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In vitro cleavage of hepatitis B virus C mRNA by 10-23 DNA enzyme

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Hepatobiliary & Pancreatic diseases International 2005年第4期4卷 573-576页

作者： wei Hou, Jian-Er Wo, Min-wei Li and Ke-Zhou Liu institute of infectious diseases, first affiliated hospital, zhejiang university school of medicine, hangzhou 310003, china institute of infectious diseases First Affiliated Hospital Zhejiang University School of Medicine Hangzhou 310003 China.

BACKGROUND: 10-23 DNA enzyme is one kind of de-oxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2. 2. 15 cells were demonstrated previously. The aim of this study was to further explore the cleavage activities of 10-23 DNA enzyme targeting at HBV C gene mRNA in vitro. METHODS: 10-23 DNA enzyme named Drz-HBV-C-9 specific to HBV C gene ORF A1816UG was designed and synthesized. HBV C gene mRNA was obtained by the in vitro transcription method. Cleavage activities of Drz-HBV-C-9 were observed in vitro. Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly. RESULTS: Under the certain cleavage conditions, Drz-HBV-C-9 could efficiently cleave target mRNA at specific sites in vitro. Cleavage products of 109nt plus 191nt were obtained. The kinetic parameters, Km,Kcat and Kcat/ Km for Drz-HBV-C-9, were 1.4 ×10-9 mol, 1.6 min-1 and 1.1 × 109 mol-1 · min-1, respectively. CONCLUSIONS: 10-23 DNA enzyme targeting at HBV C gene mRNA possesses specific cleavage activities in vitro. This would be a potent antiviral strategy with respect to HBV gene therapy.

关键词： 10-23 DNA enzyme hepatitis B C gene cleavage in vitro

Gene heterogeneity of hepatitis B virus isolates from patients with severe hepatitis B

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Hepatobiliary & Pancreatic diseases International 2005年第4期4卷 530-534页

作者： wei wu, Yu Chen, Bing Ruan and Lan-Juan Li hangzhou, china Department of infectious diseases, first affiliated hospital, zhejiang university school of medicine, Key Laboratory of infectious diseases of Health Ministry of china, hangzhou 310003, china Department of infectious diseases First Affiliated Hospital Zhejiang University School of Medicine Key Laboratory of Infectious Diseases of Health Ministry of China Hangzhou 310003 China.

BACKGROUND: The pathogenesis of severe hepatitis B remains unknown. Reports have indicated that hepatitis B virus (HBV) mutations are important factors in the pathogenesis of this disease. This study was to investigate the genetic heterogeneity of HBV strains from serum samples of patients with fulminant hepatitis B. METHODS: Full-length HBV genomes from 4 patients with severe hepatitis B were cloned and sequenced to observe mutations in every open reading-frame ( ORF). Serum samples of another 25 patients with severe hepatitis B, 30 patients with chronic hepatitis B, and 25 HBV carriers were collected for sequencing and comparison of mutations in preS2, preC and core promoter regions. RESULTS: Of 4 HBV full-length genome sequences, 3 had a G to A mutation at nucleotide A1896 in the preC region and 1 had double mutations of T1762-A1764 in the core promoter region. The 4 sequences showed mutations in the known B or T cell epitopes of the preS2 and C regions. For the other 3 groups, more mutations were seen in the preS2 region in the HBV isolates from the patients with severe hepatitis B than those from the patients with chronic hepatitis B and HBV carriers (P <0.01). There was a significant difference of mutations in the T cell epitope region of preS2 between the patients with severe hepatitis B and those with chronic hepatitis B or HBV carriers (P <0.01). In the preC and core promoter regions, the mutation frequencies of T1653 and C1753 were 48.0% and 24.0% respectively in the patients with severe hepatitis B, but none of these mutations were observed in the patients with chronic hepatitis B group or HBV carriers (P <0.01). The mutation frequency of T1762-A1764 was 76.0% in the patients with severe hepatitis B, 40.0% in the patients with chronic hepatitis B (P <0. 01) , and 16. 0% in the HBV carriers ( P < 0. 01). There was a significant difference in A1896 mutation between the patients with severe hepatitis B and the patients with chronic hepatitis B (P < 0. 05 ) o

关键词： hepatitis B virus severe hepatitis virus genome gene heterogeneity preS2 preC core promoter

