丙型肝炎患者肾小球病变的观察

为了明确丙型肝炎病毒（ＨＣＶ）感染与肾脏疾病的关系，采用免疫组化技术和抗－ＨＣＶＮＳ３和抗－ＨＣＶＮＳ５单克隆抗体，检测了２１例丙肝患尸解肾组织中的ＨＣＡｇ，结果１３例阳性（６１．９％），其中１１例为膜增殖性肾炎（ＭＰＧＮ），１例为系膜增殖性肾炎（ＭｓＰＧＮ），１例肾组织大致正常。８例ＨＣＡｇ检测阴性，其中膜性肾病（ＭＮ）１例，膜增殖性肾炎２例，毛细血管内增殖性肾炎１例，余４例肾组织未见明显异常。与乙型肝炎对照组（３３．０％）相比，丙肝患者肾脏病变更为多见，形态多样，以ＭＰＧＮ为主。同时对丙肝患者有肾脏病变者的临床资料进行了分析，病人可表现为高血压，蛋白尿，血尿及早期血清尿素氮（ＢＵＮ）增高，因此认为同乙型肝炎病毒（ＨＢＶ）感染后相同，也存在着丙肝相关性肾炎     

肾小球肾炎肾组织中HBV感染的标志

为了解肾组织中ＨＢＶＤＮＡ与ＨＢＶ抗原存在的关系，探讨肾组织中ＨＢＶ抗原的来源及其与病理变化的关系，我们检测２４６例肾炎肾组织中三种ＨＢＶ抗原。发现除肾小球沉积外，肾小管常有阳性表达。肾小管ＨＢｃＡｇ阳性率达２１．５４％，高于肾小球（１０．９８％）。有１８例肾组织经Ｓｏｕｔｈｅｒｎ印迹杂交检测ＨＢＶＤＮＡ，１５例阳性者中１４例肾组织ＨＢＶ抗原同时阳性，１２例血清感染标志亦阳性。结果提示某些肾炎可能与肾组织感染ＨＢＶ相关，肾组织中出现的ＨＢＶ抗原抗体免疫复合物除来自血循环外，有肾源性──原位形成的可能。     

乙型肝炎病毒(HBV)相关性肾炎患者肾活检组织中HBV DNA分布的分子生物学检测

为探讨乙型肝炎病毒（ＨＢＶ）相关性肾炎的发病机理，应用地高辛素标记ＨＢＶＤＮＡ探针原位分子杂交（ＩＳＨ）和直接原位聚合酶链反应（ＩＳ－ＰＣＲ）技术，对２０例临床诊断为ＨＢＶ相关性肾炎患者肾活检石蜡包埋组织切片检查。在ＩＳＨ中采用ＨＢＶＤＮＡ两种探针：用全长段探针，８５％（１７／２０）ＨＢＶＤＮＡ阳性，这１７例阳性肾组织切片再用ＨＢＶＤＮＡＳ加Ｃ段探针检测，１４例阳性（８２．３５％）。在ＩＳ－ＰＣＲ中本组病例８５％（１７／２０）亦阳性。发现ＨＢＶＤＮＡ阳性颗粒弥漫沉积于肾小球毛细血管袢、系膜区、肾小管，表现形式以浆核型、核型为主。同组病例肾组织免疫组化染色法（ＬＳＡＢ）显示，ＨＢｓＡｇ与ＨＢＶＤＮＡ的存在部位基本一致。提示ＨＢＶ引起的肾脏病变不仅是免疫介导的损害，亦可能有病毒侵犯参加免疫反应。     

乙型肝炎病毒感染在肾小球肾炎发病中的作用

为探讨ＨＢＶ感染于肾小球肾炎发病机制中的作用，收集５０例血清ＨＢＶ感染标志阳性或／和肾组织免疫组化证实ＨＢＡｇ阳性肾炎患者的肾穿刺组织，应用Ｓｏｕｔｈｅｒｎ印迹杂交和原位分子杂交观察ＨＢＶＤＮＡ的存在状态和定位。Ｓｏｕｔｈｅｒｎ印迹杂交阳性率为７３．９％，其中８２％属整合型；原位杂交显示肾小管和肾小球ＨＢＶＤＮＡ的阳性率分别为７２％和５６．５％。其结果提示肾组织本身感染ＨＢＶ，因而考虑沉积于肾小球上的ＨＢＡｇ除源于血循环（肝原）外，尚有原位合成的可能；认为ＨＢＶ相关性肾炎的发病除ＨＢＶ抗原、抗体在肾小球沉积导致的体液免疫损伤机制外，应考虑因肾组织感染ＨＢＶ而导致的细胞免疫机制参与作用。     

