HBV在慢性乙型肝炎患者肝细胞染色体上整合情况的临床分析

采用荧光原位杂交(FISH)技术检测慢性乙型肝炎患者肝细胞染色体,明确是否存在HBV的整合及整合情况。选择我院慢性乙型肝炎患者共16例,制备其肝细胞染色体标本,以地高辛标记的HBV探针进行原位杂交。16例患者中,有5例患者的肝细胞分裂期染色体上可观察到绿色荧光信号,占31.25%,HBV呈低频率整合(1.00%~1.50%),整合位点呈随机分布。通过研究发现,应用FISH技术可检测肝细胞染色体上整合型HBV。慢性乙型肝炎患者肝细胞染色体上存在整合型HBV,使HBV感染可能发生成为一种遗传性传染性疾病,并成为肝癌高发的重要危险因素。     

在原发性胆管上皮细胞性肝癌细胞中检出HBV DNA和HDV RNA

利用地高辛配基标记的HBV DNA及HDVcDNA探针,原位杂交检测了一例胆管细胞性肝癌及癌旁肝组织中的HBV DNA和HDV RNA。在癌巢及癌旁肝组织中均检出HBV DNA及HDV RNA阳性细胞,以核型为主。癌巢中阳性细胞数及阳性信号强度明显高于癌旁肝组织。经阴性对照、空白对照及DNase I,RNase A处理试验,证实阳性信号为HBV DNA或HDV RNA产生。进一步用逆转录一聚合酶链反应(RT-PCR)检出了HDV RNA,确证了HDV感染的存在。     

肝细胞癌和肝硬变组织中突变型P53蛋白的表达及与HBV DNA的关系

应用LSAB免疫组化技术,检测肝内突变型P53蛋白的表达;用地高辛探针原位杂交检测HBV DNA。结果显示,31例肝细胞癌(HCC)及其癌旁组织中突变型P53蛋白阳性率分别为45％和48％,而11例肝硬变中检出4例(36.4％)。HBV DNA阳性率分别为46％和86％。癌旁组织中突变型P53蛋白表达与HBV DNA存在相关关系(P<0.05)。提示P53基因突变与HBV慢性感染有关,可能是诱发肝癌的机制之一。癌旁组织和肝硬变组织中也存在P53蛋白异常表达,表明肝癌形成早期就发生了53基因的突变。     

应用双重原位杂交技术定位检测肝细胞癌中乙型丙型肝炎病毒

目的检测乙型肝炎病毒（ＨＢＶ）和丙型肝炎病毒（ＨＣＶ）是否可同时感染同一宿主细胞．方法联合应用地高辛标记探针和生物素标记探针的双标记方法，成功建立起非放射性双重原位杂交技术，并应用这一技术检测了１０例肝细胞性肝癌及癌周组织细胞内乙型肝炎病毒和丙型肝炎病毒的感染情况．结果发现ＨＢＶ、ＨＣＶ可同时感染同一宿主细胞这一独特的生物学现象．结论从而认为ＨＢＶ、ＨＣＶ双病毒感染可能并不遵循“病毒干扰”原则，并为了解这两种病毒在肝细胞癌发生中可能存在着的协同作用提供了新的线索．     

双探针原位杂交法检测慢性肝病和肝细胞癌患者肝组织TTV DNA的感染状况

目的 探讨慢性肝病和肝细胞癌患者肝组织TIV DNA的感染状况。方法采用PCR扩增法分别合成G1a、G2b两种亚型的双链TTV DNA探针。应用两型探针对45例肝组织标本进行TTV DNA原位杂交检测，巢氏PCR法检测血清TTV DNA。结果31例血清TTTV DNA阳性患者的肝组织TTV DNA均为阳性(100％)。14例血清TTV DNA阴性的患者肝组织中TTV DNA阳性者7例(50%)。慢性肝病患者的肝组织中TTV DNA散在分布在汇管区周围的肝细胞核内，肝癌患者TTV DNA则集中分布在肝癌细胞核内及癌组织周围的肝细胞核内。结论慢性肝病与肝癌患者肝组织中TTV DNA的感染状态存在一定差异。     