ROC curves in evaluation of serum fibrosis indices for hepatic fibrosis

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World Journal of Gastroenterology 2002年第6期8卷 1073-1076页

作者： Min Zheng wei-Min Cai Hong-Lei Weng Rong-Hua Liu,institute of infectious diseases,first affiliated hospital,school of medicine,zhejiang university,hangzhou 310003 zhejiang Province,china institute of infectious diseases First Affiliated Hospital School of Medicine Zhejiang University Hangzhou 310003 Zhejiang Province China. charming-hz@***

AIM: Use Receiver operating characteristic (ROC) curves to find out the relationship between serum level of hyaluronic acid (HA), type Ⅲ procollagen (PCⅢ), N-terminal procollagen Ⅲ peptide (PⅢNP), laminin (LN), type Ⅳ collagen (C-Ⅳ) and hepatic fibrosis, as well as to determine their value in clinical ***: 114 serum samples from chronic hepatitis patients were assayed for fibrosis indices including HA, PCⅢ, PⅢNP, LN and Ⅳ-C with radioimmunoassay (RIA). Liver biopsy was also performed in all these patients and the biopsy material was examined ***: ROC curves analysis showed that area under the curve (AUC) of PⅢNP, HA, PCⅢ, C-Ⅳ and LN was 0.800,0.728, 0.727, 0.583 and 0.463, respectively. The analysis also showed that PⅢNP (r=0.452), HA (r=0.497) and PCⅢ (r=0.404) have greater diagnosis performances than C-Ⅳ (r=0.238) and LN (r=0.128) according to fibrosis staging. The sensitivity of HA plus PⅢNP was 55.1%, it was the most sensitive combination. Combined three or more than three indices that based on HA, the specificity was 100 %.Using combination assays can improve the specificity, but its sensitivity was not high. Serum fibrosis indices increased as the grade of inflammation aggravated. But only PⅢNP and PCⅢ had significant difference between G1 and G2 (PⅢNP: 13.16±8.07 VS8.32±5.09; PCⅢ: 164.22±65.69 VS 138.23±77.63). The coefficient correlation of the results of inflammation grade and fibrosis staging to HA was 0.525 and 0.553 respectively, that to PCⅢ, 0.446 and 0.412, that to LN, 0.234 and 0.194, and that to Ⅳ-C, 0.363 and 0.351, ***: Serum fibrosis indices can indicate tendency of hepatic fibrosis, but it cannot replace liver biopsy. However, as diagnostic markers, more efficient serum fibrosis indices for the diagnosis of hepatic fibrosis need to be explored.

关键词：肝纤维化血清纤维化指标 ROC曲线评价

Cytomegalovirus infection in liver transplant recipients

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Hepatobiliary & Pancreatic diseases International 2002年第1期1卷 30-32页

作者： Jun Fan wei-Hang Ma Jing Qian Zhi Chen Dong--Sheng Huang wei-Lin Wang Shu-Sen Zheng From the first affiliated hospital, zhejiang university school of medicine, hangzhou 310003, china institute of infectious diseases and Key Laboratory of Viral infectious diseases Ministry of Health Zhejiang University School of Medicine Hangzhou 310003 China. fjjc@***

Objective: To explore cytomegalovirus （CMV） infec-tion in recipients of liver transplantation （LT）.Methods: 30 recipients of LT were screened for theappearance of CMV infection by using ELISA to testanti-CMV-Ab from serum samples and using im-munohistochemistry method to test CMV antigen ex-pression and nested-PCR to amplify CMV-DNA fromblood ***: Four of 243 samples taken from 30 recipientscame out positive of anti-CMV IgC; and anti-CMVIgM with a positive rate of 100% and 1.6% respec-tively. 85 samples resulted in CMV antigen expression（35.0%） with the average antigen index being 4.2±3.1/5×10;WBC. Besides, 99 samples were found tobe positive by nested-PCR with a positive rate of40.7%. 61 samples were found to be simultaneously pos-itive in test of CMV antigen and DNA, with a rate of25.1%.Conclusions: Infection of CMV is common in recipi-ents of LT. Simultaneous screening of anti-CMV-Ab, CMV-Ag and CMV-DNA after liver transplanta-tion is very important for early diagnosis of CMV in-fection.

