高迁移率族蛋白1及Toll样受体4在类风湿关节炎患者血清和外周血单个核细胞中的表达及意义

目的 通过研究高迁移率族蛋白(HMGB)1及Toll样受体(TLR)4在类风湿关节炎(RA)外周血单个核细胞(PBMC)和血清中的表达,探讨HMGB1的作用及其与疾病活动的关系.方法 选取活动期RA患者38例和非活动期RA患者36例及健康对照组26名.采用反转录-聚合酶链反应(RT-PCR)方法检测PBMC中HMGB1 mRNA的表达,酶联免疫吸附试验(ELISA)检测血清中HMGBI蛋白的表达.采用流式细胞术分析PBMC上TLR4的表达.结果 ①活动期RA组PBMC中HMGB1 mRNA相对表达量和血清中HMGB1蛋白水平均高于健康对照组和非活动期RA患者[分别为2.63与0.71.0.93和(10.2±1.2)与(7.5+1.8),(8.3±1.8)ng/ml,P＜0.01.②活动期RA患者CD14+单核细胞和CD3+淋巴细胞上TLR4蛋白相对表达量高于非活动期和健康对照组(P＜0.05或P＜0.01),非活动期患者高于健康对照组(P＜0.05或P＜0.01).③血清中HMGB1蛋白水平与红细胞沉降率(ESR)、C反应蛋白(CRP)、类风湿因子(RF)、关节压痛数、肿胀数及关节X线分期均呈正相关,亦与TLR4蛋白相对表达量呈正相关.结论 RA患者PBMC具有合成和分泌HMGB1蛋白的功能,并部分通过与TLR4结合激活炎症反应,加重骨质破坏.     

类风湿关节炎患者外周血单核细胞Toll样受体2的表达及意义

目的观察类风湿关节炎(RA)患者外周血单核细胞Toll样受体2(TLR2)的表达变化与疾病活动程度和临床指标的关系,探讨RA的发病机制。方法27例活动期RA患者、20例非活动期RA患者及18名正常对照,采用流式细胞术检测外周血单核细胞表面TLR2的表达,反转录聚合酶链反应(RT-PCR)检测细胞内TLR2 mRNA的表达。结果活动期RA患者外周血单核细胞TLR2的表达水平显著高于缓解期RA患者及正常对照,而缓解期RA患者TLR2与正常对照相比,差异无统计学意义。RA患者外周血单核细胞TLR2的表达与疾病活动评分(DAS)、血清C反应蛋白(CRP)及血沉(ESR)相关,而与类风湿因子(RF)及抗环瓜氨酸肽(抗CCP)抗体无明显相关性。结论活动期RA患者外周血单核细胞TLR2表达增高,且TLR2的表达与疾病活动指标密切相关。活动期RA患者存在着固有免疫系统的活化,TLR2的高表达可能促进了外周血单核细胞的活化。     

白细胞介素-18在类风湿关节炎外周血单个核细胞中的表达

目的　探讨类风湿关节炎 (RA)外周血白细胞介素 18(IL 18)的表达及其与Th1/Th2平衡和疾病活动的关系。方法　以 β actin为内参照 ,用半定量反转录多聚酶链反应 (RT PCR)法测定 16例RA患者及 15名正常对照外周血单个核细胞 (PBMC)中IL 18、IL 4、IFN γmRNA水平 ,观察IL 18与IL 4、IFN γ及疾病活动指标红细胞沉降率 (ESR)、C反应蛋白 (CRP)的相关性。结果　①RA患者PBMC中IL 18mRNA表达与正常组相比增高非常显著 (1 82± 0 39比 0 45± 0 30 ,P <0 0 0 1)。②RA组IFN γ、IL 4均显著高于对照组 ,但IL 4/IFN γ显著低于对照组。③相关分析显示 :RA组IL 18mRNA与IFN γ、ESR显著正相关 (16例 ,r分别为 0 836 ,0 75 3,P均 <0 0 5 )。结论　RA患者外周血IL 18mRNA表达水平显著高于正常对照组 ,且与RA患者外周血IFN γ的产生及疾病活动性相关。     

