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| 1 | 慢性病毒性肝炎发病机制的分子生物学研究显示文摘由乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)的感染引起的急性和慢性肝病目前还没有满意的治疗方法.新型治疗方法和治疗药物的研究开发,依赖于肝炎病毒致病的分子生物学机制的研究进展.关于HBV感染个体准种概念的引入,使我们对于HBV基因变异的研究,从单一病毒的基因变异上升到群体病毒的基因变异?从静态的基因突变上升到动态的基因变异,从而对HBV存在状态的看法发生了根本的改变.HBV感染肝细胞的相关受体蛋白虽然进行了多年的研究,但还没有最终确定.利用新型技术对于HBV表面抗原蛋白的结合蛋白进行筛选是一个重要的方向.HBV和HCV感染与肝细胞癌之间的关系已经得到确定,但是具体的分子生物学机制还有许多工作要做.研究这2种肝炎病毒反式激活作用的把基因是阐明其引起肝细胞癌分子生物学机制的重要途径.在肝细胞中表达的肝炎病毒蛋白不是孤立存在的,或者与其自身结合形成同二聚体,或者与病毒的其他蛋白?肝细胞蛋白结合形成异二聚体,从而对于肝细胞的生长?代谢?甚至是恶性转化产生重要影响. | 成军 | 2002 | 世界华人消化杂志2002，10，2： | 141 |
| 2 | 肝门部胆管癌外科治疗的现状与展望显示文摘 | 黄志强 | 2005 | 中国普外基础与临床杂志2005，12，4： | 28 |
| 3 | Hepatitis C virus infection of human hepatoma cell line 7721 in vitro显示文摘AIM To establish a cell culture system with long-term replication of hepatitis C virus in vitro.``METHODS Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RT-PCR, in situ hybridization and immunohistochemistry respectively.``RESULTS The intracellular HCV RNA was first detected on d 2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3, CP10antigens could be observed in cells. The fresh cells could be infected by supematant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed.``CONCLUSION The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro. | Zhi-Qiang Song~1 Fei Hao~1 Feng Min~2 Qiao-Yu Ma~2 Guo-Dong Liu~2 Department of Dermatology~1Department of Infectious Diseases~2,Southwest Hospital,Third Military Medical University,Chongqing 400038,China | 2001 | World Journal of Gastroenterology2001，7，5： | 26 |
| 4 | 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 |
| 5 | HCVC基因腺病毒表达载体骨架质粒pAd.HCV-C的构建、鉴定及表达显示文摘目的构建能表达HVC基因的腺病毒表达载体的重组骨架质粒,为进一步包装能高效表达HCVC基因的腺病毒载体做准备。方法用分别含有BgⅡ及HindⅢ酶切位点的HCVC区基因上、下游引物,以含有HCV H株基因序列的质粒pBRTM/HCV1-3011为模板,通过PCR扩增获得HCVC区基因片段,基因片段回收后,以BglⅡ及HindⅢ双酶切,定向插入到腺病毒骨架质粒pAd,CMV-Link.1中CMV启动子下游BglⅡ与HindⅢ位点之间,获得重组表达质粒pAd.HCV-C通过BglⅡ/HindⅢ双酶切、PCR及插入片段序列测定对质粒进行了鉴定,以抗HCVC单克隆抗体为一抗,利用间接免疫荧光法检测了pAd.HCV-C在人肝癌细胞7721中的瞬时表达。结果酶切、PCR及测序鉴定证实,pAd,HCV-C插入片段为HCVC区基因片段,免疫荧光法检测表明其可以在7721细胞中瞬时表达。结论构建的质粒pAd.HCV-C可以在7721细胞中瞬时表达HCVC区基因,为包装表达HCVC基因的腺病毒载体奠定了基础。 | 郝春秋 周永兴 冯志华 李谨革 贾战生 王平忠 | 2001 | 世界华人消化杂志2001，9，6： | 15 |
| 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 | High frequencies of HGV and TTV infections in blood donors in Hangzhou显示文摘ALM To determine the frequencies of HGV and TTVinfections in blood donors in Hangzhou.METHODS RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed.``RESULTS Thirty-two ( 15.8%) and 30 (14.8%) of the 203serum samples were positive for HGV RNA and TTV DNA,respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV RNA and TTV DNA.Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3serum samples compared with the reported HGV and TTV sequences was 89.36%. 