【作者】 武其文；

【导师】 胡丽华；

【作者基本信息】 华中科技大学 ， 临床免疫学， 2009， 博士

【摘要】 变应性哮喘是一种与免疫相关的复杂疾病，受遗传和环境等多种因素的共同影响而发病。个体的遗传因素在哮喘发病中起着十分重要的作用。应用全基因组扫描或定位克隆技术已发现多个染色体区域与哮喘连锁。其中以5q31-33区最受研究者的关注，因为该区域包含众多细胞因子的基因以及一些与免疫相关的基因，且在多个人群的关联研究中重复表现与哮喘高度的连锁。越来越多的研究表明T_H1／T_H2细胞及其分泌的细胞因子的失衡与哮喘的发病相关。最近研究显示表达在T细胞表面的Tims基因家族蛋白在调节T_H1和T_H2细胞介导的免疫应答中起重要的作用。在人类，Tim-1和Tim-3是Tims基因家族重要的两个成员，它们均位于与哮喘高度连锁的染色体5q31-33区域。Tim-1主要表达在T_H2细胞表面，调节T_H2细胞的免疫应答。而Tim-3主要表达在T_H1细胞表面，对T_H1细胞起负性免疫调节作用。基于Tim-1和Tim-3在T_H1／T_H2免疫调节中重要的功能，Tim-1和Tim-3基因已成为哮喘重要的易感候选基因。针对Tim-1和死Tim-3多态性与哮喘的关联研究虽已在多个人群中开展，但受多种因素的影响，这些研究的结果未能得到统一和重复。对Tim-1与Tim-3基因多态性与哮喘的关系，仍需进一步研究。本研究我们首先探讨了Tim-1与Tim-3基因的表达与变应性哮喘的关系，然后采用基于家系和单体型的关联研究方法，应用TDT和HRR检验来分析Tim-1和Tim-3基因的多态性及单体型与中国湖北地区变应性哮喘及相关表型的相关性。研究分为3个部分。第一部分Tim-1及Tim-3在变应性哮喘患者PBMC中的表达目的：探讨Tim-1和Tim-3 mRNA在变应性哮喘患者外周血单个核细胞的表达水平，为进一步阐明Tims基因与哮喘的关系奠定基础。方法：随机收集的尘螨过敏性哮喘患者，共计12例，哮喘诊断全部符合2003年中华医学会呼吸学分会哮喘学组制定的标准。同时选取性别和年龄匹配的健康体检者12例作为对照。实验分3组，分别为哮喘组，尘螨刺激组和正常对照组。Ficoll分离PBMC后，取24孔培养板，哮喘组和尘螨刺激组每孔分别加入10⁶个病人分离的PBMC，正常对照组加入10⁶个健康体检者的PBMC，对尘螨刺激组再加入终浓度为20μg／m1的尘螨抗原。然后置37℃5％CO₂培养箱中培养72 h，收集细胞，抽提总RNA。参照文献Tim-1、Tim-3引物序列，合成引物，采用实时荧光定量RT-PCR检测3组Tim-1和Tim-3的mRNA表达。结果：1．Tim-1 mRNA在尘螨过敏性哮喘患者外周血PBMC中相对表达值为0．27±0．09，显著高于健康对照组的0．13±0．03（P＜0．05）；患者PBMC在终浓度为20μg／m1的尘螨抗原刺激后，Tim-1表达明显升高，达0．59±0．22，与对照组和未刺激的哮喘组相比有显著差异（P＜0．01）；2．Tim-3 mRNA在哮喘患者外周血PBMC中相对表达值为0．39±0．09，明显高于健康对照组的0．18±0．06，差异有显著性（P＜0．05），同样患者PBMC在尘螨抗原刺激后，Tim-3 mRNA的表达也出现明显的升高，达0．66±0．22，与对照组和未刺激组相比差异均有显著性（P＜0．01）；结论：Tim-1和Tim-3 mRNA在尘螨变应性哮喘患者中表达明显升高，Tim-1和Tim-3可能参与人类变应性哮喘的发生和发展。第二部分Tim-3基因多态性及其与变应性哮喘易感性的关系第一节Tim-3基因序列的测定及其多态性的确定目的：探讨湖北地区汉族人群Tim-3基因启动子区和编码区的多态性及其构成的单体型特征，为后续基于Tim-3基因多态性与相关疾病的关联研究奠定基础。方法：选取湖北地区30例正常汉族人和30例变应性哮喘患者作为研究对象。采用分段PCR扩增直接测序法检测这60名研究对象Tim-3基因的启动子区、全部的外显子区及部分内含子区的多态性变异。将测序结果与NCBI及HapMap数据库进行对比，确定湖北汉族人群Tim-3基因多态性的分布特征。采用Pearsonχ²检验或Fisher’s确切概率法比较各位点的基因型和等位基因的频率在哮喘组和正常人组的分布。采用Haploview软件对各位点进行LD分析、单体型的构建和tSNPs的筛选。结果：1．Tim-3基因区域共检出13个多态位点，其中有5个为新发现的位点。各位点基因型和等位基因频率在30例正常组和30例哮喘组中未见明显的统计学差异（P＞0．05）。2．经与HapMap数据库比较，4个位点rs4704853、rs10515746、rs4704846、rs9313439等位基因的频率在湖北汉族人和北方汉族及日本人中的分布差异无统计学意义（P＞0．05），而与欧洲人及非洲人的分布有明显的不同（P＜0．01）。3．对7个频率大于5％的SNPs进行两两配对的LD分析显示其中6个位点之间呈现较强的连锁不平衡（D′和r²均大于0．3）。单体型分析共发现有5种频率大于1％的常见单体型。4．在该单体型域中选出3个tSNPs：rs10053538、rs13170556和rs9313441用来代表该区域常见多态性变异。结论：湖北汉族人群Tim-3基因的SNPs分布有别于其他人种，基于这些SNPs构建的单体型和筛选出的tSNPs为研究湖北汉族人群Tim-3基因多态性与相关疾病的关系奠定了基础。第二节基于家系的Tim-3基因多态性与变应性哮喘的关联研究目的：探讨Tim-3基因的多态性及其单体型与湖北地区汉族人群变应性哮喘及相关表型的关系。方法：收集湖北地区118个完整的由一名患者及其双亲组成的变应性哮喘核心家系，共354名成员，均为汉族。应用PCR-RFLP方法，对118个核心家系Tim-3基因3个标签SNPs，rs10053538、rs13170556和rs9313441及1个启动子区的多态性rs10515746进行基因分型；采用基于单体型．标签SNP和家系资料的关联检验的方法（TDT，HRR，FBAT），分析Tim-3基因的多态性及其单体型与湖北地区汉族人群变应性哮喘及相关表型的关系。结果：1．Tim-3基因的4个SNPs单个位点TDT和HRR分析结果显示，Tim-3基因的这4个多态性与湖北汉族人变应性哮喘之间不存在连锁不平衡关系，P＞0．05。2．应用TRANSMIT软件进行单体型的TDT分析结果显示，Tim-3基因的单体型与变应性哮喘有关联，Globalχ²检验，χ²=10．83，P=0．013。单个单体型TDT结果显示，父母传递给患病子女G-G-G单体型的观察值小于期望值，差异有显著性（χ²=8．24，P=0．004）。单体型HRR分析显示G-G-G，具有较低的发病风险（HRR=0．49）。3．