关键词： cytomegalovirus anti-CMV-Ab cytomegalovirus antigen cytomegalovirus DNA liver transplantation

Effects of Salvia miltiorrhiza on intestinal microflora in rats with ischemia/reperfusion liver injury

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Hepatobiliary & Pancreatic diseases International 2005年第2期4卷 274-280页

作者： Hui-Chun Xing, Lan-Juan Li, Kai-Jin Xu, Tian Shen, Yun-Bo Chen, Yu Chen, Su-Zhen Fu, Ji-Fang Sheng, Chun-Lei Chen, Jian-Guo Wang, Dong Yan, Fang-wei Dai and Xiao-Ying Sha hangzhou, china Key Laboratory of infectious diseases, Ministry of Public Health of china, and institute of infectious diseases, Department of infectious diseases, first affiliated hospital, zhejiang university school of medicine, hangzhou 310003, china zhejiang Academy of Medical Sciences, hangzhou 310012, china Department of infectious diseases First Affiliated Hospital Zhejiang University School of Medicine Hangzhou 310003 China.

BACKGROUND: Hepatic ischemia/reperfusion injury may induce intestinal microflora imbalance. Salvia miltiorrhiza is effective in promoting blood circulation and counteracting peroxidation in tissues. The aim of the present study was to determine the effects of Salvia miltiorrhiza on intestinal mi- croflora, endotoxemia, and bacterial translocation in rats with hepatic I/R injury. METHODS: Sprague-Dawley rats in specific pathogen free grade were divided into 3 groups: group I(n =6) for sham operation: groups ( n = 7) for liver ische- mia for 20 minutes and reperfusion for 22 hours. Group was also pretreated with 4 ml/day of Salvia miltiorrhiza solu- tion (250 mg/kg) by daily gavage for 7 days. The levels of serum alanine aminotransferase (ALT), aspartate amino- transferase (AST), malondialdehyde ( MDA) and supero- xide dismutase ( SOD ) in liver tissues, serum endotoxin, intestinal bacterial counts, intestinal mucosal histology and bacterial translocation were studied. RESULTS: The levels of ALT, AST, plasma endotoxin and MDA in liver tissues were decreased more markedly in group (57.57 ± 18.08 U/L, 147.57 ±40.84 U/L, 0.42 ± 0.144 EU/ml and 0. 52 ±0.19 nmol/mg-prot respectively) in group 295.9±216.92 U/L, 0.80± 0.262 EU/ml and 0.72±0.12 nmol/mg-prot; P <0.05-0.01 respectively). Liver SOD activity was increased more sig- nificantly in group (318.47±64.62 U/mg-prot) than in group U/mg-prot, P<0.05). The counts of Bifidobacteria and Bacteroides increased more significantly in group than in group but were similar to those in group I. Bacterial translocation to the kidney in group was 50% (5/10), whereas no bacterial translocation to the kidney occurred in the other two groups (P <0. 01). Ileal mucosal structure was markedly ameliorated in group as compared with group CONCLUSIONS: Salviae miltiorrhiza could partially restore intestinal microflora balance, improve intestinal mucosal integrity, and reduce bacterial translocation and plasma en- dotoxin in rats with hepatic is

关键词： Salviae miltiorrhiza hepatic ischemia/reperfusion microflora endotoxin bacterial translocation

Influence factors of serum fibrosis markers in liver fibrosis

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World Journal of Gastroenterology 2003年第11期9卷 2497-2500页

作者： Jun Tao Hui-Qin Peng wei-Min Cai Feng-Qin Dong Hong-Lei Weng Rong-Hua Liu, institute of infectious diseases, first affiliated hospital, school of medicine, zhejiang university, hangzhou 310003 zhejiang Province, china institute of infectious diseases First Affiliated Hospital School of Medicine Zhejiang University Hangzhou 310003 Zhejiang Province China. taojun20001@***

