基因多态性与自身免疫性疾病的相关性研究

【作者】 张庆镐；

【导师】 孟繁平；

【作者基本信息】 延边大学 ， 生理学， 2007， 博士

【副题名】FOXJ1与系统性红斑狼疮，IL-27与哮喘

【摘要】 系统性红斑狼疮（systemic lupus erythematosus，SLE）是一种较为常见的自身免疫性疾病，其特征为多种自身抗体的产生及免疫复合物的形成，最终由于针对自身的免疫反应造成几乎全身各个系统的损害如循环系统、泌尿系统、消化系统以及中枢神经系统的病变。SLE的病因和发病机制尚未完全明了，目前认为该病属于多基因遗传病，遗传因素及环境因素共同参与本病的发生、发展过程。大量的研究已经证明SLE与遗传有着密切的关系，如SLE有家族聚集现象，10％～12％的SLE患者有患该病的一级亲属，而正常对照的一级亲属患病率只有0.4％。单卵双生子SLE的患病一致率为24％，而双卵双生子仅为2％。遗传学研究已经定位了一些与SLE相关的染色体区域，其中至少在两个以上的不同研究中得到证实的区域有：1q23、1q41、2q37、4p16、6p21、11p13、12q24、16q13。与SLE易感性相关的基因包括MHC基因（人类为HLA基因）和非MHC基因。在人类HLA基因中，与SLE关联最密切的是HLA-DR2，；HLA-Ⅲ类基因产物如补体C2、C4和肿瘤坏死因子（TNF）等也被证实与SLE相关，而且，补体C2、C4的单基因变异即可引起SLE的发生。除MHC基因外，许多非MHC基因如补体C1q、FcγR、甘露糖结合凝集素（MBL）、CTLA-4以及程序性死亡基因-1（PDCD-1）也被证实参与SLE的发生。辅助性T细胞（Th）及细胞因子失衡可能是SLE发病机制中的重要组成部分。Th根据其分泌的细胞因子的不同，主要分为两类，即Th1和Th2。Th1主要分泌IL-2、IFN-γ、TNF-α等，参与细胞免疫，清除细胞内病原体；Th2主要分泌IL-4、IL-5、IL-10、IL-13等，参与体液免疫，清除细胞外病原体。Th1与Th2型细胞因子之间的平衡对维持机体的健康起着重要的作用，当两者之间的平衡被破坏时就会导致疾病的发生。当Th1亢进时，可以引起器官特异性自身免疫性疾病如类风湿性关节炎、Ⅰ型糖尿病、GRAVE病等；而Th2亢进时与变态反应性疾病及全身性免疫疾病的发生有关，如哮喘、过敏性鼻炎等。Th1与Th2型细胞因子可以互相调控相对方的分泌。如Th1型细胞因子IFN-γ可以抑制Th2型细胞因子IL-4的分泌，反过来，IL-4也可以抑制IFN-γ的分泌。大量的动物实验及临床资料证实了SLE存在Th1／Th2细胞因子失衡，而SLE的不同阶段、不同的临床表现可能是与不同的Th1／Th2细胞因子失衡状态有关。所以，影响Th1／Th2平衡的基因多态性可能与SLE的易感性相关。Forkhead-box j1（Foxj1）是叉头框（Forkhead-box，Fox）转录因子家族的一员。Fox家族的成员都含有一个由约100个氨基酸构成的DNA结合基序，目前为止已在不同种属中发现了100多个Fox家族成员，其中人类至少有43个成员，分属于17个亚族，即FoxA～FoxQ。Fox家族成员参与许多生物过程，如胚胎发育、细胞周期调控、糖类和脂类代谢、生物老化过程以及癌症的发生等。一些成员也参与免疫反应的调节过程，如Foxn1与胸腺上皮细胞的发育有关，Foxo参与维持T、B细胞的免疫耐受，Foxp3与调节T细胞的功能密切相关，等等。Fox j1又名HFH-4、FKHL-13，主要在含有纤毛上皮的组织如肺、脉络丛、睾丸及卵巢等组织中表达，对其功能的研究最初也集中在Fox j1对含有纤毛上皮组织的发育的影响及胚胎左右轴的确立等方面。但是，最近Stanford peng领导的研究小组发现Fox j1还可作用于免疫系统。他们发现在倾向于发生SLE的小鼠淋巴细胞中Fox j1的表达减少，而Fox j1^-／-嵌合体小鼠也表现类似狼疮样的全身多个系统的免疫炎症反应。体外研究表明Fox j1^-／-嵌合体小鼠中分离的Th细胞在受到CD3特异性抗体刺激时会生成比野生型多的IL-2和IFN-γ，另外Th1特异性的转录因子T-bet表达也增多，表明Fox j1^-／-嵌合体小鼠出现Th1偏移。Fox j1对T细胞活性的抑制作用一部分是通过诱导NFκB的抑制剂IκBβ亚单位表达增多而实现的，IκBβ可以阻断NFκB转位到细胞核，从而抑制NFκB介导的转录活动，使NFκB的下游产物如IL-2、IFN-γ等生成抑制，进而T细胞的活化受到抑制。而NFκB本身可以增强促炎细胞因子的分泌，因此与炎症反应有密切的关系。Fox j1也可以通过类似的机制抑制B细胞的活化，另外，Fox j1还可以抑制胸腺细胞流出到外周的过程。综合上述研究结果，Fox j1在预防自身免疫性疾病发生方面起着重要的作用。人类的FOXJ1基因位于染色体17q22-25上，本研究提取了204名SLE患者及418名正常对照组的基因组DNA，所有样本均来自韩国全北、全南和忠北地区没有血缘关系的人群。采用聚合酶链反应（polymerase chain reaction，PCR）扩增了FOXJ1基因。采用直接测序法扫描了其中16个正常人和16个系统性红斑狼疮患者的整个FOXJ1基因包括其启动子区约2.2kb。共发现了7个单核苷酸多态性（SNP）位点，其中，g．-460C＞T和g．-342G＞C位于启动子区域；g．1164G＞C，g．1805G＞T，g．1824C＞G和g．1849G＞C位于第一内含子区域；g．3375G＞C在3’-UTR区域。有意思的是，g．-460C＞T正好位于推测的转录因子MZF1结合位点上。通过两两配对连锁不平衡（LD）分析发现g．-460C＞T和-342G＞C两个位点完全连锁，g．1805G＞T、g．1824C＞G和g．1849G＞C三个位点完全连锁（D’=1，r²=1），所以根据LD结果以及SNP所在位置，选择启动子区的g．-460C＞T，第一内含子区的g．1805G＞T和3’-UTR区的g．3375G＞C进行大样本的基因分型。基因分型采用单碱基延伸法（single base extension，SBE）对SNP位点进行微测序，记录所有样本的基因型。通过x²检验验证了所有基因型分布符合Hardy-Weinberg遗传平衡定律，表明样本收集没有偏移，可以代表人群。采用2×2或2×3列联表x²检验或Fisher’s exact test比较了SLE患者和正常对照组各基因型及等位基因分布频率的差异，结果表明病例组和对照组g．-460C＞T和g．1805G＞T的基因型频率和等位基因频率无显著性差异，但是3’-UTR区的g．3375G＞C显示了明显的差异（p=0.0072和0.0042），该位点的CC基因型频率分布在正常人为1.7％，而SLE患者为0.5％，OR值为1.71，表明CC基因型可能对SLE具有保护作用。采用最大期望值算法进行了单体型构建。FOXJ1基因的3个多态性位点共构成了8种单体型，其中，对照组有3种常见单体型（占94％），而病例组只有两种常见单体型（占92.1％）。单体型-460C-1805T-3375G在对照组构成频率为5.1％，而SLE组为1.5％，显示了显著性差异（p=0.01），表明单体型-460C-1805T-3375G可能对SLE的发生具有保护作用。采用单因素方差分析比较了基因型与SLE患者抗核抗体（ANA）水平之间的关系，结果表明FOYJ1基因的g．