乙型肝炎病毒（HBV）相关性肾炎患者肾活检组织中HBVDNA分布…

为探讨乙型肝炎病毒（ＨＢＶ）相关性肾炎的发病机理，应用地高辛素标记ＨＢＶＤＮＡ探针原位分子杂交（ＩＳＨ）和直接原位聚合酶链反应（Ｓ－ＰＣＲ）技术，对２０例临床诊断为ＨＢＶ相关性肾炎患者肾活检石蜡包埋组织切片检查。在ＩＳＨ中采用ＨＢＶ ＤＮＡ两种探针；用全长段探针；８５％（１７／２０）ＨＢＶＤＮＡ阳性，这１７例阳肾组织切片再用ＨＢＶＤＮＡＳ加Ｃ段探针检测，１４例阳性（８２．３５％）。在ＩＳ－ＰＣＲ中     

慢性乙型肝炎重叠HGV感染对HBV复制的影响

目的观察慢性乙型肝炎重叠ＨＧＶ感染对ＨＢＶ复制的影响．方法采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）检测患者血清ＨＢＶ－ＤＮＡ．ＨＢＶ－ＤＮＡ定量采用荧光信号引物能量转换法．以单纯慢性乙型肝炎患者作对照研究．结果２３例ＨＧＶ重叠感染的慢性乙型肝炎患者 ＨＢＶ－ＤＮＡ、ＨＢｅＡｇ和抗－ＨＢｅ的阳性率分别为５６．５％、１７．４％和６０．９％而３４例单纯慢性乙型肝炎患者三项指标的阳性率分别为９１．２％、８５．３％和１１．８％．定量分析显示,前组ＨＢＶ－ＤＮＡ量为２．０２、后组为４．１２．经统计学处理,两组各项指标均有显著性差异．结论慢性乙型肝炎重叠ＨＧＶ感染可能会抑制ＨＢＶ的复制．     

用聚合酶链反应检测人肝细胞癌乙型肝炎病毒DNA和丙型肝炎病毒RNA

为研究乙型肝炎病毒ＤＮＡ（ＨＢＶＤＮＡ）和丙型肝炎病毒ＲＮＡ（ＨＣＶＤＮＡ）与肝细胞癌的关系，用聚合酶链反应（ＰＣＲ）和巢式ＰＣＲ（ｎｅｓｔｅｄ－ＰＣＲ）分别检测４２例肝肿瘤组织中ＨＢＶＤＮＡ和ＨＣＶＲＮＡ。结果：１例胆管细胞癌组织ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性，１例胆管囊腺瘤ＨＢＶＤＮＡ阳性。４０例肝细胞癌组织中，单纯ＨＢＶＤＮＡ阳性１９例，单纯ＨＣＶＲＮＡ阳性３例，二者均阳性１０例。ＨＢＶＤＮＡ阳性率７２．５％，ＨＣＶＲＮＡ阳性率３２．５％。ＨＢＶＤＮＡ和ＨＣＶＲＮＡ感染与肝癌组织学分型无关；且肝细胞癌中ＨＣＶ感染与ＨＢＶ未见相关。结果提示，我国ＨＢＶ感染仍是引起肝细胞癌的主要原因。但由于肝细胞癌患者中ＨＣＶ的感染率也较高，且有上升趋势，因此ＨＣＶ可能也是肝细胞癌发生的重要原因之一。     

