中国汉族人类风湿关节炎易感基因及Th17细胞的研究
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摘要
类风湿关节炎(Rheumatoid Arthritis,RA)是一种系统性自身免疫性疾病,以关节的炎症为主要特点,主要的病理变化为慢性滑膜炎和血管炎,当累及关节软骨和骨质时,逐渐形成关节畸形和强直,导致关节功能丧失。在我国的患病率约为0.4%。RA的病因尚不明确,遗传、环境和免疫因素共同发挥作用。
RA作为一种具有遗传倾向的多基因性疾病,遗传率高达53-65%,同卵双胞胎患病的相对危险度为2-12,而MHC在整个遗传效应中作用约占30%。目前已知的RA的易感基因包括:HLA-DR,PTPN22,TNFAIP3和STAT4等,但还有一半以上的易感基因和多态性位点未被发现。此外,RA的遗传特征具有明显的异质性,在主要的种族尤其是白种人和亚洲人之间,遗传学研究的结果常常不一致。之前高加索人的全基因组扫描发现了大量潜在的危险等位基因,需要在不同的种族中进行验证。
Th17细胞作为一个新型的辅助性T细胞亚群,其分化、发育和功能均受其特定的细胞因子调控,能产生IL-17A,IL-17F和IL-22等细胞因子。动物实验发现,Th17细胞在清除病原体感染和诱导自身免疫性炎症中发挥着重要作用,同时有资料显示一些自身免疫病患者如炎性肠病、多发性硬化和银屑病等,存在着Th17细胞数量和(或)功能异常。
本研究拟通过大样本的病例-对照相关性研究,在中国汉族人群中对之前已报道的与高加索人RA相关的基因位点和可能存在种族差异的易感基因的多态性进行鉴定;此外,观察RA患者外周血Th17细胞数量和功能的变化,以及抗TNF-α治疗对该细胞亚群的影响,探讨RA的发病机制和治疗机理。
第一部分中国汉族人类风湿关节炎易感基因的大样本研究
目的:在中国汉族人群中对已报道过的与高加索人RA相关的基因变异体或可能存在种族差异的易感基因的多态性进行鉴定,包括了IL2RA、PADI4、IL2RB、CDK6、TRAF1、STAT4、IL2、IL21、CTLA4、MMEL1、PTPN22等16个基因的142个SNPs位点。
方法:以821例中国汉族RA患者和1000例种族相匹配的健康正常人为研究对象。应用多重PCR和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术,对142个SNPs位点进行了基因分型。应用Haploview 4.1软件进行SNPs与疾病的关联分析,连锁不平衡和单倍体相关性分析。
结果:单个SNPs位点与RA关联分析显示:在中国汉族RA患者中,STAT4的2个位点(rs11685878和rs7574070)等位基因频率及基因型频率与对照组有显著性差异(P=0.0459和0.0045、0.0007和0.0005);CDK6的rs42041等位基因频率及基因型频率在RA组和对照组有显著性差异(P=0.0199,0.018);位于PADI4的4个位点(rs1886302、rs2477131、rs11203326和rs1635561)等位基因频率和基因型频率与对照组均无显著性差异(P>0.05);IL2RB的rs228979等位基因频率及基因型频率在病例组和对照组存在显著差异(P=0.0043,0.0112); IL21的rs2221903等位基因频率及其基因型频率与健康对照组有显著性差异(P=0.0273,0.0478)。连锁不平衡和单倍体关联性分析发现,CDK6基因rs42041/rs42042组成的单倍体CT、单倍体GT在RA患者和对照组间存在显著性差异(P=0.0322,0.0263);STAT4基因rs11685878/rs7574070组成的单倍体TA、单倍体CC在RA患者和对照组之间存在显著性差异(P=0.0167,0.0019)。此外结果还显示,MMEL1基因与RA具有很强的关联性,PTPN22基因与RA无相关性。
结论:IL-21很有可能是中国汉族人RA的易感基因。CDK6、STAT4和MMEL1是不同种族中RA共同的非MHC易感基因。在中国汉族人群中PTPN22基因与RA无相关性。
第二部分Th17细胞与类风湿关节炎相关性的研究
第一节RA患者外周血Th17细胞相关细胞因子的表达
目的:Th17细胞的分化发育以及功能的发挥依赖于特定的细胞因子。通过检测血清中IL-17、IL-23、IL-1β、IL-6以及TGFβ-1的蛋白含量,以及PBMC相应细胞因子mRNA的表达水平,研究RA患者外周血Th17细胞的功能状况。
方法:选择RA患者74例并按照疾病活动性分组,正常对照者82例。运用酶联免疫吸附测定以及实时荧光定量PCR方法,分别从蛋白和mRNA水平对外周血中IL-17、IL-23、IL-1β、IL-6以及TGFβ-1进行检测。
结果:①活动期RA患者血清中IL-17含量明显高于稳定期RA和对照组(41.33±1.40 vs27.54±2.81 vs 17.66±1.40pg/ml,P<0.001),活动期RA患者PBMC的IL-17mRNA表达明显高于正常对照组(P<0.05);②活动期RA患者血清中IL-23含量与稳定期RA和对照组无显著性差异(116.60±23.30 vs128.00±43.72 vs140.85±27.32 pg/ml,P>0.05),活动期RA患者PBMC的IL-23mRNA表达与对照组无显著性差异(P>0.05);③活动期RA和稳定期RA患者血清中IL-1β含量均明显高于对照组(23.65±2.89 vs20.68±5.61 vs 6.82±2.76 pg/ml,P<0.05),活动期RA患者PBMC的IL-1βmRNA表达明显高于正常对照组(P<0.001);④活动期RA患者血清中IL-6含量明显高于稳定期RA和对照组(32.43±4.92 vs3.43±1.75 vs 3.37±0.70 pg/ml,P<0.001),活动期RA患者PBMC的IL-6mRNA表达明显高于正常对照组(P<0.001);⑤活动期RA、稳定期RA和对照组3组间血清中TGFβ-1含量均有显著性差异(1117.04±232.25 vs403.59±93.35 vs 134.92±71.38 pg/ml,P<0.05);活动期RA患者PBMC的TGFβ-1mRNA表达明显高于正常对照组(P<0.001)。