87.94%, 88.65% and 63.51%.65.77% and 67.12%. respectively.``CONCLUSION The infection rates of HGV and/or TTV inblood donors are relatively high. and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas. | Jie Yan~1 Li-Li Chen~2 Yi-Hui Luo~1 Ya-Fei Mao~1 Meng He~1 1 Department of Pathogenic Biology,Medical School of Zhejiang University,Hangzhou 310006,Zhejiang Province,China2 Department of Stomatology,The Second Affiliated Hospital,Medical School of Zhejiang University,Hangzhou 310009,Zhejiang Province,China | 2001 | World Journal of Gastroenterology2001，7，5： | 11 |
| 8 | Distribution of nitric oxide synthase in stomach myenteric plexus of rats显示文摘AIM: To study the distribution of nitric oxide synthase (NOS) in rat stomach myenteric plexus.METHODS: The distribution of NOS in gastric wall was studied in quantity and location by the NADPH-diaphorase (NDP) histochemical staining method and whole mount preparation technique.RESULTS: NOS was distributed in whole stomach wall, most of them were located in myenteric plexus, and distributed in submucosal plexus. The shape of NOS positive neurons was basically similar, most of them being round and oval in shape. But their density, size and staining intensity varied greatly in the different parts of stomach. The density was 62 -± 38 cells/mm2(antrum), 43 ± 32 cells/mm2(body), and 32 ± 28 cells/mm2 (fundus), respectively. The size and staining intensity of NOS positive neurons in the fundus were basically the same, the neurons being large and dark stained, while they were obviously different in antrum. In the body of the stomach, the NOS positive neurons were in an intermediate state from fundus to antrum. There were some beadlike structures which were strung together by NOS positive varicosities in nerve fibers, some were closely adherent to the outer walls of blood vessels.CONCLUSION: Nitric oxide might he involved in the modulation of motility, secretion and blood ciroulation of the stomach, and the significant difference of NOS positive neurons in different parts of stomach myenteric plexus may be related to the physiologic function of stomach. | Xi Peng Jin-Bin Feng Hong Yan Yun Zhao Shi-Liang Wang Institute of Burn Research,Southwest Hospital,Third Military Medical University,Chongqing 400038,China | 2001 | World Journal of Gastroenterology2001，7，6： | 11 |
| 9 | 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 |