慢性乙型肝炎肝内HBVDNA和HBsAg的双标记定位研究

应用原位杂交和免疫组化ＰＡＰ法双标记技术，结合病人乙型肝炎（乙肝）病毒血清学标志物检测结果，研究了３１例慢性乙肝病人肝穿刺组织中乙型肝炎病毒ＤＡＮ和ＨＢｓＡｇ的分布及意义。结果显示，肝辔内检ＨＶＤＮＡ２３例，ＨＢｓＡｇ２６例，二者同时检出者２１例。从同组病人肝组织的ＨＢＶＤＮＡ和ＨＢｓＡｇ双标检测结果与其乙肝病毒血清学标志物检测结果的比较来看，肝组织内ＨＢＶＤＮＡ和ＨＢｓＡｇ同时很可能表明ＨＢＶ正     

肝细胞内HBcAg和HBsAg分布的演变特点

本文报道用免疫组化（ＡＢＣ）法检测２９例慢性乙型肝炎患者肝活检组织中ＨＢｃＡｇ和ＨＢｓＡｇ分布的动态演变，结合血清ＨＢｅＡｇ和ＨＢＶ ＤＮＡ的演变探讨其病毒学意义。ＨＢｃＡｇ、ＨＢｅＡｇ和ＨＢＶ ＤＮＡ全阳性反映ＨＢＶ高复制相，全阴性为非复制相，不一致为两相之间的过渡型（低复制相）。肝细胞内ＨＢｃＡｇ由核浆型为主演变为核型或浆型为主乃至阴性意味着ＨＢＶ从高复制相转入低复制相乃至非复制相，同时伴有血     

乙型肝炎患者HBV M和HBV DNA的相关性研究

目的 探讨乙型肝炎患者的乙型肝炎病毒（ＨＢＶ）血清学标志（ＨＢＶ Ｍ）与ＨＢＶ ＤＮＡ检测结果的相关性与临床意义。方法 对４１４例乙型肝炎的ＨＢＶ Ｍ和ＨＢＶ ＤＮＡ检测结果进行比较。ＨＢＶ Ｍ用ＥＬＩＳＡ定量分析法检测,ＨＢＶ ＤＮＡ用斑点杂交法检测。结果 急性、慢性乙型肝炎患者中ＨＢＶ ＤＮＡ的阳性率与乙型肝炎肝硬化患者的ＨＢＶ ＤＮＡ阳性率比较,差异有显著性;ＨＢｓＡｇ、抗－ＨＢｅ、抗－ＨＢｃ阳性和ＨＢｓＡｇ、ＨＢｅＡｇ、抗－ＨＢｃ阳性组的ＨＢＶ ＤＮＡ阳性率比较,差异无显著性;ＨＢｓＡｇ和／或ＨＢｅＡｇ的滴度与ＨＢＶ ＤＮＡ阳性率呈正相关关系。结论 ＨＢＶ ＤＮＡ是评价ＨＢＶ活动最理想的标志;抗－ＨＢｅ的出现不能作为ＨＢＶ复制停止的指标;ＨＢｓＡｇ的滴度和ＨＢｅＡｇ的滴度变化可作为临床评价病毒复制程度和     

Sequencing of human-viral DNA junctions in hepatocellular carcinoma from patients with HCV and occult HBV infection

DNA of free hepatitis B viruses (HBV) has been detected in the liver of patients infected with hepatitis C virus (HCV). It is unknown whether HBV DNA is integrated into such livers; if so, it may affect hepatocarcinogenesis. Hepatocellular carcinomas (HCCs) from 34 patients without HBV surface antigen (HBsAg) and with anti-HCV, and from 7 patients with HBsAg and without anti-HCV as controls, were examined, using the cassette-ligation-mediated polymerase chain reaction and primers based on HBV DNA sequence. In the controls, HBV DNA had been integrated into human DNA of all HCCs. On the basis of HBV DNA in tumor tissue, 23 of the 34 patients with anti-HCV had occult infection. Junctions between human DNA and HBV DNA were detected in 10 of the 34 patients without HBsAg and with anti-HCV. HBV DNA was integrated into chromosome 11q in 4 of the 10 HCCs with junctions. The DNA to either side of the human-viral junctions was sequenced. Clinically, the mean tumor size of these 10 HCCs was 39 mm; that of the 24 HCCs without integrated HBV was 25 mm. The surrounding tissue was cirrhotic in 2 of the 10 former HCCs and in 16 of the latter 24 HCCs. In conclusion, integrated HBV was detected in some patients with HCV infection; in these patients, the integrated DNA was associated with accelerated hepatocarcinogenesis.     