结论:在活动期RA患者体内,Th17细胞的主要效应性细胞因子IL-17水平明显升高,并与疾病活动性相关;促进初始CD4+T细胞向Th17细胞分化的细胞因子IL-1β、IL-6以及TGFβ-1的水平也明显升高,这些结果均提示活动性RA患者外周血中Th17细胞功能活跃。而IL-23主要是维持Th17细胞的存活和扩增,其功能的发挥无“剂量依赖性”。
第二节RA患者外周血Th17细胞的比例以及在体外不同微环境中的分化状态
目的:IL-17是Th17细胞标志性的细胞因子。通过检测CD4+IL-17+T细胞的比例研究RA患者外周血Th17细胞的数量。同时,观察在体外不同的细胞因子微环境对Th17细胞分化的影响。
方法:选择活动性RA患者10例,缓解期RA患者8例,正常对照10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标记,流式细胞仪检测CD4+IL-17+T细胞的比例。将分离出的一部分PBMC加入不同的细胞因子微环境(IL-1β+IL-6、IL-1β+IL23、IL-6+IL23、IL-1β+IL23+IL-6、TGF-β)体外培养5天,抗CD4-FITC和抗IL-17-PE标记,流式细胞仪检测CD4+IL-17+T细胞的比例。
结果:①活动期RA、稳定期RA患者和正常对照外周血CD4+IL-17+细胞的百分率分别为0.52±0.04%,0.28±0.03%,0.06±0.02%,RA患者外周血CD4+IL-17+细胞比例升高(P<0.001),且在不同疾病活动性的RA患者之间存在差异(P<0.001)。②分离出的PBMC在体外不同的细胞因子微环境中培养后,流式细胞术检测出的CD4+IL-17+T细胞的比例存在差异,其中经细胞因子IL-1β加IL-6共培养后CD4+IL-17+T细胞的比例最高。
结论: RA患者外周血Th17细胞的数量增多,并且与疾病活动性有关。在体外,细胞因子IL-1β加IL-6可有效的刺激人Th17细胞分化,诱导IL-17的表达。
第三节抗TNF-α治疗对Th17细胞数量和功能的影响
目的:观察活动期RA在接受人源化抗TNF-α单克隆抗体(adalimumab)治疗后,其外周血Th17细胞数量和功能的变化情况。研究抗TNF-α抗体发挥治疗作用的机制。
方法:选取参加Adalimumab药物临床试验的活动期RA患者7例,随访6个月。应用荧光定量PCR检测治疗前后外周血Th17相关的细胞因子mRNA的变化,流式细胞术检测治疗前后外周血中CD4+IL-17+T细胞比例的改变。
结果:荧光定量PCR结果显示adalimumab治疗6月以后,活动期RA患者PBMC的IL-23mRNA ( 0.0873±0.01589 vs0.0225±0.00445 )、IL-1βmRNA( 22.9281±3.95391 vs0.7859±0.44970 )和IL-6mRNA ( 395.9832±110.8752 vs102.2152±22.5869 )表达水平较治疗前明显降低( P<0.05 ),而IL-17和TGFβ-1mRNA表达水平无明显变化( 0.0061±0.00262 vs0.0038±0.00212 ,1.0547±0.33605 vs0.6669±0.10660,P>0.05)。流式结果显示,adalimumab治疗后RA患者外周血中CD4+IL-17+细胞的比例无明显降低(0.5286±0.00516% vs 0.4857±0.04592%,P>0.05)。
结论:Adalimumab能够直接中和TNF-α,使得位于TNF-α下游的IL-1β和IL-6水平降低,同时adalimumab有效地降低了RA患者PBMC中IL-23mRNA的表达水平,这可能是抗TNF-α抗体的又一种治疗机制。Adalimumab对Th17细胞的数量和效应因子IL-17的水平没有直接的影响。由此推测,TNF-α抑制剂和以Th17细胞为靶点的治疗在治疗机制上并没有重叠,两种生物制剂的合用可能是治疗RA的一条新的途径。
Rheumatoid arthritis (RA) is a chronic autoimmune arthritis characterized by progressive joint destruction. In China the occurrence of RA is somewhat lower (about 0.4 percent), whereas it is substantially higher in other ethnic groups. Although the etiology of RA remains a mystery, a variety of studies suggest that a blend of environmental , immunological and genetic factors is responsible.