| 10 | Investigation of HGV and TTV infection in sera and saliva from non-hepatitis patients with oral diseases显示文摘AIM: To determine the frequencies of HGV and TTV infectionsin serum and saliva samples of non-hepatitis patients withoral diseases in Hangzhou area, and to understand thecorrelation between detected results of HGV RNA and/or TTVDNA in sera and in saliva from the same patientsMETHODS: RT-nested PCR for HGV RNA detection andsemi-nested PCR for nv DNA detection were performed inthe serum and saliva samples from 226 non-hepatitis patientswith oral diseases, and nucleotide sequence analysis.RESULTS: Twenty-seven (11.9 %) and 21 (9.3 %) of the226 serum samples were only positive for HGV RNA andlrv DNA, respectively. 10 (4.4 %) and 9 (3.9 %) of the226 saliva samples were only positive for HGV RNA andTTV DNA, respectively. And 7 (3.1%) of the serum samplesand 2 (0.9 %) of the saliva samples showed the positiveamplification results for both HGV RNA and Irv DNA. 12saliva samples from the 34 patients (35.3 %) with HGV orHGV/TTV viremia and 11 saliva samples from the 28 patients(39.3 %) with TTV or HGV/TTV viremia were HGV RNAdetectable, respectively, including two patients positive forboth HGV RNA and TTV DNA in serum and saliva samples.No saliva samples from the 226 patients were found to beHGV RNA or nv DNA detectable while their serum sampleswere negative for HGV or TTV. Homologies of the nucleotidesequences of HGV and TTV amplification products from theserum and saliva samples of the two patients comparedwith the reported sequences were 88.65-91.49 % and65.32-66.67 %, respectively. In comparison with thenucleotide sequences of amplification products betweenserum and from saliva sample from any one of the twopatients, the homologies were 98.58 % and 99.29 % forHGV, and were 98.65 % and 98.20 % for rTV, respectively.CONCLUSION: Relatively high carrying rates of HGV and/or TTV in the sera of non-hepatitis patients with oral diseasesin Hangzhou area are demonstrated. Parts of the carriersare HGV and/or TTV positive in their saliva. The results ofthis study indicate that dentists may be one of the populationswith high risk for HGV and/or TTV infection, and by way of saliva HGV and TTV may be transmitted among individuals. | Jie Yan Department of Pathogenic Biology,College of Medical Science,Zhejiang University,Hangzhou 310031,Zhejiang Province,China Li-Li Chen Department of Stomatology,The Second Affiliated Hospital,College of Medical Science,Zhejiang University,Hangzhou 310009,Zhejiang Province,China Yong-Liang Lou Xiao-Zhi Zhong Wenzhou Medical College,Wenzhou 325000,Zhejiang Province,China | 2002 | World Journal of Gastroenterology2002，8，5： | 6 |
| 11 | 复制缺陷型HCV C基因腺病毒表达载体的构建包装及鉴定显示文摘目的:构建能表达HCVC基因的复制缺陷型腺病毒表达载体.方法:将HCVH株C区基因定向插入到腺病毒穿梭质粒pAd.CMV-Link.1中,获得重组质粒pAd.HCV-C,再与pJM17共转染293细胞,包装腺病毒表达载体.通过酶切、PCR及测序对穿梭质粒进行了鉴定.对腺病毒载体进行了感染性鉴定、电镜鉴定及双引物PCR鉴定.利用间接免疫荧光法和Westernblot检测了腺病毒载体在人肝癌细胞HepG2中的表达.结果:酶切、PCR及测序鉴定证实,穿梭质粒插入片段为HCVC区基因.包装的腺病毒载体具有良好的感染性,可以在293细胞中形成病毒颗粒,腺病毒载体内携带HCVC区基因,并可以在HepG2细胞中表达HCVC抗原.结论:包装成功的复制缺陷型腺病毒载体可以在HepG2细胞中表达HCVC抗原,为丙型肝炎的基因治疗及疫苗的进一步研究奠定了基础. | 郝春秋 冯志华 周永兴 聂青和 李谨革 贾战生 梁雪松 谢玉梅 曹义战 康文臻 | 2003 | 世界华人消化杂志2003，11，2： | 6 |
