类风湿关节炎中尿激酶型纤溶酶原激活物及其受体蛋白和基因的表达

目的　检测尿激酶型纤溶酶原激活物 (uPA)及其受体 (uPAR)蛋白和mRNA在类风湿关节炎 (RA)的表达 ,探讨uPA、uPAR基因在RA细胞外基质降解中的作用。方法　采用免疫组化和cDNA mRNA原位分子杂交技术分别检测了 2 4例RA、18例骨关节炎 (OA)和 6例正常滑膜组织中uPA、uPAR蛋白和mRNA的分布及表达情况。结果　 2 4例RA滑膜组织均呈uPA、uPAR蛋白和mRNA的阳性表达 ,uPA、uPAR蛋白的强阳性率高于mRNA。uPA、uPAR蛋白和mRNA阳性信号主要分布在RA滑膜衬里细胞、滑膜下层单核细胞、巨噬细胞样细胞及血管内皮细胞 ;18例OA滑膜组织中 ,uPA、uPAR蛋白和mRNA的表达部位类似于RA ,但阳性率、阳性程度及分布范围均明显低于RA滑膜组织 ,两组之间蛋白和mRNA表达的差异均有显著性 (P <0 0 1或P <0 0 0 1)。 6例正常滑膜组织呈阴性反应。结论　RA滑膜组织存在高水平uPA、uPAR蛋白和mRNA的表达 ,提示在RA的发生发展过程中 ,uPA和uPAR基因起着重要作用 ;RA和OA中uPA、uPAR基因表达水平的差异 ,可能与这两种疾病软骨和骨基质降解的程度及进程等临床表现密切相关     

Reactivity of monoclonal anti-type II collagen antibodies with cartilage and synovial tissue in rheumatoid arthritis and osteoarthritis

Monoclonal antibodies to 3 different epitopes on native type II collagen were used for immunohistochemical analysis of antigenic determinants that are exposed in the cartilage and synovial tissue obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Two of the monoclonal antibodies reacted with cartilage from both OA and RA joints, but not with that from normal joints. The third monoclonal did not stain any of the cartilage sections. The 2 positive antibodies also reacted with cartilage fragments in the synovial tissue of both RA and OA joints, and in RA pannus tissue, the antibodies showed intracellular staining in many class II transplantation antigen-expressing synovial cells lying close to the damaged cartilage.     

Comparative analysis of cathepsin l,cathepsin d,and collagenase messenger rna expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis,by in situ hybridization

Objective. To compare the expression of cathepsin L, cathepsin D, and collagenase messenger RNA (mRNA) in synovial specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Methods. The expression of cathepsins L and D as well as collagenase mRNA in synovial tissues from 8 patients with RA, 6 patients with OA, and 2 patients with noninflamed joints was evaluated using in situ hybridization with digoxigenin-labeled RNA probes. Results. Both RA and OA synovial tissue expressed cathepsins L and D as well as collagenase mRNA. The expression of the cathepsins was markedly higher in interstitial regions and, to some extent, in perivascular infiltrates of RA synovial tissue compared with OA specimens. Conclusion. Cathepsins L and D mRNA are expressed differently in RA and OA synovial tissues, supporting the concept that these enzymes may contribute to the influx of mononuclear cells into RA synovium. Moreover, the data reveal that the expression of collagenase and cathepsins in RA and OA synovial lining is otherwise largely similar, and suggest that the adhesion of synovial cells to cartilage mediates the invasive destructive process in RA.     

EXPRESSION OF bcl-2 IN RHEUMATOID ARTHRITIS

Since defective apoptosis has been suggested to play a rolein the development of autoimmune diseases, we have investigatedthe expression of the proto-oncogene bcl-2 in patients withrheumatoid arthritis (RA). The expression of bcl-2 was studiedin peripheral blood (PB) and synovial fluid (SF) lymphocytesand synovial tissues (ST) from patients with RA using immunohistochemistry,flow cytometry and nucleic acid hybridization. Patients withreactive arthritis (ReA) or osteoarthritis (OA) and healthyindividuals were used as controls. The expression of bcl-2 proteinin PB lymphocytes and the expression of bcl-2 mRNA in PB mononuclearcells (PBMC) was similar in healthy controls and patients withRA. However, bcl-2 protein expression was significantly reducedin SF lymphocytes when compared to PB lymphocytes. Similar resultswere observed with lymphocytes from patients with ReA, and irrespectiveof whether total lymphocytes, T cells or different T-cell subsetswere studied. In the synovial sections, the expression of bcl-2was restricted to lymphocytes, and bcl-2+ cells were observedin the majority of samples from patients with RA, OA and ReA.These data indicate that the expression of bcl-2 is not increasedin the lymphocytes or ST derived from patients with RA. Instead,decreased expression of bcl-2 protein in SF lymphocytes comparedto PB lymphocytes was demonstrated. We suggest that bcl-2 doesnot play a significant role in the pathogenesis of RA. KEY WORDS: bcl-2, Rheumatoid athritis, Lymphocytes     

Expression of basic fibroblast growth factor in synovial tissues from patients with rheumatoid arthritis: detection by immunohistological staining and in situ hybridisation.

