【作者】 罗媛；

【导师】 郭刚；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 幽门螺杆菌(Helicobacter pylori,H. pylori)是一种螺旋状、微需氧革兰阴性杆菌,主要定植于人胃粘膜,已造成全球50%以上人口感染。Hp感染是慢性胃炎、消化性溃疡及胃粘膜相关淋巴组织淋巴瘤(MALT)等疾病的重要致病因子,与胃癌的发生密切相关,已被WHO列为Ⅰ类致癌因子。自然感染Hp后的免疫反应并不能清除细菌,反而由于持续感染导致胃粘膜免疫病理损害。目前,Hp慢性持续性感染的机理仍不十分清楚。在不同人群中Hp感染率不同,感染Hp后也并非全都发病,且表现的疾病类型及临床结局也多种多样,从无症状携带、慢性萎缩性胃炎、胃、十二指肠溃疡到胃癌及胃粘膜相关淋巴组织淋巴瘤(MALT),形成了复杂的疾病谱。Hp感染相关胃部疾病的发生和发展是环境,菌株和宿主相互作用的结果。现在很多研究认为宿主因素,尤其是宿主遗传背景,影响Hp感染和疾病转归。几乎所有复杂疾病均为多基因疾病,在疾病发生过程中起作用的是一系列基因。由于技术条件的限制和认识上的差距,目前为止,疾病的遗传研究大多从单个基因入手,以生物学功能相关基因作为候选基因进行病例-对照关联研究,是当前对复杂疾病进行遗传易感性研究的重要策略。癌症等复杂疾病是多个遗传变异位点与环境因子共同作用的结果,由于发病原因复杂,涉及的基因数量多,已成为国际上疾病基因组学研究的重点。并且复杂疾病的早期诊断不是靠完全单一的方法来完成的,必定是多种诊断方法的互补。随着人类基因组计划的完成,有关个体遗传变异与疾病的关系日益受到重视,截至2010年3月份NCBI PubMed数据库中,SNPs相关的文章数为16933篇,仅2009年发表的就为7131篇(占SNP相关文章总数的42.11%),2009年SNPs相关文献中与癌症有关的1114篇,占2009年SNPs文献的15.62%,说明了SNPs在复杂疾病研究中的重要性。寻找复杂疾病风险因子的一个补充方法就是通过比较患病组与未患病的对照组来寻找遗传变异与疾病之间的相关。单核苷酸多态性(single nucleotide polymorphism, SNP)是人类基因组中遗传学变异最常见的一种类型,也是决定不同个体临床表型多样性的重要因素。因此探讨Hp感染相关胃部疾病的宿主遗传易感性,将为我们从另一角度认识Hp感染相关胃部疾病的发病机制提供新的线索,并为临床防治提供新的、合理的策略。白介素17(interleukin 17,IL-17)家族是最近发现的一类与适应性及固有性免疫应答相关的细胞因子,不同于Th1、Th2种类的一群细胞因子,通过释放促炎症和嗜中性粒细胞细胞因子在局部组织炎症中扮演重要角色。IL-17家族至少存在有6个家族成员(IL-17A-F)和5个受体(IL-17RA-RD和SEF)。目前研究多集中在IL-17基因多态性与少数自身免疫性疾病、慢性炎症性疾病、如溃疡性结肠炎、哮喘、IBD等疾病关联研究上,对IL-17多态性与慢性萎缩性胃炎的关联研究还未见报道。Luzza等研究发现IL-17mRNA的翻译和蛋白水平在Hp感染者较非感染者明显增高,这些结果提示我们IL-17参与了Hp定植的胃部疾病的发生发展。本课题前期选择了IL-17A与-17F基因的6个SNP位点,通过大量文献及预实验筛选了IL-17A与-17F基因的3个SNP位点,拟在中国人群中分析其基因多态性与Hp感染相关胃部疾病的关系,从遗传学角度探讨基因多态性与Hp感染相关胃部疾病的易感性及发病机制的关系。本课题收集研究所需的病例对照研究的临床资料和DNA样本,采用限制性片段长度多态性(PCR-RFLP)、TaqMan MGB探针方法对我们收集的基于医院的病例(如慢性萎缩性胃炎、胃溃疡、胃癌)-对照(如健康体检者)人群中对选择的IL-17A与-17F基因的3个SNPs (IL-17A(rs2275913, G-197A)、IL-17F(rs763780 , 7488T/C)、IL-17F(rs766748,6400A/G))位点进行基因分型和关联分析研究,同时所有样本均经组织快速尿素酶检测(RUT)和血清酶联免疫吸附法(ELISA)抗Hp-IgG抗体检测,以测定人群Hp感染状态,进而对Hp感染患者罹患胃部疾病的易感性进行分析。主要研究结果如下:1.收集了共432余份健康与疾病患者的样本和病例资料,完成了所有样本的整理和疾病表型的归类。2.对230名健康体检者和151例慢性萎缩性胃炎患者、27名胃溃疡患者、24名胃癌患者的IL-17A与-17F基因的3个SNPs (IL-17A(rs2275913, G-197A)、IL-17F(rs763780,7488T/C)、IL-17F(rs766748,6400A/G))位点成功进行基因分型。①结果发现慢性萎缩性胃炎组中7488T/C C等位和6400A/G A等位的等位频率显著高于健康对照组(P =0.026、P =0.029)。经非条件logistic回归校正年龄、性别、饮酒指数等因素后,在显性模式下,IL-17F 7488T/C和6400A/G两个位点与慢性萎缩性胃炎仍显著关联。与携带6400A/G GG或AG基因个体相比,携带有AA基因型的个体对慢性萎缩性胃炎易感性显著升高(显性模式:OR =1.987, 95% CI =1.137-3.472, P=0.016)。与携带7488T/C TT或CC基因个体相比,携带有CC基因型的个体对慢性萎缩性胃炎易感性显著升高(显性模式:OR =0.603, 95% CI =0.375-0.971, P =0.037)。②发现胃癌组中G-197A G等位和6400A/G A等位的等位频率显著高于健康对照组(P =0.013,P =0.023)。3.对所有疾病对照标本均经组织快速尿素酶检测(RUT)和血清酶联免疫吸附法(ELISA)抗Hp-IgG抗体检测,以测定人群Hp感染率。结果发现慢性萎缩性胃炎组、胃溃疡组、胃癌组Hp感染率比健康对照组(34.3%) Hp感染率高,尤其是胃癌组。慢性萎缩性胃炎组、胃溃疡组、胃癌组Hp感染率分别为51.7%,55.6%,66.7%。经与SNPs多态性位点结果综合分析,发现:①IL-17F 6400A/G位点的基因型和等位分布在Hp感染阳性的慢性萎缩性胃炎组及胃癌组中的等位频率均高于健康对照组(P =0.024,P =0.041)。与未携带有6400A/G A等位的个体相比,携带有6400A/G A等位的个体显著的增加了Hp感染患者罹患慢性萎缩性胃炎及胃癌的易感性。然而,IL-17A G-197A、IL-17F 7488T/C位点的基因型和等位分布在Hp感染阳性的慢性萎缩性胃炎组、胃溃疡组及胃癌组和健康对照组中均无明显差异(P>0.05)。②在慢性萎缩性胃炎组、胃溃疡组及胃癌组和健康对照组中,对各个组中Hp感染阳性及阴性的群体分别进行比较,IL-17A G-197A、IL-17F 7488T/C、IL-17F 6400A/G位点基因型和等位分布均无明显差异(P>0.05)。总之,我们通过样本的病例对照研究在群体水平和流行病学角度首度证实了IL-17A与-17F基因基因多态性与Hp感染相关胃部疾病(慢性萎缩性胃炎和胃癌)的关联,这些结果对我们在以后的研究起了很大的铺垫作用,从另一角度提示宿主基因因素在Hp感染及其疾病进程中发挥重要作用,为Hp感染及发病机制的基础研究提供新的线索,并为临床医生针对Hp感染相关胃部疾病的个性化防治策略提供新的思路。更多还原

