我国幽门螺杆菌cagA/CagA分布、多态性及其与胃十二指肠疾病关系分析
|
摘要
目前，幽门螺杆菌（Helicobacter pylori，H pylori，Hp）菌型差异导致感染
结局不同已得到普遍共识。研究显示，多数欧美国家CagA阳性菌株感染
与非贲门部胃癌和十二指肠溃疡发生相关，而亚洲地区菌株的CagA阳性
率很高，且在不同胃肠疾病间的分布无差异。我国相关的研究结果很不一
致。由于cagA/CagA具有显著的多态性，不同菌株cagA基因序列、CagA
分子大小、抗原性及磷酸化修饰间的差异，可导致其致病性不同。日本、
德国、南非的研究显示存在与胃癌或消化性溃疡相关的cagA/CagA亚型。
由此可见，各种cagA/CagA亚型菌株在各地分布不同，可能是造成各地研
究结果不一致的重要原因，而检测手段敏感性的差异，也可能导致研究结
果的偏倚。为了明确cagA/CagA及其亚型在我国Hp菌株中的分布情况，
以及有无与疾病相关的特定cagA/CagA亚型，我们进行了如下研究。
1、通过PCR扩增cagA基因，结合SDS-PAGE检测CagA蛋白表达的
结果，分析我国四省市不同疾病分离株cagA/CagA的分布情况。
2、采用免疫印迹杂交的方法，分析38株国内Hp菌株及2株国际标
准株的CagA抗原对三种不同菌株兔血清的反应性，比较CagA抗原性多
态性在不同地区及胃十二指肠疾病的分布差异。
3、采用PCR方法分析77株国内Hp菌株cagA基因的3'端可变区变
异及其与胃十二指肠疾病发生的关系。
4、克隆4株背景清楚的Hp菌株（国际标准菌株SS1和国内2株胃癌
分离株和1株十二指肠溃疡分离株）的全长cagA基因，测序后与GenBank
中已知的8个cagA基因核苷酸序列和推定氨基酸序列进行了同源性分析；
分析了它们之间的磷酸化位点差异
5、构建了上述4株菌的CapA基因的重组原核表达质粒。
我们的结果显示：l）我国菌株的 cagA儿agA阳性率极高，在不同地区
的分布无差别，与疾病发生无显著相关，因此，cagA／CagA不能作为我国
Hp的毒力标志。2）我国菌株 CagA的抗原性具有显著多态性，CagA抗原
性与致病性存在一定的关系，与抗 NCTC 63 7的 CagA抗原性相近的
CagA，可能与消化性溃疡关系较为密切。检测CagA抗原或抗体时，应联
合使用至少三种抗 CagA抗体或 CagA抗原。3）根据 3’端可变区长度，我。
国Hp菌株的cagA基因至少可分为l、11、Ill三型，不同地区、不同疾
病来源菌株的cagA基因均以1型为主，11型和皿型cagA基因阳性菌株感
染是否与临床结局有特殊关联，有必要对目前病例进行随访观察和更大规
模的人群调查研究。4）所测的国际标准菌株％ 和国内2株胃癌分离株和
1株十二指肠溃疡分离株的全长cagA基因序列已登录GenBank，序列号分
别为AF249275、AF247651、AF367250、AF367251。同源性分析表明，我
国菌株与日本菌株的cagA基因核音酸序列及推定CagA氨基酸序列间聚类
关系明显，但 GenBank中 12株 Hp菌间未发现明显的疾病聚类关系。CagA
磷酸化位点不同可能与临床感染结局间存在某种联系，但需进一步研究明
确。5）获得了上述 4株 Hp菌株 CapA融合蛋白的重组表达质粒，为进一
步进行CagA的功能和致病性研究和人群血清学调查研究奠定了基础。
Numerous previous studies in Western countries have highlighted some Helicobacter pylon (H pylon) strains are more pathogenic than others, and that CagA-positive If pylon strains are associated with severe gastroduodenal diseases. However, this is not the case in Asian countries where there are high prevalence of cagA-positive strains infection. Those studies conducted in China had revealed conflicting results.
The paradox that CagA-positive H. pylon infection correlates to two mutually exclusive diseases: gastric cancer and duodenal ulcer, and results in various clinical outcomes in different populations, may be due to the heterogeneity of cagA/CagA, which had been suggested by studies conducted in Japan, Germany and South Africa. Discriminatory techniques detecting cagA or CagA employed in different studies may be another contributory factor. Recent data have suggested that the tyrosine phosphorylation of CagA may also play a role.
cagA/CagA is highly divergent, either at gene level or protein level, which is most marked in the 3?region where there are variable number of repeat sequences. Previous studies showed that CagA protein in larger size and with stronger antigen are more likely to correlate to gastric adenocarcinoma.
Therefore, we made an attempt to examine the heterogeneity of cagA/CagA of Chinese H. pylon isolates, and determine whether there are distinct cagA/CagA types circulating in different areas in China, so as to character gastroduodenal diseases-specific cagAlCagA types.
