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| 1 | Methodologic research on TIMP-1,TIMP-2 detection as a new diagnostic index for hepatic fibrosis and its significance显示文摘AIM: To set up a new method to detect tissue inhibitors ofmetalloproteinase1 and -2(TIMP-1 and TIMP-2) in sero ofpatients with hepatic cirrhosis, and to investigate theexpression and location of TIMP-1 and TIMP-2 in liver tissueof patients with hepatic cirrhosis, and the correlationbetween TIMPs in liver and those in sera so as to discusswhether TIMPs can be used ss a diagnosis index of hepaticfibrosisMETHODS: The monoclonal antibodies (McAbs) of TIMP-1and TIMP-2 were used to sensitize erythrocytes, and solid-phase absorption to sensitized erythrocytes (SPASE) wasused to detect TIMP-1 and TIMP-2 in the sera of patients withhepatic cirrhosis. Meanwhile, with the method of in situhybridization and immunohistochemistry, we studied themRNA expression and antigen location of TIMP-1 and TIMP-2in the livers of 40 hepatic cirrhosis patients with pathologicdiagnosis.RESULTS: With SPASE, they were 16.4 % higher in theacute hepatitis group, 33.3 % higher in the chronic hepatitisgroup, and the positive rates were 73.6 % and 61. 2 %respectively in sero of hepatic cirrhosis patients, which wereremarkably higher than those in chronic hepatitis and acutehepatitis group ( P < 0. 001 ). In 40 samples of hepaticcirrhosis tissues, all of them showed positive expression ofTIMP-1 and TIMP-2 mRNA detected withimmunohistochemistry or in situ hybridization (positive ratewas 100 % ). Expression of TIMPs in different degrees couldbe found in liver tissue with cirrhosis. TIMPs were located incytoplasm of liver cells of patients with hepatic cirrhosis.There was a significant correlation between serum TIMPslevel and liver TIMPs level.CONCLUSION: SPASE is a useful method to detect the TIMP-1 and TIMP-2 in sera of patients with hepatic cirrhosis, andTIMP-1 and TIMP-2 can be considered as a useful diagnosticindex of hepatic fibrosis, especially TIMP-1. | Oing-He Nie Yong-Oian Cheng Yu-Mei Xie Yong-Xing Zhou Bai-Xian Guang Yi-Zhan Cao,The Centre of Diagnosis and Treatment for Infectious Disease of Chinese PLA,Tangdu Hospital,Fourth Military Medical University,Xi’an 710038,Shanxi Province,China | 2002 | World Journal of Gastroenterology2002，8，2： | 51 |
| 2 | Hepatoprotective role of ganoderma lucidum polysaccharide against BCG-induced immune liver injury in mice显示文摘AIM: To examine the effect of ganoderma lucidumpolysaccharide (GLP) on the immune liver injuryinduced by BCG infection, and investigate therelationship between degrees of hepatic damage andNO production in mice.METHODS: Immune hepatic injury was markedlyinduced by BCG-pretreatment (125 mg.kg-1, 2-week, iv)or by BCG-pretreatment plus lipopolysaccharide (LPS,125 μg.kg-1, 12-hour, iv) in mice in vivo.Hepatocellulardamage induced by BCG-pretreated plus inflammatorycytokines mixture (CM), which was included TNF-α, IL1β, IFN-γ and LPS in culture medium in vitro.Administration of GLP was performed by oral orincubating with culture medium at immune stimulisimultaneity. Liver damage was determined by activityof alanine aminotransferase (ALT) in serum and inhepatocytes cultured supernatant, by liver weightchanges and histopathological examination. NOproduction in the cultured supematant was determinedby the Griess reaction. Moreover, inducible nitric oxidesynthase (iNOS) protein expression was alsoexaminated by immunohistochemi1cal method.RESULTS: Immune hepatic injury was markedly inducedby BCG or BCG plus inflammatory cytokines in BALB/cmice in vivoand in vitro. Under BCG-stimulated condition,augment of the liver weight and increase of the serum/supernatant ALT level were observed, as well asgranuloma forming and inflammatory cells soakage wereobserved by microscopic analysis within liver tissues.Moreover, NO production was also increased by BCG or/and CH stimuli in the culture supernatant, and a lot ofiNOS positive staining was observed in BCG-prestimulated hepatic sections. Application of GLPsignificantly mitigated hepatic tumefaction, decreasedALT enzyme release and NO production in serum/supernatant, improved the pathological changes ofchronic and acute inflammation induced by BCG-stimuliin mice. Moreover, the immunohistochemical resultshowed that GLP inhibited iNOS protein expression inBCG-immune hepatic damage model.CONCLUSION: The present study indicates that NOparticipates in immune liver injury induced byMycobacterium bovis BCG infection. The mechanismsof protective roles by GLP for BCG-induced immune liverinjury may be due to influence NO production in mice. | Guo-Liang Zhang Ye-Hong Wang Wei Ni Hui-Ling Teng Zhi-Bin Lin Department of Pharmacology,School of Basic Medical Sciences,Beijing University,Beijing 100083,China | 2002 | World Journal of Gastroenterology2002，8，4： | 47 |
| 3 | Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry显示文摘AIM: To screen human single chain Fv antibody (scFv)against hepatitis C virus E2 antigen and identify its applicationin immunohistochemistry.METHODS: The phage antibody library was panned by HCVE2 antigen, which was coated in microtiter plate. After fiverounds of biopanning,56 phage clones were identified specificto HCV E2 antigen. The selected scFv clones were digestedby SfiI/NotI and DNA was sequenced. Then it was subclonedinto the vector pCANTABSE for expression as E-taggedsoluble scFv. The liver tissue sections from normal personand patients with chronic hepatitis B and chronic hepatitis Cwere immunostained with HCV E2 scFv antibody.RESULTS: The data of scFv-E2 DNA digestion and DNAsequencing showed that the scFv gene is composed of 750bp. ELISA and immunohistochemistry demonstrated that thehuman single chain Fy antibody against hepatitis C E2 antigenhas a specific binding character with hepatitis virus E2 antigenand paraffin-embedded tissue, but did not react with liver tissuesfrom healthy persons or patients with chronic hepatitis B.CONCLUSION: We have successfully screened andidentified HCV E2 scFv and the scFv could be used in theimmunostaining of liver tissue sections from patients withchronic hepatitis C. | Yan-Wei Zhong Jun Cheng Gang Wang Shuang-Shuang Shi Li Li Ling-Xia Zhang Ju-Mei Chen Gene Therapy Research Center,Institute of Infectious Diseases,302 Hospital of PLA,26 Fengtai Road,Beijing 100039,China | 2002 | World Journal of Gastroenterology2002，8，5： | 21 |