Detection of hepatitis B virus genome in hepatocellular carcinoma tissues with PCR-in situ hybridization

The detection is described of hepatitis B virus (HBV)-DNA in preserved hepatocellular carcinoma tissues, which were derived from 14 HBV-seropositive patients. Detection was by polymerase chain reaction (PCR) amplification of the target sequence, followed by specific localization of the PCR product with in situ hybridization. PISH (PCR-in situ hybridization) yielded strong positive signals in most of the tumor tissues despite very low copy numbers of chromosome-integrated HBV genome, whereas no signal was detected in control samples, indicating that the signals were specific for HBV. Positive signals were sometimes detected in cirrhotic nodules surrounding the tumor regions, indicating that HBV had infected non-transformed liver cells. HBV-DNA was detected in both nucleus and cytoplasm in some specimens, possibly representing HBV at different stages of the life cycle. In one case, a gradient of viral DNA was revealed, with the highest DNA signal centered at the site of viral antigen expression. Taken together, PISH is shown to be a highly sensitive molecular detection method that is capable of detecting the presence of a low copy number viral genome in situ.     

Detection of hepatitis B virus genome in hepatocellular carcinoma tissues with PCR-in situ hybridization.

The detection is described of hepatitis B virus (HBV)-DNA in preserved hepatocellular carcinoma tissues, which were derived from 14 HBV-seropositive patients. Detection was by polymerase chain reaction (PCR) amplification of the target sequence, followed by specific localization of the PCR product with in situ hybridization. PISH (PCR-in situ hybridization) yielded strong positive signals in most of the tumor tissues despite very low copy numbers of chromosome-integrated HBV genome, whereas no signal was detected in control samples, indicating that the signals were specific for HBV. Positive signals were sometimes detected in cirrhotic nodules surrounding the tumor regions, indicating that HBV had infected non-transformed liver cells. HBV-DNA was detected in both nucleus and cytoplasm in some specimens, possibly representing HBV at different stages of the life cycle. In one case, a gradient of viral DNA was revealed, with the highest DNA signal centered at the site of viral antigen expression. Taken together, PISH is shown to be a highly sensitive molecular detection method that is capable of detecting the presence of a low copy number viral genome in situ.     

HBVcccDNA在乙型肝炎患者血清、外周单核细胞及肝组织中的分布

目的 观察乙肝病毒共价闭合环状DNA(HBVcccDNA)在乙肝患者血清、外周单核细胞(PBMC)及肝组织中的分布情况.方法 选取血清HBVDNA＞105拷贝/ml的乙肝患者50例,血清HBVDNA＜105拷贝/ml的乙肝患者30例,脂肪肝患者(非乙型肝炎)20例,同时采用实时荧光定量聚合酶链反应检测患者血清、PBMC及肝组织中HBVcccDNA的存在情况.结果 50份血清HBVDNA＞105拷贝/ml的标本中血清HBVcccDNA检出28例,检出率56%,PBMC HBVcccDNA检出29例,检出率58%,肝组织中HBVcccDNA检出44例,检出率88%,血清、PBMC的检出较肝组织检出差异有统计学意义P＜0.005,血清较PBMC检出差异无统计学意义P＞0.005.30份血清HBVDNA＜105拷贝/ml的标本中血清、PBMC HBVcccDNA检出均为2例,检出率6.67%,肝组织中HBVcccDNA检出6例,检出率20%,血清、PBMC、肝组织三者之间检出差异无统计学意义,P＞0.005.20份脂肪肝患者的血清、PBMC及肝组织中均未检出HBVcccDNA.结论 HBVcccDNA主要存在于乙型肝炎患者的肝脏中,乙肝患者血清及PBMC中也有HBVcccDNA的存在但较肝组织中要少的多.     

Occult hepatitis B virus infection does not affect liver histology or response to therapy with interferon alpha and ribavirin in intravenous drug users with chronic hepatitis C.

