溶质相关载体26A3基因多态性和溃疡性结肠炎的相关性(硕士)

溶质相关载体26A3基因多态性和溃疡性结肠炎的相关性(硕士)(论文25000字)
中文摘要
研究背景和目的 炎症性肠病(IBD)包括溃疡性结肠炎(UC)和克罗恩病(CD)，是病因尚不完全明确的肠道慢性非特异性炎症性疾病。目前的研究表明遗传易感人群在环境因素作用下，肠道细菌引起不恰当并且难以控制的炎症反应从而导致疾病的发生发展。UC作为IBD的一种表现形式，以慢性复发性肠道炎症为特点，主要临床表现为腹痛、腹泻及粘液脓血便。大量研究显示遗传因素和UC密切相关，分子生物学领域的不断发展为研究UC相关基因的多态性检测提供了强大的技术支持。腹泻是UC最常见的症状，近年有学者提出肠道溶质载体的功能受损所导致水和电解质的吸收分泌失衡与UC相关性腹泻关系密切，故溶质载体可能成为治疗UC的靶点。SLC26A3是一种肠道Cl-/HCO3-转运体，主要在结肠和 Na+ /H+交换体亚型3(NHE3)共同调节NaCl的吸收。学者们发现在UC的发展过程中，SLC26A3与炎症因子和肠黏膜免疫系统相互作用，并且SLC26A3的转运功能障碍与肠黏膜的屏障损害平行。故而提出改善SLC26A3的功能有利于控制UC疾病的发生和发展。来自亚洲人群的研究结果显示rs7810937, rs7785539和 rs2108225是SLC26A3基因上最常见的SNP位点，均位于SLC26A3基因启动子上游区域。目前研究表明这3个SNP发生基因突变后可能会影响SLC26A3的转录和表达，造成肠黏膜细胞中Cl-的重吸收和HCO3-的分泌功能障碍，从而导致腹泻的发生。本研究拟在浙江籍汉族人群中探讨SLC26A3基因中3个位点(rs2108225,rs7810937和rs7785539)的多态性与 UC 易感性的关系，为进一步阐明 UC 的遗传学发病机制提供线索。

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方法收集291例 UC 患者和380名年龄、性别相匹配的健康对照者，采用多重 Snapshot 技术检测SLC26A3(rs7810937, rs7785539和rs2108225) 3种单核苷酸多态性(SNP)，并进行连锁不平衡和单倍型分析。在UC组和对照组中采用χ2检验分析SLC26A3(rs7810937, rs7785539和rs2108225) 3个SNP的基因型分布是否符合Hardy-Weinberg平衡定律。UC组和对照组的性别、年龄比较分别采用χ2检验和两独立样本t检验。在UC组和对照组之间采用非条件Logistic回归分析比较SLC26A3基因多态性的分布差异及其对UC患者临床病理特征的影响。采用Haploview 4.2软件进行连锁不平衡及单倍型分析，并在UC组和对照组之间采用χ2检验比较各单倍型频率差异。P<0.05认为有差异有统计学意义。以上数据均输入SPSS 17.0统计软件处理。
结果 UC组中rs2108225突变等位基因(G)和基因型(AG+GG)的频率均高于对照组(65.46%比58.68%，P=0.011；87.29%比81.58%，P=0.045)。与轻中度UC患者相比，重度UC患者中rs7785539突变等位基因(C)和基因型(GC+CC)的频率均显著增高(15.79%比6.13%，P=0.003；26.32%比12.25%，P=0.020)。(rs7810937,rs7785539和rs2108225) 3个SNP位点彼此连锁，但UC和对照组中各单倍型无显著差异(P>0.05) 。
结论SLC26A3 (rs2108225) 基因突变可能增加UC发病风险，rs7785539基因多态性与UC疾病严重程度相关，(rs7810937,rs7785539和rs2108225) 构建的单倍型与UC发病风险无关。

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关键词：溶质相关载体26A3；溃疡性结肠炎；单核苷酸；多态性；单倍型
 
A Study on the Associations of Ulcerative Colitis with    Solute-linked Carrier Family 26 Member A3 Gene Polymorphisms

Abstract
Background and Aims  Inflammatory bowel diseases (IBD) is a chronic inflammatory condition of the gastrointestinal tract consists of two main clinical entities whichare ulcerative colitis (UC) and Crohn’s disease (CD). Although the exact etiology andpathogenesis of IBD still remain unknown, most of the viewspresume that combinedgenetic and environmental factors that cause dysregulated mucosalimmune responses to the gut flora in genetically susceptible individuals. As oneoftheprincipalformsofIBD,the main symptomsof UC include abdominal pain and diarrhea mixed withblood.In recent years, some scholars have proposed that the impaired function of the intestinal ion transporters and channels caused the imbalance of the absorption and secretion withUC. These studies have shown that the predominant mechanismof diarrhea in UC involves impairment of water and electrolyte absorption, with very little role if any played by anion secretion. An understanding of the mechanisms of solute-linked carrier family (SLC) dysregulation isimportant for identification of novel therapeutic targets for thetreatment of IBD-associated diarrhea. Thetransport properties of solute-linked carrier family 26 member A3 (SLC26A3)have been studied in detail, revealing that it is a Cl-/HCO3-exchangerthat is functionally coupled to NHE3 (Na+/H+exchanger isoform 3) in the ileum and colon.During the development of UC, SLC26A3, inflammatory cytokines and intestinal mucosal immune system interact with each other, and the transport dysfunction of SLC26A3 is parallel to the intestinal mucosal barrier damage. Therefore, improving the function of SLC26A3 is beneficial to control the occurrence and development of UC.The results of the study from Asian populations showed that rs7810937, rs7785539 and rs2108225 are all located in the upstream region of SLC26A3 gene promoter, and are the most common SNP sites on SLC26A3 genes. The present study shows that the mutation of these three SNPsmay affect thetranscription and expression of SLC26A3, resulting in Cl- reabsorption and HCO3- secretion dysfunction in intestinal mucosal cells, leading to the occurrence of diarrhea.In this study, we aimed to find the association of ulcerative colitis (UC) with the polymorphisms of SLC26A3 gene of Han nationalitypatients in Zhejiang province and to provide clues to further elucidate the genetic pathogenesis of UC.