【Abstract】 Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic bacterium thatresides extracellularly in the gastric mucosa and infects more than 50% of the populationworldwide. H. pylori-induced chronic inflammation is the cause of gastritis and peptic ulcerand a risk factor for gastric cancer. H. pylori infection causes severe local inflammation inthe gastric mucosa. H.pylori infection in different populations lead to different outcomes, also incidence of H.pylori infection is not all.and it causes a broad spectrum of stomach diseases ranging from asymptomatic carrier (AsC), Chronic atrophic gastritis (CAG), Gastrointestinal ulcer (GU), Gastric cancer (GC), mucosal associated lymphoid tissue lymphoma (MALT). Persistent H.pylori infection and H.pylori -related stomach diseases have been considered as a multifactorial and polygenic disorder with bacterial, environmental and host genetic components.Host factors, especially host gene, played an important role in the pathology of H.pylori -related diseases. Almost all complex diseases are polygenic diseases,a series of genes may affect the disease process. Due to technical constraints and knowledge gaps, so far, most genetic studies of diseases start from a single gene to biological function of genes. Currently an important strategy on the genetic susceptibility of complex diseases is to choose candidate genes for case-control association study. Cancer and other complex diseases are the outcome of genetic variation and environmental factors, for the complex etiology and large numberof genes involved in these diseases, it has become the international focus of genomics research. And the early diagnosis of complex diseases can not be fully completed by a single approach, it depends on a variety of complementary diagnostic methods. With the completion of the human genome project, attention is now rapidly shifting towards the study of individual genetic variation., as of March 2010,in NCBI PubMed database, the number of articles related to SNPs is 16933, articles published in 2009 is up to 7131 (accounting for 42.11% of total), SNPs associated with cancer-related literature is 1114, accounting for 15.62% in 2009 published literature, these show the importance of SNPs research in complex disease. Search for complex disease risk factors in a complementary way is by comparing the disease group and control group without the disease to find genetic variation and the correlation between the disease. The most abundant source of genetic variation in the human genome is represented by single nucleotide polymorphisms (SNPs), which can account for heritable inter-individual differences in complex phenotypes. Identification of SNPs that contribute to susceptibility