-460C＞T、g．1805G＞T和g．3375G＞C多态性与ANA水平没有相关性。采用2×3列联表x²检验比较了基因型与anti-Sm，anti-Ro，anti-La，anti-RNP抗体的相关性，结果表明FOXJ1基因的g．-460C＞T、g．1805G＞T和g．3375G＞C多态性与anti-Sm，anti-Ro，anti-La，anti-RNP抗体没有相关性。采用2×3列联表x²检验比较了基因型与狼疮性浆膜炎、狼疮性关节炎、狼疮性肾炎的相关性，结果表明FOXJ1基因的g．-460C＞T、g．1805G＞T和g．3375G＞C多态性与狼疮性浆膜炎、狼疮性关节炎、狼疮性肾炎没有相关性。综上所述，本研究在韩国人群的FOXJ1基因中查找到了7个SNP位点。其中，g．-460C＞T和g．-342G＞C位于启动子区域；g．1164G＞C，g．1805G＞T，g．1824C＞G和g．1849G＞C位于第一内含子区域；g．3375G＞C在3’-UTR区域。而g．-460C＞T正好位于推测的转录因子MZF1结合位点上。位于3’-UTR区域的g．3375G＞C多态性可能与SLE的易感性相关，而单体型C-T-G可能对SLE具有保护性作用，但是FOXJ1基因的多态性与ANA、抗Sm抗体、抗RNP抗体、抗Ro抗体及抗La抗体，以及与狼疮性肾炎、狼疮性关节炎、狼疮性浆膜炎没有相关。本研究结果将有助于进一步揭示FOXJ1的生物学功能，并为系统性红斑狼疮的基因诊断及预防提供新的依据。哮喘是以Th2型免疫占优势的呼吸道疾病，主要表现为气道反应性增高，及嗜酸粒细胞增多，IgE水平升高等。全世界约有1.5亿患者，在我国约有2000万人罹患此病，且其发病率、死亡率有逐年上升的趋势。因此，哮喘已成为严重的公共卫生问题，受到世界各国的极大关注。目前大多数学者认为，哮喘是一种具有多基因遗传倾向的疾病，其发病机制和病因相当复杂，受遗传因素和环境因素双重影响。寻找和确定哮喘易感基因或哮喘相关基因成为近年研究热点。目前的研究发现与哮喘相关的异常基因主要位于5q31-33（如IL-3、IL-4、IL-5、IL-13）、6p（如TNF、MHC-Ⅱ）、11q（如IgE高亲和受体β亚单位）、12q（如IFN-γ）等。其中已经确认的和哮喘相关的基因有IL-4、TNF-α、IL-4R、IL-10、β2肾上腺素能受体等。但因为哮喘属于复杂的多基因遗传病，是多个基因微效作用的累加及环境因素的作用，所以哮喘相关基因的研究还有大量的工作需要去做。白介素-27（IL-27）是是一种新近发现的属于IL-6／IL-12家族的异二聚体细胞因子，由Epstein-Barr virus induce gene 3（EBI3，与IL-12p40同源）和p28（与IL-12p35同源）构成。IL-27主要由活化的抗原提呈细胞如树突状细胞、巨噬细胞等分泌，其信号转导是由IL-27ra（又名WSX-1，TCCR）和gp130共同构成的IL-27受体（IL-27R）进行的，IL-27R主要表达于初始T细胞和自然杀伤细胞。IL-27与受体的结合主要激活JAK／STAT信号转导途径。而最近的研究发现，IL-27还可激活p38 MAPK和ICAM-1／LFA-1信号转导途径。IL-27在很多方面与其它两种IL-12家族细胞因子IL-12、IL-23相关联，而IL-12是初始T细胞向Th1分化的关键的细胞因子，IL-23则与记忆细胞的Th1型免疫反应的维持有关。因此，初期对IL-27的研究也集中在其对Th1型免疫反应的影响方面。体外研究发现，IL-27可以通过STAT1通路诱导初始T细胞表达T-bet转录因子，T-bet是初始T细胞向Th1分化的关键的转录因子。T-bet又通过STAT4通路诱导IL-12Rβ2的表达，IL-12Rβ2是IL-12受体的一个亚单位，在初始T细胞中没有表达，通过IL-12Rβ2的表达，可以使初始T细胞获得对IL-12的反应性，促进初始T细胞向Th1的分化。因此，IL-27在Th1分化的初期起着重要的作用。另一方面，IL-27还抑制Th2特异性转录因子GATA3的表达，使IL-4、IL-13等细胞因子分泌减少。上述体外研究结果表明，IL-27的主要作用可能是使Th1／Th2平衡向Th1漂移。而体内研究结果却发现，IL-27在体内的作用与体外研究的结果并不完全一致。在体内，当缺乏IL-4时，不需要IL-27来促进Th1细胞的分化，只有当IL-4存在时（即Th1和Th2处于平衡状态时），IL-27对Th1的分化才是必需的，此时IL-27的主要作用是拮抗IL-4，其机制可能是抑制初始T细胞GATA3的表达来实现的。GATA3是Th2特异性转录因子，与IL-4、IL-5的表达水平有密切的关系，而且GATA3还可以抑制IL-12Rβ2和STAT4的表达，从而阻断Th1的分化，使IL-12、IFN-γ分泌减少。因此，在体内IL-27对Th1／Th2的影响似乎主要是通过抑制GATA3的表达，从而使Th1／Th2平衡向Th1倾斜。IL-27在体内还对Th1型免疫起负反馈调节作用。在IL-27ra基因敲除的小鼠中发现，刚地弓形虫感染的初期IL-27ra^-／-小鼠虽然能够产生有效的Th1型免疫有效控制感染，但是在后期，却由于过多的IFN-γ的生成及T细胞的过度增殖导致致死性的炎症反应。枯氏椎虫感染的IL-27ra^-／-小鼠和注射ConA的IL-27ra^-／-小鼠也形成比野生型小鼠严重的肝脏损伤，而这种肝脏损伤是由Th1型免疫介导的。这些研究结果说明IL-27对过度的Th1型免疫具有抑制作用，其机制可能是通过抑制T细胞生长因子IL-2以及具有强效免疫刺激作用的IL-6的表达来实现的。综上所述，IL-27在初始T细胞中可以促使其向Th1分化，而在已经活化的Th1细胞中则抑制其增殖从而限制其强度和持续时间，防止过度免疫反应造成组织损伤。因此，IL-27的异常表达可能与自身免疫性和／或变应性疾病的发生有关。而许多研究业已证明了IL-27与免疫性疾病的关系。有报道，在佐剂诱导的自身免疫性关节炎模型中，中和IL-27p28亚单位可以抑制疾病的进展。而在自身免疫性脑脊髓膜炎模型中，IL-27p28、EBI3以及IL-27ra的表达水平都明显升高。另外在模拟人类炎症性肠疾病的结肠炎模型中发现，IL-27／IL-27ra信号通路与肠道炎症的加重有关。除Th1型免疫介导的疾病外，IL-27缺陷也与Th2型免疫疾病发生、发展过程有关。在实验性哮喘模型中，IL-27ra缺陷小鼠会产生比野生型更严重的气道高反应性、嗜酸粒细胞增多以及IgE水平升高，表明IL-27能够下调哮喘的气道高反应性、肺部炎症反应等等。人类的IL-27p28基因位于16号染色体上。为了探索人类IL-27基因多态性与哮喘易感性的关系，本实验扫描了IL-27p28的启动子区、编码区以及其邻近的区域以查找SNP位点，并采用对照-分析方法评估了IL-27p28多态性是否与哮喘易感性相关。本研究提取了288名哮喘患者及444名正常人的基因组DNA，所有样本都来自韩国全北地区没有血缘关系的人群。采用聚合酶链反应（polymerase chain reaction，PCR）扩增了IL-27p28基因。采用直接测序法扫描了其中16个正常人和16个哮喘患者的IL-27p28基因包括其启动子区约1.9kb。共发现了4个单核苷酸多态性（SNP）位点，其中，g．-964A＞G在启动子区；g．