【作者】 郭欣；

【导师】 赵宝华；

【作者基本信息】 河北师范大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 幽门螺杆菌(Helicobacter pylori,H. pylori)是迄今为止科学家所知道的唯一寄生在人类胃粘膜组织的细菌,一般人群普遍易感。自1984年Marshall与Warren发表论文报告他们成功地在胃炎患者胃粘膜组织培养出H. pylori之后,H. pylori很快就被证明是引起胃肠道疾病的重要病原,而且具有高度致癌性。1994年世界卫生组织(WHO)癌症中心宣布H. pylori感染可以增加胃癌的发病率与死亡率,因此将H. pylori归入一类致癌因子,H. pylori是目前已知的第一个可致癌的原核生物。同年美国国立卫生研究院(NIH)提出常见的胃部疾病治疗过程中应加入针对H. pylori的抗生素。H. pylori感染是引起胃炎、胃溃疡、胃癌等胃肠道疾病的重要病原,但是尽管有50%的人群感染H. pylori,但是仅有少数人发展成胃癌。H. pylori感染引发疾病的临床结局与患者自身的遗传易感性、H. pylori菌株类型及其与宿主免疫系统的相互作用密切相关。基于此我们调查研究了河北健康成年人群中H.pylori的感染情况以及感染人群中H. pylori的主要流行亚型,在此基础上对H. pylori感染后宿主体内细胞免疫系统表现的极化状态以及在细胞免疫反应中与免疫逃避机制有关的两个基因的基因多态性进行了分析。目的调查研究河北省健康成年人中H. pylori的感染情况及H. pylori的主要流行亚型,分析H. pylori感染的危险因素以及细菌感染对宿主细胞免疫系统的影响及其感染后成功逃避宿主免疫清除的机制,同时探讨宿主细胞免疫状态与基因易感性与H. pylori感染及临床转归的关系,为了解不同遗传背景的个体对H. pylori感染易感性的差异以及不同基因型在人群中的分布特征,为从基因水平阐明个体感染H. pylori后的发病风险以及寻找新的免疫治疗的作用靶点提供依据。方法1、采用统一调查表,对2007年至2009年在河北医科大学第二医院接受常规体检的健康人进行了流行病学调查,对进行胃镜检测的H. pylori感染患者留取胃粘膜标本并进行H. pylori培养,根据文献选择并设计了9对引物对河北地区H. pylori的流行菌株及其基因亚型进行测定和分析。除调查表之外留取患者血清标本用于H. pyloriIgG抗体以及其他血清学指标的检测。2、利用免疫组化和流式细胞术检测H. pylori感染和未感染的胃炎、胃溃疡、胃癌等患者胃粘膜组织和外周血的细胞免疫反应的表达状态。以IL-2代表Thl型免疫反应,IL-10代表Th2型免疫反应测定H. pylori感染和未感染患者在胃粘膜局部和外周血中的T淋巴细胞反应的极化方向,同时测量患者外周血CD3+、CD4+和CD8+T淋巴细胞的数量以及CD4+/CD8+细胞的比例,分析H. pylori感染对患者胃黏膜局部以及整体细胞免疫反应的影响以及这些影响与患者临床病程的关系。3、根据NCBI中CD27和CD70的DNA序列以及SNP信息,查找CD27和CD70基因转录起点上游的SNP位点,用DNASIS v2.5 Demo查找限制性内切酶,并利用在线工具TFSEARCH查询得到与转录因子结合有关的SNP位点,最后筛查得到6个SNP位点：rs2532507、rs60982224、rs3087273、rs3136550、rs7253697和rs1808398,运用Primer Premier 5.0设计4对引物,运用聚合酶链反应-限制性片段长度多态性分析对所得到的6个SNP位点进行基因型测量和分析。结果1、流行病学调查和样本分析结果显示：河北成年人群H.pylori的感染率为54.5%,不同性别和年龄中感染率不同,男性感染率高于女性,随年龄增长H.pylori的感染率总体表现为上升趋势。河北省H. pylori感染类型以VacA阳性菌株为主,VacA菌株阳性率在胃炎人群中为80.00%,在溃疡人群中则达到100%。其次是CagA阳性菌株,CagA菌株阳性率在胃炎人群最高,为66.67%。VacA基因亚型s信号区域以VacA-s1a为主,中间m区域以VacA-m2为主。成年人VacA最常见的基因型为VacA-s1a-m2,其次为VacA-s1b-m2。H. pylori感染与颈动脉粥样硬化、高血脂、高血糖相关。2、免疫组化和流式细胞术检测结果表明：胃炎、溃疡、胃癌患者在胃粘膜局部存在免疫功能紊乱,以IL-2为代表的Th1型细胞免疫反应降低,患者粘膜组织IL-2表达与对照组相比明显降低；外周血中,各组患者IL-2浓度表达也有差异,其中溃疡组和胃癌组与正常对照相比,浓度表达显著降低。在H. pylori感染患者中胃癌患者组IL-2表达水平明显低于胃炎患者。流式细胞检测结果显示胃癌患者的CD3+、CD4+、CD8+、CD+/CD8+表达结果明显低于正常对照组,而其他各个实验组与正常对照相比无明显差异。3、聚合酶链反应-限制性片段长度多态性分析表明：在测量的CD27 4个SNP位点：rs3087273、rs3136550、rs 2532570rs、6098224,CD70 2个SNP位点：rs 11808398和rs 7253697中,病例组中rs 2532570 GG基因型、rs3087273 AG基因型、rs3136550 CC基因型和rs11808398 CC基因型的表达低于对照组,这些基因型可能与H. pylori感染有关。结论河北健康成年人中H. pylori的感染率为54.5%,感染类型以VacA阳性菌株为主,H. pylori感染最常见的基因型为VacA-s1a-m2。H. pylori感染影响患者局部和整体的细胞免疫反应状态并可能打破了宿主自身的免疫平衡状态,其表现为胃癌患者的CD3+、CD4+、CD8+、CD4+/CD8+表达结果明显低于正常对照组,同时以IL-2为代表的Th1型免疫反应显著降低,这可能有助于H. pylori在宿主体内的长期寄居。宿主的基因多态性与H. pylori感染后机体发生免疫反应的类型以及炎症反应的程度和结局密切相关,我们的结果表明H. pylori感染的病例组中rs 2532570 GG基因型、rs3087273 AG基因型、rs3136550 CC基因型和rs 11808398 CC基因型的表达低于对照组,这些SNP位点的基因多态性可能与H. pylori感染后疾病的发生或转归有关。宿主对H. pylori感染的个体易感性和H. pylori感染引发胃肠道炎症及癌变的机制以及其感染后成功避免宿主免疫系统免疫识别和免疫杀伤作用的免疫逃避机制仍需要进一步的研究。更多还原