Similar to what has been postulated for the majority of common diseases, a polygenic mode of inheritance has been proposed for RA. The heritability of RA estimated from twin studies is 53-65%, and the contribution from the MHC is estimated at ~30% of the total genetic effect. Previous genetic studies have identified and validated some risk loci for autoantibody-positive RA including HLA-DR,PTPN22,TNFAIP3 , STAT4 and so on. Less than half of genetic variation can be explained by the known RA risk alleles. However, previous results of genetic variation studies have not always been consistent between Caucasians and Asians. These divergent results suggest genetic heterogeneity of RA across the major racial groups. As such, a number of candidate risk alleles of RA found in Caucasians by genome-wide association scanning of functional SNPs should be replicated in major racial groups.
Th17 cell, which has been identified as a new subset of CD4+T cells, can produce IL-17, IL-22 and other effective cytokines. IL-17 is a proinflammatory cytokine that plays an important role in host defense against extracellular bacteria, protozoa, and fungi. It has also become recognized as a key mediator of chronic inflammation in animal models of immune-mediated inflammatory diseases such as RA, multiple sclerosis, inflammatory bowel disease and psoriasis. And now, there is growing evidence that Th17 cell is important in the human disease counterparts.
The aim of this study is to test the hypothesis that genes associated with RA in Caucasians are also associated with RA in Han Chinese. Another purpose of this study is to study on the role of Th17 cell in peripheral blood of patients with RA and observe the effect of anti- TNF-αtherapy on the number and function of Th17 cell.
Part I: Study on the genetic risk factors for rheumatoid arthritis in Chinese Han population
Objective: To test the hypothesis that genes associated with RA in Caucasians are associated with RA in Han Chinese. Specifically, we will perform case-control association testing of genes previously reported to be associated with RA in Caucasians in a large cohort of RA cases and controls, assessing the role of genes already implicated in the disease in other populations.
Methods:We genotyped the initial 821 RA cases and 1000 healthy controls for 142 SNPs covering the genes previously implicated in the disease including the SNPs in the genes PADI4, PTPN22, STAT4,CDK6, IL2RA, IL2RB, TRAF1, IL2, IL21,MMEL1, CTLA-4 and so on. For SNPs genotyping, Sequenom iPLEX and MALDI-TOF MS were used. Association tests for SNPs and haplotypes in patients and control subjects and the regional linkage disequilibrium structure for analyzed SNPs were determined using Haploview software, version 4.1.
Results: RA association tests: The STAT4 SNPs(rs11685878和rs7574070)were noted to be disease-associated, with a P value of 0.0459,0.0045 respectively. The CDK6 SNPs(rs42041),IL21 SNPs(rs2221903), IL2RB SNPs(rs228979) were also associated with disease. After analyzing the linkage disequilibrium structure of the SNPs, we found 4 blocks. CDK6 haplotype constructed by rs42041 and rs42042 is associated with RA. STAT4 haplotype constructed by rs11685878/rs7574070 is also associated with RA. Further more, the gene of MMEL1 is showed strong association with RA. In contrast, a negative result was obtained on analyzing SNPs in the gene of PTPN22.
Conclusions: IL21 is possibly a susceptible gene in Chinese Han RA patients. CDK6, STAT4 and MMEL1 are the common risk genes in both Caucasians and Asians. PTPN22 gene shown to be significantly associated with disease in Caucasians appears not play a role in Chinese patients with RA..