核酸原位杂交检测人淋巴结组织中的弓形虫

目的：探讨弓形虫感染的淋巴结组织中的病原体检测。方法：用聚合酶链反应（ＰＣＲ）和基因工程技术制备弓形虫（ＲＨ株）特异ＤＮＡ克隆片段，以地高辛随机引物法标记克隆的弓形虫ＤＮＡ片段作为探针，与淋巴结组织切片中核酸原位杂交试验（ＩＳＨ）检测病理标本中的弓形虫ＤＮＡ。结果：３２例何杰金淋巴瘤（ＨＤ）和４１例非何杰金淋巴瘤（ＮＨＬ）病理标本中各有一例阳性，４７例慢性淋巴结炎（ＣＬ）病理标本中呈ＩＳＨ阳性者２例，总阳性率为３．３％（４／１２０）。研究证明，所制备的探针能检测１０ｐｇ的ＤＮＡ，且特异性强。结论：核酸原位杂交为弓形虫病的病理标本中病原体检测提供了可行的方法。     

肝硬变患者肝胰组织中HBV DNA的分布

目的探讨HBV DNA对胰腺的致病作用.方法应用地高辛素标记HBV DNA S 577bp探针原位杂交(ISH)和免疫组织化学技术,检测12例血清HBV标志物阳性的肝硬变患者肝、胰组织石蜡包埋切片HBV DNA的存在.结果 ISH采用HBV DNA S 577bp探针检测肝组织11例(11/12)阳性,糖耐量试验(GTT)异常7例HBV DNA均阳性,GTT正常者阳性4例(4/5).胰腺组织阳性8例(8/12),GTT异常7例均阳性,GTT正常者1例阳性(1/5),HBVDNA阳性主要位于肝细胞、胰腺腺泡细胞和胰岛细胞的细胞核内.免疫组织化学法检测肝组织HBsAg均阳性,胰腺组织阳性10例(10/12),主要在胞质;而HBcAg在肝组织阳性5例(5/12),胰腺组织阳性5例(5/12)主要分布在细胞核,胞质分布量较少.结论HBV可直接侵害胰腺腺泡细胞和胰岛细胞,它可能是造成HBV感染后并发糖尿病的直接原因.     

聚合酶链反应评价原位杂交检测慢性HBV感染者肝内HBV DNA的结果

用地高辛探针原位杂交检测慢性HBV感染者肝内HBV DNA,聚合酶链反应(PCR)检测血清HBV DNA评价病毒复制状态。发现肝内HBV DNA阳性肝细胞,在14例肝组织病变不活动的HBeAg阳性慢性无症状HBV携带者(ASC)呈弥漫性分布;而在     

慢性肝病HBV DNA、HDV RNA原位杂交及HBsAg、HBcAg标记研究

目的与方法：为探明病毒性肝炎与肝癌的关系，以地高辛配基探针原位分子杂交及免疫组织化学技术对云南６８例慢性肝病患者进行ＨＢＶＤＮＡ、ＨＤＶＲＮＡ、ＨＢｓＡｇ、ＨＢｃＡｇ标记，并研究其相互关系。其中慢性迁延性肝炎２７例，慢性活动性肝炎１６例，肝细胞癌（Ｈ...     

采用原位杂交法检测肝组织中的TT病毒

目的：检测肝组织中ＴＴ病毒。并探讨其致病性。方法：采用对称和不对称ＰＣＲ法,以地高辛素分别标记ＴＴＶ双链和单链探针,原位杂交法检测５６例肝组织。结果：５１例非甲￣非庚型肝炎肝组织中ＴＴＶ总检出率２７．５％（１４／５１）;５例非肝炎肝组织杂交阴性。ＴＴＶ ＤＮＡ主要见于肝细胞核内,在急性、慢性及重型肝炎组织中均有检出。双链探针杂交结果与单链探针检测病毒基因链的结果一致,２／６例组织ＴＴＶ基因链与互补链同时存在,结论：ＴＴＶ是导致肝炎的一种新病原,并且在肝脏内有复制。     

Evaluation of the in situ assay for HBV DNA: An observational real-world study in chronic hepatitis B

