丙型肝炎病毒感染者自身抗体检测及意义

①目的 探讨丙型肝炎病毒 (HCV)感染与自身抗体之间的关系。②方法 对 4 4例抗HCV IgG阳性病人血清进行抗核抗体 (ANA)、类风湿因子 (RF)及抗可提取性核抗原 (ENA)抗体检测 ,并同时检测谷丙转氨酶(ALT)和谷草转氨酶 (AST)。③结果 抗HCV阳性病人ANA ,RF和抗ENA抗体的阳性率分别为 2 7.3% ,2 5 .0 %和 31.0 % ,总的自身抗体检出率为 4 5 .0 % ,与对照组比较差异有显著性 (χ2 =4 .2 0～ 15 .18,P <0 .0 5 ) ;HCV感染病人ANA和RF阳性与阴性者之间AST含量比较差异无显著性 (t =1.4 5 ,t′ =1.2 1,P >0 .0 5 ) ,ANA阳性者与ANA阴性者之间ALT含量比较差异亦无显著性 (t=1.18,P >0 .0 5 ) ,RF阳性和阴性者ALT含量比较差异有显著性 (t′ =4 .75 ,P <0 .0 1)。④结论 HCV感染可触发机体自身免疫反应 ,HCV感染者定期检测RF有助于监测病情的活动性。

延边地区乙型肝炎患者感染丁型肝炎病毒的抽样调查

[目的 ]了解本地区乙型肝炎患者合并感染丁型肝炎病毒的状况 .[方法 ]应用ELISA方法进行检测 .[结果 ]15 0名乙型肝炎患者的丁型肝炎病毒感染率为 13% 。

丙型肝炎患者外周血单个核细胞HCV免疫组化和电镜研究

目的 :探讨丙型肝炎患者外周血单个核细胞 (PBMCs)内丙型肝炎病毒抗原 (HCVAg)表达和 HCV颗粒感染状况及意义。方法 :采用抗 HCV核心区及 NS3区单克隆抗体 ,对 35例慢性丙肝患者 PBMCs进行免疫组化检测和电镜观察。结果 :慢性丙肝患者 PBMCs的 HCVAg阳性率为 82 .9% (2 9/ 35 ) ,其中 82 .8% (2 4/ 2 9)的病例电镜下发现了病毒颗粒 ,占全组病例的 6 8.6 % (2 4/ 35 )。同时发现部分细胞超微结构呈现一系列凋亡性改变。结论 :HCV可感染 PBMCs并在其中复制 ,这可能是导致患者 HCV持续感染的原因之一。

乙肝标志物阴性患者丙肝病毒感染检测结果分析

乙肝标志物阴性患者丙肝病毒感染检测结果分析程道胜张吉才胡秀学（郧阳医学院附属太和医院检验科十堰４４２０００）关键词肝炎表面抗原，乙型肝类病毒组，丙型中图法分类号Ｒ４４６丙肝病毒（ＨＣＶ）主要经血制品传播，已引起全世界的广泛关注。目前能够检测到ＨＣＶ感...

HCVC基因部分序列的克隆测序

选取５株ＨＣＶ共有序列合成一时５’端加端引物。上游加端为ＥｃｏＲⅠ切点，下游加端为ＢａｍＨＩ切点。以ＲＴ－ＰＣＲ扩增一长为５７８ｂｐ的ｃＤＮＡ作目的基因，相应酶切后，反向插入ＰＵＣ１８的多克隆位点，构建ｐＵＨＣ－Ｃ重组体。分别或混合应用ＨＣＶ特异内外引物和ｐＵＣ特异通用引物以ＰＣＲ扩增重组体，扩增产物分子片段均同预期一致。测序表明克隆基因与ＨＣＶ－ＣＨＮ最同源，在所测ＣＥ４９７个核苷酸及由之推导的１６０个氨基酸区域内。其核苷酸与氨基酸同源性都是９７．５％。

Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing *

AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.

Relationship between hepatitis B virus DNA levels and liver histology in patients with chronic hepatitis B