FBAT分析结果显示Tim-3基因的多态性及单体性与哮喘数量表型血清总IgE水平无关联。结论：Tim-3基因区域的多态性以单体型的形式影响哮喘的易感性，Tim-3基因本身或其附近的染色体区域内可能存在与变应性哮喘易感性相关的位点。单体型G-G-G可能作为用来对湖北汉族人群哮喘易感性进行预测为标志。第三部分基于家系的Tim-1基因多态性与变应性哮喘的关联研究目的：探讨Tim-1基因的多态性及其单体型与湖北地区汉族人群变应性哮喘及哮喘相关表型的关系。方法：收集湖北地区118个完整的由一名患者及其双亲组成的变应性哮喘核心家系，共354名成员，均为汉族。应用PCR—RFLP方法，对118个核心家系Tim-1基因3个标签SNPs（rs4704842、rs953569、rs2134230）和1个位于启动子区的多态位点-232 G＞A以及1个位于外显子4区多态位点rs45623443进行基因分型；采用基于单体型．标签SNPs和家系资料的关联检验的方法（TDT，HRR；，FBAT），检测Tim-1基因的多态性及其单体型与湖北地区汉族人群变应性哮喘及哮喘相关表型的关系。结果：1．Tim-1基因的5个SNPs单个位点TDT和HRR分析结果显示均与哮喘无关联，P＞0．05。2．单体型的TDT和HRR分析发现Tim-1基因区域的G-A-ins-C-G单体型与变应性哮喘有关联，P＜0．05。该单体型由父母传递给患病子女的观察值高于期望值，是变应性哮喘发病的一个风险因素，HRR=1．91。3．FBAT分析结果显示Tim-1基因rs45623443多态性位点Ins等位基因与哮喘表型血清总IgE水平相关，P＜0．05；4．Tim-1单体型的FBAT分析发现G-A-ins-C-G单体型与血清总IgE水平有关联，P＜0．05。结论：Tim-1基因单体型影响湖北汉族变应性哮喘的易感性和血清IgE的水平。提示Tim-1基因内或其附近的基因区域可能存在与变应性哮喘和高IgE水平相关的疾病位点。Tim-1单体型G-A-ins-C-G可能用来对湖北汉族人群哮喘易感性和IgE水平进行预测的标志。更多还原

【Abstract】 Allergic asthma is an immune- related complex disease caused by a combination ofgenetic and environmental factors.The individual genetic factors play an important role inthe development of asthma.By using genome-wide screens or positional cloningtechniques, several chromosomal regions have been found to have linkage with asthma.Among these regions, chromosome 5q31-33 has received the most attention, because itcontains a cluster of cytokines and other immune-related genes and has been repeatedlysuggested to be linked to asthma susceptibility in several populations.A growing body of evidence suggests that the imbalance of T_H1/T_H2 cells and theirsecretary cytokines is relatd with the development of asthma.It has been recently reportedthat the Tims gene family proteins are expressed on T cells and play an important role inregulating T_H1-and T_H2-cell-mediated immunity.In humans, Tim-1 and Tim-3 are the twoimportant members of Tim families.Both of them are located in the chromosome 5q31-33region.Tim-1 is expressed preferentially on T_H2 cells and involved in the development andregulation of T_H2-biased immune responses while Tim-3 is mainly expressed on T_H1 cellsand negatively regulate the T_H1 cell immune responses.In view of the important functionof Tim-1 and Tim-3 in T_H1/T_H2 immune regulation, Tim-1 and Tim-3 have become twogood candidate susceptibility genes for asthma.The association study based the Tim-1 andTim-3 polymorphisms with asthma have been conducted in several populations.However,the results are not unified and repeated because of many reasons.Therefore, therelationships of Tim-1 and Tim-3 polymorphisms and asthma need to be furtherinvestigated.In this study, we firstly investigated the expression of Tim-1 and Tim-3 mRNA inallergic asthma patients and thenby employing the TDT and HRR analysis, we conducted ahaplotype and family-based association study of Tim-1 and Tim-3 ploymorphisms in aChinese population from HuBei province with allergic asthma to examine the