类风湿关节炎滑膜组织血管内皮生长因子转录和转录后的研究

目的 研究类风湿关节炎(RA)患者血清、滑液及滑膜组织中血管内皮生长因子(VEGF)的表达水平，探讨其在RA致病中的作用。方法 应用双抗夹心酶联免疫吸附测定（ELISA)法分别测定RA患者血清、滑液中VEGF蛋白水平；采用半定量反转录-聚合酶链反应(RT-PCR)法和Northern blot检测滑膜组织VEGF mRNA表达水平。以骨关节炎(OA)病人及因外伤截肢的正常人作对照。结果 RA患者血清、滑液中VEGF蛋白水平显著高于对照组；RA滑膜组织VEGF mRNA表达水平也明显高于对照组。结论 RA患者滑膜组织VEGF的过度表达，引起血管增生，是引起RA发病的重要因素。     

类风湿关节炎患者外周血及滑液CC趋化因子配体5的表达

目的 探讨CC趋化因子配体5（CCLS）在类风湿关节炎（RA）患者外周血及滑液中的表达。方法 用酶联免疫吸附法测定28例RA患者血清、滑液、外周血白细胞和滑液炎性细胞CCL5水平，并与骨关节炎（OA）进行对照；RA患者滑膜组织CCL5表达采用免疫组织化学法。结果 RA患者血清和滑液CCL5水平均明显高于OA患者．且RA患者滑液CCL5水平又明显高于其血清。RA患者滑液炎性细胞CCL5水平较其外周血白细胞高，滑膜组织表达CCL5亦增高，并与滑膜炎程度呈显著性正相关。结论 滑液炎性细胞及滑膜组织是RA患者CCL5的主要来源．CCL5在RA滑膜炎病理过程中发挥着重要作用。     

OPG RANKL RANK等因子在类风湿关节炎患者血清及滑膜液中的变化及意义

目的检测类风湿关节炎（RA）患者血清、滑膜液中骨保护素（OPG）、核刺激因子受体配体（RANKL）、核刺激因子受体（RANK）、白细胞介素（IL）-18和前列腺素E-2（PGE2）水平，探讨其与RA发生发展的相关意义。方法采用酶联免疫吸附试验（ELISA）检测60例RA患者与60例同期入院骨外伤患者（作为正常对照组）血清、滑膜液中OPG、RANKL、RANK、IL-18和PGE2水平，分析其与RA的相关性。结果RA患者血清、滑膜液中OPG水平明显低于正常对照组（P〈0．05），RANKL、RANK、IL-18和PGE2水平明显高于对照组（p〈0．05）。结论RA患者血清、滑膜液中OPG水平降低，RANKL、RANK、IL-18和PGE2水平升高。推测上述细胞因子参与RA发病过程。     

一氧化氮合酶抑制剂调控白细胞介素1β和脂多糖对关节软骨的损害

目的：评价兔膝关节内注射白细胞介素－1β（IL-1β）和脂多糖（混合液后，一氧化氮合酶（iNOS）抑制剂S－甲基异硫脲（SMT）调控关节软骨代谢的作用。方法：实验分为3组，正常组：不使用任何干预药物；对照组：皮下注射生理盐水后，膝关节内给予白细胞介素－1（IL－1）和LPS；实验组：SMT持续皮下注射72h后，关节内注射内浓度IL－1β和LPS。经8h后通过检测硝酸盐和亚硝酸盐的含量来观察膝关节滑液、滑膜和软骨NO的释放量以及iNOS的活性；用原位杂交检测软骨iNOS mRNA的表达。48h后，通过Na2^35SO4掺入法检测关节软骨蛋白糖的合成率。结果：IL－1β和LPS混合液可诱发iNOS mRNA显著表达，使兔膝关节滑液、滑膜和软骨释放大量NO和增加NOS活性。关节软骨是关节内NO的主要来源。SMT减少了NO释放，降低了NOS活性，抑制了iNOS mRNA表达，并且部分逆转了蛋白多糖的抑制作用。结论：iNOS抑制剂SMT对关节软骨的损伤具有部分保护作用；体内高浓度药物的维持是其发挥作用的保证。     

白细胞介素-23在类风湿关节炎滑膜中的表达及意义

目的白细胞介素（IL）-23由IL-23p19和IL-12p40亚单位组成，它在一些自身免疫疾病发生过程中起着重要的作用。研究分析类风湿关节炎（RA）患者关节滑膜组织中IL-23p19蛋白及其mRNA的表达，旨在探讨其在RA发病中的意义。方法应用反转录-聚合酶链反应（RT—PCR）和免疫组织化学方法检测RA患者关节滑膜组织中IL-23p19基因和蛋白的表达。并与骨关节炎（OA）患者和健康人作对照研究。结果IL-23p19mRNA和蛋白在RA患者关节滑膜组织中表达明显增高．而在OA患者中低表达，正常对照组中无表达。结论IL-23p19在RA关节滑膜组织中高表达．提示IL-23可能直接参与了RA的发病过程。     