2905T＞G在第二外显子区；g．4603G＞A在第三内含子区，g．4730T＞C第四外显子区。在g．2905T＞G多态性中，T转换为G导致编码的氨基酸由丝氨酸转变为丙氨酸；而在g．4730T＞C多态性中，T转换为C导致编码的氨基酸由赖氨酸转变为脯氨酸。通过两两配对连锁不平衡（LD）分析发现g．4603G＞A和g．4730T＞C两个位点完全连锁（D’=1，r²=1）。所以根据LD结果以及SNP所在位置，选择启动子区的g．-964A＞G，第二外显子区的g．2905T＞C和第四外显子区的g．4730T＞C进行大样本的基因分型。基因分型采用单碱基延伸法（single base extension，SBE）对SNP位点进行微测序，记录所有样本的基因型。通过x²检验验证了所有基因型分布符合Hardy-Weinberg遗传平衡定律，表明样本收集没有偏移，可以代表人群。采用2×2或2×3列联表x²检验或Fisher’s exact test比较了哮喘患者和正常对照组各基因型及等位基因分布频率的差异，结果表明病例组和对照组启动子区的多态性g．-964A＞G的基因型频率和等位基因频率有显著性差异（p=0.006，p=0.003），但是第二外显子区的g．2905T＞G和第四外显子区的g．4730T＞C没有明显的差异，表明g．-964A＞G的多态性可能与哮喘的易感性相关。采用最大期望值算法进行了单体型构建。IL-27p28基因的3个多态性位点共构成了8种单体型，对照组和哮喘组都有四种常见单体型，其中对照组常见单体型占所有单体型的95.3％，而哮喘组常见单体型占所有单体型的94.7％。单体型-964A-2905T-4730T在对照组构成频率为65.8％，而哮喘组为71.2％，显示了显著性差异（p=0.035），表明单体型-964A-2905T-4730T可能与哮喘的易感性有关。而单体型-964G-2905G-4730T在对照组构成频率为8.4％，而哮喘组为5.4％，显示了显著性差异（p=0.035），表明单体型-964G-2905G-4730T可能对哮喘具有保护性作用。本研究采用单因素方差分析比较了基因型与哮喘患者血清总IgE水平之间的关系，结果表明IL-27p28基因的g．-964C＞T、g．2905G＞T和g．4730G＞C多态性与哮喘患者血清总IgE水平没有相关性。综上所述，本研究在韩国人群的IL-27p28基因中查找到了4个SNP位点。其中，g．-964A＞G位于启动子区域；g．2905T＞G在第二外显子区；g．4603G＞A在第三内含子区，g．4730T＞C第四外显子区。在g．2905T＞G多态性中，T转换为G导致编码的氨基酸由丝氨酸转变为丙氨酸；而在g．4730T＞C多态性中，T转换为C导致编码的氨基酸由赖氨酸转变为脯氨酸。启动子区的多态性g．-964A＞G的多态性可能与哮喘的易感性相关；单体型-964A-2905T-4730T可能与哮喘的易感性有关，而单体型-964G-2905G-4730T可能对哮喘具有保护性作用。但是IL-27p28基因的g．-964C＞T、g．2905G＞T和g．4730G＞C多态性与哮喘患者血清总IgE水平没有相关性。本研究结果将有助于进一步揭示IL-27的生物学功能，并为哮喘的基因诊断及预防提供新的依据。更多还原

【Abstract】 Systemic lupus erythematosus （SLE） is a prototypic autoimmune diseasecharacterized by the production of autoantibodies and deposition of immune complex,eventually leading to the damage of almost any organ systems by the autoimmunereactions. Though the exact etiology of SLE is unknown up to now, it is considered tobe a polygenic disease that involves both genetic and environment factors in the onsetand development of the disease.Great deals of data have shown that SLE represents strong familial aggregation,with a much higher frequency among first-degree relatives of patients. Theconcordance of the disease in identical twins is approximately 24%, and that indizygotic twins is about 0.4%. Genetic studies have mapped some susceptible loci ofchromosome, among them, 1q23、1q41、2q37、4p16、6p21、11p13、12q24、16q13loci have been reported to be associated with SLE at least in two independent studies.Both MHC （or HLA） and non-MHC regions included in the susceptibility to SLE.The most associated region in human HLA is HLA-DR2. HLA-Ⅲgene products suchas complement C2、C4 and tumor necrosis factor（TNF） have been confirmed to beassociated with SLE, moreover, C2 and C4 alone can result in the onset of SLE.Besides MHC genes, many non-MHC genes also take part in the development of SLE,such as complement C1q、FcγR、mannose-binding lectin （MBL）、CTLA-4 andprogrammed cell death-1 （PDCD-1）.Helper T cells （Th） play critical roles in the development of SLE. Th can beclassified into Th1 and Th2 subsets according to their different cytokine secretion.Th1 cells mainly secrete IL-2、IFN-γand TNF-α, involved in cell immunity toeliminate intracellular pathogens; Th2 secrete IL-4、IL-5、IL-10 and IL-13, taking partin humoral immunity to defense