hypothesisthat polymorphisms in these two genes are associated with asthma.The total study may bedivided into the following three parts. PartⅠ.Expression of Tim-1 and Tim-3 mRNA in PBMC from allergicasthma patientsAIM: To explore the expression of Tim-1 and Tim-3 mRNA in PBMC from allergicasthma patients, for luther clarifying the relationship between Tims genes and asthma.METHOD: A total of 12 patients with mite allergic asthma were collected in random.Asthma was diagnosed according to the criteria formulated by the Respiratory Branchasthma group of Chinese Medical Association in 2003.Another 12 gender and age-matchedhealthy people were selected as the controls.Experiments were divided into 3 groups forasthma, mite stimulation and control.PBMC were separatated by Ficoll separating medium,then taking a 24-hole plate, added 10⁶ PBMC separatated from the patients to the hole ofthe asthma group and the mite stimulation group; Added 10⁶ PBMC from the healthycontrol to the hole of the control group.Additionally, added a final concentration of 20μg/m1 dust mite antigen to the hole of the mite stimulation group.Place the plate in 37℃5% CO₂ incubator to foster for 72 h, then collected the cell and extracted the total RNA.According to the primers sequence from the references, synthesized the Tim-1 and Tim-3primers and then detected the expression of Tim-1 and Tim-3 mRNA by real-timequantitative RT-PCR.RESULT: 1.The relative expression of Tim-1 mRNA in the PBMC of patients with dustmite allergic asthma was 0.27±0.09, significantly higher than the healthy control group,0.13±0.03 （P<0.05）; The Tim-1 expression was significantly higher for 0.59±0.22 afterthe PBMC from patients were Stimulated at a final concentration of dust mite antigen for 20μg/m1 for 72 hours.Compared with the control group and asthma group, there aresignificant difference （P<0.01）; 2.The Tim-3 mRNA relative expression in the patientswas 0.39±0.09 which is obviously higher than the expression value 0.18±0.06 in thehealthy control （P<0.05）.Coincidently, the Tim-3 expression levels also appearedsignificantly increased after stimulation of 20μg/m1 dust mite antigen for 72 hours,reached 0.66±0.22, Which compared with the control group and asthma group, there arealso significant difference （P<0.01）;CONCLUSION: The expression Tim-1 and Tim-3 mRNA in the patients with dust miteallergic asthma was significantly increased.Tim-1 and Tim-3 may be involved in theoccurrence and development of human allergic asthma. PartⅡ.Tim-3 gene polymorphism and the relationship with thesusceptibility to allergic asthmaSectionⅠ.Tim-3 sequence determination and identification of polymorphismsAIM: To explore Tim-3 gene promoter and coding region polymorphisms and haplotypecharcteristics in a Chinese Han population from Hubei province and lay a foundation forthe following association study based Tim-3 polymorphisms and