哮喘患者外周血单个核细胞不同细胞亚群上Notch分子的表达

支气管哮喘是一种由多种炎症细胞和炎症介质参与的慢性气道炎症性疾病,其中肥大细胞、嗜酸性粒细胞、T淋巴细胞为主要效应细胞[1].哮喘的发病机制迄今尚不完全明了,研究发现免疫功能异常是重要的致病因素[2].近来研究表明,Notch信号通路在中枢和外周免疫系统中各种细胞发育成熟的不同阶段,尤其是外周T细胞的分化过程中发挥重要作用[3],推测其可能与哮喘中细胞免疫功能异常有关.因此本研究应用实时荧光定量PCR和流式细胞染色的方法,对哮喘患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)不同细胞亚群上Notch分子的表达进行分析,以期为哮喘的发病的分子机制提供新的思路.     

Notch信号通路在活动性类风湿关节炎中的作用

Notch信号通路在细胞发育、分化、凋亡等方面扮演重要角色,对免疫疾病也发挥广泛的调控作用。类风湿关节炎(RA)的病理特征包括滑膜炎和血管翳,反复发作的活动性RA可致成、破骨细胞失衡,形成骨破坏。研究发现Notch信号在活动性RA中影响滑膜样成纤维细胞增殖与迁移、巨噬细胞释放炎症因子、血管新生、骨破坏等多项病理环节,是RA治疗的重要靶点。综述近二十年Notch信号通路在活动性RA中作用的研究进展。     

类风湿关节炎患者外周血单个核细胞中PADI4 mRNA的表达及其意义

目的检测类风湿关节炎(RA)患者外周血单个核细胞(PBMCs)肽酰基精氨酸脱亚氨酶4(PADI4)mRNA的表达,分析其与RA患者的临床指标的相关性,探讨PADI4在RA发病机制中的作用及意义。方法实时定量PCR检测RA组(60例)、正常对照组(40例)PBMCs中PADI4 mRNA表达,并分析其与抗环瓜氨酸肽(CCP)抗体、疾病活动指数DAS28评分、C反应蛋白(CRP)、红细胞沉降率(ESR)、类风湿因子(RF)及病程等指标的关系。结果 RA组PADI4 mRNA相对表达[34.6(16.7,70.8)],明显高于正常对照组[20.6(11.1,51.8)](P<0.05)。RA组中PADl4 mRNA表达与抗CCP抗体、DAS28评分水平、ESR、RF呈正相关(r=0.527,P<0.001;r=0.416,P=0.001;r=0.371,P=0.004;r=0.287,P=0.030),与CRP、病程无相关性(r=0.015,P=0.919;r=0.064,P=0.625)。结论 RA病人外周血PBMCs PADI4 mRNA表达显著增高,并与抗CCP抗体水平、DAS28评分、ESR、RF呈正相关,可能是RA病情活动的一个有用的指标,PADI4可能在RA的发病和病理过程中起重要作用。     

类风湿关节炎患者外周血单个核细胞Sonic Hedgehog信号通路表达的初步研究

目的: 初步探讨类风湿关节炎(RA)患者外周血单个核细胞(PBMCs)和滑膜组织中Sonic Hedgehog(Shh)信号通路相关因子表达及意义。方法: 收集符合1987年美国风湿病学会(ACR)RA分类标准、28个关节疾病活动度评分(DAS28)≥3.2,病情活动RA患者(35例)及年龄、性别相匹配的健康志愿者(35例)外周血2 mL,分离PBMCs,提取总RNA,采用实时荧光定量PCR(real-time PCR)检测Shh信号通路中信号肽Shh、膜受体Ptch1和核转录因子Gli1 mRNA的表达。收集10例病情中度活动(DAS28≥3.2)RA患者的滑膜组织,同时收集5例外伤或半月板损伤(无关节炎)者滑膜组织作为对照组,免疫组化检测Shh、Ptch1和Gli1的蛋白表达情况。结果: RA患者PBMCs中Shh和Gli1 mRNA的相对表达量分别为1.36±1.48和1.15±0.68,对照组上述信号分子的mRNA表达量分别为0.47±0.25和0.49±0.05,2组差异有统计学意义(P<0.05),Ptch1 mRNA表达在2组间的差异无统计学意义(P>0.05);免疫组化示Shh和Gli1蛋白表达的阳性细胞百分率均高于对照组(P<0.05),Ptch1蛋白表达阳性细胞百分率2组无显著差异(P>0.05)。结论: RA患者PBMCs与滑膜组织中检测到Shh通路相关信号分子Shh和Gli1的表达上调,提示RA患者中可能存在Shh信号通路的激活,其在RA发病机制中的作用值得进一步研究。     