HLA-B27与强直性脊柱炎临床表现的关系

目的了解人类白细胞A系统(HLA)-B27在强直性脊柱炎(AS)诊断和病情活动中的作用.方法将2000～2002年病案资料中,38例HLA-B27阴性的AS患者与92例HLA-B27阳性患者进行临床表现和实验室及特殊检查结果分析. 结果 B27阴性组的严重的髋关节病变、窦缓和/或传导阻滞、合并类风湿性关节炎及其它合并症(颈椎病、骨关节炎、腰椎间盘突出症等)的比率均明显低于B27阳性组;疾病活动性指标如血沉(ESR)、C-反应蛋白(CRP)在B27阴性组明显低于B27阳性组.结论 B27阴性的AS患者病情较轻,预后良好,B27检查结果对于AS疑似患者有助于明确诊断.     

肝硬化患者外周血单核细胞内毒素受体表达与慢性炎症反应关系的临床意义

背景:内毒素受体在内毒素、细胞因子等介导的炎性信号转导过程中具有重要作用。目的:了解肝硬化患者外周血单核细胞(PBMC)内毒素受体的表达变化与慢性炎症反应关系的临床意义。方法:应用荧光素标记的抗人Toll样受体(TLR)4/抗人CD14单抗,以流式细胞术检测40例肝硬化患者和15名健康志愿者PBMC内毒素受体TLR4和CD14蛋白的表达;以逆转录聚合酶链反应(RT-PCR)检测内毒素受体TLR4 mRNA、CD14 mRNA和信号转导分子MyD88 mRNA的表达;以酶联免疫吸附测定(ELISA)检测血清白细胞介素(IL)-1β、IL-10和肿瘤坏死因子(TNF)-α水平;以偶氮基质显色法测定血浆内毒素水平。结果:肝硬化患者PBMC内毒素受体TLR4和CD14蛋白的表达水平显著高于对照组(P<0.05),但不同Child-Pugh分级组间差异无统计学意义;与对照组相比,PBMC内毒素受体TLR4 mRNA、CD14 mRNA和信号转导分子MyD88 mRNA的表达水平也有增高的趋势,但差异无统计学意义(P>0.05);肝硬化患者血清细胞因子IL-1β、IL-10和TNF-α水平,以及血浆内毒素水平均较对照组显著升高(P<0.05),且高内毒素水平患者内毒素受体的表达显著高于低内毒素水平患者(P<0.05),细胞因子水平也较低内毒素水平者增高。结论:肝硬化患者PBMC内毒素受体的表达明显上调,与患者的慢性炎症反应状态一致。     

实时荧光定量聚合酶链反应检测外周血趋化因子受体3 mRNA对类风湿关节炎的临床意义

目的 检测趋化因子受体3(CXCR3)在类风湿关节炎(RA)患者外周血单一核细胞(PBNC)中的表达,并分析其与疾病活动性的相关性.方法 研究建立实时荧光定量(RFQ)-聚合酶链反应(PCR)法测定外周血单个核细胞CXCR3 mRNA含量的方法;检测CXCR3在51例RA患者PBMC中的表达水平,并和32名健康人群进行对照.计量资料采用t检验、单因素方差分析,计数资料采用x2检验或Fisher's精确概率法.结果 RA患者在外周血的CXCR3 mRNA的表达水平明显高于健康对照组(8.8±1.5和7.3±1.9,P<0.05).在活动期与缓解期RA组的比较中发现活动期RA患者CXCR3 mRNA的表达水平也明显高于缓解期RA患者(9.4±1.0和8.0±1.6,P<0.05).而且活动期RA患者中外周血CXCR3 mRNA 表达水平与红细胞沉降率(ESR)、C反应蛋白(CRP)有密切的相关性,且存在正相关关系(r=0.824和0.765,P<0.05).RA患者中外周血CXCR3 mRNA表达水平与类风湿因子(RF)、抗角蛋白抗体(AKA)、抗核周因子(APF)抗体及抗环瓜氨酸多肽(CCP)抗体无显著相关性(P>0.05).结论 RFQ-PCR检测CXCR3 mRNA含量的方法具有较好的灵敏度和可重复性;RA患者CXCR3 mRNA表达含量升高,提示CXCR3可能参与了RA的发病过程.活动期RA患者CXCR3水平明显升高,并且和ESR、CRP呈正相关,提示CXCR3可作为观察病情活动性的指标和疗效、评价预后的指标.     