【Abstract】 Helicobacter pylori(H.pylori)is the only bacteria that parasites in human gastric tissue. The populations are generally susceptible to it.After Marshall and Warren reported their success in culturing the H. pylor from gastric mucosa in patients in 1984, soon H. pylor was proved to be an important cause of gastrointestinal disease and was highly carcinogenic. World Health Organization Cancer Center announces that H. pylori infection can increase morbidity and mortality of gastric cancer and classify it as carcinogen in 1994. H. pylori is the first prokaryotic carcinogenic proved. In the same year the U.S.National Institutes of Health propose that antibiotics for H. pylori should be included in the course of curing common stomach disease.H. pylori infection is the major causes of gastritis, gastric ulcer, gastric cancer and other gastrointestinal diseases. However, only a small fraction of colonized individuals, representing at least half of the world’s population, develop gastric cancer. The interplay between bacterial and host gene polymorphisms may explain why gastric cancer only occurs in a small fraction of H. pylori-infected individuals.Here we analyse host immune respone and genetic factors in the H. pylori infection,comparise those profiles with clinical and pathological data,we think those data may contribute to a better understanding of the pathophysiological mechanisns of the H. pylori infection and the identification of gene associated with the infection.Objective To invest the prevalence and the main subtypes of H. pylori in Hebei and explore the effects that the H. pylori infection contributing to the host immune system,then analyse the host immune respone and gene polymorphism in the H.pylori infection. Establish association between gene allele and the risk for H.pylori infection. Single-nucleotide polymorphism analysis may identify those individuals who would suffer gastric cancer after H. pylori infection and find new genes may associate with the infection.Methods 1.Cllecting the pathological data by a unified questionnaire in the Second Hospital of Hebei Medical University between 2007～2009, H. pylori IgG antibody is examinated by enzyme linked immunosorbent assay (ELISA), gastric biopsy are used for the H. pylori culture and H. pylori gene subtype determination. PCR is used to determine the popular subtype of H. pylori in Hebei by 9 pairs of primers. Serum specimens were used for anti-H. pylori IgG test and other serum tests.2.Using immunohistochemistry and flow cytometry to measure H. pylori antibody, IL-2,IL-10 and the number of CD3+,CD4+,CD8+ T cells of gastritis, gastric