BACKGROUND: the frequency and the impact of occult HBV infection in patients with chronic hepatitis C infection is still a matter of some controversy. OBJECTIVES: our aim was to evaluate the prevalence of occult HBV infection and assess its impact on liver biochemistry, HCV viral titre, liver histology and on outcome of therapy in patients with chronic hepatitis C. STUDY DESIGN: paired liver biopsies and serum samples were collected from 51 patients (84% IVDUS) with HBsAg negative chronic hepatitis C, and tested for HBV-DNA with nested PCR. Liver biopsies were further studied histologically, with morphometric analyses and immunostaining techniques. Twenty-five were treated with alpha Interferon and ribavirin and followed for at least 18 months. RESULTS: HBV DNA was detected in 29.4% of liver tissue specimens and in only one (1.9%) serum sample. Three liver specimens were positive for surface gene, nine for core gene, three for both and none for the X gene. No significant difference in mean transaminase values, HCV viral titre, HCV genotype, or grading and staging and morphometric analysis was observed in patients with or without HBV DNA. Moreover, all 51 liver specimens were negative for both HBsAg and HBcAg. Sustained response to combination therapy was achieved in 40% of patients with and in 53% of patients without HBV DNA in the liver specimens (P=NS). CONCLUSIONS: HBV DNA is frequently found in the liver of patients with chronic hepatitis C. However, the lack of any significant impact on HCV viral titre, liver enzymes, histological parameters and response to therapy, suggests that in most cases HBV DNA detected in the liver by PCR may be either an integrated or low level replicative form.     

Real-time quantitation of hepatitis B virus (HBV) DNA in tumorous and surrounding tissue from patients with hepatocellular carcinoma

Few data are available on the levels of HBV DNA in liver tissue of patients with hepatocellular carcinoma. In this study, HBV DNA was quantitated by a TaqMan real-time PCR method and results were normalised to an endogenous reference gene. The assay could detect reproducibly viral sequences from over 10(7) to less than 50 copies/microg of liver DNA. The HBV DNA content in liver samples from 11 HBsAg-positive patients (median: 10(5) copies/microg of DNA) was significantly higher (P < 0.001) compared to the viral DNA concentration detected in liver samples from 15 of 25 HBsAg-negative patients (median: 2.6 x 10(2) copies/microg). A liver DNA amount > or =1 HBV DNA copy per cell was detected in half of tissue samples from HBsAg-positive patients, and in none from HBsAg-negative ones. Liver tissue HBV DNA content was significantly higher in anti-HCV-negative than in anti-HCV-positive cases (P < 0.001). These results show that the quantitation of liver HBV DNA by real-time PCR can be useful to understand HBV state in hepatocellular carcinoma and viral interplay in patients with multiple viral infections.     

Detection of supercoiled hepatitis B virus DNA and related forms by means of molecular hybridization to an oligonucleotide probe

A novel assay for supercoiled and other fully double-stranded forms of hepatitis B virus (HBV) DNA in blood is presented that utilizes molecular hybridisation to a radiophosphorous-labeled oligonucleotide probe. The probe [5'-d(ACGTGCAGAGGTGAAGCGA)] is complementary to the S(+)-strand sequence furthest downstream, at the end of the gap. We examined blood specimens from 137 healthy HBsAg-positive individuals, applying the probe to dots representing 2-3.5 ml serum or plasma. We found that supercoiled HBV is present in many HBV DNA-positive blood specimens albeit in small quantities. Of the 104 specimens that were positive for HBV DNA of any form, 53 annealed to the probe. Serial specimens from the same subject taken over a period of months showed that the proportion of supercoil to other HBV DNA forms was variable. The presence of supercoil HBV DNA was not closely correlated with the level of serum HBV DNA polymerase. The supercoil is an HBV DNA form that can persist in the liver in the presence or absence of other replicative intermediates. This assay may enable further characterization of the status of HBV infection.     