Part II: Study on the role of Th17 cell in peripheral blood of patients with rheumatoid arthritis
1. Expression of cytokines related with Th17 cell in peripheral blood of patients with rheumatoid arthritis
Objective: To find out the effectiveness of Th17 cell in the RA pathogenic mechanism.
Methods: 74 RA patients are collected and dived into two groups based on their disease activity. Normal controls are 82 samples. The levels of cytokines including IL-17、IL-23、IL-1β、IL-6, and TGFβ-1 in serum were measured by ELISA. The expression levels of these cytokines mRNA were evaluated by real-time quantitative PCR.
Results: Quantitative realtime PCR and ELISA analysis demonstrated elevated mRNA expression levels and protein concentrations of four cytokines including IL-17(41.33±1.40 vs 17.66±1.40pg/ml,P<0.001), IL-1β(23.65±2.89 vs 6.82±2.76 pg/ml,P<0.05)、IL-6(32.43±4.92 vs 3.37±0.70 pg/ml,P<0.001)and TGFβ-1(1117.04±232.25 vs 134.92±71.38 pg/ml,P<0.05). In addition, ELISA analysis showed that the concentration of IL-17(41.33±1.40 vs 27.54±2.81 pg/ml)、IL-1β(23.65±2.89 vs 20.68±5.61 pg/ml)and TGFβ-1(1117.04±232.25 vs 403.59±93.35 pg/ml)in the serum of active patients were much higher than that in stable patients(P<0.05).
Conclusions: As the main effect cytokine induced by Th17 cell, the level of IL-17 is remarkably elevated in active RA patients and correlated with disease activity. As IL-1β,IL-6 and TGFβ-1 can promote na?ve CD4+T cells to differentiate into Th17 cell, the levels of these cytokines were also elevated in RA patients. The function of IL-23 is to maintain the survival and expansion of Th17 cell, which did not depend on the dose of IL-23.
2. The proportion of Th17 cell in PBMC in RA patients and effects of different cytokine environments on Th17 cell differentiation
Objective: To investigate the proportion of CD4+IL-17+T cells in PBMC in RA patients and effects of different cytokine environments on Th17 cell differentiation.
Methods: 10 active RA patients, 8 stable RA patients and 10 normal controls were recruited for study. PBMC was isolated by Ficoll desity gradient centrifugation methods first, then the PBMC was stimulated 5 hours by phorbol ester and calcium ionophore. The cells were stained by CD4-FITC and anti IL-17-PE, and CD4+IL-17+T cell was detected by Flow Cytometry. Part of PBMC has been cultured in different cytokine environment: such as IL-1β+IL-6、IL-1β+IL23、IL-6+IL23、IL-1β+IL23+IL-6 for five days. Then the cells were stained by CD4-FITC and anti IL-17-PE and CD4+IL-17+T cell was detected by Flow Cytometry.
Results: The proportion of CD4+IL-17+ lymphocyte in the peripheral blood of active RA patients is higher than in stable RA patients and normal controls(0.52±0.04% vs 0.28±0.03% vs 0.06±0.02%,P<0.001).IL-1βand IL-6 are stronger stimulator for Th17 differentiation.
Conclusions: The proportion of CD4+IL-17+ lymphocyte in the peripheral blood of active RA patients is much higher. IL-1βand IL-6 are stronger stimulator for Th17 differentiation..
3. The effect of anti- TNF-αtherapy on the number and function of Th17 cell Objective: To analyze the effect of of anti- TNF-α(adalimumab)therapy on the number and function of Th17 cell.
Methods: seven patients with RA were recruited and administrated adalimumab 40mg once two weeks for more than six months. The proportion of Th17 cell and mRNA expression levels of cytokines including IL-17, IL-23, IL-1β, IL-6 and TGFβ-1 at the baseline and at 6 months were evaluated.
Results: A significant decrease in mRNA expression levels of IL-23(0.0873±0.01589 vs 0.0225±0.00445 ) ,IL-1β( 22.9281±3.95391 vs 0.7859±0.44970 ) and IL-6 (395.9832±110.8752 vs 102.2152±22.5869)was observed at 6 months after initial treatment of adalimumab(P<0.05). The expression levels of IL17 mRNA and TGFβ-1 mRNA did not show a significant change at 6 months after the initial injection compared with pretreatment levels, respectively(0.0061±0.00262 vs0.0038±0.00212,1.0547±0.33605 vs0.6669±0.10660,P>0.05).The flow cytometry. analysis also showed that the number of CD4+IL-17+ lymphocyte in the peripheral blood of RA patients did not decrease significantly after treatment(0.5286±0.00516% vs 0.4857±0.04592%,P>0.05). Conclusions: This study demonstrated that the reduction of IL-23 production in RA patients was a newly determined function of adalimumab. IL-17 production in RA patients in vivo may not depend on the direct stimulation by TNF-α. Combination use of TNF-αinhibitor and anti-IL17 may control the disease effectively in future.