| 4 | RNA interference:Antiviral weapon and beyond显示文摘RNA interference (RNAi) is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA. The term encompasses related pathways found in a broad range of eukaryotic organisms, including fungi, plants, and animals. RNA interference is part of a sophisticated network of interconnected pathways for cellular defense, RNA surveillance, and development and it may become a powerful tool to manipulate gene expression experimentally. RNAi technology is currently being evaluated not only as an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop specific dsRNA based gene-silencing therapeutics.Several laboratories have been interested in using RNAi to control viral infection and many reports in Nature and in Cell show that short interfering (si) RNAs can inhibit infection by HIV-1, polio and hepatitis C viruses in a sequence-specific manner. RNA-based strategies for gene inhibition in mammalian cells have recently been described, which offer the promise of antiviral therapy. | Quan-Chu Wang Qing-He Nie Zhi-Hua Feng, The Center of Diagnosis and Treatment for Infectious Diseases of Chinese PLA, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province, China | 2003 | World Journal of Gastroenterology2003，9，8： | 14 |
| 5 | HCV replication in PBMC and its influence on interferon therapy显示文摘AIM: To study hepatic virus C (HCV) RNA and HCV proteinexpression in peripheral blood mononuclear cells (PBMCs)of patients with HCV infection, and explore the relationshipbetween the HCV RNA in the PBMCs and response tointerferon (IFN) therapy.METHODS: Type-specific primers were designed and RT-nested PCR was used to detect the plus- and minus- strandsof HCV RNA in PBMCs of 54 patients with HCV infection;Indirect immunofluorescence assay was applied to identifyHCVNS5 protein expression in PBMCs; 6 month-, 3 MU-IFNregiment was administrated to observe the responses toIFN in 35 chronic hepatitis C patients with different HCVRNA status in PBMCs.RESULTS: HCV plus strand RNA was found in 10 of 19(52.6 %) acute hepatitis C patients and 22 of 35 (62.9 %)chronic hepatitis C patients. HCV minus strand RNA wasdetected in 14 of 35 (40.0 %) chronic hepatitis C patients,but only one patient (5.3 %) with acute HCV infection wasfound to be minus HCV RNA positive. Though no HCV NS5protein expression was found in the examined 10 cases ofacute HCV infection, it was positive in 17 of 20 (85.0 %)chronic hepatitis C patients by indirect immunofluoresenceassay. There are significant differences of positive rate of theminus-strand and HCVNS5 protein between acute and chronichepatitis C groups(u=2.07, P<0.05and u=4.43, P<0.01respectively). The patients with minus-strand HCV RNAshowed a significantly lower 6-month sustained response (SR-6) to IFN compared to those without minus-strand HCVRNAin PBMCs (biologically 14.3 % vs 42.8 %, X2=4.12, P<0.05and virologically 7.1% vs23.9 %, X2=4.24, P<0.05).CONCLUSION: HCV is capable of infecting and replicatingin PBMCs, and HCVNS5 protein was expressed in PBMCs.The patients with minus strand HCV RNA in PBMCs showeda significantly lower 6-month sustained response to IFN,suggesting that minus-strand HCV RNA in PBMCs may beone of the factors influencing response to IFN therapy. | Guo-Zhong Gong Li-Ying Lai Yong-Fang Jiang Yan He Xian-Shi Su Center for Liver Diseases, Second Xiangya Hospital, Central South University, Changsha 410011, Hunan Province, China | 2003 | World Journal of Gastroenterology2003，9，2： | 14 |