some related diseases.METHOD: Randomly collected 30 cases of healthy people and 30 cases of allergicasthma patients from Hubei province as the research subjects.By using the PCRamplification and direct sequenceing technology, detected the polymorphisms in Tim-3promoter, all exons and parts of intron region from the 60 subjects.The sequencing resultswere compared with the HapMap and NCBI database to determinate the Tim-3ploymorphisms characteristic in Hubei Han population.The genotype and allele frequencyfor each locus were compared between the asthma group and control group by Pearsonχ²test or Fisher’s exact test.The LD analysis, haplotype construction and selection of tSNPswere conducted by the Haploview software.RESULT: 1.A total of 13 polymorphisms loci were detected in Tim-3 gene region and 5 ofthem were newly discovered.No statistically significant difference were found for thegenotype and allele frequency of each locus between the 30 cases of asthma patients and 30cases heathy people （P>0.05）.2.By Comparing with HapMap database, the allelefrequency for rs4704853, rs10515746, rs4704846, rs9313439 between Hubei Hanpopulation and China North Han population and Japanese is not significantly different （P>0.05）.However, there is significant difference for the 4 loci allele frequency between theHubei Han population and the Europeans or Africans （P<0.01）.3.The LD analysis for 7SNPs with frequency greater than 5% showed that 6 of them has strong linkagedisequilibrium with D’ and r² greater than 0.3.The haplotype analysis for the 6 SNPs found5 common haplotypes with frequency greater than 1% in this region.4.rs10053538,rs13170556 and rs9313441 were selected as tSNPs to represent the common variability inthe haplotype block region.CONCLUSION: The distribution of Tim-3 ploymorphisms in Hubei Han population isdifferent from other ethnics.The constructed haplotypes and selected tSNPs can be used forthe next association studies based Tim-3 ploymorphisms with related diseases in Hubei Hanpopulation. SectionⅡ.Family-based association study of Tim-3 polymorphisms and allergicasthmaAIM: To investigate the relationship between Tim-3 polymorphisms and haplotypes andallergic asthma and asthma-related phenotype in Hubei Han population.METHOD: 118 complete trios of allergic asthma composed of a patients and theirbiological parents participated were collected from Hubei province, a total of 354 members.By using the PCR-RFLP technology, 3 tSNPs rs10053538, rs13170556 and rs9313441 anda promoter polymorphism rs10515746 were genotyped for the 118 trios; Thehaplotype-tSNPs-based method and family-based association test （TDT, HRR, FBAT） wereemployed to investigate the relationship between Tim-3 polymorphisms and haplotypes andallergic asthma and asthma-related phenotype in Hubei Han population.RESULT: 1.The 4 SNPs of Tim-3 gene single locus TDT and HRR analysis showed