浙江地区幽门螺杆菌临床分离株中cagA、vacA、iceA基因的分布及分析
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摘要
背景和目的 现已明确，幽门螺杆菌感染(Helicobacter pyroli，Hp)是慢性活动性胃炎和消化性溃疡的主要病因，与胃癌和粘膜相关淋巴组织(MALT)淋巴瘤的发生密切相关，1994年WHO已将Hp列为Ⅰ级致癌原。据初步统计，全世界超过50％的人群有Hp感染。其中西方国家的感染率为25-50％，发展中国家有的高达80％。然而，Hp感染人群大都无症状，只有少数感染者可出现消化性溃疡，极少数感染者最终发展成胃癌，其中的原因尚不清楚。目前认为除与宿主基因易感性，环境因素有关外，更重要的是Hp菌株的毒力差异对临床结果的影响。目前，Hp的cagA、vacA、iceA基因受到研究者的重视。许多流行病学调查资料显示，cagA基因阳性的Hp菌株感染与消化性溃疡、肠化生、萎缩性胃炎和胃癌的发生密切相关。编码空泡毒素的vacA基因存在于所有的Hp菌株中，其信号序列(s)和中间序列(m)变异较大，s和m均存在许多亚型，其中s有sla，slb，slc，s2亚型，m有mla，mlb，mlb-m2，和m2亚型。不同s和m亚型组成的嵌合体使Hp产生的空泡毒素的量和活性也不同。研究发现vacA s1／m1型的Hp产生大量毒素，可能与消化性溃疡相关，vacA s1／m2型的Hp产生中等量毒素，而vacA s2／m2型的Hp则产生少量毒素或不产生毒素。Hp的iceA基因是近来发现的一个新基因，有iceA1和iceA2两个等位基因，来自荷兰和美国的几项研究显示iceA1与消化性溃疡和胃粘膜IL—8水平有显著相关性，被认为可能是Hp
扣们扯；。x穿。。。。。。＠
的又一致病因子。本研究的目的是研究浙江地区幽门螺杆菌临床分离株中cagA、
vacA、iceA各基因的存在情况并分析各基因与胃十二指肠疾病的关系。
材料和方法 共从浙江地区门 例胃十二指肠疾病患者的胃劲膜活检组织
中分离培养出 Hp。其中浙医H院 49例，岱山县人民医院 67例。116例患者中
慢性浅表性胃炎31例，慢性萎缩性31例，糜烂性胃炎18例，消化性溃疡30
例，胃癌6例。将胃十H指肠疾病患者的胃猫膜活检组织研磨后涂布于ECY选
择性培养基上，37℃，微需氧（5％OZ、10％COZ和 85％N。）环境中培养 5天后，
经系统鉴定（菌落特征／快速尿素酶实验、氧化酶实验、形态及染色特性）确认为
Hp者，进一步传代培养 2天扩增细菌。用标准酚／氯仿抽提法提取 Hp的基因组
DNA，根据文献报道的引物，进行PCR扩增（95OC预变性3min后，94℃变性
45s，54”C退火45s，72”C延伸45s，共30个循环，再于72’C延伸7min），用琼脂
糖凝胶电泳法将 PCR产物进行电泳以检测 HP临床分离株的 cagA、vacA、iceA
各基因的阳性情况。各基因与疾病的关系的分析采用 X‘检验，P＜0刀5认为有统
计学意义。
结果和讨论 Hp菌株各基因 PCR扩增产物大小均与报道的一致。116例临床
分离的 Hp中，cagA基因的阳性率为 100％（if6／11卜 vacAsla基因的阳性率为
gi．4％（106／116），vacAinZ基因型 Hp占 59．5％（69／if6），vacAInlb基因型 Hp占
12．l％（14／if6），vacAsla／InZ基因型组合的 Hp占 54．3％（63／if6），vacAsla／mlb基
因型组合的彻占12．lO（l／116），另外，vacAnllb和vacAInZ 均阳性的菌株占ZI．6
％（25／116），还有 6．9％（8／116）的菌株不能用 vacAJnlb和 vacAInZ分型。iceAl
基因的阳性率为66．4％（7／11 6），iceAZ基因的阳性率为6．9％（／11 6），另有15．5％
（18／116）的菌株iceAl和iceAZ基因均阳性，而iceAl和iceAZ基因均阴性的菌
株占 11．2％（13／116人 因此，浙江地区 Hp 临床分离株的基因型以
cagAvacAs a／InZ，iceAl为主。在慢性浅表性胃炎、慢性萎缩性胃炎、糜烂性胃炎、
消化性溃疡和胃癌各疾病组中，cagA、vacA、iceA各基因的阳性情况均无统计学
差异。提示 cagA、vacA、iceA基因不能作为浙江地区 Hp菌株毒力强弱的指标。
结论 浙江地区幽门螺杆菌的基因型以 cagA，vacAslJmZ，iceAl为主。用gA、
vacA、iceA各基因不能作为浙江地区幽门螺杆菌菌株毒力强弱的指标。
Background and Aims H. pylori is identified as the chief cause of chronic active gastritis and peptic ulcer diseases (PUD) in human beings and is considered to be a risk factor for the development of gastric adenocarcinoma and MALT lymphoma. So it has been categorized as a group I carcinogen by WHO.By estimated, there are over 50% people infected with H. pylori around the world. But many people remain asymptomatic, only a minority of patients carrying H. pylori develop severe diseases, such as atrophic gastritis, peptic ulcer disease, even gastric adenocarcinoma. The certain pathogenicity mechanism is unknown. One explanation for different clinical outcomes is the diversity of H. pylori strains, otherthan host factors and environmental factors. Now, several genes have been identified that may play a role in the pathogenicity of