类风湿关节炎外周血单个核细胞和滑膜中人类软骨糖蛋白-39的研究

目的　通过观察人类软骨糖蛋白 39(HCgp 39)mRNA在类风湿关节炎 (RA)外周血单个核细胞 (PBMC)和滑膜中的表达水平 ,寻找反映RA早期骨破坏的敏感指标。方法　采集 31例RA、6例骨关节炎 (OA)、10例血清阴性脊柱关节病 (SpA)、5例系统性红斑狼疮 (SLE)以及 10例健康人新鲜抗凝静脉血 ,分离单个核细胞 ,并采集 7例RA、5例OA的滑膜 ,行半定量逆转录 (RT) PCR。结果　RA患者PBMC中HCgp 39mRNA行RT PCR与内对照tubulin共扩增后吸光度比值为0 86 90± 0 5 2 4 0 ,与OA(P =0 0 2 4 )、SpA(P =0 0 4 9)、SLE(P =0 0 4 3)以及健康对照 (P =0 0 33)相比差异均有显著性。而OA、SpA、SLE与健康对照之间差异无显著性。RA滑膜HCgp 39mRNA行RT PCR与tubulin共扩增后吸光度比值为 1 986 3± 1 9397,与OA相比差异有显著性 (P =0 0 4 )。在RA和OA中 ,同一患者的PBMC与滑膜HCgp 39mRNA表达差异无显著性。 结论　HCgp 39mRNA在RA患者PBMC和滑膜的表达明显高于其他炎症性关节病 ,提示HCgp 39可能是RA发病机制中重要的自身抗原 ,它的异常表达可能是造成T细胞介导自身免疫反应的机制之一。     

强直性脊柱炎患者血清ASO检测及临床意义

目的探讨强直性脊柱炎（AS）患者血清中抗链球菌溶血素O（ASO）的临床意义。方法采用胶乳凝集法测定159例AS患者血清中ASO水平,检测ASO阳性（〉200IU／ml）者（67例）和阴性（≤200IU／ml）者（92例）的白细胞抗原-B27（HLA-B27）、血沉（ESR）、C-反应蛋白（CRP）等实验室指标并记录体温,观察临床症状、体征及影像学分级。结果ASO阳性者发热、ESR、外周关节疼痛、“4”字试验及Schober试验阳性的发生率均显著高于阴性者（P均〈0．05）。影像学表现：骶髂关节炎Ⅱ级和Ⅲ级者明显高于阴性者（P〈0．05）。结论溶血性链球菌感染在AS病情发展中起重要作用,ASO可作为判断AS严重程度和病情活动的重要指标。     

天然免疫分子TLR2 TLR4在类风湿关节炎的表达模式研究

目的 探讨天然免疫分子TLR2、TLR4在类风湿关节炎(RA)发病中的作用。方法 采用多色免疫荧光流式细胞仪技术，观察RA患者和正常对照外周血CD14阳性单核细胞上TLR2、TLR4分子的表达率和平均荧光强度。结果 ①TLR2和TLR4在RA患者外周血CDl4阳性单核细胞阳性率和平均荧光强度显著高于对照组；②二者表达模式在早期RA组和晚期RA组差异无统计学意义：③TLR2平均荧光强度与C反应蛋白(CRP)水平有关联。结论天然免疫分子TLR2、TLR4在RA的表达模式，提示其在RA发病和病情进展中有重要作用。     

流式细胞术分析外周血辅助性T17细胞在强直性脊柱炎患者外周血的表达及其意义

目的 检测强直性脊柱炎(AS)患者外周血辅助性T17(Th17)细胞的表达,探讨其在AS发病机制中的作用.方法 用流式细胞术检测20例AS患者和15名健康对照者外周血Th17[白细胞介素(IL)-17]、Th1[干扰素(IFN)-γ]、Th2(IL-4)细胞及人类白细胞抗原(HLA)-B27,同时检测红细胞沉降率(ESR)、C反应蛋白(CRp)等,并分析Th17细胞与Th1、Th2、HLA-B27及AS活动性指标的相关性.采用t检验和Spearman相关分析.结果 AS患者外周血Th17细胞[(2.6±0.8)%]高于对照组[(1.1±0.4)%](P＜0.01);Th1(IFN-γ)细胞[(3.9±0.8)%]低于对照组[(5.1±1.3)%](P＜0.05);Th2(IL-4)细胞[(4.1±1.6)%]比对照组[(3.1±1.4)%]升高,但差异无统计学意义(P＞0.05);Th17细胞与HLA-B27百分数及与HLA-B27平均荧光强度、ESR、CRP无相关性(P＞0.05);但是,Th17细胞表达水平有随HLA-B27百分数及HLA-B27平均荧光强度增加升高的趋势.结论 AS患者外周血Th1细胞减少,而Th17细胞增加,AS患者体内存在Th细胞的失衡,Th17比例变化是反映AS整个复杂免疫反应异常的一个重要环节.

