肝癌组织中HBV核心基因启动子突变的研究
作者：方钟燎 杨进业 王学燕 庄辉 Roger Ling，Tim J Harrison
单位：方钟燎 杨进业 王学燕(广西壮族自治区卫生防疫站，南宁市 530021)；庄辉(北京医科大学，北京 100083)；Roger Ling，Tim J Harrison(Royal Free Hospital School of Medicine, University of London,Rowland Hill St., London NW3 2PF, UK)
关键词：乙肝病毒；核心基因启动子；突变；原发性肝癌；整合
中国病毒学000307 摘要：为深入了解乙肝病毒(HBV)致癌机理，用套式PCR对广西14例血清HBV DNA阳性的原发性肝癌患者癌组织、10例乙型病毒性肝炎患者血清及10例乙肝病毒无症状携带者血清HBV核心基因启动子进行扩增，阳性者用Sanger氏双脱氧法做序列分析，发现肝癌组织10例PCR阳性，阳性率71.4%，所有PCR阳性标本的整合体均有乙肝病毒核心基因启动子双突变序列(nt 1 762 A→T, 1 764 G→A)，并且在其周围各序列都有不同部位的点突变，标本C14核苷酸的缺失突变高达10个。乙肝患者6例PCR阳性，其中3例乙肝病毒核心基因启动子发生双突变。无症状携带者中4例PCR阳性，其中仅1例发生双突变。结果提示乙肝病毒核心基因启动子双突变在肝癌患者中较常见，肝炎患者次之，无症状携带者居最后。
, http://www.100md.com
中图分类号：R735.7 文献标识码：A
文章编号：1003-5125(2000)03-0243-06
The Discovery of HBV Core Promoter Mutations in Tumour
Tissue of Patients with Hepatocellular Carcinoma
FANG Zhong-liao，YANG Jin-ye，WANG Xue-yan
(Health and Antiepidemic Station of Guangxi, Nanning 530021, China)
ZHUANG Hui
, http://www.100md.com
(Beijing Medical University, Beijing 100083, China)
Roger Ling，Tim J Harrison
(Royal Free Hospital School of Medicine, University of London,Rowland Hill St., London NW3 2PF, UK)
Abstract: In order to understand the mechanisms of hepatitis B virus (HBV) in the development of hepatocellular carcinoma, the nested PCR was used to amplify HBV core promoter from tumour tissues of 14 HBV DNA positive patients with hepatocellular carcinoma, and 10 serum samples of hepatitis B patients and asymptomatic carriers respectively. The positive samples were sequenced by Sanger method. Ten of the fourteen tumour tissue samples (71.4%) were positive for HBV core promoter. Each sequence of all positive samples had the double mutations at nt 1762(A→T), 1764(G→A) in HBV core promoter and some point mutations around them. The case C14 had more extensive mutations, including deletion of 10 nucleotides. 6 of 10 serum samples of hepatitis B patients were positive for HBV core promoter and 3 of them had the double mutations. four of the ten serum samples of asymptomatic carriers were PCR positive while only 1 of them had the double mutations. These data implicate that the double mutations is most common in HCC patients, the next is hepatitis B patients and the last is asymptomatic carriers.→T), 1764(G→A) in HBV core promoter and some point mutations around them. The case C14 had more extensive mutations, including deletion of 10 nucleotides. 6 of 10 serum samples of hepatitis B patients were positive for HBV core promoter and 3 of them had the double mutations. four of the ten serum samples of asymptomatic carriers were PCR positive while only 1 of them had the double mutations. These data implicate that the double mutations is most common in HCC patients, the next is hepatitis B patients and the last is asymptomatic carriers.