H. pylori, such as cagA, vacA, and iceA genes. Many epidemiological studies have found that infection with a cagA-positive strains was both highly associated with peptic ulcer disease and with the risk of developing intestinal metaplasia, atrophic gastritis and gastric carcinoma compared to persons harboring cagA-negative strains. vacA, encoding the vacuolating toxin ,is present in all strains, and comprises two variable parts. The s-region (s) exists as sla, sib, sic, and s2 allele, and the m-region (m) occurs as mla, mlb,mlb-m2,and m2 allele. The
mosaic combination of s- and m-region allelic types determines the quantity and activity of cytotoxin and thereby is associated with pathogenicity of the bacterium, vac A si/ml strains produce a large amount of toxin, and is associated with peptic ulceration.vacAsl/m2 strains produce moderate amounts, vacAs2/m2 strains produce very little or no toxin. Recently, a novel gene iceA of H. pylori was identified following transcriptional upregulation on contact with gastric epithelial cells. iceA exists as two distinct genotypes, iceA1 and iceA2. And in vivo carriage of H. pylori iceA1 strains has been reported to be associated with peptic ulceration and enhanced acute neutrophilic infiltration, so iceA is suspected to be another virulence factor of H. pylori. The present study aim to investigate Helicobacter pylori cagA, vacAi iceA status of Zhejiang province and relationship to gastric duodenal diseases.
Materials and Methods A total of 116 patients with different gastric duodenal diseases, living in Zhejiang province, with culture-proven H. pylori infection were recruited in this study.49 patients were from the Second Affiliated Hospital of Zhejiang University School of Medicine, and the others were from Renming Hospital of Daishan County Zhejiang province. The biopsy specimens from patients were grinded and then spreaded on ECY solid media. After cultured for 5 days at 37 in a microaerobic atmosphere containing 5% oxygen, 10% carbon dioxide, 85% nitrogen, H. pylori strains were identified by the following criteria: characteristic of colony, rapid urease test, oxidase test, morphology on Gram stain. And H. pylori strains were transfer of culture for amplification bacteria. The H. pylori DNA was extracted using the standard method of phenol/chloroform. The primers were according to the references. The cagA, vacA, iceA genotypes were determined by PCR reactions. The reaction conditions were 3 minutes pregeneration at 95 ,followed by 30 cycles of 45 seconds at 94 ,45 seconds at 54 , and 45 seconds at 72 . Final extension was performed for 7 minutes at 72 . The relationship between individual genotypes and the different gastric diseases was assessed with Chi-square ( x 2 ) test, a P value of <0.05 was defined as significant.