| 12 | HCVNS3蛋白对正常人源肝细胞生长及MAPK磷酸化的影响显示文摘目的:研究丙型肝炎病毒非结构区3(HCVNS3)蛋白对正常人源肝细胞的转化及MAPK磷酸化调节的作用.方法:利用脂质体介导转染技术和G418筛选得到稳定表达NS3蛋白的正常人源性肝细胞QSG7701,PCR和免疫组化S-P法检测细胞中NS3的表达;细胞记数和软琼脂实验鉴定其生物学性质;抗磷酸化MAPK抗体和抗MAPK抗体Westernblot检测转染细胞MAPK活性及表达.结果:HCVNS3转染的QSG7701肝细胞,其NS3蛋白过度表达于细胞质;真核表达质粒pRcHCNS3-5’转染细胞倍增时间为12h,较pRcHCNS3-3’、pRcCMV转染细胞和未转染的QSG7701明显缩短(分别24h,26h和28h).pRcHCNS3-5’、pRcHCNS3-3’和pRcCMV转染细胞及正常肝细胞在软琼脂中的克隆形成率分别为33%、1.33%、1.46%、1.11%,pRcHCNS3-5’形成的克隆明显高于其他三种细胞(P<0.01).pRcHCNS3-5’转染细胞MAPK磷酸化程度明显高于其他三种细胞(分别为8858±877,5612±656,2212±245和989±188).(P<0.01),而MAPK的表达量没有差异(P>0.05).结论:人源性正常肝细胞QSG7701是研究HCVNS3蛋白致病机制的较好的细胞系;HCVNS3蛋白N端多肽具有促进细胞增生和改变细胞表型的作用;HCVNS3蛋白N端多肽能上调MAPK活性,不影响MAPK的表达量. | 孙意 程瑞雪 冯德云 欧阳小明 郑晖 | 2003 | 世界华人消化杂志2003，11，2： | 4 |
| 13 | 基因芯片技术检测HBV HCV及HBV YMDD变异株显示文摘目的:探讨基因芯片技术在乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)及HBVYMDD变异株检测中的应用.方法:采用HBV、HCV联合基因芯片检测40份血清标本,并与HBV,HCV基因定量检测方法比较;采用HBVYMDD变异株芯片检测20份血清标本,并与错配PCR及DNA序列测定方法比较.结果:HBV,HCV联合基因芯片与HBVDNA定量检测的符合率为85%(34/40),HBV检出率为83%(19/23),假阳性率为12%(2/17);与HCVRNA定量检测的符合率为85%(34/40),HCV检出率为58%(7/12),假阳性率为4%(1/28).HBVYMDD变异株芯片与本室设计的错配PCR符合率为70%(14/20),5份芯片检测的标本与DNA序列测定结果一致,该芯片尚可检测出不同病毒体或变异体的混合感染.结论:HBV,HCV联合基因芯片对HBV的检测有较高的敏感性,但存在一定程度的非特异性;对HCV的检测敏感性偏低,但特异性较高.HBVYMDD变异株芯片敏感性和特异性均较高,同时能检测出病毒变异体的共生现象. | 李刚 舒欣 马会慧 陈伟 陈文思 陈青 江元森 姚集鲁 | 2003 | 世界华人消化杂志2003，11，2： | 4 |
| 14 | 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 |
| 15 | Effects of cholesterol on the phenotype of rabbit bile duct fibroblasts显示文摘AIM: To investigate how cholesterol (Ch) can affect thephenotype of bile duct fibroblasts of New Zealand rabbits.METHODS: 16 rabbits were divided randomly into twogroups: the control group and the experiment group. Therabbits in experiment group were fed with hypercholesteroldiet for 8 weeks. Bile duct was dissociated from rabbits andprepared for transmission electron microscopy. The purifiedbile duct fibroblasts were cultured and divided randomlyinto there groups: control group, Ch smiddle concentrationgroup (0.6 g/L), Ch high concentration group (1.2 g/L). Afterincubated for 72 h, the fibroblasts were made into specimensfor transmission electron microscopy. The expression of α-actin in bile duct fibroblasts was measured by means oflaser scanning confocal microscopy.RESULTS: With the transmission electron microscopy, thenormal bile duct fibroblasts were shuttle-shaped, and therewere abundant rough endoplasmic reticulums (RER), butfew mitochondria or microfilaments in cytoplasm. This isthe typical phenotype of fibroblasts. Bile duct fibroblasts ofhypercholesterolemic rabbits