【作者】 吴歆；

【导师】 徐沪济；

【作者基本信息】 第二军医大学 ， 内科学， 2008， 博士

【摘要】 强直性脊柱炎（Ankylosing Spondylitis,AS）是一种致病机理不明的慢性炎症性疾病,在人群中的发病率约千分之三,仅次于类风湿关节炎。该病具有明显的家族聚集现象,并与HLA-B27密切相关。其主要侵犯中轴骨骼,以骶髂关节炎和附着点炎为特征,炎症累及滑膜关节和软骨关节以及肌腱、韧带附着于骨的部位（肌腱端）,常引起纤维性和骨性强直。其他器官亦常累及,如25%～30%的患者可在病程中出现急性前色素膜炎。AS的致残率极高,给患者、家庭及社会带来了沉重的精神和经济负担。众所周知,HLA-B27是AS最主要的易感基因,但孪生子及家族调查研究显示,这种疾病是寡基因型的,且HLA-B27只能解释该病的遗传度中小于50%的部分。有研究报道IL-23R基因的多态性与AS高度相关,在该病的人群归因危险度中发挥了近12%的作用。尽管目前该病的致病机制不明,但普遍认为AS患者体内免疫功能失衡与其病理、临床表现密切相关,其中T细胞免疫功能异常在其发病中起着尤为重要的作用。越来越多的证据显示,细胞因子的分泌特点也可以影响脊柱关节病的临床进程。Th17细胞是近年新发现的一种辅助性T细胞,其各种特性与Th1和Th2细胞不同,产生的细胞因子包括IL-17、IL-21和IL-22等等。已有研究证实,Th17细胞在自身免疫性疾病如实验性自身免疫性脑脊髓炎（Experimental autoimmune encephalomyelitis,EAE）、炎性肠病（Inflammatory bowel disease,IBD）等的致病机制中都发挥了重要作用,其分化和功能受各种细胞因子的调控。而IL-23R对一个Th17细胞来说是一个关键的作用因子。本研究拟在中国汉族人群中鉴定与AS相关的IL-23R基因的变异体区域,同时研究IL-23R基因的遗传变异体是否能影响Th17细胞数量和功能,进而导致AS发病。具体分为以下三个部分:第一部分IL-23R单核苷酸多态性在强直性脊柱炎疾病中国汉族人群的研究目的:在中国人群中对IL-23R的8个SNP位点（rs11209026、rs1004819、rs10489629、rs11465804、rs1343151、rs10889677、rs11209032、rs1495965）进行检测,研究IL-23R单核苷酸多态性与AS易感性的关系。方法:参考1984年修订的纽约标准,从门诊及住院病人中收集AS患者109例,正常对照者150例。运用TagMan MGB探针法对AS患者及正常对照IL-23R的8个SNP位点进行检测。多态性及其单倍型与AS相对危险度等数据处理均采用SPSS软件系统进行。结果:IL-23R SNPs相关的8个位点（rs11209026、rs1004819、rs10489629、rs11465804、rs1343151、rs10889677、rs11209032、rs1495965）在中国汉族人群中与AS具有显著相关性（P＜0.001）。其中,相关性最强的SNP为rs11209032(优势比1.3,95%CI 1.2-1.4,P=3.5×10^-8)。本部分针对这一基因结合临床AS病例分析,发现rs11209032A/G基因型、rs11209032A/A基因型和rs11209032G/G基因型在强直性脊柱炎患者中有62例（56.1%）、32例（29.4%）和15例（13.8%）,正常对照组分别有71例（47.3%）、45例（30%）和34例（22.7%）,两组间比较无显著性差异（P＞0.05）。第二部分Th17细胞相关细胞因子检测及功能的初步探讨第一节Th17细胞相关细胞因子检测目的:通过检测外周血中IL-17、IL-23、IL-1β、IL-6以及TGFβ-1的水平,明确AS患者与正常人之间、不同病情分级AS患者之间Th17细胞相关细胞因子的差异,以期探寻Th17细胞在AS致病机理中发挥的作用。方法:参考1984年修订的纽约标准,从门诊及住院病人中收集AS患者87例,正常对照者91例。运用酶联免疫吸附测定（ELISA）以及实时荧光定量PCR（Real-timePCR）的方法分别从蛋白水平和mRNA水平在AS患者和正常人中对IL-17、IL-23、IL-1β、IL-6以及TGFβ-1进行检测。同时将AS患者根据BASDAI≤4静止期和BASDAI＞4活动期分为两组,进行AS患者组间各细胞因子水平的比较。运用独立样本t检验以及SPSS11.5统计学软件对结果进行统计分析。结果:①AS患者IL-17蛋白水平和mRNA水平检测均明显高于正常对照组（75.45±6.46 vs 41.97±2.27,P＜0.001;0.003240±0.000872 vs 0.007643±0.001656,P＜0.05）;将AS患者根据BASDAI≤4静止期和BASDAI＞4活动期分为两组,进行AS患者组间IL-17mRNA水平比较,活动期AS患者IL-17mRNA水平高于静止期AS患者（0.001213±0.000765 vs 0.004561±0.001404,P＜0.05）。②AS患者IL-23蛋白水平和mRNA水平检测均明显高于正常对照组（61.85±2.61 vs 54.45±2.54,P＜0.05;0.038562±0.013772 vs 0.078789±0.014030,P＜0.05）;AS患者分级比较IL-23 mRNA水平,活动期AS患者IL-23mRNA水平高于静止期AS患者（0.010229±0.001813 vs0.067947±0.027626,P＜0.05）。③AS患者IL-1β蛋白水平和mRNA水平检测均明显高于正常对照组（28.45±0.87 vs 22.25±0.60,P＜0.001;0.324742±0.128426 vs1.426351±0.464371,P＜0.05）;AS患者分级比较IL-1βmRNA水平,活动期AS患者水平与静止期AS患者无显著性差异（P＞0.05）。④AS患者IL-6蛋白水平和mRNA水平检测均明显高于正常对照组（5.10±0.52 vs 2.79±0.31,P＜0.001;0.013524±0.005065vs 0.058685±0.015024,P＜0.05）;AS患者分级比较IL-6 mRNA水平,活动期AS患者IL-6mRNA水平高于静止期AS患者（0.003831±0.001475 vs 0.044567±0.013595,P＜0.05）。⑤AS患者TGFβ-1蛋白水平和mRNA水平检测均明显高于正常对照组（14.09±1.76 vs 3.59±0.60,P＜0.001;0.200738±0.048957 vs 1.098782±0.318909,P＜0.05）;AS患者分级比较TGFβ-1 mRNA水平,活动期AS患者TGFβ-1 mRNA水平高于静止期AS患者（0.058109±0.017780 vs 0.256579±0.075089,P＜0.05）。