AIM： To study the relationship between hepatitis B virus （HBV） DNA levels and liver histology in patients with chronic hepatitis B （CHB） and to determine the prevalence and characteristics of hepatitis B e antigen （HBeAg） negative patients.METHODS： A total of 213 patients with CHB were studied, and serum HBV DNA levels were measured by the COBAS Amplicor HBV Monitor test. All patients were divided into two groups according to the HBeAg status.The correlation between serum HBV DNA levels and liver damage （liver histology and biochemistry） was explored.RESULTS： Of the 213 patients with serum HBV DNA levels higher than 10^5 copies/mL, 178 （83.6%） were HBeAg positive, 35 （16.4%） were HBeAg negative. The serum HBV DNA levels were not correlated to the age,history of CHB, histological grade and stage of liver disease in either HBeAg negative or HBeAg positive patients. There was no correlation between serum levels of HBV DNA and alanine aminotransferanse （ALT）,aspartate aminotrans-ferase （AST） in HBeAg positive patients. In HBeAg negative patients, there was no correlation between serum levels of HBV DNA and AST,while serum DNA levels correlated with ALT （r = 0.351, P = 0.042）. The grade （G） of liver disease correlated with ALT and AST （P 〈 0.05, r = 0.205, 0.327 respectively）in HBeAg positive patients. In HBeAg negative patients,correlations were shown between ALT, AST and the G （P 〈 0.01, and r = 0.862, 0.802 respectively）. HBeAg negative patients were older （35 ± 9 years vs 30 ±9 years, P 〈 0.05 ） and had a longer history of HBV infection （8 ± 4 years vs 6 ± 4 years, P 〈 0.05） and a lower HBV DNA level than HBeAg positive patients （8.4± 1.7 Log HBV DNA vs 9.8 ± 1.3 Log HBV DNA, P 〈0.001）. There were no significant differences in sex ratio,ALT and AST levels and liver histology between the two groups.CONCLUSION： Serum HBV DNA level is not correlated to histological grade or stage of liver disease in CHB patients with HBV DNA more than 10^5 copies/mL.Compared to HBeAg positive patients, HBeAg negative patients are older and have a lower HBV DNA level and a longer HBV infection history. There is no significant difference in sex ratio, ALT and AST levels and liver histology between the two groups.

Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-γat 0.1 to 5μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5μg/L, IFN-γalso suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γshowed no additive effect, sequential treatment first with lamivudine and then IFN-γwas found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2μmol/L lamivudine for two days, followed by 1μg/L IFN-γfor another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2μmol/L lamivudine for two days, followed by 5μg/L IFN-γfor six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γand lamivudine, especially in IFN-αnon-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.

Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma

AIM:Only a minority of patients carrying a defined viral aetiologic agent develop cirrhosis and ultimately hepatocellular carcinoma(HCC),the mechanism underlying the worsening is still undefined.Experimental infection by Helicobacter hepaticus in mice causes chronic hepatitis and HCC and recently,more Helicobacterspecies(Helicobacter spp.)have been detected in the liver of patients suffering from cholestatic diseases and HCC arising from non-cirrhotic liver.We investigated whether Helicobacterspp.sequences could be detected in the liver of patients with cirrhosis and HCC compared to subjects with metastasis to liver from colon cancer. METHODS:Twenty-three liver samples from patients operated upon for HCC superimposed on hepatitis C virus (HCV)-related cirrhosis and 6 from patients with resected metastases from colorectal cancer,were tested by polymerase chain reaction for presence of genomic 16S rRNA of Helicobacter genus using specific primers.DNA sequencing and cag A gene analysis were also performed. RESULTS:Genornic sequences of Helicobacter spp.were found in 17 of 20(85%)liver samples from patients with HCC and in 2 of 6 samples from patients with liver metastasis. In three samples of the first group the result was uncertain. Hpyloriwas revealed in 16 out of 17 positive samples and Helicobacter pullorum in the other. CONCLUSION:Helicobacter spp.,carcinogenic in mice, were found at a higher frequency in the liver of patients with HCV-related cirrhosis and HCC than those in patients without primary liver disease.

Sequential algorithms combining non-invasive markers and biopsy for the assessment of liver fibrosis in chronic hepatitis B

AIM： To assess the performance of several noninvasive markers and of our recently proposed stepwise combination algorithms to diagnose significant fibrosis （F ≥ 2 by METAVIR） and cirrhosis （F4 by METAVIR） in chronic hepatitis B （CHB）.METHODS： One hundred and ten consecutive patients （80 males, 30 females, mean age： 42.6 ± 11.3） with CHB undergoing diagnostic liver biopsy were included. AST-to-Platelet ratio （APRI）, Forns＇ index, AST-to-ALT Ratio, Goteborg University Cirrhosis Index （GUCD, Hui＇s model and Fibrotest were measured on the day of liver biopsy. The performance of these methods and of sequential algorithms combining Fibrotest, APRI and biopsy was defined by positive （PPV） and negative （NPV） predictive values, accuracy and area under the curve （AUC）. RESULTS： PPV for significant fibrosis was excellent （100%） with Forns and high （〉 92%） with APR1, GUCI, Fibrotest and Hui. However, significant fibrosis could not be excluded by any marker （NPV 〈 65%）. Fibretest had the best PPV and NPV for cirrhosis （87% and 90%, respectively）. Fibrotest showed the best AUC for both significant fibrosis and cirrhosis （0.85 and 0.76, respectively）. Stepwise combination algorithms of APR1, Fibrotest and biopsy showed excellent performance （0.96 AUC, 100% NPV） for significant fibrosis and 0.95 AUC, 98% NPV for cirrhosis, with 50%-80% reduced need for liver biopsy. CONCLUSION： In CHB sequential combination of APRI, Fibrotest and liver biopsy greatly improves the diagnostic performance of the single non-invasive markers. Need for liver biopsy is reduced by 50%-80% but cannot be completely avoided. Non-invasive markers and biopsy should be considered as agonists and not antagonists towards the common goal of estimating liver fibrosis.