were observed, by thetransmission electron microscopy Rough endoplasmicreticulums were significantly reduced, with a lot ofmicrofilament bundles or stress fibers appeared in cytoplasm,especially under plasma membrane. Dense bodies werescattered within these bundles. Macula densas anddiscontinuous sarcolemma were found under plasmamembrane. It suggested that the bile duct fibroblasts ofhypercholesterolemic rabbits presented the phenotype ofsmooth muscle cell. The cultured bile duct fibroblasts alsohad typical phenotype of fibroblasts. After stimulated bymiddle concentration cholesterol (0.6 g/L) for 72 h, thereappeared lots of microfilaments in cytoplasm, but withoutdense body, macula densa and discontinuous sarcolemma.Observed with confocal microscopy, there were many regularbundles of microfilaments in fibroblasts treated with middleconcentration ch (0.6 g/L) and the expression of α-actinwas signifiantly increased. The average fluorescence valueof middle concentration group was 1 628+189 (P<0.01 vscontrol group). Microfilaments and the expression of α-actinwere greatly decreased in fibroblastes of high concentrationgroup (1.2 g/L). The average fluorescence value of highconcentration group was 1 427±153 (P<0.05 vs middleconcentration group). There were a lower expression of α-actin and few microfilaments in bile duct fibroblasts of controlgroup with an average fluorescence value of 1 224±138.CONCLUSION: Cholesterol can make bile duct fibroblastshave the phenotypic characteristics of smooth muscle cellboth in vitro andin vivo and this effect is more significant invivo. The effect is probably associated with some otherfactors besides cholesterol. | Bao-YingChen Jing-GuoWei Yao-ChengWang Chun-Meiwang JunYu Xiang-XinYang | 2003 | World Journal of Gastroenterology2003，9，2： | 3 |
| 16 | HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro显示文摘瞄准:与丙肝的长期的复制建立一个房间文化系统病毒(HCV ) 染色体和病毒的抗原的表示在试管内。方法:HepG2 房间线被孵化与长期的丙肝从一个病人与浆液为它的危险性测试到 HCV。房间和上层清液在文化期间在各种各样的时间点被收获。文化上层清液为它感染天真的房间的能力被测试。存在减(反感觉) 在房间的核心和 E1 抗原的 RNA 海滨,和察觉被 RT-PCR 和免疫学的技术(流动血细胞计数和西方的污点) 分别地检验。结果:细胞内部的 HCV RNA 首先在 d 上被检测 3 在感染以后然后能一致地在至少三个月的一个时期上在房间和上层清液被检测。新鲜房间能从有教养的感染的房间感染上层清液。流动 cytometric 分析证明表面和在房子里使用的细胞内部的 HCV 抗原表示使 polyclonal 成为了抗体(反核心,和 anti-E1 ) 。西方的污点分析证明在分子量的产生免疫性的肽的簇的表示在一个月内在 31 和 45 kDa 之间延长了感染的房间的旧文化而这簇在 uninfected HepG2 房间是无法发现的。结论:HepG2 房间线产生 HCV 感染而且支持它的复制在试管内不仅。HCV 结构的蛋白质的表示能在感染的 HepG2 房间被检测。这些房间也能够流病毒的粒子进接着对 uninfected 房间变得传染的培养基。 | Mostafa K El-Awady Ashraf A Tabll Yasmine S El-Abd Mahmoud M Bahgat Hussein A Shoeb Samar S Youssef Noha G Bader El Din El-Rashdy M Redwan Maha El-Demellawy Moataza H Omran Wael T El-Garf Said A Goueli | 2006 | World Journal of Gastroenterology2006，12，30： | 2 |