VEGF与TGF-β1在胶原诱导的RA大鼠滑膜组织中的表达及意义

目的探讨血管内皮生长因子（VEGF）和转化生长因子（TGF）-β1在类风湿关节炎（RA）发病中的作用,为RA的靶点治疗提供依据。方法采用皮下多点注射牛Ⅱ型胶原方法对18只Wistar大鼠建立RA模型（实验组）,同时选择10只Wistar大鼠皮下注射等量生理盐水作对照组,实验28d处死大鼠、收集滑膜组织,用免疫组化法检测两组VEGF蛋白与TGF-β1蛋白表达,RT—PCR技术检测VEGF mRNA、TGF-β1 mRNA水平。结果VEGF蛋白及TGF-β1蛋白阳性染色均主要位于滑膜衬里层和滑膜下层的滑膜细胞及血管内皮细胞,实验组滑膜VEGF、TGF-β1蛋白及mRNA表达均显著高于对照组（P均〈0．01）;VEGF mRNA与TGF-β1 mRNA表达呈正相关,r=0．481（P〈0．05）。结论VEGF和TGF-β1在RA滑膜增生、浸润及关节破坏中起协同作用,以两者为靶点的药物有望为RA治疗提供新思路。     

白细胞介素-17在胶原诱导性关节炎大鼠血清水平及滑膜组织表达

目的探讨胶原诱导性关节炎（CIA）大鼠不同时期血清白细胞介素（IL）-17水平及在滑膜组织的表达。方法用酶联免疫吸附试验（ELISA）方法检测造模组和正常对照组不同时期血清IL—17水平，用免疫组织化学过氧化物酶标记的链霉卵白素（S—P）法检测两组晚期关节滑膜组织IL—17表达。结果对照组和造模组均有一定水平IL—17表达，同期成模组较对照组和造模失败组明显升高（P〈0．01），成模组IL—17在早期就开始升高，到中晚期达到较高浓度，中晚期与早前相比，差异有统计学意义（P〈0．01）。晚期成模组滑膜组织表达IL-17阳性率亦高于对照组和造模失败组（P〈0．01）。对不同时期血清IL—17水平与关节病理破坏程度进行直线相关性分析呈正相关（早期P〈0．01；中期P〈0．01；晚期P〈0．05）。结论IL—17在CIA大鼠滑膜组织及不同时期血清中高表达，与关节滑膜的病理破坏程度有关，可作为判断类风湿关节炎（RA）预后的重要指标。     

骨桥蛋白介导类风湿关节炎发病机制的初步研究

目的研究骨桥蛋白(OPN)介导类风湿关节炎(RA)发病和促进炎症因子产生的作用。方法采用实时定量聚合酶链反应(PCR)的方法检测OPN在RA患者外周血、关节液和关节滑膜组织和在T淋巴细胞亚群中的表达;从蛋白水平分析RA患者关节液中促炎症细胞因子和抗炎症细胞因子的分泌格局,分析OPN与细胞因子的相关性。结果RA患者关节滑液和关节滑膜组织的OPN表达远远高于外周血单个核细胞的OPN基因表达,CD4 T细胞OPN基因表达均高于CD8 T细胞;RA患者关节液中OPN的表达显著高于血清,白细胞介素(IL)-10,干扰素(IFN)-γ和肿瘤坏死因子(TNF)-α水平比自身血清及对照组血清增高,IL-18和IL-12水平比正常对照组明显升高。结论OPN可能通过调节病灶部位的细胞因子的分泌来调控RA病理过程中的炎症反应。     

类风湿关节炎关节滑膜细胞中IL-18表达及意义

取类风湿关节炎（RA）患者的关节滑膜40份（RA组）及外伤后患者的关节滑膜40份（对照组）,用胶原酶消化培养法培养至3-8代,用免疫组化法检测滑膜细胞IL-18表达,用酶联免疫吸附检测细胞培养上清液中IL-18水平。结果RA组滑膜细胞及上清液中IL-18分泌量明显高与对照组（P〈0.05）。认为IL-18高表达参与RA发病,可作为临床治疗的靶点之一。     