The visualization of intrahepatic hepatitis B virus (HBV) DNA by in situ hybridization (ISH) has uncovered some interesting aspects of HBV life cycle at the single-cell level. In the current study, we intend to evaluate the reliability and robustness of this assay in the real-world clinical scenario and its relationship with currently available clinical biomarkers in chronic hepatitis B (CHB) patients.In this cross-sectional study, 94 CHB patients and 10 patients with non-HBV related liver diseases were enrolled. Liver biopsies and routine histopathology analysis were performed. Intrahepatic HBV DNA and viral antigens (HBsAg and HBcAg) were detected by ISH and immunohistochemistry (IHC), respectively. The basic biochemical and virological parameters such as alanine transaminase, serum HBV DNA, and serum HBsAg were measured.The HBV DNA-ISH assay showed 55.8% (53/94 cases) positive rate in CHB patients, no false positive was found in non-HBV related hepatitis. The IHC of HBsAg and HBcAg showed a positive rate of 94.7% (89/94 cases) and 19.5% (17/87 cases), respectively. Quantification of HBV DNA-ISH signal showed a significant correlation with serum HBV DNA (r_s = 0.6223, P < .0001). In addition, the staining pattern of HBV DNA in situ in the context of collagen deposition informed the histopathological progression of chronic liver disease.The application of this ISH assay in evaluating intrahepatic viral replication in real-world CHB patients showed favorable performance. It can be a complementation to conventional liver histopathology examination and IHC detection of viral antigens. This methodology provides an intuitive assessment of virological and pathological state of CHB patients, and further supports clinical diagnosis and management.     

原发性肝癌内HBV DNA及HBAg的分布与意义

采用原位分子杂交和免疫组化及双标记技术,对44例原发性肝细胞肝癌及癌旁肝组织进行了乙型肝炎病毒DNA及其表达产物X抗原,核心抗原检测。癌组织中HBV DNA,HBxAg,HBcAg检出率分别为70．5％,65．9％及20．5％,癌旁组织依次为76．5％,76．5％,29．4％。HBV DNA及HBxAg主要位于肝细胞浆中,在HBcAg阳性组中的检出率高于HBcAg阴性组（P＜0．05）。9例枯否细胞胞浆有HBxAg检出。HBV DNA在癌及癌旁组织中的检出有“伴随现象”。HBcAg多位于核内,部分为核浆型,且总是伴随HBV DNA和（或)HBxAg检出,无一例单独检出HBcAg者。此外,HBxAg可由肝内游离型HBV DNA所表达;肝内核型或浆型HBc Ag均可反映HBV的复制。因此,肝内HBxAg检测可作为HBV感染的灵敏指标之一,进一步证实HBV感染与HCC的发生有密切关系。     

Role of occult hepatitis B virus infection in chronic hepatitis C

The development of sensitive assays to detect small amounts of hepatitis B virus(HBV) DNA has favored the identification of occult hepatitis B infection(OBI), a virological condition characterized by a low level of HBV replication with detectable levels of HBV DNA in liver tissue but an absence of detectable surface antigen of HBV(HBs Ag) in serum. The gold standard to diagnose OBI is the detection of HBV DNA in the hepatocytes by highly sensitive and specific techniques, a diagnostic procedure requiring liver tissue to be tested and the use of non-standardized non-commercially available techniques. Consequently, in everyday clinical practice, the detection of anti-hepatitis B core antibody(antiHBc) in serum of HBs Ag-negative subjects is used as a surrogate marker to identify patients with OBI. In patients with chronic hepatitis C(CHC), OBI has been identified in nearly one-third of these cases. Considerable data suggest that OBI favors the increase of liver damage and the development of hepatocellular carcinoma(HCC) in patients with CHC. The data from other studies, however, indicate no influence of OBI on the natural history of CHC, particularly regarding the risk of developing HCC.     

Occult and previous hepatitis B virus infection are not associated with hepatocellular carcinoma in United States patients with chronic hepatitis C

Previous studies have suggested that prior exposure to hepatitis B virus (HBV) infection may increase the risk of development of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C. The aim of this study was to compare the prevalence of previous or occult HBV infection in a cohort of hepatitis B surface antigen-negative patients with histologically advanced chronic hepatitis C in the United States who did or did not develop HCC. Stored sera from 91 patients with HCC and 182 matched controls who participated in the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial were tested for hepatitis B core antibody (anti-HBc), hepatitis B surface antibody, and HBV DNA. Frozen liver samples from 28 HCC cases and 55 controls were tested for HBV DNA by way of real-time polymerase chain reaction. Anti-HBc (as a marker of previous HBV infection) was present in the serum of 41.8% HCC cases and 45.6% controls (P=0.54); anti-HBc alone was present in 16.5% of HCC cases and 24.7% of controls. HBV DNA was detected in the serum of only one control subject and no patients with HCC. HBV DNA (as a marker of occult HBV infection) was detected in the livers of 10.7% of HCC cases and 23.6% of controls (P=0.18). CONCLUSION: Although almost half the patients in the HALT-C Trial had serological evidence of previous HBV infection, there was no difference in prevalence of anti-HBc in serum or HBV DNA in liver between patients who did or did not develop HCC. In the United States, neither previous nor occult HBV infection is an important factor in HCC development among patients with advanced chronic hepatitis C.     