外周血单个核细胞中mRNA表达与类风湿关节炎活动度的相关性研究

目的 以28个关节疾病活动度评分(disease activity score in 28 joints,DAS28)为基础对不同活动度群组的类风湿关节炎(rheumatoid arthritis, RA)病人和健康人群进行分析,挑选出RA活动度相关基因,为RA的诊断治疗提供新思路与线索。方法 收集苏州大学附属第一医院风湿免疫科门诊28个不同活动度的RA病人和18个健康人的基本信息并对它们外周血单个核细胞中mRNA水平进行检测,通过生物信息学分析寻找活动度相关基因。结果 筛选出80个单纯与RA活动度相关的基因,36个与RA活动度和发病都有关系的基因。在这些基因中,12个单纯与活动度相关的基因(SOCS3、C12orf44、KIFC1、MRPS18A、GYG1、RAB35、B4GALT5、LRRC41、LILRB4、LILRA5、CR1、PDIA6)与DAS28显著相关(r值分别为0.55、0.53、0.52、0.49、0.44、0.42、0.42、0.41、0.41、0.41、0.40、0.39,P值均<0.05)且呈趋势性下降,与发病和活动度都与相关基因PPIH与DAS28显著相关(r=-0.38,P<0.05)且呈趋势性上升。生物功能聚类发现有四项功能和甲基化相关,一项功能和应激反应相关。蛋白质关联分析显示HIST1H3E、HIST1H4I、HIST1H4F、HIST1H3D和HIST1H3F这五个基因不仅出现在与甲基化相关的功能中,而且在实验、文本挖掘和共表达三个方面都存在相互关系。结论 以DAS28为基础挑选出80个单纯与RA活动度相关的基因,功能分析表明他们大多受到甲基化调控作用,部分与免疫应激反应有关。     

类风湿关节炎患者单个核细胞IL-23的表达及意义

目的:通过分析比较类风湿关节炎(Rheumatoid Arthritis,RA)患者血清和外周血单个核细胞(Peripheral bloodmononuclear cell,PBMC)IL-23的表达,探讨其在RA发病过程中的作用,以期为治疗开辟新途径。方法:应用ELISA方法检测RA、骨性关节炎(Osteoarthritis,OA)及正常对照组血清中IL-23和TNFα-蛋白水平,采用RT-PCR法测定各组PBMC中IL-23p19mRNA的表达,并作血清IL-23含量、PBMC IL-23p19mRNA的表达与血清TNFα-水平的相关分析。结果:RA组血清IL-23水平高于正常对照组(P=0.001),其他组间无统计学差异(分别为P=0.122,P=0.127);而各组血清TNFα-的水平均具有统计学差异,RA和OA组均高于对照组(P=0.000,P=0.042),RA组也高于OA组(P=0.013);RA组IL-23p19mRNA的表达高于OA和正常对照组(P=0.000,P=0.000),而OA组与正常对照组差异无统计学意义(P=0.628);血清中IL-23和TNFα-的相关性无统计学差异(r=0.212,P=0.262),而PBMC中IL-23p19mRNA的表达与血清TNFα-呈正相关关系(r=0.392,P=0.032)。结论:IL-23在RA患者PBMC中高表达,在RA的炎性发展过程起到了重要作用。     