The prevalence of cagAlCagA of Chinese H. pylon strains was first
determined by detecting the cagA gene using polymerase chain reaction and
CagA protein using SDS-PAGE simu1taneously, Which revealed very high. So
the anigenic heterogeneity of CagA of 38 strains was then deterrnined by
aligning and comparing their reactivity to thIee different rabbit sera immunized
by H Pylori strains CAPM N62, NCTCl1637 and CAPM Z-2 respectively
using western blots. To examine whether distinct cagA alleles may circulate in
different areas and correlate to different clinical outcomes, the variable
segment of 3' region of cagA gene of different clinical origin were amplified
by PCR, and the length of PCR products were compared. In order to fully
understand the relationship between heterogeneity of CagA and clinical
presents, the full-length cagA genes of two gastric cancer-associated and one
duodena1 ulcer-associated H PylOri strains and strain SSl were cloned and
sequenced, and the homology of their nucleotide and deduced amino acid
sequences as wel1 as phosphorylation motifS preseni in CagA were analyzed.
Considering it is difficult to get 1ots of CagA from H Pylori culture for studies
of the function and pathogenesis of CagA, cagA of these fOur strains were
cloned inio PinPointTM Xa-l T-vector to constructed recombinant plasmids
expressing CagA fused to PinPointTM biotinylation segment.
Our resu1ts showed: (l) There is a high prevalence of cagAjCagA of
Chinese H Pylori isolates irrelevant geograPhic or clinical origin, Which agrees
well with those of other Asian couniTies, and suggests that cagA/CagA is not
the virulent mark of H Pylori strains in China. (2) None of the three
immunized sera cou1d recognize CagA of all isolates, howeveT, each CagA
could be recognized by at least one serum. The antigenic heterogeneity of
CagA was grouPed as I -V, group II which reacted most strong1y with
serum immunized by NCTC11637 was more likely but not significantly
correlated to peptic ulcer' (3) cagA genes were designed to three types I, II,
and III according to the amplified PCR product length of variable segment of
3' region, of which was about 825bp, 900bp or 950bp respectively. Type I
cagA gene predominates in China, both the rare two tyPes II and Ill cagA
gene present in only two iso1ates respectively. (4) The complete sequences of
IV
cagA genes of strain SS1, CAPM N62, CAPM N93 and CAPM Nih have been deposited in GenBank under accession number AF247651, AF249275, AF36
结局不同已得到普遍共识。研究显示，多数欧美国家CagA阳性菌株感染
与非贲门部胃癌和十二指肠溃疡发生相关，而亚洲地区菌株的CagA阳性
率很高，且在不同胃肠疾病间的分布无差异。我国相关的研究结果很不一
致。由于cagA/CagA具有显著的多态性，不同菌株cagA基因序列、CagA
分子大小、抗原性及磷酸化修饰间的差异，可导致其致病性不同。日本、
德国、南非的研究显示存在与胃癌或消化性溃疡相关的cagA/CagA亚型。
由此可见，各种cagA/CagA亚型菌株在各地分布不同，可能是造成各地研
究结果不一致的重要原因，而检测手段敏感性的差异，也可能导致研究结
果的偏倚。为了明确cagA/CagA及其亚型在我国Hp菌株中的分布情况，
以及有无与疾病相关的特定cagA/CagA亚型，我们进行了如下研究。
1、通过PCR扩增cagA基因，结合SDS-PAGE检测CagA蛋白表达的
结果，分析我国四省市不同疾病分离株cagA/CagA的分布情况。
2、采用免疫印迹杂交的方法，分析38株国内Hp菌株及2株国际标
准株的CagA抗原对三种不同菌株兔血清的反应性，比较CagA抗原性多
态性在不同地区及胃十二指肠疾病的分布差异。
3、采用PCR方法分析77株国内Hp菌株cagA基因的3'端可变区变
异及其与胃十二指肠疾病发生的关系。
4、克隆4株背景清楚的Hp菌株（国际标准菌株SS1和国内2株胃癌
分离株和1株十二指肠溃疡分离株）的全长cagA基因，测序后与GenBank
中已知的8个cagA基因核苷酸序列和推定氨基酸序列进行了同源性分析；
分析了它们之间的磷酸化位点差异
5、构建了上述4株菌的CapA基因的重组原核表达质粒。
我们的结果显示：l）我国菌株的 cagA儿agA阳性率极高，在不同地区
的分布无差别，与疾病发生无显著相关，因此，cagA／CagA不能作为我国
Hp的毒力标志。2）我国菌株 CagA的抗原性具有显著多态性，CagA抗原
性与致病性存在一定的关系，与抗 NCTC 63 7的 CagA抗原性相近的
CagA，可能与消化性溃疡关系较为密切。检测CagA抗原或抗体时，应联
合使用至少三种抗 CagA抗体或 CagA抗原。3）根据 3’端可变区长度，我。
国Hp菌株的cagA基因至少可分为l、11、Ill三型，不同地区、不同疾
病来源菌株的cagA基因均以1型为主，11型和皿型cagA基因阳性菌株感
染是否与临床结局有特殊关联，有必要对目前病例进行随访观察和更大规
模的人群调查研究。4）所测的国际标准菌株％ 和国内2株胃癌分离株和
1株十二指肠溃疡分离株的全长cagA基因序列已登录GenBank，序列号分
别为AF249275、AF247651、AF367250、AF367251。同源性分析表明，我
国菌株与日本菌株的cagA基因核音酸序列及推定CagA氨基酸序列间聚类
关系明显，但 GenBank中 12株 Hp菌间未发现明显的疾病聚类关系。CagA
磷酸化位点不同可能与临床感染结局间存在某种联系，但需进一步研究明
确。5）获得了上述 4株 Hp菌株 CapA融合蛋白的重组表达质粒，为进一
步进行CagA的功能和致病性研究和人群血清学调查研究奠定了基础。
Numerous previous studies in Western countries have highlighted some Helicobacter pylon (H pylon) strains are more pathogenic than others, and that CagA-positive If pylon strains are associated with severe gastroduodenal diseases. However, this is not the case in Asian countries where there are high prevalence of cagA-positive strains infection. Those studies conducted in China had revealed conflicting results.