结论:这些结果提示AS患者外周血中IL-17、IL-23、IL-1β、IL-6以及TGFβ-1细胞因子水平均显著高于正常对照,且活动期AS患者外周血中IL-17、IL-23、IL-6以及TGFβ-1细胞因子水平均显著高于静止期AS患者,说明Th17细胞相关细胞因子可能在AS发病机制中发挥作用。第二节Th17细胞功能的初步探讨目的:检测AS患者和正常对照外周Th17细胞分布状态,并比较其差异;同时,观察不同细胞因子刺激对Th17分化的影响,探讨AS患者及正常人Th17细胞功能的异同。方法:收集AS患者10例,正常对照15例。Ficoll密度梯度离心法分离外周血单个核细胞（PBMC）,用佛波酯和钙离子载体刺激5小时后,抗CD4-FITC和抗IL-17-PE、抗CD8-FITC和抗IL-17-PE标记,流式细胞仪检测CD4⁺IL-17⁺T、CD8⁺IL-17⁺T细胞的比例。将一部分PBMC加入不同的细胞因子组合IL-1β+IL-6、IL-1β+IL23、IL-6+IL23、IL-1β+IL23+IL-6进行培养5天,抗CD4-FITC和抗IL-17-PE标记,流式细胞仪检测IL-17的相对含量。结果:①AS患者外周血中CD4⁺IL-17⁺淋巴细胞高于正常人群（2.79±0.20 vs0.06±0.01 P＜0.01）,而且IL-17主要是由CD4⁺T淋巴细胞分泌,CD8⁺T淋巴细胞表达较少。②不同细胞因子对T细胞刺激培养后,结果显示IL-1β和IL-6细胞因子组合对细胞表达IL-17刺激作用较为明显。结论:AS患者外周血IL-17⁺调节性T细胞外周血分布高于正常对照组,且以CD4⁺IL-17⁺细胞群为主。IL-1β和IL-6可有效的刺激人Th17细胞分化,诱导IL-17的表达。更多还原

【Abstract】 Ankylosing spondylitis（AS） is a chronically inflammatory disease,which the pathogenic mechanism has not been clarified yet.The incidence of disease is about three in a thousand,which is a little bit inferior to rheumatoid arthritis.AS has obvious familial aggregation phenomenon which is related to HLA-B27 tightly.AS majorly affects axial skeleton,and its major characters are sacroiliitis which involved synovial joint,gristle joint and entheses that cause fibrosis and bone ankylosis often.Some other organs could be affected as well,for example,25%～30%patients may appear anterior uveitis.AS has very high disability rate,which brings extraordinary heavy psychological and economical burden.It is well known that HLA-B27 is the key susceptibility gene of AS.With the twins and familiy investigation we can see that this disease is oligo-genotype,and less than 50% part in the heritability can be explained by HLA-B27.Some report shows the polymorphism of IL-23R gene is highly related to AS,because it has 12%affection in the attributable risk in the patients group.Although the pathogenic mechanism has not been clarified,the common sense is the imbalance of immune system in the AS patient that has tight relationship with its pathology and clinic,in which the abnormity of T cell immune system plays an important role on its onset.More and more evidences show that the characters of cell excretion also affect the clinic process of spinal disease.Th17 cell is a helper T cell which is found in the recent years.All its characters are different from Th1 and Th2,and it produces cytokines include IL-17、IL-21和IL-22,etc.Some research proved that Th17 cell plays a vital roll in the pathogenic mechanism of autoimmune disease,such as experimental autoimmune encephalomyelitis,and inflammatory bowel disease and the polarization and functions are adjusted by different cytokines.IL-23R is a key gene to Th17 cell.The research will identify IL-23R gene variant or areas that related to AS in Han nationalities in China.At the same time,whether the IL-23R gene variant may affect Th17 cell’s amount and functions and then cause AS will also be researched.The details are shown as the following three parts:PartⅠ:Research of single nucleotide porlimorphism in the