【作者】 李继荣；

【导师】 陈嵩；

【作者基本信息】 第三军医大学 ， 内科学， 2011， 硕士

【摘要】 T-bet是Th1特异性转录调节因子,属T-box蛋白家族成员。T-bet上调IFN-γ转录和IL-12Rβ2表达,提高T细胞对IL-12信号传递的敏感性,从而促进初始Th细胞分化为Th1细胞[1]。目前已发现在人体CD4+ T细胞、NK细胞、CD8+效应T细胞中表达T-bet,且与宿主炎症反应、抗病原体免疫应答关系密切[2, 3]。对孪生子研究还发现,宿主遗传因素对TBX21基因(其编码T-bet)表达及其调控的Th1细胞分化起主要作用;Th2应答不受遗传因素影响,而受T-bet诱导的Th1应答所调控[4]。人类基因组中存在广泛的多态性,基因组序列的差异构成了不同个体与群体对疾病的易感性、对药物与环境因子不同反应的遗传基础。单核苷酸多态性(single nucleotide polymorphism,SNP)是人类基因组中分布广泛、密度高、易于批量检测且相对稳定的第三代遗传标记,在复杂疾病的基因定位、关联分析、个体和群体对环境致病因子与药物的易感性研究中得到广泛应用[5]。启动子核苷酸序列的遗传变异可导致一些复杂遗传疾病关联基因表达量的改变。宿主遗传因素决定的IFN-γ水平降低可促进机体免疫耐受形成。对TBX21基因外显子和启动子的扫描显示,T-1993C SNP为日本人和高加索人群中最常见多态。T-1993C多态性与日本人和高加索人群的哮喘发病有关[6],且体外功能实验证实T-1993C多态性可影响TBX21基因的转录活性[7]。我们对中国人群的TBX21基因SNP分析显示,T-1993C多态性与慢性HBV感染[8]、自身免疫性肝炎和系统性红斑狼疮关联[9]。这些免疫性疾病发生可能与免疫耐受的缺失或免疫反应受到抑制的分子遗传机制有关。遗传关联研究不能说明TBX21基因功能是直接受T-1993C多态性影响,还是受与T-1993C SNP连锁不平衡的其他多态性位点影响。为更好地弄清T-bet表达的调控机制及其关联的自身免疫性疾病、变应性和感染性疾病的宿主易感性,本研究采用电泳迁移率分析(electrophoretic mobility shift assay, EMSA)、染色质免疫沉淀分析(chromatin immunoprecipitation, ChIP),证实-1993位点的等位特异性顺式调控作用;同时,以健康体检者外周血为研究对象,采用流式细胞检测技术观察-1993位点T/C等位变异对正常人群CD4+ T淋巴细胞TBX21表达量(蛋白水平)及Th1/Th2应答的影响,以期深入认识TBX21基因调节性SNP(regulatory SNP,rSNP)在感染性疾病、自身免疫性疾病及变应性疾病中的作用。1.转录因子YY1与TBX21基因-1993T/C位点区域结合的确定生物信息分析显示,TBX21基因启动子-2211至-1712区域包含一个转录因子YY1结合的保守序列,T-1993C SNP相邻YY1结合的保守序列(AAATGG)。采用-1993T和-1993C等位寡核苷酸探针进行竞争抑制实验显示,YY1与T和C等位均特异性结合,但其与C等位亲和力明显高于T等位。T、C等位探针交叉抑制实验进一步证实,同等浓度时C等位可以将T等位结合条带完全抑制,而T等位却不能将C等位结合条带完全抑制; YY1冷探针可以将T、C等位结合的慢迁移率结合带完全抑制,而突变YY1冷探针则不能抑制慢迁移率结合带。采用YY1抗体进行超漂移实验证实,与T和C等位结合的慢迁移率条带包含转录因子YY1。同时,我们利用人CD4+ T淋巴细胞株jurkat进行ChIP分析,进一步确定YY1与TBX21基因启动子区包含-1993位点序列发生结合。2.重庆人群TBX21基因T-1993C多态性的分布状况我们对收集的370例健康献血员的全血标本基因组DNA进行PCR-限制性内切酶长度多态分析,结果显示:370例标本中,-1993TT基因型为279例(76%), -1993TC基因型为86例(23%), -1993CC基因型为5例(1%),-1993T/C等位频率为13%。3. -1993T/C基因型与TBX21表达量和CD4+ T细胞应答的关系在-1993位点分型标本中,我们随机选取17例,其中,TT基因型和TC基因型各7例,CC基因型3例。再次采集全血,采用五色流式细胞检测技术对T-bet、IFN-γ及IL-4水平进行检测,结果显示,T-bet和IFN-γ的表达量从TT→TC→CC基因型呈递减趋势,而IL-4表达量则呈递增趋势,且不同基因型个体的各种因子之间存在显著差异(P<0.05)。结论:TBX21基因启动子-1993位点区域与转录因子YY1特异性结合,后者与C等位结合量明显大于T等位;TBX21-1993TT纯合子人群的CD4+ T细胞T-bet表达量明显高于-1993CC纯合子人群,前者外周血主要表现Th1细胞应答,后者则表现Th2应答。转录因子YY1对TBX21基因表达的负性调控作用,直接影响初始Th细胞分化为Th1细胞。更多还原