, http://www.100md.com
Key words: Hepatitis B virus; Core promoter; Mutation; Hepatocellular carcinoma; Integration
广西科技厅(桂科回字9817136)和卫生厅资助课题(9836)
作者简介：方钟燎(1963年-)，男，壮族，广西省人，副主任医师，医学硕士，研究方向：HBV的分子生物学。
参考文献
[1] Robinson WS. Hepadnaviruses and hepatocellular carcinoma (HCC) [J]. Cancer Detect Prev, 1989,14:245-252
[2] Buckwold V, Xu Z, Chen M, et al. Effects of a naturally occuring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication [J]. J Virol, 1996,5845-5851
, 百拇医药
[3] 方钟燎，王树声，麦威，等.广西原发性肝癌患者HBV DNA核心基因启动子突变的研究[J].广西预防医学，1997，3(4)：197-200
[4] Koshy R, Maupas PH, Muller R, et al. Detection of hepatitis B virus-specific DNA in the genome of human hepatocellular carcinoma and liver cirrhosis tissue [J]. J Gene Virol, 1981,57:95-102
[5] Ogston CW, Jonak GJ, Rogler CE, et al. Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks [J]. Cell, 1982,29:385-394
, 百拇医药
[6] Marion PC, Pavelaar MJ, Knight SS, et al. Hepatocellular carcinoma in groud squirrels persistently infected with groud squirrel hepatitis virus [J]. Proceedings of National Academy of Sciences USA, 1986,83:4543
[7] Yokosuka O, Omata M, Zhou YZ, et al. Duck hepatitis B virus DNA in liver and serum of Chinese ducks: Integration of viral DNA in hepatocellular carcinoma [J]. Proceedings of National Academy Sciences USA, 1985,82:5180
, 百拇医药
[8] Koshy R, Koch S, Kahmann R, et al. Integration of hepatitis B virus DNA: Evidence for integration in the single-stranded gap. Cell, 1983,34:21
[9] Mizusawa H, Taira M, Yaginuma K, et al. Inversely repeating integrated hepatitis B virus DNA and cellular flanking sequences in the human hepatoma-derived cell line huSP. Proceedings of National Academy Sciences USA, 1985,82:208-212
[10] Tim J Harrison, Arie J Zuckerman. The Molecular Medicine of Viral Hepatitis [M]. England: John Wiley & Sons Ltd, 1997. 99-103
[11] Chu Chieh Hsia, Hao Yuwen, Edward Tabor. Hot-spot mutations in hepatitis B virus x gene in hepatocellular carcinoma [J]. The Lancet, 1996,348:625-626
收稿日期：1999-09-07，修回日期：2000-01-07, http://www.100md.com
单位：方钟燎 杨进业 王学燕(广西壮族自治区卫生防疫站，南宁市 530021)；庄辉(北京医科大学，北京 100083)；Roger Ling，Tim J Harrison(Royal Free Hospital School of Medicine, University of London,Rowland Hill St., London NW3 2PF, UK)
关键词：乙肝病毒；核心基因启动子；突变；原发性肝癌；整合
中国病毒学000307 摘要：为深入了解乙肝病毒(HBV)致癌机理，用套式PCR对广西14例血清HBV DNA阳性的原发性肝癌患者癌组织、10例乙型病毒性肝炎患者血清及10例乙肝病毒无症状携带者血清HBV核心基因启动子进行扩增，阳性者用Sanger氏双脱氧法做序列分析，发现肝癌组织10例PCR阳性，阳性率71.4%，所有PCR阳性标本的整合体均有乙肝病毒核心基因启动子双突变序列(nt 1 762 A→T, 1 764 G→A)，并且在其周围各序列都有不同部位的点突变，标本C14核苷酸的缺失突变高达10个。乙肝患者6例PCR阳性，其中3例乙肝病毒核心基因启动子发生双突变。无症状携带者中4例PCR阳性，其中仅1例发生双突变。结果提示乙肝病毒核心基因启动子双突变在肝癌患者中较常见，肝炎患者次之，无症状携带者居最后。
, http://www.100md.com
中图分类号：R735.7 文献标识码：A
文章编号：1003-5125(2000)03-0243-06
The Discovery of HBV Core Promoter Mutations in Tumour
Tissue of Patients with Hepatocellular Carcinoma
FANG Zhong-liao，YANG Jin-ye，WANG Xue-yan
(Health and Antiepidemic Station of Guangxi, Nanning 530021, China)
ZHUANG Hui
, http://www.100md.com
(Beijing Medical University, Beijing 100083, China)
Roger Ling，Tim J Harrison
(Royal Free Hospital School of Medicine, University of London,Rowland Hill St., London NW3 2PF, UK)