The paradox that CagA-positive H. pylon infection correlates to two mutually exclusive diseases: gastric cancer and duodenal ulcer, and results in various clinical outcomes in different populations, may be due to the heterogeneity of cagA/CagA, which had been suggested by studies conducted in Japan, Germany and South Africa. Discriminatory techniques detecting cagA or CagA employed in different studies may be another contributory factor. Recent data have suggested that the tyrosine phosphorylation of CagA may also play a role.
cagA/CagA is highly divergent, either at gene level or protein level, which is most marked in the 3?region where there are variable number of repeat sequences. Previous studies showed that CagA protein in larger size and with stronger antigen are more likely to correlate to gastric adenocarcinoma.
Therefore, we made an attempt to examine the heterogeneity of cagA/CagA of Chinese H. pylon isolates, and determine whether there are distinct cagA/CagA types circulating in different areas in China, so as to character gastroduodenal diseases-specific cagAlCagA types.
The prevalence of cagAlCagA of Chinese H. pylon strains was first
determined by detecting the cagA gene using polymerase chain reaction and
CagA protein using SDS-PAGE simu1taneously, Which revealed very high. So
the anigenic heterogeneity of CagA of 38 strains was then deterrnined by
aligning and comparing their reactivity to thIee different rabbit sera immunized
by H Pylori strains CAPM N62, NCTCl1637 and CAPM Z-2 respectively
using western blots. To examine whether distinct cagA alleles may circulate in
different areas and correlate to different clinical outcomes, the variable
segment of 3' region of cagA gene of different clinical origin were amplified
by PCR, and the length of PCR products were compared. In order to fully
understand the relationship between heterogeneity of CagA and clinical
presents, the full-length cagA genes of two gastric cancer-associated and one
duodena1 ulcer-associated H PylOri strains and strain SSl were cloned and
sequenced, and the homology of their nucleotide and deduced amino acid
sequences as wel1 as phosphorylation motifS preseni in CagA were analyzed.
Considering it is difficult to get 1ots of CagA from H Pylori culture for studies
of the function and pathogenesis of CagA, cagA of these fOur strains were
cloned inio PinPointTM Xa-l T-vector to constructed recombinant plasmids
expressing CagA fused to PinPointTM biotinylation segment.
Our resu1ts showed: (l) There is a high prevalence of cagAjCagA of
Chinese H Pylori isolates irrelevant geograPhic or clinical origin, Which agrees
well with those of other Asian couniTies, and suggests that cagA/CagA is not
the virulent mark of H Pylori strains in China. (2) None of the three
immunized sera cou1d recognize CagA of all isolates, howeveT, each CagA
could be recognized by at least one serum. The antigenic heterogeneity of
CagA was grouPed as I -V, group II which reacted most strong1y with
serum immunized by NCTC11637 was more likely but not significantly
correlated to peptic ulcer' (3) cagA genes were designed to three types I, II,
and III according to the amplified PCR product length of variable segment of
3' region, of which was about 825bp, 900bp or 950bp respectively. Type I
cagA gene predominates in China, both the rare two tyPes II and Ill cagA
gene present in only two iso1ates respectively. (4) The complete sequences of
IV
cagA genes of strain SS1, CAPM N62, CAPM N93 and CAPM Nih have been deposited in GenBank under accession number AF247651, AF249275, AF36
引文
1． Tummuru MK, Cover TL, Blaser MJ. Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect Immun 1993; 61(5) : 1799-809
2． Covacci A, Censini S, Bugnoli M, et al. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci USA 1993;90(12) : 5791-5
3． Blaser MJ, Perez-Perez GI, Kleanthous H., et al. Infection with Helicobacter pylori strains processing cagA is associated with an increased risk of developing adenocarcinoma of stomach. Cancer Res 1995;55:2111-2115．
4． Parsonnet J, Friedman GD, Orentreich N, et al. Risk for gatric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. Gut 1997;40:297-301．
5． Rudi J, Kolb C, Maiwald M, et al. Serum antibodies against Helicobacter pylori proteins VacA and CagA are associated with increased risk for gastric adenocarcinoma. Dig Dis Sci, 1997;42(8) : 1652-59．