| 6 | 丙型肝炎病毒包膜蛋白E2抗独特型人源单链可变区抗体的筛选与鉴定显示文摘目的:制备抗丙型肝炎病毒(HCV)包膜蛋白E2(E2)的抗独特型单链可变区抗体scFv(抗-IdscFv),为研制HCVE2的抗-IdscFv疫苗奠定基础.方法:采用噬菌体表面展示技术,将抗HCVE2单克隆抗体固相包被于Nunc板,从噬菌体单链可变区抗体库中经过5轮“黏附-洗脱-扩增”筛选过程,随机挑选出53个克隆,利用酶联免疫黏附法、交叉反应和竞争抑制实验,对其进行免疫学检测,获得与HCVE2单克隆抗体结合活性较强的抗独特型抗体单链可变区片段(抗-IdscFv)的阳性克隆,并对HCVE2特异性抗-IdscFv的编码序列进行序列测定分析.结果:对噬菌体单链可变区抗体库经过5轮“黏附-洗脱-扩增”的筛选后,结合到包被平皿的噬菌体与第一轮相比,富集了12倍.用酶联免疫黏附实验(ELISA)方法测定第五轮筛选后上清液中含有的抗-IdscFv与HCVE2单克隆抗体结合活性.其中有18株克隆ELISA的吸光度(A450nm)值较高(E11A450nm0.928,E14A450nm1.152,E17A450nm1.136,E28A450nm1.163,E53A450nm0.965).对这些噬菌体抗体进行与牛血清白蛋白(BSA)的交叉反应后,确定其中有5株交叉反应较弱(E11A450nm0.044,E14A450nm0.062,E17A450nm0.166,E28A450nm0.012,E53A450nm0.069),结合2次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株(E28)阳性克隆.提取质粒,进行DNA序列测定,DNA大小为768bp.结论:用噬菌体抗体库技术能够成功地获得单抗HCVE2的抗-IdscFv,本实验结果为开展用抗-IdscFv防治丙型肝炎的研究创造了条件. | 钟彦伟 成军 蔡炯 王刚 洪源 王琳 李莉 张玲霞 陈菊梅 | 2002 | 世界华人消化杂志2002，10，8： | 12 |
| 7 | Construction of HCV-core gene vector and its expression in cholangiocarcinoma显示文摘AIM: To establish an experimental model for exploring therole of hepatitis C vivus (HCV) in the development ofcholangiocarcinoma.METHODS: Recombinant plasmid of HCV-core gene wesconstructed with molecular cloning technique andtransfected into QBC939 cells with lipofection. After it wesselected with G418, resistant colonies were obtained. Thecolonies were analysed by immunocytochemistry andWestern blotting. The morphology was observed undertransmission electron microscope (TEM) and microscope.RESULTS: The recombinant plasmid wes proved to carry thetarget gene by PCR and restriction enzymed mapping.Moreover, it could express HCV-C protein efficiently inQBC039 cells. The HCV-like particles were found in thecytoplasm by EM, which were spherical with a diameter of50nr-80nm possessing outer membrane. The transfectedcells had lower differentiation and higher malignant degreeunder microscope.CONCLUSION: Because HCV-core gene could expresssteadily in cholangiocarcinoma cells, the transfected tumorcells(QBC939-HCVC) could be used to study the effect ofHCV in the development of cholangiocarcinoma. | Xiao-Fang Liu Sheng-Quan Zou Fa-Zu Qiu Department of General Surgery of Tongji Hospital,Wuhan 430030,Hubei Province,China | 2002 | World Journal of Gastroenterology2002，8，1： | 10 |
| 8 | Epstein-Barr virus in hepatocellular carcinogenesis显示文摘AIM: In recent years, studies have suggested that EpsteinBarr virus (EBV) is associated with HCC. The present study was to determine the prevalence of EBV in HCC patients,and whether EBV acted synergistically with hepatitis viruses in HCC carcinogenesis.METHODS: Liver tissue 115 HCC patients and 26 noncarcinoma patients were studied, Polymerase chain reaction (PCR) was performed to detect EBV BarnHI W DNA, EBV LMP1 DNA, HI3V X DNA, and HBV S DNA, Reverse transcription PCR (RT-PCR) was performed to detect HCV RNA and HDV RNA, Immunohistochemistry was performed to detect LMP1,HBsAg, HBcAg and HCV, The positive ratios were compared between HCC group and control group by X^2 test,RESULTS: Totally, 78 HCC samples whose 13-globulin DNA was positively detected by amplified PCR were selected,PCR was performed in all cases for EI3V DNA and HBV DNA,RT-PCR was performed in 18 cases for HCV RNA and HDV RNA. EBV BarnHI W and EBV LMP1 were positive in 18 and 6 cases, respectively. HBV X gene and HBV S gene were positive in 42 and 27 cases respectively. HCV was positive in one of the 18 cases, and none was positive for HDV. The positive rates were 28.2% (22 of 78) for EBV DNA (BarnHI W and/or LMP1) and 56.4% (44 of 78) for HBV DNA (X gene and/or S gene) respectively. In addition, 12 cases were positive for both EBV DNA and HBV DNA. Among the 26 cases in the control group, 2 cases were positive