against extracellular pathogens. Keeping balancebetween Th1 and Th2 is very important for health, once the balance destroyed, manwill suffer from some diseases. Predominance of Th1 cytokines is associated with organ-specific autoimmune diseases, such as rheumatoid arthritis、type 1 diabetes andGRAVE disease, while, predominance of Th2 cytokines is associated with allergicdiseases and systemic autoimmune diseases like asthma、allergic rhinitis. Cytokinesproduced by Th1 and Th2 can cross-regulate partner subset’s development andactivity. For instance, IFN-γ, a Th1-cytokine, can inhibit the production of a Th2cytokine, IL-4. And vice versa, IL-4 inhibits the secretion of IFN-γ.Animal experiments and clinical data confirmed that SLE is associated withTh1/Th2 bias, and it seems different stage and different manifestations of SLEresulted from different Th1/Th2 bias. Therefore, gene polymorphisms affectingTh1/Th2 balance might be associated with susceptibility to SLE.Forkhead-box jl（Foxj1） belongs to Forkhead-box （Fox） transcription factorfamily. All members in this family contain an about 100 amino-acid DNA bindingdomain. Up to now, more than 100 members included in this family, among them, atleast 43 members belong to human FOX gene family, these members classified into17 subfamilies, FoxA to FoxQ. Fox members take part in diverse biologicalprocesses, including development, metabolism, aging and cancer. Some members alsoparticipate in the regulation of immune responses, for example, Foxnl is associatedwith development of thymic epithelial cells; Foxo is important for immune toleranceof T and B lymphocytes; Foxp3 is closely related to regulatory T cells’ function, andso on. Fox j1, also named HFH-4、FKHL-13, is mainly expressed in ciliated cells oflung, choroids plexus, testis and ovary, it’s functions mainly linked to ciliated celldevelopment and left-right axis determination. Recently, Peng and his coworkersfound that Fox j1 expression was reduced in lupus-prone mice, and Fox j1^-/- chimeramice represented lupus-like systemic autoimmune inflammation. In vitro studyrevealed that Th cells isolated from Fox j1^-/- chimera mice produce more IL-2、IFN-γas well as Th1 specific transcription factor T-bet than wild type when stimulated withCD3 specific antibodies, indicating a Th1 bias state. The inhibition of T cell activity ispartially through the induction of IκBβwhich inhibited the nuclear translocation ofNFκB and reduced the production of IL-2 and IFN-γ, subsequently inhibited T cellactivation. Fox j1 still inhibit B cell activity through a similar mechanism. Furthermore, Fox j1 can also inhibit thymocyte egress. Therefore, Fox j1 plays criticalroles in preventing autoimmune diseases.Human FOXJ1 gene lies on chromosome 17q22-25, consisting of 2 exons and 1intron. 204 SLE patients and 418 controls were recruited from cheonbuk, cheonnamand chungbuk area in South Korea. Polymerase chain reaction （PCR） was performedto amplify FOXJ1 gene. Direct sequencing method was used to scan the wholeFOXJ1 gene including it’s promoter（～2.2kb）. Total of 7 single nucleotidepolymorphisms （SNPs） were found, among them, g.