AIM: To analyze the factors which influence the serum levels of hyaluronic acid (HA), type Ⅲ pro-collagen (PCIII), laminin (LN) and type Ⅳ collagen (C-Ⅳ) in liver ***: The serum specimens from 141 chronic hepatitis patients were assayed for fibrosis indexes including HA, PCIIT,LN and C-Ⅳ with radioimmunoassay (RIA) and liver function indexes by an automatic biochemistry analyzer. The patients were then divided into consistent group and inconsistent group. The patients'clinical manifestations were recorded,routine blood pictures were done by a blood counter and analyzer (AC-900). Liver biopsy specimens were examined path-morphologically. The inner diameters of portal vein,splenic vein and thickness of spleen were all measured by ***: Sixteen patients (14.16 %) had serum fibrosis indexes inconsistent with histological stage of their hepatic fibrosis. Their serum fibrosis indexes did not correlate with the stage of hepatic fibrosis (P>0.05), but were positively correlated with the grade of inflammation (X2=12.07, P0.05).CONCLUSION: Serum fibrosis indexes can be influenced by the grade of inflammation, some liver function indexes and clinical manifestations. Comprehensive analysis is necessary for its proper interpretation.

关键词：肝纤维化血清标志物放射免疫分析透明质酸胶质原

Diagnostic value of platelet derived growth factor-BB,transforming growth factor-131,matrix metalloproteinase-1,and tissue inhibitor of matrix metalloproteinase-1 in serum and peripheral blood mononuclear cells for hepatic fibrosis

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World Journal of Gastroenterology 2003年第11期9卷 2490-2496页

作者： Bin-Bin Zhang wei-Min Cai Hong-Lei Weng Zhong-Rong Hu Jun Lu Min Zheng Rong-Hua Liu, institute of infectious diseases, first affiliated hospital, school of medicine, zhejiang university, hangzhou 310003, zhejiang Province, china Bin-Bin Zhang Wei-Min Cai Hong-Lei Weng Zhong-Rong Hu Jun Lu Min Zheng Rong-Hua Liu Institute of Infectious Diseases First Affiliated Hospital School of Medicine Zhejiang University Hangzhou 310003 Zhejiang Province China

AIM: Noninvasive diagnosis of hepatic fibrosis has become the focus because of the limited biopsy, especially in the surveillance of treatment and in screening hepatic fibrosis. Recently, regulatory elements involved in liver fibrosis, such as platelet derived growth factor-BB (PDGF-BB), transforming growth factor-β1(TGF-β1), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), have been studied extensively. To determine whether these factors or enzymes could be used as the indices for the diagnosis of hepatic fibrosis, we investigated them by means of receiver operating characteristic (ROC) curve. METHODS: Serum samples from sixty patients with chronic viral hepatitis B and twenty healthy blood donors were assayed to determine the level of PDGF-BB, TGF-β1, MMP-1, and TIMP-1 with ELISA, and HA, PCIII, C-IV, and LNlevel with RIA. The message RNA (mRNA) expression of TIMP-1 and MMP-1 in peripheral blood mononuclear cells (PBMCs) was detected by RT-PCR and Northern blot hybridization. Liver biopsy was performed in all patients. The biopsy samples were histopatholocjically examined. The trial was double-blind controlled. RESULTS: The serum level of PDGF-BB, TIMP-1, the ratio of TIMP-1 and MMP-1 (TIMP-1/MMP-1), mRNA expression of TIMP-1 (TIMP-lmRNA), and the ratio of TIMP-lmRNA and MMP-lmRNA (TIMP-lmRNA/MMP-lmRNA) in patients was significantly higher than those in the healthy blood donors (t=2.514-11.435, P=0.000-0.016). The serum level of PDGF-BB, TIMP-1, TIMP-1/MMP-1, and TIMP-lmRNA was positively correlated with fibrosis stage and inflammation grade (r=0.239-0.565, P=0.000-0.033), while the serum level of MMP-1 was negatively correlated with fibrosis stage and inflammation grade, and TIMP-lmRNA/MMP-lmRNA was positively correlated with inflammation grade. Through the analysis by ROC curve, serum PDGF-BB was the most valuable marker, and its sensitivity was the highest among the nine indices. The markers with the highest spe

关键词：外周血基质多发蛋白酶-1 单核细胞肝纤维化转化生长因子血小板衍化生长因子

Association of cytomegalovirus infection with human leukocyte antigen genotypes in recipients after allogeneic liver transplantation

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Hepatobiliary & Pancreatic diseases International 2006年第1期5卷 34-38页