Abstract：

Objective To detect the expression of T helper 17 (Th17) cells in the peripheral blood of patient with ankylosing spondylitis (AS),and discuss its role in thc pathogenesis of AS.Methods Twenty AS patients and fifteen healthy controls were enrolled in the study.Th17 (IL-17),Th1 (IFN-γ),Th2 (IL-4)cells and HLA-B27 of their peripheral blood were analyzed by flow cytometry,at the same time,the levels of ESR and CRP were also measured in order to analyze thc relation of Th17 and HLA-B27,ESR as well as the CRP level.The statistical analysis was carried out with single-sample t-test and Speraman's correlation test.Results The level of Th17 cells was significantly higher in the perpipheral blood of AS patients[(2.6±0.8 )%] than those in healthy controls[ (1.1±0.4)% ] (P＜0.01).The level of Th 1 cells was significantly lower in the peripheral blood of AS patients[(3.9±0.8)%] than those in healthy controls[(5.1±1.3)%] (P＜0.01)and the level of Th2 cells was not different in the peripheral blood of AS patients[(4.1±1.6)%] when compared to healthy controls[ (3.1±1.4)% ] (P＞0.05).Th 17 cells was not significantly correlated with the percentage and mean fluorescent intensity of HLA-B27,ESR,CRP(P＞0.05); but there was a tendency that increased expression level of Th17 cells was associated with elevated percentage and mean fluorescent intensity of HLA-B27.Conclusion The level of Th1 cells is decreased,but Th17 is increased in the peripheral blood of AS patients.Th cells are imbalance in AS,patients.The change of Th17 cells may be an important part of the pathogenesis of AS.     

类风湿关节炎患者外周血高迁移率族蛋白1表达

目的通过检测类风湿关节炎（RA）患者外周血单个核细胞（PBMC）、血浆中高迁移率族蛋白1（HMGB1）表达,为寻找治疗RA的新靶点提供依据。方法采集38例活动期RA患者、24例相对稳定期RA患者和20例健康对照者外周血。RT．PCR检测PBMC HMGB1mRNA表达,Western blot检测PBMC、血浆HMGB1蛋白表达。结果与相对稳定期RA患者、健康对照者相比,活动期RA患者PBMC HMGB1mRNA表达水平差异无统计学意义（F=1．23,P〉0．05）,而HMGB1蛋白表达水平下降（F=70．91,P〈0．01）,血浆HMGB1水平显著增高（P〈0．001）。相对稳定期RA患者与健康对照者之间差异无统计学意义（P〉0．05）。活动期RA患者血浆HMGB1水平与ESR（r．=0．478。P〈0．001）、C-反应蛋白（rs=0．574,P〈0．05）呈正相关。结论HMGB1与RA发病密切相关,并可能成为新的治疗靶点。     

Prevalence of hepatitis B surface antigen in patients with ankylosing spondylitis and its association with HLA-B27: a retrospective study from south China