Prealbumin is predictive for postoperative liver insufficiency in patients undergoing liver resection

AIM:To investigate the risk factors for postoperative liver insufficiency in patients with Child-Pugh class A liver function undergoing liver resection.METHODS:A total of 427 consecutive patients undergoing partial hepatectomy from October 2007 to April 2011 at a single center(Department of Hepatic SurgeryⅠ,Eastern Hepatobiliary Surgery Hospital,Shanghai,China) were included in the study.All the patients had preoperative liver function of Child-Pugh class A and were diagnosed as having primary liver cancer by postoperative histopathology.Surgery was performed by the same team and hepatic resection was carried out by a clamp crushing method.A clamp/unclamp time of 15 min/5 min was adopted for hepatic inflow occlusion.Patients' records of demographic variables,intraoperative parameters,pathological findings and laboratory test results were reviewed.Postoperative liver insufficiency and failure were defined as prolonged hyperbilirubinemia unrelated to biliary obstruction or leak,clinically apparent ascites,prolonged coagulopathy requiring frozen fresh plasma,and/or hepatic encephalopathy.The incidence of postoperative liver insufficiency or liver failure was observed and the attributing risk factors were analyzed.A multivariate analysis was conducted to determine the independent predictive factors.RESULTS:Among the 427 patients,there were 362 males and 65 females,with a mean age of 51.1 ± 10.4 years.Most patients(86.4%) had a background of viral hepatitis and 234(54.8%) patients had liver cirrhosis.Indications for partial hepatectomy included hepatocellular carcinoma(391 patients),intrahepatic cholangiocarcinoma(31 patients) and a combination of both(5 patients).Hepatic resections of ≤ 3 and ≥ 4 liver segments were performed in 358(83.8%) and 69(16.2%) patients,respectively.Seventeen(4.0%) patients developed liver insufficiency after hepatectomy,of whom 10 patients manifested as prolonged hyperbilirubinemia unrelated to biliary obstruction or leak,6 patients had clinically apparent ascites and prolonged coagulopathy,1 patient had hepatic encephalopathy and died on day 21 after surgery.On univariate analysis,age ≥ 60 years and prealbumin < 170 mg/dL were found to be significantly correlated with postoperative liver insufficiency(P = 0.045 and P = 0.009,respectively).There was no statistical difference in postoperative liver insufficiency between patients with or without hepatitis,liver cirrhosis and esophagogastric varices.Intraoperative parameters(type of resection,inflow blood occlusion time,blood loss and blood transfusion) and laboratory test results were not associated with postoperative liver insufficiency either.Age ≥ 60 years and prealbumin < 170 mg/dL were selected on multivariate analysis,and only prealbumin < 170 mg/dL remained predictive(hazard ratio,3.192;95%CI:1.185-8.601,P = 0.022).CONCLUSION:Prealbumin serum level is a predictive factor for postoperative liver insufficiency in patients with liver function of Child-Pugh class A undergoing hepatectomy.Since prealbumin is a good marker of nutritional status,the improved nutritional status may decrease the incidence of liver insufficiency.

Clinical relevance and public health significance of hepatitis B virus genomic variations

Ten hepatitis B virus （HBV） genotypes （A-J） and 34 HBV subgenotypes have been identified so far. HBV genotypes and subgenotypes have distinct geographical distributions, and have been shown to differ with regard to clinical outcome, prognosis, and response to interferon treatment. Infection with subgenotype A2 is frequently associated with high viral load, resulting in acute infection via horizontal transmission. Genotypes A and B are more sensitive to interferon treatment than genotypes D and C, respectively. Genotype B is more frequent in acute hepatitis than genotype C, whereas genotype C （C2） is more frequently associated with an increased risk of hepatocellular carcinoma （HCC）, mostly cirrhotic, as compared with genotype B （B2）. Genotype mixture is associated with high viral load and worse outcome of HBV infection. HBV mutations in the S genes, especially amino acids substitution at position 145 （G145R）, are associated with immune escape, whereas mutations in the PreS or S genes which impair HBsAg secretion could present a risk to blood safety. HBV variants harboring mutations in the viral polymerase gene that confer resistance to nucleoside analogs may be selected during antiviral therapy. Different genotypes have distinct mutation patterns in the PreS and EnhH/BCP/Precore regions. PreS deletions, C1653T, T1753V, and A1762T/G1764A are associated with an increased risk of HCC. HCC- associated HBV mutants may not transmit via motherto-child transmission, and are likely generated during HBV-induced pathogenesis. Examination of HBV mutations alone or in combination and host genetic suscep-tibility will be helpful in classifying the HBV-infected subjects who will develop HCC and need active antiviral treatments.

