【作者】 范秀杰；

【导师】 马春红；

【作者基本信息】 山东大学 ， 免疫学， 2008， 硕士

【摘要】 [研究背景]TIM(T-cell immunoglobulin domain and mucin domain)家族成员的编码蛋白是近年来新发现的免疫分子。TIM家族位于人染色体5q32．2，共包括3个基因，分别是TIM-1，TIM-3及TIM-4。TIM基因编码的蛋白是一类具有共同基序的跨膜糖蛋白，其基本结构包括一个信号肽、免疫球蛋白样区、粘蛋白样区、跨膜区和胞内区。TIM-3最初是作为Th1／Th2的特异性表面标志而被克隆的，TIM-3仅表达于分化后的Thl细胞上，现已证明TIM-3参与Th细胞的分化及免疫应答调节，并在自身免疫病发生中具有重要作用，已有文献证实TIM分子与多种自身免疫性疾病易感性有关。系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种典型的自身免疫病，其特征是产生大量自身抗体和多系统受累。该病病因不明，以T细胞依赖性B细胞克隆活化和产生自身抗体、形成多种免疫复合物而导致组织损害为特征。Th细胞及细胞因子在SLE等多种自身免疫病发病机制中有着非常重要的作用，但具体机制尚未阐明。TIM家族成员作为重要免疫调节分子参与多种免疫性疾病发生，但其在SLE发生中的作用尚不明确。本研究室前期研究发现SLE患者外周血中TIM-3与参与SLE发生的IFN-γ的表达有良好的正相关关系，且TIM-3配体表达显著升高，提示TIM-3通路在SLE发生中发挥重要作用。本研究旨在研究TIM-3多态性位点与SLE相关性，以期寻找新的SLE易感基因，并为证明TIM-3参与SLE，揭示SLE发生机制提供更多实验依据。[研究目的]利用病例-对照研究方法探讨TIM-3基因内的3个多态位点与中国北方汉族人群系统性红斑狼疮之间的相关性，这些多态位点分别是TIM-3基因启动子区域两个SNP(T-882C和T-574G)及编码区一个SNP(A+2398G)。观察这三个位点的等位基因在人群中是否达到遗传平衡，比较它们在正常人群和SLE患者中的分布是否存在差异，分析各基因型和等位基因频率与SLE感性的关系，以期筛选与SLE易感性有关的多态性位点，探索SLE易感的遗传因素。[研究方法]1．SLE病例组与对照组样本收集：在中国北方汉族人群中收集209例SLE患者及245例正常对照血样并提取基因组DNA。所收集的SLE患者均符合系统性红斑狼疮诊断标准，同时排除慢性炎症等疾病。正常对照均排除自身免疫性病、慢性炎症等疾病。2．基因分型，基因型及等位基因频率统计及分布差异检测：采用聚合酶链反应-限制性片断长度多态性(polymerase chain reaction and restriction fragment lengthpolymorphism，PCR-RFLP)法，分析SLE病例组与对照组所有个体中TIM-3基因启动子区域两个SNP(T-882C和T-574G)及编码区一个SNP(A+2398G位点)基因型，根据琼脂糖凝胶电泳结果确定个体基因型，计算各位点在SLE病例组与对照组中的基因型频率及等位基因频率。利用卡方检验比较各多态位点在SLE病例组与对照组间的基因型频率及等位基因型频率差异，以P<0．05表示差异具有统计学意义。若某多态位点的基因型频率及等位基因型频率在组间具有统计学差异，说明该多态位点与SLE具有相关性，反之则与SLE无相关性。3．测序验证分型结果：选取三个多态位点各种不同基因型个体的DNA样本，分别用相应引物进行扩增，2％琼脂糖凝胶电泳检测PCR扩增效果后，送上海生工公司测序。4．Hardy-Weinberg平衡吻合度检验：根据Hardy-Weinberg平衡定律计算各多态位点在SLE病例组与对照组中的预期基因型频率，利用卡方检验比较观察到的基因型频率与预期基因型频率间的差异，以P<0．05表示差异具有统计学意义。以确定选取的SLE病例组与对照组在所研究的各个多态位点均处于遗传平衡状态，具有群体代表性。5．采用酶联免疫吸附试验(ELISA)检测抗双链DNA抗体，抗核小体抗体，抗组蛋白抗体，抗核糖体P蛋白抗体，抗心磷脂抗体IgA、IgG、IgM:检测这五项指标的浓度，采用t检验与SLE不同基因型的比较，以P<0．05为差异有显著性。[结果]1．无论是SLE病例组还是在对照组，T-882C、T-574G和A+2398G三个位点的等位基因在人群中均已达到遗传平衡。(T-882C对照组：x~2=0 P=1病例组：x~2=0P=1；T-574G对照组：x~2=0 P=1病例组：x~2=0 P=1；A+2398G对照组：x~2=0P=1病例组：x~2=0 P=1)。2．T-882C三种基因型TT、TC、CC在病例组中的分布频率为0、0．96％、99．04％，在对照组中为0、2．45％、97．55％，此位点的基因型频率以及等位基因频率在病例组及对照组之间均无统计学差异(P=0．397，P=0．399)，说明该位点的基因多态性与SLE易感性无关。携带CT杂合子基因型会使SLE发病的风险降低(OR：0．385，95％可信区间：0．077-1．927)。3．T-574G三种基因型TT、TG、GG在病例组中的分布频率为0、0．96％、99．04％，在对照组中为0、1．63％、98．37％，此位点的基因型频率以及等位基因频率在病例组及对照组之间均无统计学差异(P=0．829，P=0．829)，说明该位点的基因多态性与SLE易感性无关。携带GT杂合子基因型也会使SLE发病的风险降低(OR：0．582，95％可信区间：0．106-3．211)。4．A+2398G三种基因型AA、AG、GG在病例组中的分布频率为0、0、100％，在对照组中为0、0、100％，在中国北方汉族人群中未发现有AA、AG基因型。5．T-882C与T-574G两位点抗双链DNA抗体，抗核小体抗体，抗组蛋白抗体，抗核糖体P蛋白抗体，抗心磷脂抗体IgA、IgG、IgM水平在不同基因型之间差异无显著意义(P值均大于0．05)。[结论]T-882C、T-574G和A+2398G三个位点的等位基因在人群中达到遗传平衡。T-882C、T-574G和A+2398G三个位点的SNPs在正常人群和SLE患者中的分布无差异，与SLE易感性无关。T-882C位点的CT基因型，T-574G的GT基因型，可降低SLE的发病风险。T-882C、T-574G位点变异不影响抗双链DNA抗体，抗核小体抗体，抗组蛋白抗体，抗核糖体P蛋白抗体，抗心磷脂抗体IgA、IgG、IgM水平。更多还原