泌乳素促进类风湿关节炎患者外周血单个核细胞分泌白细胞介素-6

目的:探讨类风湿关节炎(RA)患者血清泌乳素(PRL)水平与疾病活动程度的关系,以及PRL促进外周血单个核细胞(PBMCs)分泌白细胞介素-6(IL-6)的机制。方法:收集我院2015年3月至9月40例初治RA患者临床及实验室资料。采用化学发光免疫分析法(CLIA)检测血清PRL水平,ELISA检测IL-6水平,RT-q PCR检测泌乳素受体(PRLR)mRNA的表达,Western blot法检测MAPK通路相关蛋白p-p38的蛋白水平。结果:RA患者血清PRL水平明显升高(P0.01),活动期RA患者PRL水平明显高于非活动期RA患者(P0.01)。PRL水平与DAS28评分、ESR和CRP呈正相关(P0.01)。RA患者PBMCs中PRLR水平明显升高(P0.01)。PRL可诱导PBMCs分泌IL-6,siRNA沉默PRLR或采用MAPK通路抑制剂可抑制IL-6的产生。结论:RA患者血清PRL升高与DAS28评分、ESR和CRP呈正相关,PRL可作为预测RA严重程度的指标。PRL通过与PRLR相互作用,激活p38 MAPK通路,从而促进IL-6分泌。     

类风湿关节炎患者外周血淋巴细胞的凋亡及其意义

目的研究类风湿关节炎(RA)患者外周血淋巴细胞的凋亡及其意义。方法采用流式细胞术和激光共聚焦显微镜,检测35例RA患者和30例健康志愿者的外周血淋巴细胞抵抗凋亡的能力,包括分析新鲜分离和培养的淋巴细胞凋亡率及其与临床病情严重度的相关性。结果两组间新鲜分离的淋巴细胞凋亡率差异无统计学意义[(2.63±1.47)%比(3.53±2.04)%,P>0.05]。活化淋巴细胞检测的结果显示,RA组的凋亡率明显低于对照组[(12.08±6.06)%比(18.66±7.27)%,P<0.05],并与DAS28评分呈负相关(r=-0.617,P=0.008)。结论RA患者活化的外周血淋巴细胞出现凋亡缺陷,并与RA病情活动度相关。提示这些细胞募集到组织损害和自身免疫炎性反应进程异常的部位并对炎性反应进程的发展和维持起重要作用。     

类风湿关节炎患者外周血单个核细胞中TRAIL、c-FLIP 等表达水平及临床意义

目的:分析类风湿关节炎(RA)患者外周血单个核细胞中肿瘤坏死因子相关凋亡诱导配体TRAIL、c-FLIP、Caspase-8等基因表达情况,探讨其在RA中的临床意义,为RA的临床诊治寻找更有效的途径和方法提供实验基础。方法:采用实时荧光定量PCR的方法检测外周血单个核细胞肿瘤坏死因子相关凋亡诱导配体TRAIL、c-FLIP、caspase-8 mRNA 表达水平;采用蛋白免疫印迹法检测PBMC 中TRAIL、c-FLIP、caspase-8 蛋白表达水平。结果:各组RA 患者PBMC 的TRAIL、c-FLIP、Caspase-8 的mRNA 表达水平:中活动组(M)中TRAIL的mRNA为(3.26±0.78),高于健康对照(N)(1.30±0.20,P=0.028)。低活动组(L)和高活动组(H)(1.56±0.37、1.83±0.26),略高于健康对照(N)(P=0.048、0.043);L、M、H 组c-FLIP mRNA相对表达量分别为1.27±0.28,1.32±.34,1.93±0.40,均高于N组1.08±0.12(P 分别为0.035、0.034、0.030),差异有统计学意义;caspase-8 mRNA L、M、H 组中分别为(2.77±0.97)、(4.52±0.85)、(2.13±0.44),均高于N 组(1.04±0.13,P=0.023,0.012,0.032)。RA 患者PBMC 中TRAIL、c-FLIP、caspase-8 蛋白表达:M 组TRAIL 蛋白表达明显高于N 组(P=0.013 ),H 组稍高于N 组(P=0.043),L组与N组无明显统计学差异(P=0.058),M 组明显高于L、H 组(P=0.011,0.021);c=FLIP 蛋白表达中M组明显高于N 组(P<0.01),而L、H 组与N 组无显著差异(P =0.062,0.053);L 组表达与N 组无显著差异(P>0.05),而M、H组caspase-8 蛋白表达明显高于N 组(P=0.003,0.001)。结论:在RA 的不同活动期中,外周血单个核细胞TRAIL、c-FLIP 和Caspase-8 等表达水平总体趋势增加,可为RA 的临床诊治及后续研究提供实验参考。     