Chinese group of Ankylosing Spondylitis.Objective:8 SNP sites（rs11209026、rs1004819、rs10489629、rs11465804、rs1343151、rs10889677、rs11209032、rs1495965） of IL-23R will be inspected in the Chinese group.The purpose is to get the relationship of IL-23R single nucleotide porlimorphism with AS affectability.Patients and Methods:Refer to 1984 amended New York standard.109 AS patients are collected from clinic and in-patients,normal collators are 150 samples.8 SNP sites of IL-23R will be inspected by TagMan MGB probe methods on the patients.Porlimorphism, its hyploit,AS relative dangerous rate and date analysis are implemented by SPSS sys.Results:IL-23R SNPs related 8 sites rs11209026、rs1004819、rs10489629、rs11465804、rs1343151、rs10889677、rs11209032、rs1495965) has obvious correlation with AS in Chinese（P＜0.001）.The strongest pertinent SNP is rs11209032(OR 1.3,95% CI 1.2-1.4,P=3.5×10^-8).According to this gene related to the analysis of AS clinic,we find rs11209032A/G gene,rs11209032A/A gene,and rs11209032G/G gene in the patients of AS stands separately are 62 samples（56.1%）,32 samples（29.4%）,and 15 samples（13.8%）.Regular comparison group stands separately are 71 samples（47.3%）,45 samples（30%）,and 34 samples（22.7%）.No obvious difference between these two groups （P＞0.05）.PartⅡ:Th17 cell related cytokines inspection and functions initial discuss1.Inspection of Th17 cell related cytokinesObjective:Through inspecting IL-17、IL-23、IL-1β、IL-6,and TGFβ-1 level in the peripheral blood,identify Th17 cell related cytokines difference between AS patient and normal people and between different levels AS patients,in order to find out the effectiveness of Th17 cell in the AS pathogenic mechanism.Patients and Methods:Refer to 1984 amended New York standard.87 AS patients are collected from clinic and in-patients,normal collators are 91 samples.ELISA and Real-timer RT-PCR are used for inspection of IL-17、IL-23、IL-1β、IL-6,and TGFβ-1 from protein level and mRNA level to AS patients.AS patients are separated into two groups based on BASDAI≤4 in-active period and BASDAI＞4 active period for a comparison of all the cytokine level in AS patients.Independent sample t analysis and SPSS11.5 statistic software are used for statistic and analysis.Results:①IL-17 protein level and mRNA level of AS patients is much higher than normal control group（75.45±6.46 vs 41.97±2.27,P＜0.001;0.003240±0.000872 vs 0.007643±0.001656,P＜0.05）;AS patients are separated into two groups based on BASDAI≤4 in-active period and BASDAI＞4 active period,for the comparison of IL-17 mRNA level between AS patients.IL-17mRNA level of active period AS patients is higher than in-active patients（0.001213±0.000765 vs 0.004561±0.001404,P＜0.05）.②IL-23 proteinlevel and mRNA level of AS patients is much higher than normal control group （61.85±2.61 vs 54.45±2.53,P＜0.05;0.038562±0.013772 vs 0.078789±0.014030, P＜0.05）;Classified comparison of IL-23mRNA level of AS patients shows,IL-23mRNA level of active period AS patients is higher than in-active patients（0.010229±0.001813 vs 0.0679466±0.027626,P＜0.05）.