RA作为一种具有遗传倾向的多基因性疾病,遗传率高达53-65%,同卵双胞胎患病的相对危险度为2-12,而MHC在整个遗传效应中作用约占30%。目前已知的RA的易感基因包括:HLA-DR,PTPN22,TNFAIP3和STAT4等,但还有一半以上的易感基因和多态性位点未被发现。此外,RA的遗传特征具有明显的异质性,在主要的种族尤其是白种人和亚洲人之间,遗传学研究的结果常常不一致。之前高加索人的全基因组扫描发现了大量潜在的危险等位基因,需要在不同的种族中进行验证。
Th17细胞作为一个新型的辅助性T细胞亚群,其分化、发育和功能均受其特定的细胞因子调控,能产生IL-17A,IL-17F和IL-22等细胞因子。动物实验发现,Th17细胞在清除病原体感染和诱导自身免疫性炎症中发挥着重要作用,同时有资料显示一些自身免疫病患者如炎性肠病、多发性硬化和银屑病等,存在着Th17细胞数量和(或)功能异常。
本研究拟通过大样本的病例-对照相关性研究,在中国汉族人群中对之前已报道的与高加索人RA相关的基因位点和可能存在种族差异的易感基因的多态性进行鉴定;此外,观察RA患者外周血Th17细胞数量和功能的变化,以及抗TNF-α治疗对该细胞亚群的影响,探讨RA的发病机制和治疗机理。
第一部分中国汉族人类风湿关节炎易感基因的大样本研究
目的:在中国汉族人群中对已报道过的与高加索人RA相关的基因变异体或可能存在种族差异的易感基因的多态性进行鉴定,包括了IL2RA、PADI4、IL2RB、CDK6、TRAF1、STAT4、IL2、IL21、CTLA4、MMEL1、PTPN22等16个基因的142个SNPs位点。
方法:以821例中国汉族RA患者和1000例种族相匹配的健康正常人为研究对象。应用多重PCR和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术,对142个SNPs位点进行了基因分型。应用Haploview 4.1软件进行SNPs与疾病的关联分析,连锁不平衡和单倍体相关性分析。
结果:单个SNPs位点与RA关联分析显示:在中国汉族RA患者中,STAT4的2个位点(rs11685878和rs7574070)等位基因频率及基因型频率与对照组有显著性差异(P=0.0459和0.0045、0.0007和0.0005);CDK6的rs42041等位基因频率及基因型频率在RA组和对照组有显著性差异(P=0.0199,0.018);位于PADI4的4个位点(rs1886302、rs2477131、rs11203326和rs1635561)等位基因频率和基因型频率与对照组均无显著性差异(P>0.05);IL2RB的rs228979等位基因频率及基因型频率在病例组和对照组存在显著差异(P=0.0043,0.0112); IL21的rs2221903等位基因频率及其基因型频率与健康对照组有显著性差异(P=0.0273,0.0478)。连锁不平衡和单倍体关联性分析发现,CDK6基因rs42041/rs42042组成的单倍体CT、单倍体GT在RA患者和对照组间存在显著性差异(P=0.0322,0.0263);STAT4基因rs11685878/rs7574070组成的单倍体TA、单倍体CC在RA患者和对照组之间存在显著性差异(P=0.0167,0.0019)。此外结果还显示,MMEL1基因与RA具有很强的关联性,PTPN22基因与RA无相关性。
结论:IL-21很有可能是中国汉族人RA的易感基因。CDK6、STAT4和MMEL1是不同种族中RA共同的非MHC易感基因。在中国汉族人群中PTPN22基因与RA无相关性。
第二部分Th17细胞与类风湿关节炎相关性的研究
第一节RA患者外周血Th17细胞相关细胞因子的表达
目的:Th17细胞的分化发育以及功能的发挥依赖于特定的细胞因子。通过检测血清中IL-17、IL-23、IL-1β、IL-6以及TGFβ-1的蛋白含量,以及PBMC相应细胞因子mRNA的表达水平,研究RA患者外周血Th17细胞的功能状况。
方法:选择RA患者74例并按照疾病活动性分组,正常对照者82例。运用酶联免疫吸附测定以及实时荧光定量PCR方法,分别从蛋白和mRNA水平对外周血中IL-17、IL-23、IL-1β、IL-6以及TGFβ-1进行检测。
结果:①活动期RA患者血清中IL-17含量明显高于稳定期RA和对照组(41.33±1.40 vs27.54±2.81 vs 17.66±1.40pg/ml,P<0.001),活动期RA患者PBMC的IL-17mRNA表达明显高于正常对照组(P<0.05);②活动期RA患者血清中IL-23含量与稳定期RA和对照组无显著性差异(116.60±23.30 vs128.00±43.72 vs140.85±27.32 pg/ml,P>0.05),活动期RA患者PBMC的IL-23mRNA表达与对照组无显著性差异(P>0.05);③活动期RA和稳定期RA患者血清中IL-1β含量均明显高于对照组(23.65±2.89 vs20.68±5.61 vs 6.82±2.76 pg/ml,P<0.05),活动期RA患者PBMC的IL-1βmRNA表达明显高于正常对照组(P<0.001);④活动期RA患者血清中IL-6含量明显高于稳定期RA和对照组(32.43±4.92 vs3.43±1.75 vs 3.37±0.70 pg/ml,P<0.001),活动期RA患者PBMC的IL-6mRNA表达明显高于正常对照组(P<0.001);⑤活动期RA、稳定期RA和对照组3组间血清中TGFβ-1含量均有显著性差异(1117.04±232.25 vs403.59±93.35 vs 134.92±71.38 pg/ml,P<0.05);活动期RA患者PBMC的TGFβ-1mRNA表达明显高于正常对照组(P<0.001)。