原位杂交法检测肝组织中丁型和乙型肝炎病毒核酸

利用国外引进的重组质粒获得纯化基因片段，分别以随机引物法和ＰＣＲ法制备地高辛素标记的ＨＢＶＤＮＡ探针和ＨＤＶｃＤＮＡ探针。用原位杂交法检测了石蜡包埋的肝组织切片ＢＶＤＮＡ和ＨＤＶＲＮＡ。４９例感染肝组织分为两组：丁肝组２３例；单纯乙肝组２６例，ＨＢＶＤＮＡ的检出率丁肝组（７８．２６％）与乙肝组（７６．９２％）无统计学差异；而ＨＤＶＮＡ的检出率丁肝组（６０．８７％）明显高于乙肝组（１５．３８％）。ＨＢＶＤＮＡ可见于受染肝细胞的胞核或胞浆内，而ＨＤＶＲＮＡ绝大部分见于肝细胞胞核。两种病毒核酸阳性细胞在肝组织中的分布特点大致相同：弥漫或散在地分布于肝小叶或假小叶内，或局灶性分布于小叶周边。ＨＤＶＲＮＡ阳性的肝组织都或多或少地同时存在ＨＢＶＤＮＡ。同一例肝组织中，ＨＢＶＤＮＡ阳性细胞从数量和颗粒密度上似略高于ＨＤＶＲＮＡ。将乙肝组和丁肝组两组病人肝内ＨＢ－ｓＡｇ、ＨＢｃＡｇ和ＨＢＶＤＮＡ及血清ＨＢｅＡｇ作了比较，各指标阳性率虽有差异，但均无统计学意义。因此，未发现ＨＤＶ感染对ＨＢＶ的复制有明显抑制作用。此结果对以往用血清学或免疫组化方法对ＨＤＶ的研究有所补充和深入，亦可为研究其它类型病毒性肝炎之间的重叠感染所借鉴。     

Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma

To search for events underlying reduction of peripheral viremia and integration of hepatitis B virus (HBV) DNA into the liver cell genome in long-term virus carriers with hepatocellular carcinoma, paired samples of liver and tumor tissue were analyzed by molecular hybridization and immunological methods. Most tumor tissues contained integrated viral DNA; in none was extrachromosomal HBV DNA detected. Integrated HBV DNS was also found in peritumor liver tissue in the majority of patients. However, liver of patients either with or without peripheral viremia also contained free HBV DNA and replicative intermediates. In three nonviremic patients with replicative HBV DNA in liver, viral core antigen expression was markedly reduced or absent, whereas viral envelope protein (surface antigen) expression was normal. In one case, replicative intermediates in liver were sensitive to DNase I digestion, indicating that viral DNA was not encapsidated in normal viral core particles. These results suggest that decreased or defective core antigen production can lead to reduced viremia associated with blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell. Accumulation of such HBV DNA molecular forms in the liver may lead to an increased propensity for HBV DNA to integrate into the host genome, which has been found with high frequency in hepatic neoplasms from patients infected with hepatitis B virus.     

Frequent integration of hepatitis B virus DNA in noncancerous liver tissue from hepatocellular carcinoma patients

To investigate the relationship between hepatocarcinogenesis and integration of hepatitis B virus (HBV) DNA in the cellular DNA of the liver, we studied the integration of HBV DNA in various noncancerous regions of the liver from 31 patients using Southern blot analysis. Of 13 patients without hepatocellular carcinoma (HCC), 4 had heterogeneously integrated HBV DNA. Of the latter four patients, two had chronic liver disease, and two had nonspecific histological changes. In contrast, integration of HBV DNA was found in noncancerous tissue from 11 of 18 patients with HCC. In eight patients, homogeneous integration was found in noncancerous tissue, and restriction fragments of integrated HBV DNA were different from those found in cancerous tissue. Moreover, integration of HBV DNA was found in all portions examined from the same liver, and homogeneously integrated HBV DNA showed different restriction patterns in different areas. These results suggest that integration of HBV DNA may occur in heterogeneous sites of cellular DNA before hepatocarcinogenesis. Subsequently, multi-focal clonal populations develop from these hepatocytes, especially frequently in the case of HCC. Integrated HBV DNA may play an important role in the clonal growth of hepatocytes, although the development of HCC requires additional factors.