Prevalence of hepatitis C antibody in patients with chronic liver disease and hepatocellular carcinoma in Korea

Summary. To investigate the contribution of hepatitis C virus (HCV) to chronic liver disease and hepatocellular carcinoma (HCC) in Korea, antibodies to HCV (anti-HCV) were tested by enzyme immunoassay in 1759 patients with chronic liver disease and HCC, and in 808 healthy adults. The prevalence of anti-HCV was 1.6% in 808 controls. Anti-HCV was present in 32 (7.7%) of 418 hepatitis B surface antigen (HBsAg)-positive and 128 (53.1%) of 241 HBsAg-negative patients with chronic hepatitis, 16 (6.0%) of 265 HBsAg-positive and 90 (30.5%) of 295 HBsAg-negative patients with liver cirrhosis, and 16 (4.8%) of 330 HBsAg-positive and 61 (29.0%) of 210 HBsAg-negative patients with HCC. Antibodies to hepatitis B core antigen (anti-HBc) were present in 80–88% of patients who were seropositive for anti-HCV and seronegative for HBsAg. Among the sera from 114 patients with HBsAg-negative and anti-HCV-positive chronic liver diseases, HBV DNA and HCV RNA were detected by polymerase chain reaction (PCR) in 54 (47.4%) and 61 (53.3%), respectively. Both HBV DNA and HCV RNA were detected in 4 (4.4%) samples. The mean age of the patients with both HBsAg and anti-HCV was not different from that of patients who were seropositive for HBsAg alone. These findings indicate that current and/or past HBV infection is still the main cause of chronic liver disease in Korea. Although multivariate analysis showed that anti-HCV is a risk factor for chronic hepatitis, cirrhosis of the liver and HCC, PCR data for HBV DNA and HCV RNA indicate that HCV infection plays only a minor role in HBsAg-positive as well as in HBsAg-negative liver disease and does not accelerate the development of HCC in HBV carriers.     

聚合酶链反应评价原位杂交检测慢性HBV感染者肝内HBVDNA的结果

用地高辛素探针原位杂交检测慢性ＨＢＶ感染者肝内ＨＢＶＤＮＡ，聚合酶链反应（ＰＣＲ）检测血清ＨＢＶＤＮＡ评价病毒复制状态。发现肝内ＨＢＶＤＮＡ阳性肝细胞，在１４例肝组织病变不活动的ＨＢｅＡｇ阳性慢性无症状ＨＢＶ携带者（ＡｓＣ）呈弥漫性分布；而在ＨＢｅＡｇ阳性或阴性活动性肝病病人均聚合分布于肝细胞坏死区。上述慢性感染者９８％血清ＨＢＶＤＮＡ阳性。说明ＨＢＶ复制与肝组织病变有关但非肝损伤直接原因。１６例ＨＢｅＡｇ阴性ＡｓＣ中，９例肝内仅见局灶性ＨＢＶＤＮＡ阳性肝细胞，其中４例血清ＨＢＶＤＮＡ阳性，说明部分ＨＢｅＡｇ阴性ＡｓＣ也存在极低水平病毒复制。     

不明原因持续肝功能异常患者肝组织和外周血单个核细胞中HBV DNA表达

采用原位分子杂交技术分别检测随机选择的40例不明原因持续肝功能异常患者外周血单个核细胞和其肝组织中HBV DNA,肝组织中HBV DNA阳性24例,阳性率为58%;外周血单个核细胞中HBV DNA阳性18例,阳性率为45%;两者比较,差异无显著性(x2=2.10,P＞0.05).对肝组织和外周血单个核细胞进行HBVDNA检测,有助于诊断隐性HBV感染.