【作者】 刘海燕；

【导师】 方平楚；

【作者基本信息】 浙江大学 ， 病原生物学， 2004， 硕士

【摘要】 背景和目的 幽门螺杆菌(Helicobacter pylori，H.pylori)是一种螺旋状、微需氧的革兰阴性杆菌，定植于人胃粘膜。1983年澳大利亚学者Warren和Marshall在人胃粘膜活检组织中首次分离到该菌。H.pylori被认为是慢性胃炎、消化性溃疡的主要致病因子，并与胃腺癌、胃粘膜相关淋巴组织淋巴瘤以及原发性胃非何杰金淋巴瘤相关。1994年WHO将H.pylori列为Ⅰ类致癌因子。全球约有超过一半的人口感染H.pylori，西方国家H.pylori的感染率为25-50％；发展中国家，儿童期H.pylori的感染率为60％左右，成人期可高达80—90％。虽然H.pylori感染可持续终身，但大部分感染者(80％)均为无症状的带菌者，只有10—20％的感染者可发生慢性胃炎和消化性溃疡，极少数的感染者(0.1—4％)则可发展为胃癌。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H.pylori菌株的毒力差异对临床疾病严重程度的影响。目前，H.pylori的cag致病岛(cag pathogenicity island，cag PAI)受到研究者的重视。许多流行病学调查资料显示，cag PAI阳性的H.pylori感染与严重胃炎、消化性溃疡、萎缩性胃炎和胃癌的发生密切相关。现已明确，cag PAI编码一个Ⅳ型分泌系统，该Ⅳ型分泌系统具有两个功能：一、将CagA从H.pylori转运到宿主细胞中；二、诱导胃上皮细胞合成和分泌细胞因子如IL—8等。cag PAI中的cagM、cagT以及ORF13等基因所表达的蛋白被认为是组成Ⅳ型分泌系统的重要结构成分，缺失cagM、cagT或ORF13的突变株则不能将CagA转运至宿主细胞内，也不能诱导宿主细胞合成和分泌IL—8。ORF10编码的蛋白与Ⅳ型分泌系统的形成无关，而是作为引导CagA进入Ⅳ型分2004户斤歹，次掌硕士学位论文馨泌系统的通道，缺失ORF10的H Pylori突变株不能转运CagA，但仍能诱导胃上皮细胞合成和分泌细胞因子IL一8。由于cagA、cagM、cagT、O盯13和ORF10具有重要的功能，且在cagl叭l内的分布具有一定的问距，可能可以作为检测完整cagPAI的依据。为此，本实验用PCR方法分别检测了cagl上的cagA、cagM以及cagH上的cagT、oRF 13和oRF10基因，以了解胃癌高发区浙江省岱山县H Pylori cagI〕AI的分布并分析各基因与胃十二指肠疾病类型的关系及与杭州市H Pylori的差〔己少十。 材料和方法收集因胃部不适接受胃镜检查患者的胃粘膜标本，进行H.Pylori分离培养，其‘卜岱山县分离培养得到H. Pylori124株，患者中男性90例，女性34例，年龄16一71岁，平均42.7岁，经胃镜及病理检查确诊为慢性胃炎60例，消化性溃疡59例，胃腺癌5例;杭州市分离培养得到厅Pylori47株，患者中男性25例，女性22例，年龄24一67岁，立「均43.3岁，经胃镜及病理检查证实为’漫性胃炎41例，消化性溃疡6例。将患者胃粘膜组织研磨后涂布在ECY选择性培养基上，37℃微需氧(5%02，10%cOZ，85%N:)培养5天，选择有典型H Pylori菌落生长、快速尿素酶试验阳性、氧化酶试验阳性、革兰染色镜检为革兰阴性、形态呈螺杆状或飞鸟状者为H Pyl、i，进一步传代培养2天扩增细菌。用标准酚/氯仿抽提法提取HPylori的基因组DNA，根据文献报道的引物，进行PCR扩增，反应参数为95℃预变性3min，随后94OC305、56oC305、72oC455共30个循环，最后72oC延伸7min。用琼脂糖凝胶电泳法将PCR产物进行电泳以检测H. Pyzor川击床分离株cagA、eagM、eagT、ORF13和ORF10的阳性情况。实验结果采用才检验(SpSSll.5)，凡0.05认为具有统计学差异。 结果和讨论H刃zori cagA、eagM、eagT、oRF13和oRFlo的阳性率:岱111县分别为100.0%、82.3%、96.8%、94.4%和99.2%;杭州市分别为100.0%、93.6%、97.9%、957%和100%。岱山县cagA阳性的菌株中，82.3%可能具有完整cag PAI，完整cag队I在消化性溃疡、’净性胃炎和胃癌患者的检出率分别为84.7%、80.0%和80.0%;杭州市cagA阳性的菌株中，91 .5%的菌株可能有完整的cag PAI，完整cag PAI在消化性溃疡和慢性胃炎患者的检出率分别为1 00.0%、90.2%。cag PAI内单个基因在消化胜溃疡、’隆胜胃炎和胃癌的检出率:岱山县，cagA的检出率均为100.0%;cagM分别为84.7%、80.0%和80.0%;cagT分别为%.6%、%.7%和2004户亏j’次掌硕士学位论文嘟100.0%;ORF13分别为94.9%、95.0%和80.0%;O即10分别为100.0%、98.3%和100.0%。杭州市来自消化性溃疡患者的6株H Pylori中，各基因检出率均为100.0%;自慢性胃炎患者分离的H Pylori，cagM的阳性率为92.7%，cagT为97.6%，o好13为95.1%，ORF10为100.0%。cagA不能作为完整cag PAI存在的标志;cag pAI的完整性及对其中单个基因的检测都不能用来预测H Pylori相关的胃、十二指肠疾病的类型;岱11)县与杭州市相比，厅pylori eagA、eagM、eagT、O盯13和ORFlo的阳性率及可能有完整cag pAI的H刀lori的比例未见明显统计学差异。 结论浙江省胃癌高发区岱11一I县临床分离H州ori菌株，其cagA不能作为完整cag pA】存在的标志;cag PAI的完整性及对其中的单个基因的检测都不能用来预测H Pylori相关的胃、十一二指肠疾病的类型;来自胃癌高发区岱山县的HPylori?更多还原