-460C＞T and g.-342G＞C lie onpromoter region; g. 1164G＞C, g. 1805G＞T, g. 1824C＞G and g. 1849G＞C lie on intronregion; and g.3375G＞C lies on 3’-UTR. Interestingly, g.-460C＞T lies on a bindingsite of a speculated transcription factor MZF1. Pairwise linkage disequilibriumbetween each pair of SNP loci was evaluated, the result indicate that g.-460C＞T and-342G＞C are in complete LD, g.1805G＞T、g.1824C＞G and g.1849G＞C are incomplete LD （D’=1,r²=1）. According to LD and locations of SNPs, 3 of them werechosen to perform large sample genotyping, that is, g.-460C＞T in promoter region,g.1805G＞T in intron and g.3375G＞C in 3’-UTR. Genotyping was carried out bysingle base extension （SBE） method, a kind of minisequencing method. All genotypeswere recorded and were statistically analyzed.Hardy-Weinberg equilibrium test was performed by chi-square test, the resultindicates all genotypes were in Hardy-Weinberg equilibrium, indicating no samplebias.Chi-square test from 2×2 or 2×3 contingency table or Fisher’s exact test wereperformed to compare the difference of the genotyping or allele frequency, the resultshowed that genotyping and allele frequency of g.-460C＞T and g.1805G＞Tpolymorphisms did not have significant difference between SLE and the controlgroup. But polymorphism g.3375G＞C in 3’-UTR showed significant differencebetween two groups （p=0.0072和0.0042）. The genotype frequency of CC consistsof 1.7% in the control group, but only 0.5% in SLE group, indicating this genotypemay be a protective factor.Expectation maximization （EM） algorithm was used to construct haplotypes. Polymorphisms of g.-460C＞T、g. 1805G＞T和g. 3375G＞C in FOXJ1 constructed totalof 8 haplotypes. There are 3 main haplotypes, explaining 94% of distribution incontrols, and 2 major haplotypes in SLE accounting for 92.1% of distribution.Haplotype -460C-1805T-3375G consists of 5.1% in the control group, but only 1.5%in SLE group, represented significant difference（p=0.01）, indicating this haplotype ispossibly a protective factor.ANOVA was used to evaluate the relationship between each genotype and ANAlevel in SLE patients, the result indicating no association between each genotype inthese 3 polymorphisms with ANA levels.Chi-square test from 2×3 contingency table was used to evaluate the associationbetween each genotype and anti-Sm, anti-Ro, anti-La, anti-RNP antibodiesrespectively in SLE patients, the results indicating no association between eachgenotype in these 3 polymorphisms with anti-Sm, anti-Ro, anti-La, anti-RNPantibodies.Chi-square test from 2×3 contingency table was used to evaluate the associationbetween each genotype and serositis, arthritis and lupus nephritis respectively, theresults indicating no association between each genotype in these 3 polymorphismswith serositis, arthritis and lupus nephritis.In a word, total of 7 SNPs were found in this study. Among them, g.-460C＞Tand g.-342G＞C lie on promoter region; g.1164G＞C,g.1805G＞T,g.1824C＞G andg.1849G＞C lie on intron; g.3375G＞C lies on 3’-UTR. g.-460C＞T lies on aspeculated transcription factor MZF1 binding site. Polymorphism g.3375G＞C mightbe associated with SLE susceptibility, and haplotype -460C-1805T-3375G might be aprotective factor on SLE. Polymorphisms of g.