Results and Discussion Every product of PCR was coincident to the
references. In all 116 patients, the prevalence of cagA was 100%, vacAsla was 91.4%, vacAm2 was 59.5%, vacAmlb was 12.1%, vacA sla/m2 was 54.3%, vacA sla/mlb was 12.1%, vacAmlb and vacAm2 were both found in 21.6%, there were 6.9% strains carrying neither vacAmlb nor vacAm2 genes. The prevalence of iceAl was
扣们扯；。x穿。。。。。。＠
的又一致病因子。本研究的目的是研究浙江地区幽门螺杆菌临床分离株中cagA、
vacA、iceA各基因的存在情况并分析各基因与胃十二指肠疾病的关系。
材料和方法 共从浙江地区门 例胃十二指肠疾病患者的胃劲膜活检组织
中分离培养出 Hp。其中浙医H院 49例，岱山县人民医院 67例。116例患者中
慢性浅表性胃炎31例，慢性萎缩性31例，糜烂性胃炎18例，消化性溃疡30
例，胃癌6例。将胃十H指肠疾病患者的胃猫膜活检组织研磨后涂布于ECY选
择性培养基上，37℃，微需氧（5％OZ、10％COZ和 85％N。）环境中培养 5天后，
经系统鉴定（菌落特征／快速尿素酶实验、氧化酶实验、形态及染色特性）确认为
Hp者，进一步传代培养 2天扩增细菌。用标准酚／氯仿抽提法提取 Hp的基因组
DNA，根据文献报道的引物，进行PCR扩增（95OC预变性3min后，94℃变性
45s，54”C退火45s，72”C延伸45s，共30个循环，再于72’C延伸7min），用琼脂
糖凝胶电泳法将 PCR产物进行电泳以检测 HP临床分离株的 cagA、vacA、iceA
各基因的阳性情况。各基因与疾病的关系的分析采用 X‘检验，P＜0刀5认为有统
计学意义。
结果和讨论 Hp菌株各基因 PCR扩增产物大小均与报道的一致。116例临床
分离的 Hp中，cagA基因的阳性率为 100％（if6／11卜 vacAsla基因的阳性率为
gi．4％（106／116），vacAinZ基因型 Hp占 59．5％（69／if6），vacAInlb基因型 Hp占
12．l％（14／if6），vacAsla／InZ基因型组合的 Hp占 54．3％（63／if6），vacAsla／mlb基
因型组合的彻占12．lO（l／116），另外，vacAnllb和vacAInZ 均阳性的菌株占ZI．6
％（25／116），还有 6．9％（8／116）的菌株不能用 vacAJnlb和 vacAInZ分型。iceAl
基因的阳性率为66．4％（7／11 6），iceAZ基因的阳性率为6．9％（／11 6），另有15．5％
（18／116）的菌株iceAl和iceAZ基因均阳性，而iceAl和iceAZ基因均阴性的菌
株占 11．2％（13／116人 因此，浙江地区 Hp 临床分离株的基因型以
cagAvacAs a／InZ，iceAl为主。在慢性浅表性胃炎、慢性萎缩性胃炎、糜烂性胃炎、
消化性溃疡和胃癌各疾病组中，cagA、vacA、iceA各基因的阳性情况均无统计学
差异。提示 cagA、vacA、iceA基因不能作为浙江地区 Hp菌株毒力强弱的指标。
结论 浙江地区幽门螺杆菌的基因型以 cagA，vacAslJmZ，iceAl为主。用gA、
vacA、iceA各基因不能作为浙江地区幽门螺杆菌菌株毒力强弱的指标。
Background and Aims H. pylori is identified as the chief cause of chronic active gastritis and peptic ulcer diseases (PUD) in human beings and is considered to be a risk factor for the development of gastric adenocarcinoma and MALT lymphoma. So it has been categorized as a group I carcinogen by WHO.By estimated, there are over 50% people infected with H. pylori around the world. But many people remain asymptomatic, only a minority of patients carrying H. pylori develop severe diseases, such as atrophic gastritis, peptic ulcer disease, even gastric adenocarcinoma. The certain pathogenicity mechanism is unknown. One explanation for different clinical outcomes is the diversity of H. pylori strains, otherthan host factors and environmental factors. Now, several genes have been identified that may play a role in the pathogenicity of H. pylori, such as cagA, vacA, and iceA genes. Many epidemiological studies have found that infection with a cagA-positive strains was both highly associated with peptic ulcer disease and with the risk of developing intestinal metaplasia, atrophic gastritis and gastric carcinoma compared to persons harboring cagA-negative strains. vacA, encoding the vacuolating toxin ,is present in all strains, and comprises two variable parts. The s-region (s) exists as sla, sib, sic, and s2 allele, and the m-region (m) occurs as mla, mlb,mlb-m2,and m2 allele. The