To investigate the prevalence of hepatitis B surface antigen (HBsAg), a seromarker for current infection of hepatitis B virus, in patients with ankylosing spondylitis (AS) from south China and to evaluate its association with human leukocyte antigen (HLA)-B27. The prevalence of HBsAg was retrospectively investigated in 439 patients with AS, 606 age- and sex-matched general individuals, 172 patients with other spondyloarthropathy (SpA), 698 patients with rheumatoid arthritis (RA), and 220 patients with osteoarthritis (OA). The positive rate of HBsAg in AS group was compared with those of the general population group and other disease groups, respectively, and the prevalence of HBsAg was compared between HLA-B27-positive and HLA-B27-negative patients with AS. The positive rate of HBsAg in AS patients, general population, other-SpA, RA, and OA patients were 25.39, 12.87, 14.53, 9.60, and 8.18%, respectively. The HBsAg prevalence of AS group was statistically higher than those of any other groups (P?<?0.05). The prevalence of HBsAg in HLA-B27-positive and HLA-B27-negative AS patients were 26.68 and 14.49%, respectively, the positive rate of HBsAg in HLA-B27-positive AS patients was statistically higher than that of HLA-B27-negative AS patients (P?<?0.05). The prevalence of HBsAg in AS patients was higher than those in general population, patients with other-SpA, RA, and OA. The high HBsAg prevalence in AS patients might be associated with their high frequency of HLA-B27 gene.     

TLR4 mutations (Asp299Gly and Thr399Ile) are not associated with ankylosing spondylitis

BACKGROUND: Immunoregulatory genes and Gram negative gut bacteria are thought to be important in disease expression in ankylosing spondylitis (AS). OBJECTIVE: To compare the frequency of two common and functional TLR4 mutations (Asp299Gly, and Thr399Ile) between patients with AS and HLA-B27 healthy controls. METHODS: The TLR4 genotypes of patients and healthy HLA-B27 controls were determined using allele-specific PCR and restriction fragment length polymorphism analysis. Asp299Gly genotype was determined in 193 patients and 125 HLA-B27 positive controls and Thr399Ile genotype in 184 patients and 113 HLA-B27 controls. Allele frequencies were compared using a chi(2) test of association. RESULTS: 29/193 (15%) patients with AS had a polymorphism in the Asp299 site compared with 18/125 (14.4%) healthy HLA-B27 controls. Of the patients genotyped for the Thr399Ile allele, 29/184 (15.8%) carried the polymorphism compared with 19/113 (16.8%) HLA-B27 controls. No significant difference between the frequencies of the Asp299Gly genotype or the Thr399Ile genotype between patients with AS and healthy HLA-B27 controls was found. No significant difference in allele frequency was found at either site. CONCLUSION: Two common TLR4 polymorphisms, which cause a functional deficiency in host immune response to Gram negative bacteria, are not overrepresented in patients with AS.     

强直性脊柱炎患者外周血CD4^＋CD25^＋CD127^low/-T细胞检测及其意义△

目的检测CD4^＋CD25^＋CD127^low/-T细胞在强直性脊柱炎（ankylosing spondylitis，AS）患者外周血的表达，并分析其与CD4^＋CD25^＋FOXP3^＋T细胞的相关性。方法 以21例AS患者外周血为研究组，20例类风湿关节炎（rheumatoid arthritis，RA）患者和24名正常人外周血作为对照组，采用三色直接荧光素标记法和多参数流式细胞仪检测外周血CD4^＋CD25^＋CD127^low/-T细胞、CD4^＋CD25^high细胞及CD4^＋CD25^＋FOXP3^＋T细胞的比例，同时检测外周血C反应蛋白（C-reactive protein，CRP）、血沉（ESR）、免疫球蛋白、补体等水平以及HLA—B27并进行相关性分析。结果与正常对照组相比，AS患者外周血CD4^＋CD25^＋CD127^low/-T及CD4^＋CD25^＋FOXP3＋T细胞百分率降低（均P〈0．05）。CD4^＋CD25^＋细胞百分率升高（P〈0．05）；CD4^＋CD25^＋CD127^low/-T细胞与CD4^＋CD25^＋FOXP3^＋T细胞呈正相关（r=0．589，P=0．021），与CRP、ESR、血小板及免疫球蛋白（IgG、IgA、IgM）和补体（C3、C4）、BASDAI、HLA-B27等均无明显相关性（均P〉0．05）。AS患者外周血CD4^＋CD25^＋CD127^low/-T细胞、CD4^＋CD25^＋FOXP3^＋T细胞和CD4＋CD25^highT细胞的百分率与RA患者外周血比较差异均无统计学意义（P〉0．05）。结论AS患者外周血CD4^＋CD25^＋CD127^low/-T细胞降低，且与CD4^＋CD25^＋FOXP3^＋T细胞呈正相关，提示在调节性T细胞研究中CD127具有替代FOXP3的可能性。     