外周血单个核细胞CCL5表达水平作为类风湿关节炎与骨性关节炎潜在鉴别诊断标志物的研究

目的 探讨类风湿关节炎(RA)与骨性关节炎(OA)患者外周血单个核细胞(PMBC)中C-C模式趋化因子配体5(CCL5)表达水平差异,以及其作为类风湿关节炎与骨性关节炎潜在鉴别诊断标志物的可能性。方法 收集临床RA与OA患者外周血样本,以健康人做对照,提取PMBC中mRNA,利用Q-PCR方法检测CCL5表达水平。比较RA、OA患者PMBC中CCL5表达差异,并对比类风湿因子(RF)与抗环瓜氨酸肽抗体(ACPA)在RA与OA鉴别诊断中的作用,利用ROC曲线明确CCL5表达水平对RA与OA的区分情况,对CCL5表达水平与RA严重程度进行相关性分析。结果 收集RA与OA患者外周血样本各30例,RA患者PMBC中CCL5表达水平显著高于OA患者(P<0.05),其在RA与OA鉴别诊断中的灵敏度、特异性、阳性预测值、阴性预测均高于RF,ROC曲线表明,CCL5表达水平可以有效区分RA与OA患者(AUC=0.823),RA患者CCL5表达水平与TJC(关节压痛指数)、SJC(肿胀关节指数)、DAS28评分呈显著正相关(P<0.05),其相关系数分别为0.60、0.75、0.79。结论...     

Rheumatoid factor production by mononuclear cells derived from different sites of patients with rheumatoid arthritis.

To investigate the origin of circulating rheumatoid factor (RF) and the relation between RF production at different sites in patients with rheumatoid arthritis (RA), mononuclear cells derived from bone marrow, synovium and peripheral blood of patients with RA were examined for the presence of plasma cells and for their capacity to produce RF and other immunoglobulins in vitro. Analysis of culture supernatants for the presence of immunoglobulins demonstrated that cells derived from bone marrow, synovium and peripheral blood were all found to be capable of producing every immunoglobulin and RF isotype investigated. No significant correlations were found between concentrations of immunoglobulin isotypes produced by cells derived from different sites of one individual. Significant correlations were found, however, between concentrations of RF isotypes produced by cells derived from the three sites. These results indicate that the production of RF in the different compartments is not an autonomously regulated process. Mononuclear cells derived from bone marrow were found to be able to produce RF in similar quantities to cells dissociated from synovial tissue. In combination with the fact that circulating immunoglobulins are produced mainly in the bone marrow, this observation suggests that bone marrow is also a major source of circulating RF.     

Regulation of IL-2 production by mononuclear cells from rheumatoid arthritis synovial fluids.

Products of polyamine oxidation down-regulate IL-2 production by peripheral blood T cells. We show here that the production of IL-2 by rheumatoid arthritis synovial fluid mononuclear cells is inversely correlated with the concentrations of polyamines in these cells. In addition, the inhibition of polyamine biosynthesis or oxidation in cultures of these cells enhances their ability to produce IL-2. Our findings suggest that polyamine oxidation plays an important role in the suppression of T cell function characteristic of rheumatoid arthritis synovial fluids.     

Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic,pathogenic and treatment features

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared-epitope, HLA-SE), autoimmune response [anti-cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS-28) and treatment (disease-modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3-labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3-monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA-SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety-one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti-CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty-eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.     