ulcer and gastric cancer patinets.Immune responses are assessed by T cell counts and the cytokines.3.According to the DNA sequence of CD27 and CD70 and SNP informations published from NCBI, Searches restriction enzyme with DNASIS v2.5 Demo, use online tools TFSEARCH to define the transcription factor binding sites,at last, We select six SNPs of CD27 and CD70,such as:rs3087273,rs3136550, rs2532507, rs60982224, rs7253697, rs1808398, measure those SNPs in patients with or without H. pylori infection,analyse these genes polymorphism in both groups.Results 1.Epidemiological investigation and clinical characteristics of the sample results show that H. pylori infection rate was 54.5% in Hebei adult population and the infection rate is various, it infects more man than woman, older than the young.H.pylori infection is associated with atherosclerosis, hyperlipidemia and hyperglycemia.H. pylori VacA-positive strains is the main genetype in Hebei, VacA-positive strain is 80.00% in gastritis people and 100% in ulcers.CagA strain is 66.67% in gastritis people.The most common strain of H. pylori is VacA-s1a-m2, then VacA-s1b-m2.2. Immunohistochemistry and flow cytometry results show that Comparing with control people,IL-2 is lower in serum specimens and gastric biopsy of gastritis, ulcers and gastric people, the gastric people which H. pylori is positive express lower IL-2 than gastritis,and the expression of CD3+,CD4+, CD4+/CD8+ also significantly lower than the control group in gastric people.3.Polymerase chain reaction-restriction fragment length polymorphism results show that the rs3087273 AG genotype, rs3136550 CC genotypes, rs 2532570 GG genotype, rs 11808398 CC genotype are lower in the H. pylori infection patients than control group. The carriage of that allele may be associated with a decreased risk for H.pylori-related gastric cancer development in Hebei.Conclusion In this study we find that H. pylori infection rate was 54.5% and VacA-positive strains is the main genetype in Hebei,and the most common strain of H. pylori is VacA-sla-m2 subtype. H. pylori infection affects the patients immune respone and breaks the balance between Thl and Th2 cells, which may contribute to persistence of the bacteria and increase the risk of gastric in H. pylori infection people.Host gene polymorphisms are also associated with H. pylori infection.Our results suggest the polymorphism of rs3087273, rs3136550, rs 2532570 and rs 11808398 may be involved in the H. pylori infection diease. In addition, our results shed light on the interplay between H. pylori and host susceptibility factors in the induction of gastric cancer.More studies are needed to invest the inflammatory mechanisms involved in the H. pylori-host interaction.更多还原