【Abstract】 Background and Aims Helicobacter pylori (H. pylori) is a Gram-negative spiral-shaped microaerophilic bacterium that colonizes the gastric mucosa of human stomach.. Australian scientists named Warren and Marshall for the first time isolated this bacterium from the human gastric biopsy specimens in 1982. Infection of H. pylori has been implicated as the major cause of gastritis and peptic ulcer, and also appears to be associated with gastric adenocarcinoma, gastric mucosa-associated lymphoid tissue lymphoma and primary gastric non-Hodgkin’s lymphoma. In 1994, H. pylori was declared group I carcinogen for gastric cancer by WHO. More than 50% of the world’s population is infected with H. pylori and the prevalence of H. pylori infection is 25-50% in Western nations. In the developing countries, the prevalence of H. pylori infection is 60% in children and 80-90% in adults. In most patients (80%) H. pylori does not cause clinical symptoms, but 10-20% of infected patients will develop gastritis and peptic ulcer. A smaller percentage (0.1-4%) of infected patients will develop gastric adenocarcinoma. The certain pathogenicity mechanism is unknown. One explanation for different clinical outcomes is the diversity of H, pylori strains, other than host factors and environmental factors. There is growing evidence that genetic differences among strains help determine the clinical outcome of infection.The cag pathogenicity island (cag PAI) is considered to be one of the major virulence factors of H, pylori. Many epidemic logical studies have found that infection with cag PAI positive strains was highly associated with peptic ulcer, atrophic gastritis and gastric cancer. The cag PAI encode a type IV secretion system, which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin-8 (IL-8). The genes of cagM, cagT and ORF13 in cag PAI were found to be absolutely essential for translocation of CagA into host cells and for the ability of H , pylori fully to induce transcription of IL-8. The product of ORF10 is absolutely essential for the translocation of CagA, but not for the induction of IL-8. However, the distribution of cag PAI in H. pylori from Daishan County where gastric carcinoma has a high prevalence in Zhejiang Province and their relationship with the gastroduodenal diseases remain unclear. Because of having representative spacing sequences along the cag PAI and playing an important role in pathogenicity of cag PAI, the genes of cagA, cagM, cagT, ORF13 and ORF10 were selected and amplified by polymerase chain reaction (PCR) to evaluate the distribution in H. pylori from Daishan County where gastric carcinoma has a high prevalence in Zhejiang Province, their relationship with the gastroduodenal diseases and the difference of the cag PAI of H, pylori from Daishan County and Hangzhou City.Materials and Methods A total of 124 H. pylori isolates was obtained from H pylori-infected adults who had undergone upper gastrointestinal endoscopy at the Hospital of Daishan County in Zhejiang Province. The patients consisted of 90 men and 34 women with a mean age of 42.7 years (ranging from 16 to 71 years) and classified into 3 groups with chronic gastritis (n=60), peptic ulcer (n=59) and gastric adenocarcinoma (n-5). A total of 47 H. pylori isolates was obtained from H. pylori-infected adults who had undergone upper gastrointestinal endoscopy at the Second Affiliated Hospital of Zhejiang University. The patients consisted of 25 men and 22 women with a mean age of 43.3 years (ranging from 24 to 67 years) and classified into 2 groups with chronic gastritis (n=41), peptic ulcer (n=6). The classification of patients was based on the results of endoscopic and histologicalexamination. Biopsy specimens were cultured on ECY-selective agar plates at 37 for 5 days, under 100% humidity and microaerophilic conditions (5%O2, 10%CO2, and 85% N2). H. pylori was identified by the following criteria: characterist更多还原