HLA-B60 and B61 are strongly associated with ankylosing spondylitis in HLA-B27-negative Taiwan Chinese patients

OBJECTIVES: Carriage of HLA-B60 has been shown to increase the risk of ankylosing spondylitis (AS) in B27-positive Caucasian patients, but the association in B27-negative cases is less certain. This study assessed HLA class I gene associations in Chinese HLA-B27-negative AS patients. METHODS: Forty-one Chinese HLA-B27-negative AS patients fulfilling the modified New York diagnostic criteria for AS were recruited, and 11 383 HLA-B27-negative blood donors were used for comparison. HLA-A and -B typing was done with the microlymphocytotoxicity assay. RESULTS: Among the B27-negative AS patients, 21 were male and 20 were female. Of HLA-B alleles, only B60 and B61 significantly increased susceptibility to AS in HLA-B27-negative patients (P<0.001). CONCLUSIONS: In Taiwan Chinese, carriage of B60 is increased in HLA-B27-negative AS patients. The association between B61 and HLA-B27-negative AS patients has not been reported previously. Whether the gene involved is HLA-B60 or B61 or another gene in linkage disequilibrium with these genes is unknown.     

活化的类风湿关节炎患者外周血单个核细胞Toll样受体4诱导Th17细胞分化

目的 研究类风湿关节炎(RA)患者外周血单个核细胞(PBMCs) Toll样受体4(TLR4)通过诱导Th17细胞分化参与RA疾病发生发展的意义.方法 采集22例RA患者和20名健康对照外周血,流式细胞术检测Th17细胞频率,脂多糖刺激PBMCs 2 d,实时荧光定量聚合酶链反应(RT-qPCR)检测PBMCs TLR4 mRNA水平,酶联免疫吸附试验(ELISA)法检测上清液白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α的含量.收集上清液与CD4+脐带血单个核细胞(CBMCs)培养3d,RT-qPCR检测CBMCsIL-17 mRNA水平.ELISA检测上清液IL-17含量.统计学处理采用成组t检验.结果 与健康对照组比较,RA患者Th17细胞频率[(0.53±0.14)％与(1.57±0.68)％]、TLR4 mRNA显著升高(P均＜0.01);PBMCs经脂多糖刺激后,RA患者TLR4 mRNA升高3.5倍,而健康对照组下降到0.11倍;RA患者PBMCs产生的IL-6、TNF-α较健康对照组明显增加(P＜0.01);RA患者PBMCs上清液诱导CD4+CBMCs IL-17 mRNA水平、IL-17含量与健康对照组相比均显著升高(P＜0.01);而未经脂多糖刺激,2组差异无统计学意义(P＞0.05).结论 RA患者PBMCs的TLR4表达及对脂多糖的刺激的反应性增加,且具有较强的诱导Th17细胞分化能力.     

Methotrexate attenuates the Th17/IL-17 levels in peripheral blood mononuclear cells from healthy individuals and RA patients

To investigate whether the inhibition of Th17/interleukin (IL)-17 contributes to the beneficial effects of methotrexate (MTX) in the treatment of rheumatoid arthritis (RA). Peripheral blood mononuclear cells (PBMCs) from healthy donors and RA patients were collected. The cells were stimulated with monoclonal antibodies to CD3 and CD28 in the absence or presence of MTX. After coincubation, IL-17 production was detected at both the mRNA and protein levels, and the percentage of cells positive for both CD4 and IL-17 in PBMCs was analyzed by flow cytometry. PBMCs of healthy donors and RA patients were stimulated with CD3 and CD28 monoclonal antibodies to produce high levels of IL-17. The augmentation of IL-17 at the mRNA and protein levels was significantly inhibited when PBMC cultures were preincubated with MTX. Compared with PBMCs of healthy donors, PBMCs of RA patients produced higher levels of IL-17, and this increase in IL-17 levels was more inhibited by MTX pretreatment. MTX inhibited IL-17 at the mRNA level in a dose-dependent manner, but not at the protein level, in both PBMCs of healthy donors and RA patients. MTX did not affect the percentage of CD4- and IL-17-positive cells in PBMCs. MTX dose dependently suppressed the production of IL-17 at the mRNA level by PBMCs from healthy donors and RA patients. Suppression of IL-17 by MTX may contribute to its potent anti-inflammatory role in RA therapy.