6． Miehlke S, Kirsch C, Agha-Amiri K, et al. The Helicobacter pylori vacA s1, m1 genotype and cagA is associated with gastric carcinoma in Germany. Int J Cancer 2000;87(3) :322-327
7． Rudi J, Kolb C, Maiwald M, et al. Diversity of Helicobacter pylori vacA and cagA genes and relationship to VacA and CagA protein expression, cytotoxin production, and associated diseases. J Clin Microbiol 1998;36(4) :944-8
8． Bach S, Makristathis A, Pinto A, et al. Helicobacter pylori type I strains among Austrian and Portuguese patients with gastritis, peptic ulcer or
gastric cancer. Eur J Clin Microbiol Infect Dis 1999;18(11) :807-10
9． Vorobjova T, Nilsson I, Kull K, et al. CagA protein seropositivity in a random sample of adult population and gastric cancer patients in Estonia. Eur J Gastroenterol Hepatol 1998;10(l):41-6
10． Queiroz DM, Mendes EN, Rocha GA, et al. cagA-positive Helicobacter pylori and risk for developing gastric carcinoma in Brazil. Int J Cancer 1998;78(2) :135-9
11． Torres J, Perez-Perez GI, Leal Herrera Y, et al. Infection with CagA+ Helicobacter pylori strains as a possible predictor of risk in the development of gastric adenocarcinoma in Mexico. Int J Cancer 1998;78(3) :298-300
12． Maeda S., Ogura K., Yoshida H., et al. Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan. Gut 1998;42(3) :338-343．
13． Miehlke S, Kibler K, Kim JG, et al. Allelic variation in the cagA gene of Helicobacter pylori obtained from Korea compared to the United States. Am J Gastroenterol, 1996;91:1322-5
14． Mahachai V, Tangkijvanich P, Wannachai N, et al. CagA and VacA: virulence factors of Helicobacter pylori in Thai patients with gastroduodenal diseases. Helicobacter 1999;4(3) :143-7
15． Zheng PY, Hua J, Yeoh KG, et al. Association of peptic ulcer with increased expression of lewis antigens but not cagA, iceA, and vacA in helicobacter pylori isolates in an asian population. Gut 2000;47(1) : 18-22
16． Nessa J, Chart H, Owen RJ, et al. Human serum antibody response to Helicobacter pylori whole cell antigen in an institutionalized Bangladeshi population. J Appl Microbiol 2001;90(l):68-72
17． Kersulyte D, Mukhopadhyay AK, Velapatino B, et al. Differences in genotypes of Helicobacter pylori from different human populations. J
Bacteriol 2000; 182(11): 3210-8
18. Pan ZJ, van der Hulst RW, Feller M, et al. Equally high prevalences of infection with cagA-positive Helicobacter pylori in Chinese patients with peptic ulcer disease and those with chronic gastritis- associated dyspepsia. J Clin Microbiol 1997;35(6): 1344-7.
19．杜奕奇，许国铭，纪徐淮，等。中国人感染幽门螺杆菌的基因分型及其临床意义。第二军医大学学报 1998；19(5)：448-451
20．尹焱，孙兆军，张建中。北京人群感染幽门螺杆菌cagA分布特征。中华微生物学和免疫学杂志 2000；20(5)：457
21. Mitchill HM, Hazell SL, Li YY, et al. Serological response to specific Helicobacter pylori antigens: antibody against CagA antigen is not predictive of gastric cancer in a developing country. Am J Gastroenterology 1996;91(9): 1785-1788
22. Yang JC, Wang TH, Wang HJ, et al. Genetic analysis of the cytotoxin-associated gene and the vacuolating toxin gene in Helicobacter pylori strains isolated from Taiwanese patients. Am J Gastroenterol 1997; 92:1316-21
23. Yang H, Wu SV, Pichuantes S, et al. High prevalence of cagA-positive strains in Helicobacter priori-infected, healthy, young Chinese adults. J Gastroenterol Hepatol 1999; 14(5):476-80
24. Shiesh SC, Sheu BS, Yang HB, et al. Serologic response to lower-molecular-weight proteins of H. priori is related to clinical outcome of H. pylori infection in Taiwan. Dig Dis Sci. 2000;45(4): 781-8
25．韩锋产，张沥，阎小君，等。cagA阳性Hp感染与胃粘膜组织恶性化转变的初步研究。中华消化杂志 1998；18(4)：249
26．张联，马峻岭，潘凯枫，等。胃癌高和低发区人群幽门螺杆菌及cagA+和hspA+型感染的研究。中华预防医学杂志 1998；32(2)：67-69
27．赵志泉，张红杰。具有cagA、vacA基因的幽门螺杆菌感染与胃、十二指肠疾病的关系。中华消化内镜杂志 1997；14(6)：333-336
28. Wong BC, Lam SK, Ching CK, et al. Seroprevalence of cytotoxin-associated gene A positive Helicobacter pylori strains in Changle, an area with very high prevalence of gastric cancer in south China. Aliment Pharmacol Ther, 1999; 13(10): 1295-302
29. Evans DJ Jr, Queiroz DMM, Mendes EN, et al. Diversity in the variable region of Helicobacter pylori cagA gene involves more than simple repetition of a 102-nucleotide sequence. Biochem Biophys Res Commun 1998; 245: 780-784.