Abstract: In order to understand the mechanisms of hepatitis B virus (HBV) in the development of hepatocellular carcinoma, the nested PCR was used to amplify HBV core promoter from tumour tissues of 14 HBV DNA positive patients with hepatocellular carcinoma, and 10 serum samples of hepatitis B patients and asymptomatic carriers respectively. The positive samples were sequenced by Sanger method. Ten of the fourteen tumour tissue samples (71.4%) were positive for HBV core promoter. Each sequence of all positive samples had the double mutations at nt 1762(A→T), 1764(G→A) in HBV core promoter and some point mutations around them. The case C14 had more extensive mutations, including deletion of 10 nucleotides. 6 of 10 serum samples of hepatitis B patients were positive for HBV core promoter and 3 of them had the double mutations. four of the ten serum samples of asymptomatic carriers were PCR positive while only 1 of them had the double mutations. These data implicate that the double mutations is most common in HCC patients, the next is hepatitis B patients and the last is asymptomatic carriers.→T), 1764(G→A) in HBV core promoter and some point mutations around them. The case C14 had more extensive mutations, including deletion of 10 nucleotides. 6 of 10 serum samples of hepatitis B patients were positive for HBV core promoter and 3 of them had the double mutations. four of the ten serum samples of asymptomatic carriers were PCR positive while only 1 of them had the double mutations. These data implicate that the double mutations is most common in HCC patients, the next is hepatitis B patients and the last is asymptomatic carriers.
, http://www.100md.com
Key words: Hepatitis B virus; Core promoter; Mutation; Hepatocellular carcinoma; Integration
广西科技厅(桂科回字9817136)和卫生厅资助课题(9836)
作者简介：方钟燎(1963年-)，男，壮族，广西省人，副主任医师，医学硕士，研究方向：HBV的分子生物学。
参考文献
[1] Robinson WS. Hepadnaviruses and hepatocellular carcinoma (HCC) [J]. Cancer Detect Prev, 1989,14:245-252
[2] Buckwold V, Xu Z, Chen M, et al. Effects of a naturally occuring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication [J]. J Virol, 1996,5845-5851
, 百拇医药
[3] 方钟燎，王树声，麦威，等.广西原发性肝癌患者HBV DNA核心基因启动子突变的研究[J].广西预防医学，1997，3(4)：197-200
[4] Koshy R, Maupas PH, Muller R, et al. Detection of hepatitis B virus-specific DNA in the genome of human hepatocellular carcinoma and liver cirrhosis tissue [J]. J Gene Virol, 1981,57:95-102
[5] Ogston CW, Jonak GJ, Rogler CE, et al. Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks [J]. Cell, 1982,29:385-394
, 百拇医药
[6] Marion PC, Pavelaar MJ, Knight SS, et al. Hepatocellular carcinoma in groud squirrels persistently infected with groud squirrel hepatitis virus [J]. Proceedings of National Academy of Sciences USA, 1986,83:4543
[7] Yokosuka O, Omata M, Zhou YZ, et al. Duck hepatitis B virus DNA in liver and serum of Chinese ducks: Integration of viral DNA in hepatocellular carcinoma [J]. Proceedings of National Academy Sciences USA, 1985,82:5180
, 百拇医药
[8] Koshy R, Koch S, Kahmann R, et al. Integration of hepatitis B virus DNA: Evidence for integration in the single-stranded gap. Cell, 1983,34:21
[9] Mizusawa H, Taira M, Yaginuma K, et al. Inversely repeating integrated hepatitis B virus DNA and cellular flanking sequences in the human hepatoma-derived cell line huSP. Proceedings of National Academy Sciences USA, 1985,82:208-212
[10] Tim J Harrison, Arie J Zuckerman. The Molecular Medicine of Viral Hepatitis [M]. England: John Wiley & Sons Ltd, 1997. 99-103
[11] Chu Chieh Hsia, Hao Yuwen, Edward Tabor. Hot-spot mutations in hepatitis B virus x gene in hepatocellular carcinoma [J]. The Lancet, 1996,348:625-626
收稿日期：1999-09-07，修回日期：2000-01-07, http://www.100md.com