thatthere is no linkage disequilibrium between the 4 SNPs and the allergic asthma in Hubei Hanpopulation, P> 0.05.2.The haplotype TDT analysis by using TRANSMIT softwarerevealed that Tim-3 haplotypes are associated with allergic asthma, Globalχ² test,χ² =10.83, P=0.013.Single haplotype TDT results showed that G-G-G haplotype wasundertransmitted from parents to patients （χ²=8.24, P=0.004）.The haplotype HRRanalysis showed that G-G-G haplotype had a lower risk （HRR=0.49） and could be used asa protection factor for resistance to allergic asthma.3.FBAT analysis revealed that Tim-3polymorphisms and haplotypes were not associated with asthma phenotype of total serumIgE.CONCLUSION: Tim-3 gene polymorphisms could influence asthma susceptibility in theform of haplotypes, which means Tim-3 gene or the region nearby the Tim-3 may existsome SNPs locus associated with allergic asthma.As a logo the G-G-G Haplotype can beused for asthma susceptibility prediction in Hubei Han population. PartⅢ.Family-based association study of Tim-1 polymorphisms andallergic asthmaAIM: To investigate the relationship between Tim-1 polymorphisms and haplotypes andallergic asthma and asthma-related phenotype in Hubei Han population.METHOD: 118 complete families of allergic asthma composed of a patients and theirbiological parents participated were collected from Hubei province, a total of 354 members.By using the PCR-RFLP technology, 3 tSNPs （rs4704842、rs953569、rs2134230） and apromoter polymorphism -232 G>A and a exon 4 polymorphism rs45623443 weregenotyped for the 118 trios; The haplotype-tSNPs-based method and family-basedassociation test （TDT,HRR, FBAT） were employed to investigate the relationship betweenTim-1 polymorphisms and haplotypes and allergic asthma and asthma-related phenotype inHubei Han population.RESULT: 1.The 5 SNPs of Tim-1 gene single locus TDT and HRR analysis showed thatthere is no association between the 5 SNPs and the allergic asthma in Hubei Han population,P> 0.05.2.The haplotype TDT and HRR analysis revealed that a haplotype G-A-ins-C-Gin Tim-1 gene region is associated with allergic asthma （P< 0.05）.This haplotype wasovertransmitted from parents to asthma patients counld be as a risk factor for allergicasthma with a higher HRR=1.91.3.FBAT analysis revealed that the allele Ins of Tim-1rs45623443 polymorphisms was associated with asthma phenotype of total serum IgE, P<0.05.4.FBAT analysis of Tim-1 haplotypes showed that haplotype G-A-ins-C-G was alsoassociatied with total serum IgE level, P< 0.05.CONCLUSION: Tim-1 gene polymorphisms could influence asthma susceptibility in theform of haplotypes, which means Tim-3 gene or the region nearby the Tim-3 may existsome SNPs locus associated with allergic asthma and high serum total IgE level.As a logo,the G-A-ins-C-G haplotype can be used for asthma susceptibility and serum IgE levelprediction in Hubei Han population.更多还原