【作者】 陈学军；

【导师】 严杰；

【作者基本信息】 浙江大学 ， 病原生物学， 2003， 硕士

【摘要】 幽门螺杆菌(Helicobacter pylori，Hp)是慢性胃炎、消化性溃疡和胃MALT淋巴瘤的病原体，也与胃癌发病密切相关。人群中几乎有一半终身感染Hp，但只有部分感染者发展为明显的临床疾病，其中部分原因可能与Hp的致病力不同有关。 Hp空泡毒素基因(vacuolating cytotoxin gene A，vacA)的产物(VacA)能使上皮细胞产生空泡变性；细胞毒素相关蛋白基因(cytotoxin-associated gene A，cagA)的产物(CagA)被Hp排泌入胃上皮细胞后会导致细胞骨架的重排，刺激胃上皮细胞产生炎细胞因子IL-8等，两者均为该菌重要的致病因子。 所有Hp菌株均含vacA基因，但只有50％左右菌株发现有空泡毒活性，而空泡毒活性与vacA基因型有关。一般认为，vacA基因含有信号区(s区)和中间区(m区)两个主要的可变区，s区存在s1a、s1b或s2亚型，而m区存在m1或m2亚型，s1/m1型毒素活性最强，s1/m2型毒素活性中等或偏弱，s2/m2型毒素活性极弱或无，进一步的研究也已发现了一些别的s区和m区亚型变异体(s1c、m1a、m1b、m1b-m2等)。 欧美国家分离的Hp菌株中约60～70％具有cagA基因，并发现感染cagA阳性和/或vacA s1/m1型菌株可能更易导致消化性溃疡的发生。韩国、日本浙江人学顾1一学位论义 中又流多等部分亚洲国家分离的Hp菌株中90％以上携带cagA基因，主要的vacA基因型也有别于欧美国家，而且不同cagA基因状态和／或vacA基因型的菌株在消化性溃疡和慢性胃病中的分布无显著性差异。 cagA和 vacA均为单拷贝基因，同一 HP菌株只有携带或不携带 cagA基因两种情况，vacA的s区或m区各亚型不可能同时出现于同一Hp菌株中，因而多种cagA携带状态和／或vacA亚型的存在表明其来源于不同Hp菌株。近年国外文献己有报道，同一病人可感染多株不同cagA状态和／或vacA基因型的Hp，且此类混合感染与消化性溃疡的发生密切相关，但国内类似研究几乎没有。 因此，了解浙江地区患者单株式多株Hp感染、不同cagA状态和／或vacA基因型的Hp菌株在消化性溃疡和慢性胃炎中的分布，对于明确本地区Hp的致病机制、流行特点、感染来源等均有一定意义。 目的：了解浙江地区消化性溃疡和慢性胃炎患者分离的 Hp临床菌株的cagA基因状态和 vacA基因型（agA个acA优势基因型的分布情况以及是否存在不同基因型多株Hp混合感染现象。分析cagA基因和／或vacA基因型、不同基因型Hp混合感染与胃病的关系。 方法：选择胃窦、胃体双份活检标本均培养出Hp的42例慢性胃炎（CG）和 36例消化性溃疡（PU）患者作为研究对象，采用聚合酶链反应（PCR）检测分离的 156份 Hp菌株 cagA基因、vacA基因的信号区＊）和中间区 （m）亚型，分析 Hp基因型及多株Hp混合感染在 CG和 PU中的分布。部分优势基因型的扩增产物TA克隆后进行核苦酸序列测定。 结果：96．2％的分离自病人胃窦标本的 Hp菌株 （7厂8）cagA阳性，97．4％的十应胃体标本中分离的Hp菌株（76／78）为cagA阳性，有1例（1．3％）患者胃窦、胃体存在cagA状态不一的两株Hp混合感染。78例患者胃窦标本分离的Hp菌株vacA基因分型中，sla／ml、slajmZ、sla／mlb、Sla／mlb－SZ4种 vacA基因型菌株所占比例分另为 6．40（5／78）、55．lO（43／78）、26．9o ～2～浙江大学硕士学位论文 中又视要（ZI／78）和 1．3O（l／78），混合感染率为3．80（3／78）。而相应的胃体标本中，前述四种vacA基因型菌株所占比例依次为6．4％（5／78）、53．8％（42／78）、25石％（20／78）和 3．8％（／78），混合感染率为 5．l％（4／78）。cagA十一slajmZ和 cagA”－sla／mlb在胃窦标本分离的 Hp菌株中占引．3％（40／78）和 26．9％ 门1／78），而在相应的胃体标本来源的菌株中占5二．6％（41／78）和25．6％u0门8人少数Hp菌株vacA基因的 s区和 m区不能分型，未发现sib、sZ和 mla型。综合分离自胃窦、胃体标本的 Hp菌株基因型分析结果，20．5％（ 6厂8）患者标本中检出不同基因型的多株 Hp菌株，同一胃内多部位采样比单部位采样者有更高的混合感染检出率。Hp cagA基因、vacA基因型、cagA八acA基因组合及不同基因型菌株混合感染在CG和 PU中的分布差异均无显著性0＞0刀5人6株sla型Hp菌株的s区扩增产物与报道的sla型60 90株核着酸序列同源性为 93，15－94．86％，4株 mZ型 Hp菌株的 m区扩增产物与报道的mZ型87－203株核着酸序列之间同源性为93．63～97石1％。 结论：cagA＼sl a／mZ是浙江地区慢性胃炎或消化性溃疡中最主要的 HP菌株的优势基因型，其次为 cagA＼sl a／m fo，部分菌株 vacA sla型和 mZ型扩增片段的核着酸序列与国外文献报道参考菌株的序列有很高的同源性，部分患者可同时感染不同C明A携带状态和／或不同VSCA基因型的多株Hp。但cagA携带状态、各种vacA基因型、不同基因型混合感染的Hp菌株，在 CG和 PU中的分布无显著性差异。更多还原