to complexs diseases, such as H.pylori-related diseases, will provide highly accurate diagnostic information that will facilitate early diagnosis, prevention, and treatment of human diseases.Interleukin-17 (IL-17) is a newly described cytokine that bridges the adaptive and innate immune systems. To date, six IL-17 family ligands (IL-17A-F) and five receptors(IL-17RA-IL-17RD and SEF) have been identified. It was also reported that both IL-17 RNA transcripts and protein were expressed at a higher level in the whole gastric mucosal and lamina propria mononuclear cell (LPMC) samples from H. pylori-infected patients than in those from uninfected subjects. Thus an important role of IL-17 in the inflammatory response to H. pylori colonization has been indicated. Our study group have systematically screened the SNPs of IL-17A and IL-17F susceptibility candidate genes by extensive literature and pre-screened have confirmed that the polymorphisms of interleukin-17 (IL-17) gene are related with susceptibility to disease process of Chronic atrophic gastritis and Gastric Cancer diseases by the case-control association study, However, many previous studies only focused on IL-17 gene polymorphism and a small number of autoimmune diseases, chronic inflammatory diseases such as ulcerative colitis, asthma, IBD and other diseases, Association study on the positive sites with chronic atrophic gastritis has not been established yet. So it is important and necessary to carry out functional studies to identify SNPs that affect transcriptional regulation. Therefore, we select IL-17 gene as candidate genes and examine the relationship between their polymorphisms and the susceptibility to H.pylori -related stomach disease of case-control research. In addition, we also investigate the significance of the relationship between the gene polymorphism and susceptibility to H.pylori -related stomach diseases from the perspective of population genetics.In our study, The clinical date and blood samples were collected and genomic DNA was isolated from peripheral blood cells using the DNA blood minikit from Promega according to the manufacturer’s protocol. In a hospital-based case-control cohort composed of 230 unrelated health Controls, 151 Chronic atrophic gastritis patients (CAG),27 gastric ulcer patients(GU) and 24 gastric cancer patients (GC), three polymorphisms in the IL-17 gene were analyzed by TaqMan-MGB-PCR analysis, the presence of H.pylori infection was determined by rapid urea test ,histological examination, and serology using a commercial Ig-G ELISA kit.The main results of our study were listed as follow:1. The clinical date and blood samples of more than 432 patients with chronic H.pylori infection were collected and the classification of disease phenotype of all the samples previous collected was completed.2. The IL-17 gene polymorphisms (IL-17A (rs2275913, G-197A)、IL-17F(rs763780,7488T/C)、IL-17F(rs766748,6400A/G))were successfully genotyped in 230 unrelated health Controls, 151 Chronic atrophic gastritis patients and 27 gastric ulcer patients,24 gastric cancer patients.