结论:在活动期RA患者体内,Th17细胞的主要效应性细胞因子IL-17水平明显升高,并与疾病活动性相关;促进初始CD4+T细胞向Th17细胞分化的细胞因子IL-1β、IL-6以及TGFβ-1的水平也明显升高,这些结果均提示活动性RA患者外周血中Th17细胞功能活跃。而IL-23主要是维持Th17细胞的存活和扩增,其功能的发挥无“剂量依赖性”。
第二节RA患者外周血Th17细胞的比例以及在体外不同微环境中的分化状态
目的:IL-17是Th17细胞标志性的细胞因子。通过检测CD4+IL-17+T细胞的比例研究RA患者外周血Th17细胞的数量。同时,观察在体外不同的细胞因子微环境对Th17细胞分化的影响。
方法:选择活动性RA患者10例,缓解期RA患者8例,正常对照10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标记,流式细胞仪检测CD4+IL-17+T细胞的比例。将分离出的一部分PBMC加入不同的细胞因子微环境(IL-1β+IL-6、IL-1β+IL23、IL-6+IL23、IL-1β+IL23+IL-6、TGF-β)体外培养5天,抗CD4-FITC和抗IL-17-PE标记,流式细胞仪检测CD4+IL-17+T细胞的比例。
结果:①活动期RA、稳定期RA患者和正常对照外周血CD4+IL-17+细胞的百分率分别为0.52±0.04%,0.28±0.03%,0.06±0.02%,RA患者外周血CD4+IL-17+细胞比例升高(P<0.001),且在不同疾病活动性的RA患者之间存在差异(P<0.001)。②分离出的PBMC在体外不同的细胞因子微环境中培养后,流式细胞术检测出的CD4+IL-17+T细胞的比例存在差异,其中经细胞因子IL-1β加IL-6共培养后CD4+IL-17+T细胞的比例最高。
结论: RA患者外周血Th17细胞的数量增多,并且与疾病活动性有关。在体外,细胞因子IL-1β加IL-6可有效的刺激人Th17细胞分化,诱导IL-17的表达。
第三节抗TNF-α治疗对Th17细胞数量和功能的影响
目的:观察活动期RA在接受人源化抗TNF-α单克隆抗体(adalimumab)治疗后,其外周血Th17细胞数量和功能的变化情况。研究抗TNF-α抗体发挥治疗作用的机制。
方法:选取参加Adalimumab药物临床试验的活动期RA患者7例,随访6个月。应用荧光定量PCR检测治疗前后外周血Th17相关的细胞因子mRNA的变化,流式细胞术检测治疗前后外周血中CD4+IL-17+T细胞比例的改变。
结果:荧光定量PCR结果显示adalimumab治疗6月以后,活动期RA患者PBMC的IL-23mRNA ( 0.0873±0.01589 vs0.0225±0.00445 )、IL-1βmRNA( 22.9281±3.95391 vs0.7859±0.44970 )和IL-6mRNA ( 395.9832±110.8752 vs102.2152±22.5869 )表达水平较治疗前明显降低( P<0.05 ),而IL-17和TGFβ-1mRNA表达水平无明显变化( 0.0061±0.00262 vs0.0038±0.00212 ,1.0547±0.33605 vs0.6669±0.10660,P>0.05)。流式结果显示,adalimumab治疗后RA患者外周血中CD4+IL-17+细胞的比例无明显降低(0.5286±0.00516% vs 0.4857±0.04592%,P>0.05)。
结论:Adalimumab能够直接中和TNF-α,使得位于TNF-α下游的IL-1β和IL-6水平降低,同时adalimumab有效地降低了RA患者PBMC中IL-23mRNA的表达水平,这可能是抗TNF-α抗体的又一种治疗机制。Adalimumab对Th17细胞的数量和效应因子IL-17的水平没有直接的影响。由此推测,TNF-α抑制剂和以Th17细胞为靶点的治疗在治疗机制上并没有重叠,两种生物制剂的合用可能是治疗RA的一条新的途径。
Rheumatoid arthritis (RA) is a chronic autoimmune arthritis characterized by progressive joint destruction. In China the occurrence of RA is somewhat lower (about 0.4 percent), whereas it is substantially higher in other ethnic groups. Although the etiology of RA remains a mystery, a variety of studies suggest that a blend of environmental , immunological and genetic factors is responsible.