核因子κB在类风湿关节炎滑膜组织中的表达与意义

目的检测核因子κB(NF-κB)及白细胞介素(IL)-1β、基质金属蛋白酶(MMP)-9在类风湿关节炎(RA)、骨关节炎(OA)及正常滑膜组织中的表达差异。方法反转录-聚合酶链反应(RT-PCR)检测46例滑膜组织(RA17例,OA24例,正常5例)中p65、p50、NF-κB抑制因子(IκB)及IL-1β、MMP-9的mRNA水平。免疫组织化学检测39例滑膜组织(RA14例,OA21例,正常4例)中p65的表达情况。对8例RA、6例OA滑膜组织进行滑膜细胞培养,提取核蛋白进行免疫印迹,检测p65含量。结果RA组p65、IL-1β、MMP-9的mRNA水平显著高于正常对照组(分别为P<0.05、P<0.01、P<0.05),与OA组比较差异无显著性。RA、OA与正常组之间p50、IκB的mRNA水平差异无显著性。NF-κBp65免疫组织化学染色组显示RA滑膜衬里层细胞、衬里下层浸润的炎症细胞、血管内皮细胞均有着色。RA组NF-κB活性系数显著高于OA组、正常对照组(P<0.001)。免疫印迹发现RA滑膜细胞核蛋白中p65含量显著高于OA组(P<0.05)。结论RA滑膜组织中NF-κB的表达与活化水平显著高于OA及正常滑膜组织。RA组IL-1β与MMP-9的mRNA水平显著高于正常对照。     

Fibroblast-like synovial cells derived from synovial fluid

OBJECTIVE: To obtain fibroblast-like synovial cells (FLS) from synovial fluid (SF). METHODS: SF aspirated from joints of patients with rheumatoid arthritis (RA), other types of inflammatory arthritis, and osteoarthritis (OA) was centrifuged and the resulting cell pellet resuspended in growth medium. After 2 days, nonadherent cells were removed. FLS were also cultured from surgical specimens of synovial tissue (td-FLS). Phenotype characterization of fluid derived FLS (fd-FLS) was accomplished by flow cytometry and immunohistochemistry staining. Tumor necrosis factor-alpha (TNF-alpha) induced interleukin 6 (IL-6), IL-8, and cyclooxygenase 2 (COX-2) mRNA levels were assessed. RESULTS: Second and later passage fd-FLS exhibited uniform fibroblast-like morphology. Fd-FLS and td-FLS expressed a similar profile of cell surface antigens including the fibroblast marker Thy-1. Less than 2% of either cell type expressed surface markers characteristic of dendritic cells, phagocytic cells, T cells, or leukocytes. Immunohistochemistry staining revealed the presence of fibroblast products prolyl-4 hydroxylase, procollagen I, and procollagen III in both culture types. TNF-a induced increases in IL-6, IL-8, and COX-2 mRNA were suppressed by dexamethasone in both fd-FLS and td-FLS. CONCLUSION: FLS can be cultured from SF. The fibroblast phenotype was confirmed by analysis of surface antigens and intracellular proteins. Inflammatory mediators produced after stimulation of both fd-FLS and td-FLS were suppressed by dexamethasone. In addition to providing a more accessible source of FLS, fd-FLS may also facilitate study of synovial cells in early RA when tissue specimens are not readily available.     

Resistin in rheumatoid arthritis synovial tissue, synovial fluid and serum

BACKGROUND: Resistin is a newly identified adipocytokine which has demonstrated links between obesity and insulin resistance in rodents. In humans, proinflammatory properties of resistin are superior to its insulin resistance-inducing effects. OBJECTIVES: To assess resistin expression in synovial tissues, serum and synovial fluid from patients with rheumatoid arthritis, osteoarthritis and spondylarthropathies (SpA), and to study its relationship with inflammatory status and rheumatoid arthritis disease activity. METHODS: Resistin expression and localisation in synovial tissue was determined by immunohistochemistry and confocal microscopy. Serum and synovial fluid resistin, leptin, interleukin (IL)1beta, IL6, IL8, tumour necrosis factor alpha, and monocyte chemoattractant protein-1 levels were measured. The clinical activity of patients with rheumatoid arthritis was assessed according to the 28 joint count Disease Activity Score (DAS28). RESULTS: Resistin was detected in the synovium in both rheumatoid arthritis and osteoarthritis. Staining in the sublining layer was more intensive in patients with rheumatoid arthritis compared with those with osteoarthritis. In rheumatoid arthritis, macrophages (CD68), B lymphocytes (CD20) and plasma cells (CD138) but not T lymphocytes (CD3) showed colocalisation with resistin. Synovial fluid resistin was higher in patients with rheumatoid arthritis than in those with SpA or osteoarthritis (both p<0.001). In patients with rheumatoid arthritis and SpA, serum resistin levels were higher than those with osteoarthritis (p<0.01). Increased serum resistin in patients with rheumatoid arthritis correlated with both CRP (r=0.53, p<0.02), and DAS28 (r=0.44, p<0.05), but not with selected (adipo) cytokines. CONCLUSION: The upregulated resistin at local sites of inflammation and the link between serum resistin, inflammation and disease activity suggest a role for resistin in the pathogenesis of rheumatoid arthritis.     