①The allele frequencies of IL-17 -7488C and -6400A were significantly higher in Chronic atrophic gastritis patients than in health Controls (P =0.026、P =0.029). Logistic regression analysis and stratification analysis with adjustment for age and sex indicated that the polymorphisms of IL-17F 7488T/C and 6400A/G were associated with susceptibility to Chronic atrophic gastritis. The individuals carrying 6400A/G AA genotype exhibited a significantly higher susceptibility to chronic atrophic gastritis, comparing with those carrying the GG or AG genotype.(Dominant model:OR =1.987, 95% CI = 1.137-3.472, P =0.016) The individuals carrying 7488T/C CCgenotype exhibited a significantly higher susceptibility to chronic atrophic gastritis, comparing with those carrying the AA or AC genotype.(Dominant model:OR = 0.603, 95% CI = 0.375-0.971, P =0.037)②The allele frequencies of IL-17 -197G and -6400A were significantly higher in Gastritis Cancer patients than in health Controls (P =0.013、P =0.023).3. In both patients and controls, the presence of H.pylori infection was determined by rapid urea test ,histological examination, and serology using a commercial Ig-G ELISA kit. In 151 cases of patients with chronic atrophic gastritis group and 27 gastric ulcer patients group,24 gastric cancer patients group has a higher rate of H. pylori infection than healthy control group(34.3%), especially gastric cancer group. H.pylori infection rates in chronic atrophic gastritis, gastric ulcers, gastric cancer group were respectively 51.7%, 55.6%, 66.7%.There was no significant difference in allele frequencies or genotype distributions of three polymorphisms in the IL-17A and IL-17F gene between the all subjective with H. pylori infection and without with H. pylori infection. (P>0.05)。Associated with H.pylori infection in all groups compared,There was no significant difference in allele frequencies or genotype distributions of IL-17A G-197A and IL-17F 7488T/C between health Controls and all gastroenteric patients (P>0.05). However, the IL-17F 6400A/G allele frequencies were higher in CAG and GC patients than those in health Controls (P =0.024, P =0.041).In conclusion, based on population genetic association study and genetic functional analysis of the cadidate genes and loci, our study emphasizes the importance of IL-17A and -17F in the pathophysiology of H.pylori -related stomach disease (CAG and GC) on the population level. The results of our research in the future play an important role, it not only provides researchers new clues to the further basic research of pathogenesis of H.pylori infection and H.pylori -related stomach diseases, but also provides the clinicians new ideas to personalized prevention and treatment strategies for patients of H.pylori infection and H.pylori -related stomach disease.更多还原