-460C＞T,, g. 1805G＞T and g. 3375G＞Cin FOXJ1 gene seems no association with ANA levels, anti-Sm、anti-RNP、anti-Roand anti-La antibodies as well as serositis, arthritis and lupus nephritis. This studymight be helpful in uncovering the biological function of FOXJ1 gene further, andwill provide new evidence.Asthma is a Th2 predominant disease characterized by airwayhyper-responsiveness, eosinophil counting increase and IgE elevation. There are about 150 million of Asthma patients all over the world, and 20 million of that in China. Itsmorbidity and mortality are increasing year by year. Therefore, Asthma becomes acommon social problem that attracting more and more attention. It is considered thatAsthma is a kind of polygenic disease influenced by both genetic and environmentfactors. Searching and localizing the susceptible genes is a hotspot. Up to now,5q31-33、6p、、11q、12q are considered to be main susceptible loci of Asthma, andIL-4、TNF-α、IL-4R、IL-10、β2-Adrenoceptor are confirmed to be susceptible genes.But, for Asthma is a complex polygenic disease associated with many minor effectedgenes, there are still a lot of works to be done.IL-27 is a newly identified heterodimeric cytokine of IL-6/IL-12 family. It iscomposed by Epstein-Barr virus induce gene 3 （EBI3, IL-12p40 homology） and p28（IL-12p35 homology）. IL-27 is mainly secreted by activated APCs likemacrophages and dendritic cells). Its signal is conducted through IL-27 receptor（IL-27R）, a heterodimeric chain of IL-27ra （WSX-1, TCCR） and gpl30. IL-27R ismainly expressed on the surface of naive T cells and natural killer （NK） cells, it’sligation with IL-27 resulted in the activation of JAK/STAT pathways. Recently, it isreported that IL-27 also can activate p38 MAPK and ICAM-1/LFA-1 pathways.IL-27 is associated with IL-12 and IL-23 in many ways, and IL-12 is a keyplayer in Th1 differentiation of naive T cells, whereas, IL-23 is associated with Th1maintenance in memory T cells. Therefore, early researches on IL-27 are emphasizedon Th1 differentiation. In vitro studies identified that IL-27 can induce T-bet, a Th1specific transcription factor, through STAT1 pathway on naive T cells. Subsequently,T-bet induce the expression of IL-12Rβ2, a subunit of IL-12 receptor which is notexpressed on na（?）ve T cells, in this way, na（?）ve T cells can response to IL-12 stimulation,resulting Th1 differentiation. So, IL-27 is a critical player in the early stage of Th1differentiation. On the other hand, IL-27 can inhibit the expression of a Th2 specifictranscription factor-GATA3, resulted the decrease of IL-4 and IL-13. These in vitrostudies indicate that IL-27 might make the Th1/Th2 balance skewing to Th1 state.Whereas, quite confused results were obtained from in vivo studies. It isfounded that IL-27 is required for Th1 differentiation only when IL-4 exists in vivo, but is not required when IL-4 is absent. When IL-4 exists, the role of IL-27 is toantagonize IL-4 through inhibiting the expression of GATA3 on na（?）ve T cells. GATA3is a Th2 specific transcription