mosaic combination of s- and m-region allelic types determines the quantity and activity of cytotoxin and thereby is associated with pathogenicity of the bacterium, vac A si/ml strains produce a large amount of toxin, and is associated with peptic ulceration.vacAsl/m2 strains produce moderate amounts, vacAs2/m2 strains produce very little or no toxin. Recently, a novel gene iceA of H. pylori was identified following transcriptional upregulation on contact with gastric epithelial cells. iceA exists as two distinct genotypes, iceA1 and iceA2. And in vivo carriage of H. pylori iceA1 strains has been reported to be associated with peptic ulceration and enhanced acute neutrophilic infiltration, so iceA is suspected to be another virulence factor of H. pylori. The present study aim to investigate Helicobacter pylori cagA, vacAi iceA status of Zhejiang province and relationship to gastric duodenal diseases.
Materials and Methods A total of 116 patients with different gastric duodenal diseases, living in Zhejiang province, with culture-proven H. pylori infection were recruited in this study.49 patients were from the Second Affiliated Hospital of Zhejiang University School of Medicine, and the others were from Renming Hospital of Daishan County Zhejiang province. The biopsy specimens from patients were grinded and then spreaded on ECY solid media. After cultured for 5 days at 37 in a microaerobic atmosphere containing 5% oxygen, 10% carbon dioxide, 85% nitrogen, H. pylori strains were identified by the following criteria: characteristic of colony, rapid urease test, oxidase test, morphology on Gram stain. And H. pylori strains were transfer of culture for amplification bacteria. The H. pylori DNA was extracted using the standard method of phenol/chloroform. The primers were according to the references. The cagA, vacA, iceA genotypes were determined by PCR reactions. The reaction conditions were 3 minutes pregeneration at 95 ,followed by 30 cycles of 45 seconds at 94 ,45 seconds at 54 , and 45 seconds at 72 . Final extension was performed for 7 minutes at 72 . The relationship between individual genotypes and the different gastric diseases was assessed with Chi-square ( x 2 ) test, a P value of <0.05 was defined as significant.
Results and Discussion Every product of PCR was coincident to the
references. In all 116 patients, the prevalence of cagA was 100%, vacAsla was 91.4%, vacAm2 was 59.5%, vacAmlb was 12.1%, vacA sla/m2 was 54.3%, vacA sla/mlb was 12.1%, vacAmlb and vacAm2 were both found in 21.6%, there were 6.9% strains carrying neither vacAmlb nor vacAm2 genes. The prevalence of iceAl was
引文
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[2] Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet, 1984, 1(8390): 1311-1315.
[3] Nicolaas, Arents, Anton, et al. The importance of vacA,cagA, iceA genotypes of Helicobacter pylori infection in peptic ulcer disease and gastroesophageal reflux disease. Am J Gastroenterol, 2001,96(9): 2603-2608.
[4] Van Doom LJ, Henskens Y, Nouhan N, et al. The efficacy laboratory diagnosis of Helicobacter pylori infection in gastric biopsy specimens is related to bacterial density and vacA, cagA, and iceA genotypes. J Clin Microbiol, 2000, 38(1): 13-17.
[5] Leunk RD, Johnson PT, David BC, et al. Cytotoxin activity in broth-culture filtrates ofCampylobacter pylori. J Med Microbiol, 1988,26 (2): 93-99.
[6] Reyrat JM, Lanzavecchia S, Lupetti P, et al. 3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin. J Mol Biol, 1999, 290 (2): 459—470.
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