Mucin from rheumatoid arthritis synovial fluid enhances interleukin-6 production by human peripheral blood mononuclear cells

The carbohydrate chains represented by mucins (MUCs) are expressed by a variety of normal and malignant secretory epithelial cells and induce a variety of immunoreactions. To find new mucins related to the pathogenesis of rheumatoid arthritis (RA), we examined high-molecular-weight molecules inducing cytokines on human peripheral blood mononuclear cells (PBMCs) in synovial fluid from affected joints. We found a high-molecular-weight substance that induces interleukin 6 production on PBMCs in RA synovial fluid on gel filtration. MUC-1 was present in the resulting fractions, although they had been purified by CsCl density gradient centrifugation. We also found that MUC-1 was expressed on synovial cells and infiltrating inflammatory mononuclear cells on the sublining layer and lymphoid follicles in RA synovial tissues. CD68-positive superficial synovial cells colocalized with MUC-1 and CD68-positive macrophages were in contact with MUC-1-positive mononuclear cells. These findings imply that mucins, including MUC-1, may be related to immunoinflammatory reactions in the pathogenesis of RA.     

类风湿性关节炎患者外周血TH1/TH2细胞的研究

目的探讨CD4+TH1/TH2细胞在类风湿性关节炎(RA)发生发展过程中的作用.方法采用酶联免疫斑点法(ELISPOT)对15例RA患者和30例健康人外周血中T淋巴细胞亚群及CD4+TH1/TH2功能亚型进行检测.结果RA患者外周血中TH1细胞的百分率较正常对照组升高(P＜0.05).结论 TH1细胞介导的细胞免疫可能与RA的发生发展有关.     

Interleukin 4 inhibits polyclonal immunoglobulin secretion and cytokine production by peripheral blood mononuclear cells from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is associated with T- and B-cell dysfunction. Immunoglobulin (Ig) production is under the control of T cells and their derived cytokines such as interleukin 2 (IL-2) and IL-4. Herein we studied the regulation of the production of immunoglobulins and cytokines by peripheral blood mononuclear cells from RA patients and controls. In the controls, IL-4 inhibited Ig production in response toStaphylococcus aureus and pokeweed mitogen stimulation. IL-2 induced maximal Ig production in association withStaphylococcus aureus, whereas it inhibited pokeweed mitogen-induced production. In patients, levels of Ig production in response to mitogens and cytokines were reduced, particularly for the response to IL 2. The inhibitory effect of IL-4 on mitogen-induced Ig production was observed in RA patients as in the controls. Spontaneous production of IL-6 was increased in RA patients. These levels were correlated with indicators of active disease such as sedimentation rate and Ritchie index. IL-4 inhibited the production of IL-6, IL-1, and tumor necrosis factor (TNF) by both controls and rheumatoid patients. Thus as first described for the T-cell response, mononuclear cells from RA patients display a reduced response to mitogens and cytokines which induce their B-cell differentiation into Ig-screening cells. However, IL-4 was able to inhibit Ig and cytokine production, properties suggesting a potential antiinflammatory role for this cytokine.     

初发性类风湿关节炎患者外周血中Tfh细胞及其相关细胞因子的临床意义

滤泡辅助性T细胞(T follicular helper cells,Tfh细胞)是近年来新发现的,真正意义上辅助B细胞功能的T细胞亚群,其辅助B细胞活化、增殖、分化和分泌免疫球蛋白[1].在类风湿性关节炎(rheumatoid arthritis,RA)中,B细胞分泌大量高亲和力抗体可诱发炎性反应,致关节损伤[2].本实验拟通过对RA患者外周血Tfh细胞表型及相关细胞因子的检测,探讨其与初发性RA发病的关系.     

VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA)

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (V_H) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of V_H genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the V_H composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual V_H genes (eight V_H3 and three V_H4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these V_H genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of V_H3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged V_H4 genes was also observed in these patients. These data suggest that although usage of individual V_H genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.