factor, closely related to the expression levels of IL-4and IL-5. Otherwise, GATA3 can inhibit the expression of IL-12Rβ2 and STAT4,blocking Th1 differentiation and decreasing IL-12、IFN-γsecretion. Therefore, theinfluence of IL-27 in vivo seems more important in inhibiting Th2 differentiation,making the Th1/Th2 balance skewing to Th1.IL-27 also plays an important role in Th1 negative regulation in vivo. IL-27ra^-/-mice infected with T. gondii could produce efficient Th1 responses in the early stageto control infection. But these animals will develop tissue damage even lethalinflammation as a result of sustained T cell activation. T. cruzi infected and ConAinjected IL-27ra^-/- mice develop more serious liver damage than wild type, this kind ofdamage is induced by Th1 responses. These studies indicated that IL-27 could inhibitoveractivated Th1 responses. The immunosuppressive activity may be resulted byIL-2 and IL-6 suppression.Taken together, IL-27 drive naive T cells to Th1 differentiation at the early phase,whereas in activated Th1 cells, IL-27 inhibits Th1 cells’ proliferation, limit Th1 cells’strength and duration to prevent organ damage by overactivated immune responses.Therefore, abnormal expression of IL-27 might be associated with autoimmune and/or allergic diseases. Many studies have confirmed the relationship between IL-27 andimmune diseases. It is reported that neutrolizing IL-27p28 subunit could suppressongoing of adjuvant-induced arthritis. In a experimental autoimmuneencephalomyelitis （EAE） model, the expression levels of IL-27p28、EBI3 and IL-27raare highly elevated. Otherwise, IL-27/IL-27ra signaling is also related to theexacerbation of intestinal inflammation in a colitis model resembling humaninflammatory bowel disease. Besides Th1 predominant diseases, IL-27 is alsoassociated with the development of Th2 predominant diseases. In an experimentalasthma model, IL-27ra deficient mice were induced more serious airwayhyper-reactivity、increased eosinophil counting and elevated IgE level, indicating thatIL-27 can down-regulate airway hyper-reactivity and pulmonary inflammation. Human IL-27p28 lies on chromosome 16. To detect whether IL-27 genepolymorphisms associated with asthma susceptibility, IL-27p28 promoter region、exons and it’s boundary regions were scanned for searching single nucleotidepolymorphisms（SNPs）, and the relationship of these polymorphisms with asthmasuscetptibility was evaluated by a case-control study.288 asthma patients and 444 normal controls were included in this study. Allmembers were recruited from Cheonbuk area in South Korea. Polymerase chainreaction （PCR） was used to amplify IL-27p28 gene. Then, direct sequencing methodwas used to scan the IL-27p28 gene including it’s promoter（～1.9kb）、5 exons andtheir boundary regions. Total of 4 SNPs were found, among them, g.