【Abstract】 T-bet is a Th1-specific transcription factor and a member of T-box protein family. T-bet upregulates IFN-γtranscription and IL-12Rβ2 expression, enhances the sensitivity of IL-12 signal to T cell, thereby promoting the initial Th cells differentiating into Th1 cells. T-bet expression has been found in human CD4 + T cells, NK cells, CD8 + T cells, and is closely related with the host inflammatory response and anti-pathogen immune response. Twins study also found that host genetic factors play a major role in TBX21 gene (which encode T-bet) expression and Th1 cells differentiation regulated by T-bet; Th2 response is not influenced by genetic factors,but regulated by Th1 responses induced by T-bet.There is a wide range of human genome polymorphism. Differences in genome sequences of different individuals and groups constitute the susceptibility to disease, drugs and environmental factors on the genetic basis of different responses. Single nucleotide polymorphisms (single nucleotide polymorphism, SNP) is widely distributed in the human genome, high density, easy-to-volume and relatively stable detection of the third generation of genetic markers. It has been widely used in complex disease gene mapping, association analysis, individual and groups of Environmental factors and disease susceptibility to the drug.Genetic variation of Promoter sequences can lead to changes in gene expression associated with complex genetic diseases. the lower IFN-γlevel Determined by host genetic factors can promote the formation of immune tolerance. The scan Of TBX21 exons and promoter shows T-1993C SNP is the most common polymorphism for the Japanese and Caucasian populations. T-1993C polymorphism was associated with the pathogenesis of asthma in Japanese and Caucasian populations. Functional experiments in vitro confirmed that T-1993C polymorphism can affect the transcription activity of TBX21 gene. Analysis of TBX21 gene SNP of our Chinese population indicate T-1993C polymorphism was associated with chronic HBV infection, autoimmune hepatitis and systemic lupus erythematosus.These autoimmune diseases may be resulted from a molecular genetic mechanism related with the absence of immune tolerance or a suppressed immune response .Genetic association studies can not explain whether TBX21 gene function is directly affected by the T-1993C polymorphism, or the other which has a linkage disequilibrium relationship with the T-1993C polymorphism. To better understand the regulatory mechanism of T-bet expression and its associated host susceptibility of autoimmune diseases, allergic and infectious diseases, in this study, electrophoretic mobility shift analysis (electrophoretic mobility shift assay, EMSA), Chromatin immunoprecipitation analysis (chromatin immunoprecipitation, ChIP) was applied to confirm -1993 site-specific cis-allelic effect; at the same time, we analyzed the peripheral blood of healthy objects by flow cytometry to observe the affection of the -1993 site T / C allele variation on TBX21 expression (protein level) of CD4 + T lymphocytes and the Th1/Th2 response in the normal population and expected a in-depth understanding of the role of TBX21 gene regulatory SNP (regulatory SNP, rSNP) in infectious diseases, autoimmune Disease and allergic diseases.Results:1. Determination of YY1 protein binding to the region around the -1993 site of TBX21 gene.Bioinformatics analysis showed that, -2211 to -1712 region of TBX21 gene promoter contains a conserved transcription factor YY1 binding sequence, T-1993C SNP adjacent to the conserved YY1 binding sequence (AAATGG). Competitive inhibition experiments with-1993T and-1993C allele oligonucleotide probe showed that, YY1 can specifically bind to T and C allele probe, but its affinity with the C allele was significantly higher than the T allele. T, C allele probe cross-inhibition experiments further confirmed that the same concentration of T allele binding bands can be completely inhibited by the C allele, while the T allele is not able to completely inhibit the C allele binding bands; YY1 cold probe can completely inhibit the slow mobility band of T, C allele, while the mutant cold probe YY1 can not. Super shift experiment with YY1 antibody confirmed that the slow mobility band of the T allele and the C allele contains the transcription factor YY1. We use human CD4 + T lymphocyte lines jurkat for ChIP analysis, further defining YY1 binding to sequences containing -1993 site in the TBX21 gene promoter region.2. Distribution of TBX21 gene T-1993C polymorphism in Chongqing populationWe collected whole blood samples of 370 healthy blood donors for genomic DNA extraction,then analyzed its genotype by RFLP(restriction fragment length polymorphism. The result showed: of 370 samples,0.76(279 ? 370) for TT homozygotes, 0.23 (86 ? 370) for TC heterozygotes, 0.01 (5 ? 370) for CC homozygotes and 0.13 for T-1993C allele frequency.3.The relationship between -1993T / C genotype and TBX21 expression、the response of CD4 + T cells .We randomly selected 17 cases in genotyped samples, of which, 7 cases are for each TT genotype and TC genotype, 3 cases for CC genotype. We collected their whole blood again, detected the level of T-bet, IFN-γand IL-4 by five-color flow cytometry .the results indicate that T-bet and IFN-γexpression showed an order of TT〉TC〉CC, while IL-4 was just opposite, and there were a significant difference in different genotypes (P <0.05).Conclusion:Transcription factor YY1 specifically bind to TBX21 gene promoter at -1993 position, which combined with the C allele was significantly greater than the T allele; T-bet expression in CD4 + T cell of TBX21-1993TT homozygous population was significantly higher than -1993CC homozygous population, with the former mainly expressing Th1 cell responses in peripheral blood, while the latter mainly Th2 response. the negative regulation of transcription factor YY1 on TBX21 gene expression directly affect the initial Th cells differentiation into Th1 cells.更多还原