| 17 | HCV-Fc融合基因疫苗真核表达载体的构建及表达显示文摘目的:构建能表达HCV C-Fc融合基因的真核表达载体,为进一步修饰和转染树突状细胞,制备能高效表达HCV C和Fc基因的树突状细胞疫苗做准备。方法:用分别含有Kpn Ⅰ和Xhol Ⅰ酶切位点的HCV C区基因上、下游引物,以含有HCV H株基因序列的质粒pBRTM/HCV1-3011为模板,通过PCR扩增获得HCV C区基因片段回收后,以Kpn Ⅰ和Xhol Ⅰ双酶切,定向插入到含IgG1 Fc基因的质粒pIgC3双粘端位点之间,获得重组表达质粒pHCVFc。通过Kpn Ⅰ双位点酶切、PCR及插入片段序列测定对质粒进行了鉴定,以抗HCV C单克隆抗体为一抗,利用间接免疫荧光法检测了pHCV-IgFc在人肝癌细胞7721中的瞬时表达。结果:酶切、PCR及测序鉴定证实,pHCV-IgFc插入片段为HCV C区基因片段,免疫荧光法检测表明其可以在7721细胞中瞬时表达。结论:构建的质粒pHCV-IgFc可以在7721细胞中瞬时表达HCV C区基因,为研究HCV C-Fc融合基因修饰的树突状细胞的功能奠定了基础。 | 冯志华 王全楚 周永兴 郝春秋 聂青和 | 2003 | 世界华人消化杂志2003，11，6： | 2 |
| 18 | 非同位素法分析序列特异性DNA结合蛋白的三种方法显示文摘目的为在转录调控水平研究蛋白质异常表达机制,利用核转录因子与相应DNA调控序列特异结合的原理,非同位素法分析细胞内序列特异性DNA结合蛋白。方法凝胶滞留实验(EMSA)采用Dig-ddUTP末端标记DNA,核蛋白粗提或纯化产物与之液相结合后,PAGE分离核蛋白并转印至尼龙膜,化学发光法检测结果,Southwestern分析方法首先采用SDS-PAGE分离核蛋白粗提或纯化产物,蛋白转印至膜上后与Dig-dUTP末端标记的DNA杂交,酶免疫学检测结果,磁性DNA结合蛋白纯化法则将靶DNA与商品化磁性DNA粒子相连,用不同的连接、洗脱条件得到序列特异性靶DNA结合蛋白,银染显示核蛋白纯化结果。结果 EMSA中由于游离DNA与DNA-蛋白复合物有不同电泳迁移率,出现滞后DNA条带提示有识别该特定DNA序列的核蛋白存在,Southwestern分析除能提示核抽提物中有无可与DNA结合的核蛋白外,还提示其中核蛋白分子质量,核蛋白粗提物经磁性DNA亲和层析后DNA结合蛋白在银染时出现较纯的特异识别该DNA序列的核蛋白条带。结论 EMSA,Southwestern分析及磁性DNA层析是3种实用的研究DNA结合蛋白的方法,配伍应用具有简便、重复性好、敏感性和特异性高、无同位素污染等优点,是转录调控水平研究疾病相关基因异常表达的有效方法。 | 高春芳 王皓 高国华 宓庆梅 孔宪涛 | 2001 | 世界华人消化杂志2001，9，5： | 1 |
| 19 | Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus显示文摘AIM:Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes,was evaluated.METHODS:Three male patients, infected with HCV through multiple transfusions,were identified from clinical symptoms and monitored by aminotransferase for 60 months.Blood samples taken at months 0, 32, and 60 were used for viral RNA extraction. A segment of HCV NS3 region was amplified from the RNA extraction by RT-PCR and subjected to subcloning and sequencing.HLA types of these three patients were determined using complement-dependent microlymphocytotoxic assay. CTL epitopes were predicted using MHC binding motifs.RESULTS:No patient had clinical symptoms or elevation of aspartate/alanine arninotransferase. Two patients showed positive HCV PCR results at all 3 time points. The other one showed a positive HCV PCR result only at month 0.A reported HLA-A2-restricted CTL epitope had no alteration in the HLA-A2-negative carrier over 60 months.In the HLA-A2-positive individuals, all the sequences from 0 month 0 showed an amber mutation on the initial codon of the epitope. Most changes of consensus sequences in the same patient occurred on predicted cytotoxic T cell epitopes.CONCLUSION:Amber mutation and changes of consensus sequence in HCV NS3 region may be related to viral immune escape. | Hua-ZhangGuo YingYin Wen-LiangWang Chuan-ShanZhang TaoWang ZheWang JingZhang HongCheng Hai-TaoWang | 2004 | World Journal of Gastroenterology2004，10，6： | 0 |
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