for EBV BarnHI W, 4 positive for HBV X gene and 3 positive for HBV S gene. The positive rates were 8.0% (2 of 26) and 23.1% (6 of 26),respectively, for EBV DNA and HBV DNA. The result of DNA sequencing of BamHI W was 100% homologous with the corresponding sequence of B95-8. There was significant difference in EBV infection rate between HCC patients and controls (X^2 = 4.622, P<0.05). The difference in HBV infection rate was also significant (X^2 = 8.681, P<0.05). However, there was no obvious correlation between HBV and EBV in HCC patients (X^2 = 0.835,/)>0.05). LMP1, HBV (HBsAg, HI3cAg) and HCV were detected positively in 25, 45 and 6 of 78 cases of HCC tissues respectively. In the 26 control cases,the corresponding positive cases were 2, 4 and O. The difference in EBV infection rate between HCC patients and control cases was statistically significant (X^2= 6.02,P<0.05). The difference in HBV infection rate was also statistically significant (X^2 = 10.03, P<0.05). In the 25 cases with positive LMP1 expression, 6 were in the nuclei of tumor cells, 9 in the cytoplasm of tumor cells and 10 in mesenchymal lymphocyte cytoplasm.CONCLUSION: The existence of EBV infection in HCC tissues suggests that EBV may be involved in the hepatocellular carcinogenesis in China. HBV infection may be a major cause of HCC. There is no correlation between EBV and HBV in the development of HCC. The prevalence of HCV infection is low in our area, and HDV appears not to play a direct role in hepatocellular carcinogenesis. | WeiLi Bao-AnWu Yong-MingZeng Guang-CanChen Xin-XinLi Jun-TianChen Yu-WenGuo Man-HongLi YiZeng | 2004 | World Journal of Gastroenterology2004，10，23： | 5 |
| 9 | IRES特异性IRNA对HCV IRES启动蛋白翻译细胞内抑制作用显示文摘目的:研究核糖体内部进入位点(internalribosomeentrysite,IRES)特异性抑制性RNA(inhibitorRNA,IRNA)细胞内对丙型肝炎病毒(HCV)IRES介导蛋白翻译的抑制作用.方法:体外应用脂质体细胞转染法,将IRNA及其突变体mIRNA真核表达载体pcRz-IRNA/pcRz-mIRNA转染人肝癌细胞株(HHCC),经G418筛选4wk后建立IRNA及mIRNA表达株;以相同的方法构建pcHCVcluc转染株;以脂质体介导细胞转染法将pCMVNCRLuc转染IRNA及mIRNA细胞株,于转染后48h检测荧光素酶表达量;将IRNA及mIRNA真核表达体转染pcHCVcLuc表达株,于转染后不同时间检测荧光素酶表达量.结果:HCVIRES介导蛋白翻译在IRNA表达株明显受到抑制,同样IRNA对HCV翻译复制子的蛋白翻译作用有明显抑制性;突变体mIRNA表达株和空载体对照株中未见相似的抑制性.结论:pcHCVcluc在HHCC细胞中获得有效表达;IRES特异性IRNA能有效的抑制HCVIRES介导细胞内蛋白翻译作用. | 梁雪松 连建奇 周永兴 聂青和 郝春秋 | 2003 | 世界华人消化杂志2003，11，2： | 4 |
| 10 | Epstein-Barr病毒在肝细胞癌发生中的作用探讨显示文摘目的:了解肝细胞癌(hepatocellular carcinoma,HCC)患者的EpsteinBarr病毒(EBV)感染情况,探讨EBV与肝炎病毒有无协同致癌作用。方法:研究组为78例HCC石蜡标本,对照组为26例非癌症肝组织标本。用PCR检测EBVDNA(BamHIW,LMP1)、HBVDNA(S基因、Χ基因),用RTPCR检测HCVRNA和HDVRNA,用免疫组化检测EBV(LMP1)、HBV(HBsAg、HbcAg)和HCV。结果:EBVDNA在HCC组阳性率高于对照组(28.2%vs8.0%),χ2=4.622,P=0.032;HBVDNA在HCC组阳性率高于对照组(56.4%vs23.1%),χ2=8.681,P=0.008;EBV与HBV在HCC组无相关关系,χ2=0.835,P=0.375。HCVRNA、HDVRNA在18例HCC中阳性分别为1和0例。免疫组化测EBV在HCC组阳性率高于对照组(32.1%vs2.5%),χ2=6.02,P=0.012;HBV在HCC组阳性率高于对照组(57.7%vs5.1%),χ2=10.03,P=0.001。结论:EBV在HCC发生中可能起作用,与HBV无明显协同致癌作用;HCV、HDV检出率不高,与EBV关系未能确定。 | 李威 吴宝安 曾永明 陈广灿 李欣欣 陈君填 郭毓文 李曼红 曾毅 | 2005 | 肿瘤防治杂志2005，12，11： | 3 |
| 11 | HCV包膜糖蛋白E2基因的克隆、蛋白表达及纯化显示文摘目的:获得大量重组HCVE2蛋白,为研究E2蛋白的功能及制备其抗体奠定基础.方法:利用PCR方法从HCV基因组序列中扩增出831bp(384-661aa)的E2基因片段并按读框克隆到原核表达载体pET32a(+)上,得到重组质粒pET32a-HCVE2,转化大肠杆菌BL-21(DE3)菌株,IFFG诱导HCVE2蛋白表达,SDS-PAGE和Westernblot检测蛋白表达,Ni-NTA偶联的琼脂糖黏附柱纯化融合蛋白.结果:经IPTG诱导后,可见分子量约55000的融合蛋白表达;表达的蛋白主要以包涵体形式存在;经Ni-NTA偶联的琼脂糖黏附柱纯化的融合蛋白与抗His抗体及HCV阳性血清具有良好的反应原性.结论:HCVE2基因的克隆、表达及其融合蛋白的纯化为进一步开展HCVE2蛋白功能和HCV受体的研究奠定了基础. | 杜德伟 贾战生 秦鸿雁 刘秋平 周永兴 韩骅 | 2004 | 世界华人消化杂志2004，12，2： | 1 |
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