【Abstract】 Helicobacter pylori(H. pylori) infection causes chronic gastritis (CG), peptic ulceration (PU) and gastritis MALT lymphoma, and is an important risk factor for gastric adenocarcinoma. Most infections are asymptomatic, and only partial infections develop Clinical diseases with obvious symptom. Two major bacterial virulence markers have been described, expression of vacuolating cytotoxin activity encoded by vacA and the presence of cagA (cytotoxin-associated gene A), and the CagA protein is secreted by the bacteria into gastric epithelial cells where it initiates cytoskeletal rearrangements , and enhances gastric epithelial cells to express IL-8.vacA, the gene encoding the vacuolating cytotoxin, is present in nearly all H. pylori strains, and the activity of this cytotoxin is found in about half of H. pylori strains. Analysis of vacA from many strains indicates the existence of two main regions, the signal and middle regions. In general, the signal region exists in one of three subtypes (s1a, s1b or s2), and the middle region possesses one of two subtypes (ml or m2). Other variants of the signal and middle region (s1c,m1 a, m1b, m1b-m2, et al.) have been described in some populations. Production of the vacuolating cyctotoxin is related to the mosaic structure of vacA; for instance, type s1/m1 and s1/m2 strains produce high and moderate levels of toxin, respectively, whereas s2/m2 strains produce little or no toxin.cagA is present in approximately 60-70% of H. pylori strains from Western patients, Various studies have demonstrated that infection of H. pylori strains with cagA+ and /or vacA s1 /m1 are more likely to result in PU.However, in Korea, Japan, et al, about more than 90% H. pylori isolates carry cagA gene, and the distribution of vacA genetype in partial Asia countries is different from west countries.In addition, some studies from other countries have suggested that more than one H. pylori strain with different cagA and vacA status may coexist within the human stomach, and that the multiple-strains coinfections are associated with PU. However, similar studies about coinfection of H. pylori strains is seldom done in our country.Therefore, in this study, the cagA status and vacA genotypes and mixed infections of H. pylori in Zhejiang patients is analyzed, and the frequency of the genotypes with gastric disease is compared, Which is contributed to reveal pathogenic mechanism, traits of transmission, and infectious source of H.pylori in local area.Objective: To analyze the vacA status and cagA genotypes and mixed infections of H. pylori in Zhejiang patients by PCR, and compare the frequency of the genotypes with gastric disease. To determine dominant cagA-vacA genotypes of H.pylori in patients suffing from CG or PU, and to understand the correlation of different genotype H.pylori infection, coinfection and thegastroduodenal diseases.Methods: H. pylori strains were isolated from antrum and corpus samples from 42 patients with CG and 36 patients with PU. PCR was used to detect cagA and the s and m regions of vacA in 156 H. pylori isolates from both the antrum and corpus. Parts of the amplification products were sequenced after T-A cloning. The distribution of H. pylori genotypes and coinfection in CG and PU was analyzed.Results: Almost all of the isolated H. pylori strains were cagA positive. Region of vacA, only one genotype of signal region (s1a) and four genotypes of the middle region (m1, m2, m1b and m1b-m2) were found. The proportion of s1a/m1, s1a/m2, s1a/m1b, s1a/m1b-m2 and coinfection of multiple H. pylori strains in 78 isolates from antrum samples was 6.4%, 55.1%, 26.9%, 1.3% and 3.8%; and the according proportion of those from corpus samples was 6.4%, 53.8%, 25.6%, 3.8% and 5.1% , respectively. Sixteen (20.5%)of the patients had multiple H. pylori strains with different cagA and vacA genotypes, and multiple samples were better than single sample from one stomach to increase the detection positive proportion of coinfection. In comparison with the reported sequences of H更多还原