may be part of the pathophysiology of this chronic disease.     

Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C

ＨｅｐａｔｉｔｉｓＣｖｉｒｕｓＲＮＡｄｅｔｅｃｔｉｏｎｉｎｓｅｒｕｍａｎｄｐｅｒｉｐｈｅｒａｌｂｌｏｏｄｍｏｎｏｎｕｃｌｅａｒｃｅｌｓｏｆｐａｔｉｅｎｔｓｗｉｔｈｈｅｐａｔｉｔｉｓＣＺＨＯＵＰｉｎｇ，ＣＡＩＱｉｎｇ，ＣＨＥＮＹｏｕＣｈｕｎ，ＺＨＡ...     

Improved Serodiagnosis of Posttransfusion Hepatitis C Virus Infection by a Second-Generation Immunoassay Based on Multiple Recombinant Antigens

Serial serum samples from 35 patients with posttransfusion non-A, non-B hepatitis in a prospective study were tested for antibody to hepatitis C virus (HCV) by a multiple recombinant antigen based immunoassay [anti-HCV (2nd); Abbott]. Of them, 23 were positive for anti-C100, and 27 were positive for HCV RNA by polymerase chain reaction. By anti-HCV (2nd), 28 patients were positive, and all except 1 of the 28 patients were positive for HCV RNA. A total of 24 patients, who became HCV RNA positive at the acute stage and who were negative for both anti-C100 and anti-HCV (2nd) before transfusion, were considered to have posttransfusion hepatitis C. The mean time to seroconversion for anti-HCV (2nd) was 7.5 or 12.1 weeks after the onset of hepatitis or the date of transfusion, respectively, and was generally 6 weeks earlier than that detected by anti-C100. However, seroconversion was delayed in 2 hepatitis B surface antigen carriers as compared with anti-C100. The anti-C100 assay detected 20 (83%) and the anti-HCV (2nd) all 24 patients with documented posttransfusion hepatitis C. The second-generation test is, therefore, better than conventional anti-C100 for the early diagnosis of HCV infection.     

Testing for hepatitis C virus sequences in peripheral blood mononuclear cells of patients with chronic hepatitis C in the absence of serum hepatitis C virus RNA

Hepatitis C virus (HCV) is able to replicate in peripheral blood mononuclear cells (PBMC) of HCV-infected patients. Few data are available on PBMC testing for HCV RNA in serum HCV RNA negative patients, positive for anti-HCV and with histological evidence of chronic hepatitis. Twenty such patients were studied; of these, 11 were tested during interferon α (IFN) treatment, at the time of serum HCV RNA clearance and ALT normalisation: only one was found to be positive for HCV sequences in PBMC. Within 3 months of IFN withdrawal all 11 patients relapsed with high ALT and recurrence of serum HCV RNA. Of nine serum HCV RNA negative patients with chronic hepatitis C who were not receiving IFN when tested (four untreated patients and five patients who had already completed IFN schedule), PBMC HCV RNA was detected in four. Evidence of active HCV replication (presence of the minus strand genome) in PBMC was also observed in two cases. Thus, five of the 20 patients without detectable serum HCV RNA turned out to be carriers of HCV sequences in PBMC. These data indicate that: 1. PBMC are an extrahepatic replication site of HCV; this is true also in the absence of serum HCV RNA; 2. the role of PBMC as a “viral reservoir” after IFN-induced serum HCV RNA clearance is questioned; 3. the absence of both serum and PBMC HCV RNA in patients under IFN is not predictive of sustained viral loss; 4. testing for PBMC viral sequences might enhance the chances of detecting HCV infection.     

输血后丙型肝炎病毒感染的血清病毒定量研究

目的 研究血清丙型肝炎病毒（ＨＣＶ）含量与ＨＣＶ致病的关系及ＨＣＶ含量与抗－ＨＣＶ和丙氨酸转氨酶（ＡＬＴ）的相关性。方法 以逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）法对ＨＣＶ感染的受血及相关供血系列血清进行ＨＣＶ ＲＮＡ定量分析,同时检测ＡＬＴ与抗－ＨＣＶ。结果 致输血后ＨＣＶ感染的供血中,ＨＣＶ ＲＮＡ平均含量为１０＾８．６拷贝／Ｌ,抗－ＨＣＶ及ＡＬＴ的异常检出率随ＨＣＶ ＲＮＡ滴度升高而增加。结论     

丙型肝炎病毒母婴传播的研究

目的研究丙型肝炎病毒母婴传播的可能性与机率。方法采用前瞻性研究方法，使用ＰＣＲ技术检测ＨＣＶＲＮＡ，用二代ＥＩＡ试剂检测抗－ＨＣＶ。结果１３例输血后丙型肝炎母亲和她们所生的１５例婴儿，婴儿ＨＣＶ感染率为８６．７％，其中临床型１例（７．７％），亚临床型ＨＣ３例（２３．１％），ＨＣＶ隐性感染９例（６９．２％）。抗－ＨＣＶ和ＨＣＶＲＮＡ阳性率均随年龄增长而降低，至３岁时分别为７．７％和１５．４％。结论感染ＨＣＶ母亲所生婴儿，ＨＣＶ感染率高，但转归较好。观察引产胎儿肝脏和血液发现，孕妇抗－ＨＣＶ和ＨＣＶＲＮＡ阳性所引产的胎儿血清和／或肝脏中抗－ＨＣＶ和ＨＣＶＲＮＡ阳性，ＨＣＶ的母婴传播，主要发生于子宫内。