作者： Jun Fan, Xue-Qin Meng, Mei-Fang Yang, Lin Zhou, Xiao-Ming Chen, Min-Jun Hu, wei-wei Fan, wei-Hang Ma and Lan-Juan Li Key Laboratory of Viral infectious diseases, Ministry of Public Health, institute of infectious diseases Organ Transplantation Center first affiliated hospital, zhejiang university school of medicine, hangzhou 310003. china Key Laboratory of Viral infectious diseases Ministry of Public Health Institute of Infectious Diseases First Affiliated Hospital Zhejiang University School of Medicine Hangzhou 310003 China. fanjunyz@***

BACKGROUND: Cytomegalovirus (CMV) infection is the important cause affecting the survival rate and function of the transplanted organ after transplantation. The occurrence of CMV infection after liver transplantation (LT) is associated with many factors. Lots of studies suggest that genetic mutation between hosts and CMV may play a role in the occurrence and development of CMV infection. CMV exists in an incubative state, affect or destroy the expression of human leukocyte antigen (HLA) molecules in the host cell surface, and interfere antigen's submission. This mechanism is the key of CMV to avoid immune defense mechanism of the host. To detect HLA and CMV antibody (CMV-Ab), CMV antigen (CMV-Ag) of transplantation recipients, we evaluated the association of CMV infection and the particular HLA genotypes in recipients after LT. METHODS: 277 blood samples were collected from 39 LT recipients. CMV antibody and antigen were detected by ELISA or immunohistochemical methods. The HLA types of the recipients were determined by PCR. To analyze the association of HLA alleles and the occurrence of CMV antigenemia in the patients, relative risk degree (RR) was used as the parameter for the Chi-square test. RESULTS: The LT recipients were serum CMV IgG positive (100%), but none of them was CMV IgM positive (0%). Thirty-three LT recipients (84.6%) were CMV antigenic positive with 1-50 positive leukocytes per 50000 leukocytes in extent and 7.2±4.2 positive leukocytes per 50000 leukocytes on average. Thirteen patients developed CMV pneumonia, with CMV antigenic positive (100%) and 17.7±5.5 positive leukocytes per 50000 leukocytes on average. Some HIA alleles were associated with the occurrence and extent of CMV antigenemia. HLA-A2 was the higher frequency allele for patients with antigenemia (P<0.05), and 7 patients carrying HLA-DR11 allele developed antigenemia (P<0.05). In the lower antigenemia group, HLA-A11 was higher in frequency than others (P<0.05). Besides, none of the pat

关键词： cytomegalovirus human leukocyte antigen liver transplantation cytomegalovirus antigen

A proteomic analysis of allograft rejection in rats after liver transplantation

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Science china(Life Sciences) 2007年第3期50卷 312-319页

作者： ZHANG ChunChao1,2, ZHU Feng2,3, wei JianFeng2, ZHENG ShuSen2 & LI LanJuan1 1 Department of infectious diseases, The first affiliated hospital, zhejiang university school of medicine, hangzhou 310003, china 2 institute of Surgery, Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The first affiliated hospital, zhejiang university school of medicine, hangzhou 310003, china 3 Department of Hepatobiliary Surgery, The Third affiliated hospital, Suzhou university, Changzhou 213003, china 1. Department of infectious diseases The First Affiliated Hospital Zhejiang University School of Medicine Hangzhou 310003 China 2. institute of Surgery Key Laboratory of Combined Multi-organ Transplantation Ministry of Public Health The First Affiliated Hospital Zhejiang University School of Medicine Hangzhou 310003 China 3. Department of Hepatobiliary Surgery The Third Affiliated Hospital Suzhou University Changzhou 213003 China

In order to understand the allograft rejection in orthotopic liver transplantation (OLT), an allograft re- jection rat model was established and studied by proteomic approach. The protein expression profiles of liver tissues were acquired by fluorescence two-dimensional difference gel electrophoresis (2D DIGE) that incorporated a pooled internal standard and reverse fluorescent labeling method. The expression levels of 27 protein spots showed significant changes in acute rejection rats. Among these spots, 19 were identified with peptide mass fingerprinting using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) after tryptic in-gel digestion. The results of the present paper could be helpful for our better understanding of allograft rejection in organ transplantation.

关键词： A proteomic analysis of allograft rejection in rats after liver transplantation DIGE