Similar to what has been postulated for the majority of common diseases, a polygenic mode of inheritance has been proposed for RA. The heritability of RA estimated from twin studies is 53-65%, and the contribution from the MHC is estimated at ~30% of the total genetic effect. Previous genetic studies have identified and validated some risk loci for autoantibody-positive RA including HLA-DR,PTPN22,TNFAIP3 , STAT4 and so on. Less than half of genetic variation can be explained by the known RA risk alleles. However, previous results of genetic variation studies have not always been consistent between Caucasians and Asians. These divergent results suggest genetic heterogeneity of RA across the major racial groups. As such, a number of candidate risk alleles of RA found in Caucasians by genome-wide association scanning of functional SNPs should be replicated in major racial groups.
Th17 cell, which has been identified as a new subset of CD4+T cells, can produce IL-17, IL-22 and other effective cytokines. IL-17 is a proinflammatory cytokine that plays an important role in host defense against extracellular bacteria, protozoa, and fungi. It has also become recognized as a key mediator of chronic inflammation in animal models of immune-mediated inflammatory diseases such as RA, multiple sclerosis, inflammatory bowel disease and psoriasis. And now, there is growing evidence that Th17 cell is important in the human disease counterparts.
The aim of this study is to test the hypothesis that genes associated with RA in Caucasians are also associated with RA in Han Chinese. Another purpose of this study is to study on the role of Th17 cell in peripheral blood of patients with RA and observe the effect of anti- TNF-αtherapy on the number and function of Th17 cell.
Part I: Study on the genetic risk factors for rheumatoid arthritis in Chinese Han population
Objective: To test the hypothesis that genes associated with RA in Caucasians are associated with RA in Han Chinese. Specifically, we will perform case-control association testing of genes previously reported to be associated with RA in Caucasians in a large cohort of RA cases and controls, assessing the role of genes already implicated in the disease in other populations.
Methods:We genotyped the initial 821 RA cases and 1000 healthy controls for 142 SNPs covering the genes previously implicated in the disease including the SNPs in the genes PADI4, PTPN22, STAT4,CDK6, IL2RA, IL2RB, TRAF1, IL2, IL21,MMEL1, CTLA-4 and so on. For SNPs genotyping, Sequenom iPLEX and MALDI-TOF MS were used. Association tests for SNPs and haplotypes in patients and control subjects and the regional linkage disequilibrium structure for analyzed SNPs were determined using Haploview software, version 4.1.
Results: RA association tests: The STAT4 SNPs(rs11685878和rs7574070)were noted to be disease-associated, with a P value of 0.0459,0.0045 respectively. The CDK6 SNPs(rs42041),IL21 SNPs(rs2221903), IL2RB SNPs(rs228979) were also associated with disease. After analyzing the linkage disequilibrium structure of the SNPs, we found 4 blocks. CDK6 haplotype constructed by rs42041 and rs42042 is associated with RA. STAT4 haplotype constructed by rs11685878/rs7574070 is also associated with RA. Further more, the gene of MMEL1 is showed strong association with RA. In contrast, a negative result was obtained on analyzing SNPs in the gene of PTPN22.
Conclusions: IL21 is possibly a susceptible gene in Chinese Han RA patients. CDK6, STAT4 and MMEL1 are the common risk genes in both Caucasians and Asians. PTPN22 gene shown to be significantly associated with disease in Caucasians appears not play a role in Chinese patients with RA..
Part II: Study on the role of Th17 cell in peripheral blood of patients with rheumatoid arthritis
1. Expression of cytokines related with Th17 cell in peripheral blood of patients with rheumatoid arthritis
Objective: To find out the effectiveness of Th17 cell in the RA pathogenic mechanism.