30．张建中。中国协和医科大学博士学位论文。
31. Odenbreit S, Ptlls J, Sedlmaier B, et al. Translocation of Helicobacter priori CagA into gastric epithelial cells by type Ⅳ secretion. Science 2000; 287(5457): 1497-500
32. Yamaoka Y, Osato MS, Sepulveda AR, et al. Molecular epidemiology of Helicobacter pylori: separation of H. pylori from East Asian and non-Asian countries. Epidemiol Infect 2000; 124(1): 91-6
33. van der Ende A; Pan Z J; Bart A; et al. cagA-positive Helicobacter pylori populations in China and The Netherlands are distinct. Infect Immun, 1998; 66(5): 1822-6
34. Yamaoka Y, El Zimaity HM, Gutierrez O, et al. Relationship between the cagA 3' repeat region of Helicobacter pylori, gastric histology, and susceptibility to low pH. Gastroenterology 1999; 117(2): 342-9
35. Yamaoka Y, Kodama T, Kashima K, et al. Variants of the 3' region of the cagA gene in Helicobacter priori isolates from patients with different H. pylori-associated diseases. J Clin Microbiol 1998; 36(8): 2258-63
36. Kidd M, Lastovica AJ, Atherton JC, et al. Heterogeneity in the Helicobacter pylori vacA and cagA genes: association with gastroduodenal disease in South Africa? Gut 1999; 45(4): 499-502
37. Yamaoka Y, Kodama T, Graham D, et al. Comparision of four serological tests to determine the CagA or VacA status of Helicobacter pylori strains. J Clin Microbiol, 1998; 36(11): 3433-4.
38. Yamaoka Y, Graham DY. CagA status and gastric cancer unreliable serological tests produce unreliable data. Gastroenterology 1999;117(3):745
39. Vaucher C, Janvier B, Nousbaum JB, et al. Antibody response of patients with Helicobacter pylori-related gastric adenocarcinoma: significance of anti-cagA antibodies. Clin Diagn Lab Immunol 2000; 7(3): 463-7.
40．姒健敏，高敏，王杭勇，等。幽门螺杆菌CagA抗体测定价值探讨。浙江医学 1998；20(12)：720-721
41．萨姆布鲁克，费里奇，曼尼阿蒂斯著，金冬雁，黎孟枫等译。《分子克隆实验指南》。第二版，北京；科学出版社，1988
42. Peek RM Jr, Miller GG, Tham KT, et al. Detection of Helicobacter pylori gene expression in human gastric mucosa. J Clin Microbiol 1995; 33(1): 28-32
43. Lage AP, Godfroid E, Fauconnier A, et al. Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens. J Clin Microbiol 1995; 33(10): 2752-6
44. Miehlke S, Go MF, Kim JG, et al. Serologic detection of Helicobacter pylori infection with cagA-positive strains in duodenal ulcer, gastric cancer, and asymptomatic gastritis. J Gastroenterol 1998; 33 (S 10): 18-21
45. van Doom LJ, Figueiredo C, Rossau R, et al. Typing of Helicobacter priori vacA gene and detection of cagA gene by PCR and reverse hybridization. J Clin Microbiol 1998; 36(5): 1271-6
46. Busolo F, Bertollo G, Bordignon G, et al. Detection and characterization of Helicobacter pylori from patients with gastroduodenal diseases. Diagn Microbiol Infect Dis 1998; 31(4): 531-6
47. Xiang Z, Bugnoli M, Ponzetto A, et al. Detection in an enzyme immunoassay of an immune response to a recombinant fragment of the 128 kilodalton protein (CagA) of Helicobacter pylori. Eur J Clin Microbiol Infect Dis 1993; 12(10): 739-45
48．韩锋产，阎小君，苏成芝。幽门螺杆菌cagA基因的克隆表达及其产物的应用。华人消化杂志 1998；6(12)：1084-1086
49. Maeda S, Ogura K, Yoshida H, et al. Structure of cag pathogenicity island in Japan Helicobacterpylori isolates. Gut 99; 44: 336-341.
50. van Doorn LJ, Figueiredo C, Sanna R, et al. Distinct variants of Helicobacter pylori cagA are associated with vacA subtypes. J Clin Microbiol 1999,37: 2306-11.
51. Peek RM Jr, Moss SF, Tham KT, et al. Helicobacter pylori cagA+ strains and dissociation of gastric epithelial cell proliferation from apoptosis. J Natl Cancer Inst 1997; 89(12): 863-8
52. Rokkas T, Ladas S, Liatsos C, et al. Relationship of Helicobacter pylori CagA status to gastric cell proliferation and apoptosis. Dig Dis Sci 1999; 44(3): 487-93
53. Segal ED, Cha J, Lo J, et al. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori. Proc Natl Acad Sci USA 1999; 96: 14559-64.