Detection of HBsAg, HBcAg, and HBV DNA in ovarian tissues from patients with HBV infection

AIM: To investigate the presence of HBsAg, HBcAg, and HBV DNA in ovarian tissues from patients with HBV infection.METHODS: HBsAg and HBcAg were examined in ovarian biopsy tissues from 26 patients with HBV infection by immunocytochemistry, and HBV DNA was detected in ovarian tissues by PCR.RESULTS: HBsAg and HBcAg were present with the same positive rate of 34.6% (9/26). The total positive rate was 46.2% (12/26). HBsAg and HBcAg were positive in 6 (23.1%) of the 26 patients. Brown positive particles were diffusely distributed in ovarian cells. The positive rate of HBV DNA was 58.3% (7/12).CONCLUSION: HBsAg, HBcAg, and HBV DNA can be detected in ovarian tissues from patients with HBV infection. The presence of HBsAg and HBcAg in ovarian tissues does not correlate with the HBV markers in serum.

Hepatocellular carcinoma in patients with autoimmune hepatitis

AIM: To evaluate and confirm the low incidence of hepatocellular carcinoma (HCC) in patients with autoimmune hepatitis (AIH). At present only very few cases of HCC in patients with AIH and definite exclusion of chronic viral hepatitis have been published, suggesting that HCC due to AIH is rare. METHODS: In order to further investigate the incidence of HCC in patients with AIH, we reviewed our large cohort of 278 patients with AIH. RESULTS: Eighty-nine patients (32%) were diagnosed with liver cirrhosis, a preneoplastic condition for HCC. We studied a total of 431 patient years of cirrhosis in these patients, an average 4.8 years per patient. During this period none of the patients of our own study cohort developed HCC. However, three patients with HCC due to AIH associated liver cirrhosis were referred to our department for further treatment of HCC. In all three patients chronic viral hepatitis was excluded. CONCLUSION: We conclude that HCC may under rare circumstances develop due to chronic AIH dependent liver cirrhosis. Compared to other causes of liver cirrhosis such as chronic viral hepatitis, alcohol, or hemochromatosis, the incidence of HCC is significantly lower. Pathophysiological differences between AIH and chronic viral hepatitis responsible for differences in the incidence of HCC are yet to be further characterized and may lead to new therapeutic concepts in prevention and treatment of liver cancer.

Anti-HBV activity of TRL mediated by recombinant adenovirus

AIM:To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1 (-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGIox(delta)El,3Cre to acquire RAd/TRL, TR, HBVc and hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63±0.14 vs1.60±0.47, P= 0.0266, <0.05) and other control groups (0.63±0.14 vs8.50±2.78,8.25±2.26, 8.25±2.29, 8.50±1.51, 8.57±1.63, P<0.01). MTT assay suggested that there were no significant differences in cell metabolic activity between groups (P>0.05). CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.

Psychosocial stress and liver disease status

"Psychosocial stress" is an increasingly common concept in the challenging and highly-demanding modern society of today. Organic response to stress implicates two major components of the stress system, namely the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Stress is anamnestically reported by patients during the course of disease, usually accompanied by a decline in their overall health status. As the mechanisms involving glucocorticoids and catecholamines have been deciphered, and their actions on immune cell function deeper understood, it has become clear that stress has an impact on hepatic inflam-matory response. An increasing number of articles have approached the link between psychosocial stress and the negative evolution of hepatic diseases. This article reviews a number of studies on both human populations and animal models performed in recent years, all linking stress, mainly of psychosocial nature, and the evolution of three important liver-related pathological entities: viral hepatitis, cirrhosis and hepatocellular carcinoma.

Giant pedunculated sarcomatoid hepatocellular carcinoma: a case report

We report a case who was a 74-year-old female with hepatitis B virus （HBV） carrier for more than 30 years. The serum levels of AFP, CEA and CA199 were within normal range. CT scan demonstrated that a huge pedunculated mass of the right hepatic lobe presenting as Iobulated and cystic-solid appearance and heterogeneous density of the solid portion composed of multiple nodules. The patient was performed surgery, and the tumor was removed. Sarcomatoid hepatocallular carcinoma （SHC） was diagnosed by pathological and immunohistochemical test. The patient has been living for 4 months without tumor recurrence, which indicates that pedunculated SHC might have a high probability for resection and acquire better survival.