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Methods  A total of 291 UC patients and 380 healthy subjects matched for age and sex were enrolled.The genetic polymorphisms of SLC26A3 (rs7810937, rs7785539 and rs2108225) were detected by Snapshot technique.The analyses of linkage disequilibrium (LD) and haplotype were performed in all study subjects.Theχ2 test was used to analyze whether the genotype distribution of the 3 SNP(rs7810937, rs7785539 and rs2108225) conform to the Hardy-Weinberg equilibrium rulein the UC group and control. The sex and age of these twogroups were compared with theχ2 test and the independent sample test. The unconditional Logistic regression analysis was used to compare the distribution difference of SLC26A3 gene polymorphism and its effect on the clinicopathological features of UC patients.The Haploview 4.2 software was used for linkage disequilibrium and haplotype analysis, and the frequency differences of haplotypes were compared between these two groups by theχ2 test.P<0.05 was considered significant.All the data were treated with SPSS 17.0 statistical software. [来源：http://Doc163.com]
Results  The mutant allele (G) and genotype (AG+GG) of SLC26A3 (rs2108225)  were more frequent in UC patients than in the controls (65.46% vs 58.68%, P=0.010; 87.29% vs 81.58%, P=0.045, respectively). The mutant allele (C) and genotype (GC+CC) of SLC26A3 (rs7785539) were more prevalent in severe UC patients than in the other patients (15.79% vs 6.13%, P=0.003; 26.32% vs 12.25%, P=0.020). Moreover, the three SNPs (rs781093,rs7785539 and rs2108225) of SLC26A3 gene were found to be in a strong LD. However, there were no significant difference of haplotypes between UC and controls.
Conclusions The mutation ofSLC26A3 (rs2108225) might contribute to enhancing the risk of UC.SLC26A3 (rs7785539) gene polymorphisms were correlated with the severity of UC.The haplotypes formed by (rs7810937,rs7785539 and rs2108225)  were not significantly associated with the susceptibility of UC.
[Key Words] SLC26A3; Ulcerative colitis; Single nucleotide polymorphism; Haplotype

目录
英文缩略词表   
中文摘要   
英文摘要   
前言   
材料与方法   
结果   
分析与讨论   
参考文献   
附录   
致谢   
综述及参考文献   

中英文缩写对照表
英文缩写    英文名称    中文名称
3’UTR    3’Untranslated Region    3’非翻译区
5’UTR    5’Untranslated Region    5’非翻译区
ABI    Applied Biosystems    美国应用生物系统公司
CD    Crohn’s disease    克罗恩病
cDNA    complementary DNA    互补脱氧核糖核酸
CFTR    Cystic Fibrosis Transmembrane  Conductance Regulator    囊性纤维跨膜转导调控因子
CLD    Congenital chloridediarrhea    先天性失氯性腹泻 [资料来源：http://doc163.com]
CI    Confidence interval    可信区间
dNTP    deoxyribonucleoside triphosphate    三磷酸脱氧核糖核苷
ddNTP    dideoxyribonucleoside triphosphate    双脱氧核苷三磷酸
DSS    Dextransulfatesodium    葡聚糖硫酸钠
EDTA    Ethylene Diamine Tetraacetic Acid    乙二胺四乙酸二钠
ENaC    Na+Channel    钠离子通道
EPEC    enteropathogenic E. coli    肠致病型大肠埃希菌
GWAS    genome-wide association study    全基因组关联研究
HNF4A    Hepatocyte Nuclear Factor 4A    肝细胞核因子4A
Hi-Di    Highly deionized-formamide    高度去离子甲酰胺
IBD    Inflammatory bowel diseases    炎症性肠病
IFN    Interferon    干扰素 [资料来源：http://Doc163.com]
IL-1β    interleukin-1β    白细胞介素1β
LA    Lacticacid bacteria    乳酸杆菌
LD    linkage disequilibrium    连锁不平衡
LPA    lysophosphatidic acid    溶血磷脂酸
MIF    macrophage migration inhibitory factor    巨噬细胞迁移抑制因子
MAPK    mitogen-activated protein kinase    有丝分裂原活化蛋白激酶
NHE3    Na+/H+exchanger-isoform3        Na+/H+交换体亚型3
NHERF    Na + /H + exchanger regulating factor    钠氢交换子调节因子
PDZ    PDZ-domain proteins    盘状同源区域(PDZ结构蛋白) 
RAGE    receptor for advanced glycation   endproduct    晚期糖基化终末产物受体
SAP    Shrimp Alkaline Phosphatase    虾碱性磷酸酶

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SLC        solute-linked carrier family    溶质相关载体
SLC26A3    solute-linked carrier family 26 member A3        溶质相关载体26A3
SNP    single nucleotide polymorphism    单核苷酸多态性
STAS    sulfate transporter anti-sigma domain    硫酸盐转运及抗δ因子
TNF    tumor necrosis factor    肿瘤坏死因子
UC    ulcerative colitis    溃疡性结肠炎

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