③IL-1βprotein level and mRNA level of AS patients is much higher than normal control group（28.45±0.866 vs 22.25±0.603,P＜0.001; 0.324742±0.128426 vs 1.426351±0.464371,P＜0.05）;Classified comparison of IL-1βmRNA level of AS patients shows,no obvious difference between active period and in-active patients（P＞0.05）.④IL-6 protein level and mRNA level of AS patients is much higher than normal control group（5.10±0.52 vs 2.79±0.31,P＜0.001;0.013524±0.005065 vs 0.058685±0.015024,P＜0.05）;Classified comparison of IL-6 mRNA level of AS patients shows,IL-6 mRNA level of active period AS patients is higher than in-active AS patients （0.003831±0.001475 vs 0.044567±0.013595,P＜0.05）.⑤TGFβ-1 protein level and mRNA level of AS patients is much higher than normal control group（14.09±1.76 vs 3.59±0.60,P＜0.001;0.200738±0.048957 vs 1.098782±0.318909,P＜0.05）;Classified comparison of TGFβ-1 mRNA level of AS patients shows,TGFβ-1 mRNA level of active period AS patients is higher than in-active AS patients（0.058109±0.017780 vs 0.256579±0.075089,P＜0.05）.Conclusions:These results show that cell gene level of IL-17、IL-23、IL-1β、IL-6, and TGFβ-1 in the peripheral blood of AS patients is higher than normal controls.That is to say,Th17 cell related cytokines should be working in the AS pathogenic mechanism.2.Th17 cell function initial discussionObjective:Inspect Th17 cell distribution status of AS patients and normal controls and make comparison.At the same time,observe the affection of different cytokines stimulation to Th17 polarization,and discuss the difference of Th17 cell functions between AS patients and normal people.Patients and Methods:Collect 10 AS patients,and 15 normal collators.Ficoll desity gradient centrifugation methods is used for isolating PBMC first,then five hours stimulation has to be implemented by phorbol ester and calcium ionophore,anti CD4-FITC, anti IL-17-PE,anti CD8-FITC,and anti IL-17-PE has to be marked,and the rate of CD4⁺IL-17⁺T cell and CD8⁺IL-17⁺T cell has to be inspected by Flow Cytometry.Part of PBMC should be added in different cell gene IL-1β+IL-6、IL-1β+IL23、IL-6+IL23、 IL-1β+IL23+IL-6 and should be maintained for five days,anti CD4-FITC and anti IL-17-PE should also be marked,and the relative contents should be inspected by Flow Cytometry.Results:①CD4⁺IL-17⁺ lymphocyte in the peripheral blood of AS patients is higher than normal controls（2.79±0.20 vs 0.06±0.01 P＜0.01）,and IL-17 are mainly excreted by CD4⁺T lymphocyte,but CD8⁺T lymphocytes do not excrete much.②after the stimulation of different cell gene to T cell,the results show IL-1βand IL-6 cell gene have obvious result to the stimulation of IL-17.Conclusions:Peripheral blood IL-17+ and T cell peripheral blood distribution of AS patients are higher than normal collators group,and CD4⁺IL-17⁺ cell group is the majority group.IL-1βand IL-6 may effectively stimulate Th17 cell polarizeation,and lead to the appearance of IL-17.更多还原