OBJECTIVE--The distribution and production of basic fibroblast growth factor (bFGF) was examined on the synovium from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS--The localisation of bFGF was determined by an immunohistochemical staining procedure using anti-bFGF monoclonal antibody. The expression of bFGF mRNA was detected by nonradioactive in situ hybridisation using bFGF antisense oligo DNA. RESULTS--The bFGF was found in the synovial lining cell, sublining stromal fibroblast-like cells, and vascular endothelial cells from patients with RA and OA. Little or no bFGF was found in non-inflamed synovium. Immunostaining of bFGF in the synovial cells was more extensive and intense in synovium of patients with RA than that of patients with OA. The nuclei of the synovial lining cell layer were also immunostained. These nuclear staining were more intense in the lining cell layer from RA patients with moderate or severe proliferation of synovial cells than in RA patients with mild proliferation. The bFGF mRNA was also detected in the synovial lining cell layer of the inflamed synovium. CONCLUSION--The synovial lining cells produced bFGF. The proliferation of synovial cells in the inflamed joints may be the results of stimulation by the bFGF in autocrine manner.     

护骨素在强直性脊柱炎外周关节骨质破坏病理机制中的作用

目的检测护骨素(OPG)在强直性脊柱炎(AS)外周关节滑膜组织中的表达,并以类风湿关节炎(RA)、骨关节炎(OA)患者和健康志愿者外周关节滑膜组织为对照,了解OPG表达与AS患者外周关节骨质破坏病理改变的相关性。方法应用单克隆抗体,通过免疫组织化学方法检测13例AS、16例RA、17例OA及6名健康对照关节滑膜组织中OPG的表达及分布状况,并通过计算机辅助图像分析系统和半定量分析方法确定OPG在各滑膜组织中表达水平之间的差异,分析OPG表达与炎性指标及关节X线分期之间的相关性。结果OPG蛋白在所有13例AS滑膜组织中均有阳性表达,阳性细胞主要分布于滑膜衬里层、衬里下层区域,滑膜软骨交界区OPG表达明显低于滑膜衬里层和衬里下层;2名健康对照滑膜组织中有阳性表达,但明显低于AS患者组。RA及OA患者组未见OPG阳性表达。结论譹AS组滑膜组织中OPG表达水平明显高于健康对照组(P<0.01),而RA、OA滑膜组织中未见OPG表达,说明OPG的高水平表达是滑膜组织对炎症反应/关节破坏所特有的表现,OPG的局部表达是维持AS关节骨代谢稳定的重要因素,这可能是大多数AS患者外周关节受累预后好于RA的原因之一。譺AS患者组滑膜软骨交界区中OPG表达量显著减少可能是导致关节骨质破坏的重要原因,提示关节局部应用OPG治疗有可能改善受累关节的骨     

类风湿关节炎滑膜组织中细胞外基质金属蛋白酶诱导因子的研究

目的研究类风湿关节炎(RA)患者滑膜组织中细胞外基质金属蛋白酶诱导因子(EMM-PRIN)的表达,探讨其在RA致病中的作用。方法对20例RA患者膝关节滑膜组织标本采用免疫组织化学染色和半定量反转录-聚合酶联反应(RT-PCR)法,观察RA滑膜组织中EMMPRIN的蛋白及mRNA表达。10例骨关节炎OA滑膜作为对照。结果RA滑膜内衬层的单核细胞、成纤维细胞中EMMPRIN阳性表达广泛,RA组EMMPRIN的免疫反应明显强于对照组(P<0.01)。14例RA、2例OA滑膜标本EMM-PRINmRNA表达阳性,EMMPRINmRNA表达水平RA也明显高于OA组。结论滑膜中过度表达的EMMPRIN通过上调MMPs促进关节软骨的破坏。因此选择性地抑制EMMPRIN生物活性,可能是治疗RA的新途径。     

类风湿关节炎患者滑膜、滑液和血浆中PAI的检测及其临床意义

目的 检测Ⅰ型和Ⅱ型纤深酶原激活物抑制剂（ＰＡＩ－１和ＰＡＩ－２）在类风湿关节炎（ＲＡ）患者滑膜组织中的定位和表达,同时测定ＲＡ患者滑液和血浆中ＰＡＩ－１的含量和活性,并分析其临床意义。方法 运用免疫组织化学方法检测了２４例ＲＡ、１８例骨关节炎（ＯＡ）和６例正常滑膜组织中ＰＡＩ－１和ＰＡＩ－２的定位及其表达;采用ＥＬＩＳＡ双抗体夹心法测定４６例ＲＡ和８例ＯＡ患者血浆、１４例ＲＡ滑液和１２例正常对照     