-964A＞G lies onpromoter region; g.2905T＞G in the exon 2, g.4603G＞A in intron 3, and g.4730T＞Cin exon 4. Interestingly, a single nucleotide change T to G at g.2905T＞G in exon 2resulted in a amino acid change of p.Ser59Ala and a single nucleotide change T to Cat g.4730T＞C in exon 4 resulted in a amino acid change of p.Leu119Pro. Pairwiselinkage disequilibrium between each pair of SNP loci was evaluated, the resultindicate that g. g.4603G＞A and g.4730T＞C are in complete LD, g.1805G＞T、g.1824C＞G and g.1849G＞C are in complete LD （D’=1,r²=1）. According to LD andlocations of SNPs, 3 of them were chosen to perform large sample genotyping, that is,g.-964A＞G in promoter region, g.2905T＞G in exon 2 and g.4730T＞C in exon 4.Genotyping was carried out by single base extension （SBE） method, a kind ofminisequencing method. All genotypes were recorded and were statistically analyzed.Hardy-Weinberg equilibrium test was performed by chi-square test, the resultindicates all genotypes were in Hardy-Weinberg equilibrium, indicating no samplebias.Chi-square test from 2×2 or 2×3 contingency table or Fisher’s exact test wereperformed to compare the difference of the genotyping or allele frequency, the resultshowed that genotyping and allele frequency of g.2905T＞G and g.4730T＞Cpolymorphisms did not have significant difference between the asthma and the controlgroup. But polymorphism g.-964A＞G in promoter region showed a significant difference between two groups （p=0.006和0.003）, indicating this polymorphismmay be associated with susceptibility to asthma.Expectation maximization （EM） algorithm was used to construct haplotypes.Polymorphisms of g.-964A＞G、g.2905T＞G and g.4730T＞C in IL-27p28 constructedtotal of 8 haplotypes. There are 4 main haplotypes in each group, explaining 95.3% ofdistribution in controls, and 94.7% of distribution in asthma. Haplotype964A-2905T-4730T consists of 65.8% in the control group, but 71.2% in asthma group,represented significant difference （p=0.035）, indicating this haplotype is possibly asusceptible gene polymorphism; Haplotype -964G-2905G-4730T consists of 8.4% inthe control group, but 5.4% in asthma group, represented significant difference（p=0.035）, indicating this haplotype is possibly a protective polymorphism.ANOVA was used to evaluate the relationship between each genotype and serumtotal IgE level in asthma patients, the result indicating no association between eachgenotype in these 3 polymorphisms with IgE level.To summary, total of 4 SNPs were found in this study. Among them, g.-964A＞Glies on promoter region; g.2905T＞G in exon 2, g.4603G＞A in intron 3, andg.4730T＞C in exon 4. Interestingly, a single nucleotide change T to G at g.2905T＞Gin exon 2 resulted in a amino acid change of p.Ser59Ala and a single nucleotidechange T to C at g.4730T＞C in exon 4 resulted in a amino acid change ofp.Leu119Pro. Polymorphism g.-964A＞G might be associated with asthmasusceptibility, and haplotype -964A-2905T-4730T might be associated withsusceptbility to asthma, whereas, haplotype -964G-2905G-4730T in IL-27p28 mightbe a protective factor. And IL-27p28 gene polymorphisms of g.-964C＞T、g.2905G＞Tand g.4730G＞C seems no association with serum total IgE levels, anti-Sm、anti-RNP、anti-Ro and anti-La antibodies as well as serositis. This study might behelpful in uncovering the biological function of IL-27 gene further, and will providenew evidence for gene diagnosis and prevention of asthma.更多还原