54. Meyer-ter-Vehn T, Covacci A, Kist M, et al. Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun. J Biol Chem 2000; 275(21): 16064-72
55. Naumann M, Wessler S, Bartsch C, et al. Activation of activator protein 1 and stress response kinases in epithelial cells colonized by Helicobacter pylori encoding the cag pathogenicity island. J Biol Chem 1999; 274(44): 31655-62
56． Akopyants NS, Clifton SW, Kersulyte D, et al. Analyses of the cag pathogeneicity island of Helicobacterr pylori. Mol Microbiol 1998;28(1) :37-53．
57． Censini S, Lange C, Xiang Z, et al. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci USA 1996;93(25) : 14648-53
58． Spohn G, Beier D, Rappuoli R, et al. Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori. Mol Microbiol 1997;26: 361-72．
59． Odenbreit S, Gebert B, Puls J, et al. Interaction of Helicobacter pylori with professional phagocytes: role of the cag pathogenicity island and translocation, phosphorylation and processing of CagA. Cell Microbiol 2001;3(1) :21-31
60． Backert S, Ziska E, Brinkmann V, et al. Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus. Cell Microbiol 2000;2(2) : 155-64
61． Asahi M, Azuma T, Ito S, et al. Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. J Exp Med 2000; 191: 593-602．
62． Stein M, Rappuoli R, Covacci A. Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation. Proc Natl Acad Sci USA 2000; 97: 1263-8．
63． Covacci A and Rappuoli R. Tyrosine phosphorylated bacterial proteins: Trojan horses for the host cell. J Exp Med 2000;191(4) :587-92
64． Bliska JB, Guan K, Dixon JE, et al. Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci USA, 1991;88(4) ,1187-91
65． Occhialini A, Marais A, Urdaci M, et al. Composition and gene expression
of the cag pathogenicity island in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica. Infect Immun 2001; 69(3): 1902-8
66. Lee A, ORourke J, De Ungria MC, et al. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 1997; 112(4): 1386-97
67．曾志荣，胡品津，陈旻湖，等。幽门螺杆菌长期感染鼠腺胃模型的研究。中华医学杂志1998；78(7)：494-7．
68．曾志荣，胡品津，陈旻湖。幽门螺杆菌感染与胃癌形成关系的动物实验研究。第六届全国消化学术会议摘要汇编，P29，中国西安，1999年月10月。
69. Shomer NH, Dangler CA, Whary MT, et al. Experimental Helicobacter pylori infection induces antral gastritis and gastric mucosa-associated lymphoid tissue in guinea pigs. Infect Immun 1998; 66(6): 2614-8
70. Fox JG, Dangler CA, Taylor NS, et al. High-salt diet induces gastric epithelial hyperplasia and parietal cell loss, and enhances Helicobacter pylori colonization in C57BL/6 mice. Cancer Res 1999; 59(19): 4823-8
71．陈永昌，张建中，李铁，等。幽门螺杆菌对人和大鼠胃上皮细胞外信号调节激酶活性的影响。北京医科大学学报2000；32(2)：109-112
2． Covacci A, Censini S, Bugnoli M, et al. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci USA 1993;90(12) : 5791-5
3． Blaser MJ, Perez-Perez GI, Kleanthous H., et al. Infection with Helicobacter pylori strains processing cagA is associated with an increased risk of developing adenocarcinoma of stomach. Cancer Res 1995;55:2111-2115．
4． Parsonnet J, Friedman GD, Orentreich N, et al. Risk for gatric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. Gut 1997;40:297-301．
5． Rudi J, Kolb C, Maiwald M, et al. Serum antibodies against Helicobacter pylori proteins VacA and CagA are associated with increased risk for gastric adenocarcinoma. Dig Dis Sci, 1997;42(8) : 1652-59．
6． Miehlke S, Kirsch C, Agha-Amiri K, et al. The Helicobacter pylori vacA s1, m1 genotype and cagA is associated with gastric carcinoma in Germany. Int J Cancer 2000;87(3) :322-327
7． Rudi J, Kolb C, Maiwald M, et al. Diversity of Helicobacter pylori vacA and cagA genes and relationship to VacA and CagA protein expression, cytotoxin production, and associated diseases. J Clin Microbiol 1998;36(4) :944-8
8． Bach S, Makristathis A, Pinto A, et al. Helicobacter pylori type I strains among Austrian and Portuguese patients with gastritis, peptic ulcer or
gastric cancer. Eur J Clin Microbiol Infect Dis 1999;18(11) :807-10
9． Vorobjova T, Nilsson I, Kull K, et al. CagA protein seropositivity in a random sample of adult population and gastric cancer patients in Estonia. Eur J Gastroenterol Hepatol 1998;10(l):41-6
10． Queiroz DM, Mendes EN, Rocha GA, et al. cagA-positive Helicobacter pylori and risk for developing gastric carcinoma in Brazil. Int J Cancer 1998;78(2) :135-9
11． Torres J, Perez-Perez GI, Leal Herrera Y, et al. Infection with CagA+ Helicobacter pylori strains as a possible predictor of risk in the development of gastric adenocarcinoma in Mexico. Int J Cancer 1998;78(3) :298-300
12． Maeda S., Ogura K., Yoshida H., et al. Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan. Gut 1998;42(3) :338-343．
13． Miehlke S, Kibler K, Kim JG, et al. Allelic variation in the cagA gene of Helicobacter pylori obtained from Korea compared to the United States. Am J Gastroenterol, 1996;91:1322-5
14． Mahachai V, Tangkijvanich P, Wannachai N, et al. CagA and VacA: virulence factors of Helicobacter pylori in Thai patients with gastroduodenal diseases. Helicobacter 1999;4(3) :143-7
15． Zheng PY, Hua J, Yeoh KG, et al. Association of peptic ulcer with increased expression of lewis antigens but not cagA, iceA, and vacA in helicobacter pylori isolates in an asian population. Gut 2000;47(1) : 18-22
16． Nessa J, Chart H, Owen RJ, et al. Human serum antibody response to Helicobacter pylori whole cell antigen in an institutionalized Bangladeshi population. J Appl Microbiol 2001;90(l):68-72
17． Kersulyte D, Mukhopadhyay AK, Velapatino B, et al. Differences in genotypes of Helicobacter pylori from different human populations. J
Bacteriol 2000; 182(11): 3210-8
18. Pan ZJ, van der Hulst RW, Feller M, et al. Equally high prevalences of infection with cagA-positive Helicobacter pylori in Chinese patients with peptic ulcer disease and those with chronic gastritis- associated dyspepsia. J Clin Microbiol 1997;35(6): 1344-7.