骨关节炎患者软骨及滑膜高迁移率族蛋白1的表达

目的探讨高迁移率族蛋白1(HMGB1)与骨关节炎(OA)发生和发展的关系。方法采用免疫组织化学方法观察42例老年 OA 患者关节软骨、滑膜和3例未累及关节的创伤患者正常关节软骨和滑膜 HMGB1的表达规律与分布特点及其与 C 反应蛋白(CRP)的关系。结果 42例OA 中,35例软骨 HMGB1染色阳性,占83.3%;30例滑膜 HMGB1染色阳性.占71.4%,其表达以胞浆分布为主,胞核中较少,3例正常关节软骨和滑膜 HMGB1标记均为阳性。其表达以胞核分布为主,胞浆中极少分布。OA 患者关节软骨和滑膜 HMGB1细胞内分布与关节正常者有明显的不同。OA 患者 HMGB1阳性的软骨细胞数占软骨细胞总数的70.2%,HMGB1阳性的滑膜上皮细胞数占滑膜上皮细胞总数的60.5%,与关节正常者比较,差异无统计学意义(P>0.05)。OA 患者外周血CRP 含量[(20.4±16.7)ng/L]明显升高(P<0.01)。结论 OA 患者关节软骨及滑膜中核外HMGB1可能在 OA 组织损伤的病理过程中发挥作用,OA 患者关节软骨及滑膜上皮细胞中核外HMGB1作为晚期炎症因子参与了 OA 的疾病过程。     

Localisation of vitronectin receptor immunoreactivity and tartrate resistant acid phosphatase activity in synovium from patients with inflammatory or degenerative arthritis.

The influx of cells into the synovial intima in rheumatoid joints may include osteoclasts and their precursors. The distribution of osteoclast markers--namely, tartrate resistant acid phosphatase activity and the expression of vitronectin receptor (shown with monoclonal antibodies 13C2 and 23C6)--was therefore examined in synovium obtained from patients with rheumatoid (RA) or degenerative (OA) arthritis. Tartrate resistant acid phosphatase positive cells were found in frozen sections of 60% (n = 30) of RA and 69% (n = 29) of OA synovial membranes. Whereas all synovia tested (four RA, four OA) showed diffuse staining of the lining cells with 13C2, 55% (n = 11) of RA and 57% (n = 14) of OA synovial membranes contained isolated cells stained with 23C6 scattered throughout the tissue. In cultures of synovial cells, tartrate resistant acid phosphatase positive, multinuclear, and 23C6 positive cells were found; these cells did not, however, form resorption pits on bone slices. The results show that fully differentiated osteoclasts are uncommon in synovium from patients with either degenerative or inflammatory arthropathies.     

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.     

THE RELEVANCE OF CHONDROITIN AND KERATAN SULPHATE MARKERS IN NORMAL AND ARTHRITIC SYNOVIAL FLUID

This study investigated the synovial fluid concentrations ofglycosaminoglycan (GAG), keratan sulphate (KS) epitope 5D4 andchondroitin sulphate (CS) sulphation patterns in healthy volunteersand patients with osteoarthritis (OA) and rheumatoid arthritis(RA). Synovial fluids were collected from knee joints of healthyvolunteers (n = 24), and patients with OA (n = 28) and RA (n= 29). Concentrations of GAG and the keratan sulphate epitopeSEW were measured in 15 of the healthy volunteers, and all ofthe OA and RA synovial fluids. Total GAG was measured usinga dye-binding method and 5D4 by an ELISA. The unsaturated CSdisaccharides     

Dynamics of interleukin (IL)-18 in serum, synovial fluid and synovial membrane in the patients with rheumatoid arthritis]

OBJECTIVES: IL-18 is a novel cytokine that plays an important role in the Th1 response. The aim of this study is to investigate the dynamics of IL-18 in serum, synovial fluid and synovial membrane in the patients with rheumatoid arthritis. MATERIALS AND METHODS: The serum, synovial fluid and synovial membrane were obtained from RA patients at operation. The levels of IL-18 in the serum and synovial fluid were measured by ELISA. We then examined the expression of IL-18 in synovial tissues using anti-human IL-18 monoclonal antibody in immunohistochemical study. RESULTS: The levels of IL-18 in serum and synovial fluid in RA patients were 193.7 +/- 109.7 pg/ml and 258.8 +/- 238.0 pg/ml, respectively. Compared with OA patients and normal volunteers, the level of IL-18 in RA patients was higher in both serum and synovial fluid. (P < 0.05) In synovial membrane, the cells positive for anti IL-18 antibody were confirmed not only in RA (n = 26) but also in OA (n = 7) patients. The positive cells were the synovial lining cells, macrophages, fibroblasts and endothelial cells. However, a large number of positive cells were demonstrated in synovial tissues in RA compared with OA patients.