【Abstract】 [Background]T-cell immunoglobulin domain and mucin domain (TIM) family consists of three genes on syntenic human chromosome 5q33.2 with no other intervening genes. The human TIM family members are TIM -1, TIM -3 and TIM -4. TIM family members are type I membrane glycoproteins expressed on T cells and containing common structural motifs, namely Ig V domain, highly glycosylated mucin domain and cytoplasmic domain. TIM family members characterized thus far appear to have a role in the regulation of Th1 and Th2 differentiation and T cell mediated immune responses. TIM-3 is specifically expressed on Thl cells. It has been reported that the expression of TIM -3 in some autoimmune disease patients is different from that of controls and some SNPs of TIM -3 have relationship with autoimune diseases, both of which indicates that they are related to autoimmune diseases.Systemic lupus erythematosus (SLE) is characterized by B -cell hyperactivity, autoantibody production, and immune complex deposition in vital organs. SLE has been extensively studied and a large number of abnormalities in both cellular and humoral immunity have been described. Nevertheless, our understanding of the initiating and perpetuating mechanisms in SLE is still incomplete. The imbalance of Th1/Th2 cytokines plays an important role in the onset and development of SLE. Our preliminary studies showed the upregulation of galactin-9, ligand of TIM -3 in peripheral blood monocytes from SLE patients which suggests the involvement of TIM-3 pathway in SLE. However, it needs more studies to uncover the detail role of TIM -3 in SLE.[Objective]Using case-control study to determine the association between SLE and three polymorphisms in TIM-3 gene in Chinese Han population, including two SNPs T-882C and T-574G in TIM-3 promoter region, one SNP A+2398G in TIM-3 coding region. The objective is to study whether the T-882C,T-574G and A+2398G sites have reach genetic equilibrium and discover the difference of the SNPs between the normal control and SLE cases. Furthermore, we expect to screen out the relational SNPs with genetic susceptibly to SLE based this study.