19．杜奕奇，许国铭，纪徐淮，等。中国人感染幽门螺杆菌的基因分型及其临床意义。第二军医大学学报 1998；19(5)：448-451
20．尹焱，孙兆军，张建中。北京人群感染幽门螺杆菌cagA分布特征。中华微生物学和免疫学杂志 2000；20(5)：457
21. Mitchill HM, Hazell SL, Li YY, et al. Serological response to specific Helicobacter pylori antigens: antibody against CagA antigen is not predictive of gastric cancer in a developing country. Am J Gastroenterology 1996;91(9): 1785-1788
22. Yang JC, Wang TH, Wang HJ, et al. Genetic analysis of the cytotoxin-associated gene and the vacuolating toxin gene in Helicobacter pylori strains isolated from Taiwanese patients. Am J Gastroenterol 1997; 92:1316-21
23. Yang H, Wu SV, Pichuantes S, et al. High prevalence of cagA-positive strains in Helicobacter priori-infected, healthy, young Chinese adults. J Gastroenterol Hepatol 1999; 14(5):476-80
24. Shiesh SC, Sheu BS, Yang HB, et al. Serologic response to lower-molecular-weight proteins of H. priori is related to clinical outcome of H. pylori infection in Taiwan. Dig Dis Sci. 2000;45(4): 781-8
25．韩锋产，张沥，阎小君，等。cagA阳性Hp感染与胃粘膜组织恶性化转变的初步研究。中华消化杂志 1998；18(4)：249
26．张联，马峻岭，潘凯枫，等。胃癌高和低发区人群幽门螺杆菌及cagA+和hspA+型感染的研究。中华预防医学杂志 1998；32(2)：67-69
27．赵志泉，张红杰。具有cagA、vacA基因的幽门螺杆菌感染与胃、十二指肠疾病的关系。中华消化内镜杂志 1997；14(6)：333-336
28. Wong BC, Lam SK, Ching CK, et al. Seroprevalence of cytotoxin-associated gene A positive Helicobacter pylori strains in Changle, an area with very high prevalence of gastric cancer in south China. Aliment Pharmacol Ther, 1999; 13(10): 1295-302
29. Evans DJ Jr, Queiroz DMM, Mendes EN, et al. Diversity in the variable region of Helicobacter pylori cagA gene involves more than simple repetition of a 102-nucleotide sequence. Biochem Biophys Res Commun 1998; 245: 780-784.
30．张建中。中国协和医科大学博士学位论文。
31. Odenbreit S, Ptlls J, Sedlmaier B, et al. Translocation of Helicobacter priori CagA into gastric epithelial cells by type Ⅳ secretion. Science 2000; 287(5457): 1497-500
32. Yamaoka Y, Osato MS, Sepulveda AR, et al. Molecular epidemiology of Helicobacter pylori: separation of H. pylori from East Asian and non-Asian countries. Epidemiol Infect 2000; 124(1): 91-6
33. van der Ende A; Pan Z J; Bart A; et al. cagA-positive Helicobacter pylori populations in China and The Netherlands are distinct. Infect Immun, 1998; 66(5): 1822-6
34. Yamaoka Y, El Zimaity HM, Gutierrez O, et al. Relationship between the cagA 3' repeat region of Helicobacter pylori, gastric histology, and susceptibility to low pH. Gastroenterology 1999; 117(2): 342-9
35. Yamaoka Y, Kodama T, Kashima K, et al. Variants of the 3' region of the cagA gene in Helicobacter priori isolates from patients with different H. pylori-associated diseases. J Clin Microbiol 1998; 36(8): 2258-63
36. Kidd M, Lastovica AJ, Atherton JC, et al. Heterogeneity in the Helicobacter pylori vacA and cagA genes: association with gastroduodenal disease in South Africa? Gut 1999; 45(4): 499-502
37. Yamaoka Y, Kodama T, Graham D, et al. Comparision of four serological tests to determine the CagA or VacA status of Helicobacter pylori strains. J Clin Microbiol, 1998; 36(11): 3433-4.
38. Yamaoka Y, Graham DY. CagA status and gastric cancer unreliable serological tests produce unreliable data. Gastroenterology 1999;117(3):745
39. Vaucher C, Janvier B, Nousbaum JB, et al. Antibody response of patients with Helicobacter pylori-related gastric adenocarcinoma: significance of anti-cagA antibodies. Clin Diagn Lab Immunol 2000; 7(3): 463-7.