Methods: 74 RA patients are collected and dived into two groups based on their disease activity. Normal controls are 82 samples. The levels of cytokines including IL-17、IL-23、IL-1β、IL-6, and TGFβ-1 in serum were measured by ELISA. The expression levels of these cytokines mRNA were evaluated by real-time quantitative PCR.
Results: Quantitative realtime PCR and ELISA analysis demonstrated elevated mRNA expression levels and protein concentrations of four cytokines including IL-17(41.33±1.40 vs 17.66±1.40pg/ml,P<0.001), IL-1β(23.65±2.89 vs 6.82±2.76 pg/ml,P<0.05)、IL-6(32.43±4.92 vs 3.37±0.70 pg/ml,P<0.001)and TGFβ-1(1117.04±232.25 vs 134.92±71.38 pg/ml,P<0.05). In addition, ELISA analysis showed that the concentration of IL-17(41.33±1.40 vs 27.54±2.81 pg/ml)、IL-1β(23.65±2.89 vs 20.68±5.61 pg/ml)and TGFβ-1(1117.04±232.25 vs 403.59±93.35 pg/ml)in the serum of active patients were much higher than that in stable patients(P<0.05).
Conclusions: As the main effect cytokine induced by Th17 cell, the level of IL-17 is remarkably elevated in active RA patients and correlated with disease activity. As IL-1β,IL-6 and TGFβ-1 can promote na?ve CD4+T cells to differentiate into Th17 cell, the levels of these cytokines were also elevated in RA patients. The function of IL-23 is to maintain the survival and expansion of Th17 cell, which did not depend on the dose of IL-23.
2. The proportion of Th17 cell in PBMC in RA patients and effects of different cytokine environments on Th17 cell differentiation
Objective: To investigate the proportion of CD4+IL-17+T cells in PBMC in RA patients and effects of different cytokine environments on Th17 cell differentiation.
Methods: 10 active RA patients, 8 stable RA patients and 10 normal controls were recruited for study. PBMC was isolated by Ficoll desity gradient centrifugation methods first, then the PBMC was stimulated 5 hours by phorbol ester and calcium ionophore. The cells were stained by CD4-FITC and anti IL-17-PE, and CD4+IL-17+T cell was detected by Flow Cytometry. Part of PBMC has been cultured in different cytokine environment: such as IL-1β+IL-6、IL-1β+IL23、IL-6+IL23、IL-1β+IL23+IL-6 for five days. Then the cells were stained by CD4-FITC and anti IL-17-PE and CD4+IL-17+T cell was detected by Flow Cytometry.
Results: The proportion of CD4+IL-17+ lymphocyte in the peripheral blood of active RA patients is higher than in stable RA patients and normal controls(0.52±0.04% vs 0.28±0.03% vs 0.06±0.02%,P<0.001).IL-1βand IL-6 are stronger stimulator for Th17 differentiation.
Conclusions: The proportion of CD4+IL-17+ lymphocyte in the peripheral blood of active RA patients is much higher. IL-1βand IL-6 are stronger stimulator for Th17 differentiation..
3. The effect of anti- TNF-αtherapy on the number and function of Th17 cell Objective: To analyze the effect of of anti- TNF-α(adalimumab)therapy on the number and function of Th17 cell.
Methods: seven patients with RA were recruited and administrated adalimumab 40mg once two weeks for more than six months. The proportion of Th17 cell and mRNA expression levels of cytokines including IL-17, IL-23, IL-1β, IL-6 and TGFβ-1 at the baseline and at 6 months were evaluated.
Results: A significant decrease in mRNA expression levels of IL-23(0.0873±0.01589 vs 0.0225±0.00445 ) ,IL-1β( 22.9281±3.95391 vs 0.7859±0.44970 ) and IL-6 (395.9832±110.8752 vs 102.2152±22.5869)was observed at 6 months after initial treatment of adalimumab(P<0.05). The expression levels of IL17 mRNA and TGFβ-1 mRNA did not show a significant change at 6 months after the initial injection compared with pretreatment levels, respectively(0.0061±0.00262 vs0.0038±0.00212,1.0547±0.33605 vs0.6669±0.10660,P>0.05).The flow cytometry. analysis also showed that the number of CD4+IL-17+ lymphocyte in the peripheral blood of RA patients did not decrease significantly after treatment(0.5286±0.00516% vs 0.4857±0.04592%,P>0.05). Conclusions: This study demonstrated that the reduction of IL-23 production in RA patients was a newly determined function of adalimumab. IL-17 production in RA patients in vivo may not depend on the direct stimulation by TNF-α. Combination use of TNF-αinhibitor and anti-IL17 may control the disease effectively in future.
引文
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