[Methods]1. Collect blood samples from SLE patients and healthy controls in the Chinese Han population in North and then extract DNA from the white blood cells in peripheral blood.2. Genotyping: Two SNPs T-882C and T-574G in TIM-3 promoter region and one SNP A+2398G in TIM-3 coding region were genotyped using polymerase chain reaction and restriction fragment length polymorphism(PCR-RFLP) method in 209 SLE patients and 245 healthy controls.3. Evaluate each polymorphism for Hard-Weinberg equilibrium (HWE) using chi-square test and make sure all the polymorphisms were at Hard-Weinberg equilibrium in both SLE patients and healthy controls used in the study.4. Compare the distribution of different genotypes and alleles of every polymorphism in the two groups using chi-square test to determine whether the polymorphism was associated with SLE.5. The ds-DNA, AunA , AHA , rRNP and ACL- IgA ,IgG ,IgM antibodies were analyszed with ELISA. Compare the different genotypes and levels of the five antibodies to discover whether the polymorphism was associated with these antibodies. [Results]1. The alleles of both healthy controls and SLE cases have achieved genetic equilibrium in the three SNP sits (T-882C Healthy: x~2=0 P=1; SLE patients: x~2=0 P=1;T-574G Healthy: x~2=0 P=1; SLE patients: x~2=0 P=1; A+2398G Healthy: x~2=0 P=1; SLE patients: x~2=0 P=1) .2. SNPs T-882C,T-574G and A+2398G were at Hard-Weinberg equilibrium in both SLE patients and healthy controls. Frequencies of TT, TC,CC genotypes at T-882C site was 0, 0.0096, 0.9904 in the SLE patients and 0, 0.0245, 0.9755 in the healthy controls .Frequencies of TT, TG,GG genotypes at T-574G site was 0, 0.0096, 0.9904 in the SLE patients and 0, 0. 0163, 0. 9837 in the healthy controls. Frequencies of AA, AG, GG genotypes at +2398A>G site was 0, 0, 1 in the SLE patients and 0, 0, lin the healthy controls .The genotype and allele frequencies of the three SNPs in SLE patients were not different from those in the healthy controls.3. The carrier of T-882C CT genotype has an decreased risk of SLE ( OR:0.385; 95%CI: 0.077-1.927). The carrier of T-574G GT genotype has an decreased risk of SLE (OR: 0.582;95%CI: 0.106-3.211).4. No significant difference in levels of ds-DNA, AunA , AHA , rRNP and ACLIgA,IgG ,IgM antibdy respectively in different genetype of SLE patients (P >0.05).[Conclusions]SNPs T-882C,T-574G and A+2398G were at Hard-Weinberg equilibrium in both SLE patients and healthy controls. SNPs T-882C,T-574G and A+2398G in TIM-3 gene might not be associated with SLE susceptibility in a Chinese Han population in North. Otherwise, we also found several potential dangerous factors of SLE, The carriers of T-882C CT and T-574G GT genotypes have an decreased risk of SLE. These polymorphisms do not influence the level of ds-DNA, AunA, AHA , rRNP and ACL-IgA,IgG,IgM.更多还原