40．姒健敏，高敏，王杭勇，等。幽门螺杆菌CagA抗体测定价值探讨。浙江医学 1998；20(12)：720-721
41．萨姆布鲁克，费里奇，曼尼阿蒂斯著，金冬雁，黎孟枫等译。《分子克隆实验指南》。第二版，北京；科学出版社，1988
42. Peek RM Jr, Miller GG, Tham KT, et al. Detection of Helicobacter pylori gene expression in human gastric mucosa. J Clin Microbiol 1995; 33(1): 28-32
43. Lage AP, Godfroid E, Fauconnier A, et al. Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens. J Clin Microbiol 1995; 33(10): 2752-6
44. Miehlke S, Go MF, Kim JG, et al. Serologic detection of Helicobacter pylori infection with cagA-positive strains in duodenal ulcer, gastric cancer, and asymptomatic gastritis. J Gastroenterol 1998; 33 (S 10): 18-21
45. van Doom LJ, Figueiredo C, Rossau R, et al. Typing of Helicobacter priori vacA gene and detection of cagA gene by PCR and reverse hybridization. J Clin Microbiol 1998; 36(5): 1271-6
46. Busolo F, Bertollo G, Bordignon G, et al. Detection and characterization of Helicobacter pylori from patients with gastroduodenal diseases. Diagn Microbiol Infect Dis 1998; 31(4): 531-6
47. Xiang Z, Bugnoli M, Ponzetto A, et al. Detection in an enzyme immunoassay of an immune response to a recombinant fragment of the 128 kilodalton protein (CagA) of Helicobacter pylori. Eur J Clin Microbiol Infect Dis 1993; 12(10): 739-45
48．韩锋产，阎小君，苏成芝。幽门螺杆菌cagA基因的克隆表达及其产物的应用。华人消化杂志 1998；6(12)：1084-1086
49. Maeda S, Ogura K, Yoshida H, et al. Structure of cag pathogenicity island in Japan Helicobacterpylori isolates. Gut 99; 44: 336-341.
50. van Doorn LJ, Figueiredo C, Sanna R, et al. Distinct variants of Helicobacter pylori cagA are associated with vacA subtypes. J Clin Microbiol 1999,37: 2306-11.
51. Peek RM Jr, Moss SF, Tham KT, et al. Helicobacter pylori cagA+ strains and dissociation of gastric epithelial cell proliferation from apoptosis. J Natl Cancer Inst 1997; 89(12): 863-8
52. Rokkas T, Ladas S, Liatsos C, et al. Relationship of Helicobacter pylori CagA status to gastric cell proliferation and apoptosis. Dig Dis Sci 1999; 44(3): 487-93
53. Segal ED, Cha J, Lo J, et al. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori. Proc Natl Acad Sci USA 1999; 96: 14559-64.
54. Meyer-ter-Vehn T, Covacci A, Kist M, et al. Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun. J Biol Chem 2000; 275(21): 16064-72
55. Naumann M, Wessler S, Bartsch C, et al. Activation of activator protein 1 and stress response kinases in epithelial cells colonized by Helicobacter pylori encoding the cag pathogenicity island. J Biol Chem 1999; 274(44): 31655-62
56． Akopyants NS, Clifton SW, Kersulyte D, et al. Analyses of the cag pathogeneicity island of Helicobacterr pylori. Mol Microbiol 1998;28(1) :37-53．
57． Censini S, Lange C, Xiang Z, et al. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci USA 1996;93(25) : 14648-53
58． Spohn G, Beier D, Rappuoli R, et al. Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori. Mol Microbiol 1997;26: 361-72．
59． Odenbreit S, Gebert B, Puls J, et al. Interaction of Helicobacter pylori with professional phagocytes: role of the cag pathogenicity island and translocation, phosphorylation and processing of CagA. Cell Microbiol 2001;3(1) :21-31
60． Backert S, Ziska E, Brinkmann V, et al. Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus. Cell Microbiol 2000;2(2) : 155-64
61． Asahi M, Azuma T, Ito S, et al. Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. J Exp Med 2000; 191: 593-602．
62． Stein M, Rappuoli R, Covacci A. Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation. Proc Natl Acad Sci USA 2000; 97: 1263-8．
63． Covacci A and Rappuoli R. Tyrosine phosphorylated bacterial proteins: Trojan horses for the host cell. J Exp Med 2000;191(4) :587-92
64． Bliska JB, Guan K, Dixon JE, et al. Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci USA, 1991;88(4) ,1187-91
65． Occhialini A, Marais A, Urdaci M, et al. Composition and gene expression
of the cag pathogenicity island in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica. Infect Immun 2001; 69(3): 1902-8
66. Lee A, ORourke J, De Ungria MC, et al. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 1997; 112(4): 1386-97
67．曾志荣，胡品津，陈旻湖，等。幽门螺杆菌长期感染鼠腺胃模型的研究。中华医学杂志1998；78(7)：494-7．
68．曾志荣，胡品津，陈旻湖。幽门螺杆菌感染与胃癌形成关系的动物实验研究。第六届全国消化学术会议摘要汇编，P29，中国西安，1999年月10月。
69. Shomer NH, Dangler CA, Whary MT, et al. Experimental Helicobacter pylori infection induces antral gastritis and gastric mucosa-associated lymphoid tissue in guinea pigs. Infect Immun 1998; 66(6): 2614-8
70. Fox JG, Dangler CA, Taylor NS, et al. High-salt diet induces gastric epithelial hyperplasia and parietal cell loss, and enhances Helicobacter pylori colonization in C57BL/6 mice. Cancer Res 1999; 59(19): 4823-8
71．陈永昌，张建中，李铁，等。幽门螺杆菌对人和大鼠胃上皮细胞外信号调节激酶活性的影响。北京医科大学学报2000；32(2)：109-112