Fibrin and fibronectin in rheumatoid synovial membrane and rheumatoid synovial fluid

Normal synovial membranes and synovial membranes from patients with classic rheumatoid arthritis were investigated for the presence of fibrin and fibronectin by an indirect immunoperoxidase technique. In normal synovial membranes, fibronectin was found around the monolayer of the synovial lining cells. Staining was most intense on the surface and beneath the lining cells, but not detectable in the cytoplasm. Fibronectin was also found in the cytoplasm of the endothelial cells. No staining for fibrin was found in the normal synovial membrane. In synovial membranes from patients with rheumatoid arthritis, large amounts of fibronectin were found around the multilayer of synovial lining cells, in the cytoplasm of the endothelial cells, and in argyrophilic fiber-rich connective tissue. In superficial areas denuded of synovial lining cells, high amounts of fibronectin were found incorporated in fibrin. In some areas with noninjured synovial lining cells, fibrin was also found, but in this case no fibronectin was incorporated. No fibronectin was found in connective tissue in areas with infiltration of inflammatory cells. After treatment of normal and rheumatoid synovial membranes with hyaluronidase, fibronectin was still present around the lining cells but the staining was found to be more distinct. This study relates the presence of fibrin and fibronectin in the rheumatoid synovial membrane to the high amount of these proteins, recently described, in rheumatoid synovial fluid. It also suggests that fibronectin present in the synovial membrane is produced and secreted by the endothelial cells.     

Detection and initial characterization of synovial lining fragments in synovial fluid

OBJECTIVE: Free fragments of synovium have occasionally been seen in synovial fluid but have not been studied systematically. We wished to establish a method for the reliable detection of these fragments in joint and bursa effusions and begin to characterize them by histochemical and immunohistochemical methods. METHODS: Cell smears, wet drop preparations and cytospins were prepared from 39 consecutive joint and bursa effusions. Paraffin cell blocks were prepared from a subset. Analysis encompassed standard and polarized light microscopy, histochemistry, immunohistochemistry and transmission electron microscopy (EM). Synovial biopsy tissue from one different patient was examined for comparison. RESULTS: Tissue fragments were not seen in Wright-stained cell smears and only rarely in wet drop preparations. In contrast, variously sized fragments with the histological appearance of hyperplastic synovial lining were detected in ethanol-fixed, haematoxylin/eosin-stained cytospins from bursitis and all arthropathies studied [17/24 (71%) of non-inflammatory and 12/15 (80%) of inflammatory specimens]. Immunostaining revealed CD68 expression in a subset of cells in a pattern characteristic of hyperplastic synovial lining. Juxtaposed cells with morphological features of macrophage-like and fibroblast-like synoviocytes were seen by EM. CONCLUSIONS: Synovial lining fragments can be detected in effusions from diverse arthropathies and bursitis. They maintain important properties of the synovial lining and can be analysed by immunohistochemistry. They may afford the opportunity to study a relatively pure preparation of synovial lining cells without the need for cell culture, and to evaluate their possible role in augmenting or perpetuating synovitis or joint damage.     

Dickkopf-1 perpetuated synovial fibroblast activation and synovial angiogenesis in rheumatoid arthritis

Clinical Rheumatology - Dickkopf-1 (Dkk-1), a regulatory molecule of the Wnt pathway, is elevated and leads to bone resorption in patients with RA. This study is aimed to investigate the...     

Phospholipids in inflammatory synovial effusions

Summary The concentration of phospholipids and proteins was determined in 23 inflammatory synovial fluids obtained from human knee joints. The synovial fluid to plasma phospholipid ratio (0.48 and 0.37 at high and low inflammatory state) was lower than the value found for the total protein content (0.68 and 0.53, respectively) indicating that phospholipids were more discriminated than proteins in their transfer from plasma to the synovial space. Constant amounts of phosphatidylinositol were found in all synovial fluids, whereas trace amounts of lysophosphatidylethanolamine and phosphatidylserine were more frequent in the active inflammatory state. A decrease in the relative amounts of phosphatidylcholine and phosphatidylinositol with respect to plasma suggested the possibility of phospholipid hydrolysis in the synovial compartment. In agreement, determinations of phospholipase activity disclosed the presence of a phospholipase A₂ in the fluid phase of synovial effusions. Phospholipid derivatives formed in the synovial space may thus contribute to the amplification of the inflammatory response.