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The role of PRG-B which is a novel angiogenesis-related gene in rheumatoid arthritis and osteoarthritis

新型血管生成相关基因PRG-B在类风湿关节炎和骨关节炎中的作用

基本信息

批准号：
15591602
负责人：
MORIOKA Hideo
金额：
$ 2.24万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Cartilage is an avascular tissue and this avascularity may be due to the local production of angiogenesis inhibitors. During our previous studies on the expression of plasminogen in human articular cartilage, we isolated a unique cDNA fragment named plasminogen related gene-B (PRG-B) which predicts a 9kDa polypeptide designated as plasminogen-related protein-B (PRP-B). We reported that recombinant PRP-B (rPRP-B) inhibited angiogenesis and that this effect was, at least partly, mediated through the inhibition of basic fibroblast growth factor (bFGF) -induced tyrosine kinase signaling in endothelial cells. In rheumatoid arthritis (RA), activation of angiogenesis through the up-regulation of pro-angiogenic cytokines in synovium has been reported. Therefore, attempts have recently been made to employ angiogenesis-inhibitors for the treatment of RA. We also reported that rPRP-B down-regulates VEGF expression in human fibroblast-like synoviocytes (FLS). The aim of this study is to investigat … More e the expression of PRG-B and the regulation of angiogenesis by PRG-B in RA cartilage.MATERIALS AND METHODS :Recombinant protein : The isolation and characterization of rPRP-B has been described previously.Cell culture : Human Chondrocytes (HC) were purchased from CELL APPLICATIONS, INC. Human chondrosarcoma cell line (CS-1) which has phenotype as chondrocyte was also used in this study.Clinical samples : Cartilage samples were obtained at arthroplasty surgery from knee joints of patients with RA.Reverse transcription- polymerase chain reaction (RT-PCR) : PCR was performed using specific primer pairs for PRG-B or GAPDH, and the reaction products were analyzed by agarose gel electrophoresis. The expression levels of PRG-B mRNA in HC and CS-1 with or without bFGF were compared.Antibodies and immunohistochemistry : Purified rPRP-B was applied to rabbits as antigen and antibody against PRP-B was raised. Serum was collected and subject to the protein-A purification. Antibody against plasminogen (PLG) was obtained from DAKO. Immunohistochemistry for detection of PRP-B and plasminogen was performed using paraffin embedded cartilage samples using the standard technique.Real-time polymerase chain reaction (Real-time PCR) : We investigated the expression of VEGF mRNA in CS-1 with or without PRP-B treatment by Real-time PCR.RESULTS :RT-PCR : In analysis for cell lines, PRG-B expression was induced by bFGF stimulation in HC and CS-1.Immunohistochemistry : In immunohistochemical staining of cartilage using anti-PRP-B antibody, PRP-B expression was shown to be up-regulated in cartilage from RA patients.Real-time PCR : rPRP-B reduced the expression level of VEGF mRNA of CS-1.Discussion :In this study, we investigated the role of PRG-B as an anti-angiogenesis gene in the arthritic cartilage. The results of RT-PCR showed that bFGF induced up-regulation of PRG-B in vitro. In addition, we detected expression of protein derived from PRG-B in clinical samples of RA cartilages. Our results also showed that rPRP-B down-regulated VEGF expression in vitro. In arthritic joint, articular cartilage is stimulated by pro-angiogenic and inflammatory cytokines such as bFGF and IL-1β, which are produced from synovium. We reported that rPRP-B down-regulates VEGF expression in FLS in vitro at the 51^<st> ORS meeting. Therefore, our results suggest that PRG-B is expressed in articular cartilage and has autocrine and paracrine anti-angiogenesis effects on synoviocyte and chondrocyte in the arthritic joint. Therefore, PRG-B seems to be a candidate for anti-angiogenesis targeted gene in arthritic diseases. Less

软骨是一种无血管组织，这种无血管性可能是由于局部血管生成抑制剂的产生。在我们之前对人关节软骨中纤溶酶原表达的研究中，我们分离了一个独特的cDNA片段，称为纤溶酶原相关基因- b (PRG-B)，该片段预测了一个9kDa的多肽，称为纤溶酶原相关蛋白- b （PRP-B）。我们报道了重组PRP-B （rPRP-B）抑制血管生成，并且这种作用至少部分是通过抑制碱性成纤维细胞生长因子（bFGF）诱导的内皮细胞酪氨酸激酶信号传导介导的。在类风湿关节炎（RA）中，通过上调滑膜中促血管生成细胞因子激活血管生成已被报道。因此，最近已经尝试使用血管生成抑制剂来治疗RA。我们还报道了rPRP-B下调人成纤维细胞样滑膜细胞（FLS）中VEGF的表达。本研究旨在进一步探讨PRG-B在RA软骨中的表达及其对血管生成的调控作用。材料和方法：重组蛋白：rPRP-B的分离和表征已经在前面描述过。细胞培养：人软骨细胞（HC）购自Cell APPLICATIONS， INC。具有软骨细胞表型的人软骨肉瘤细胞系CS-1也被用于本研究。临床样本：关节成形术中取自RA患者膝关节的软骨样本。逆转录-聚合酶链反应（RT-PCR）：对PRG-B或GAPDH进行特异性引物PCR，反应产物琼脂糖凝胶电泳分析。比较添加或不添加bFGF时HC和CS-1中PRG-B mRNA的表达水平。抗体及免疫组化：纯化的rPRP-B作为抗原，制备抗PRP-B抗体。采集血清，进行蛋白a纯化。从DAKO获得抗纤溶酶原（PLG）抗体。免疫组织化学检测PRP-B和纤溶酶原使用石蜡包埋软骨样品采用标准技术。实时聚合酶链反应（Real-time polymerase chain reaction, Real-time PCR）：采用实时聚合酶链反应（Real-time PCR）检测PRP-B处理前后CS-1细胞中VEGF mRNA的表达情况。结果：RT-PCR：在细胞系分析中，bFGF刺激HC和CS-1可诱导PRG-B表达。免疫组化：用抗PRP-B抗体对软骨进行免疫组化染色，发现RA患者软骨中PRP-B表达上调。Real-time PCR: rPRP-B降低CS-1细胞VEGF mRNA的表达水平。讨论：在这项研究中，我们研究了PRG-B作为抗血管生成基因在关节炎软骨中的作用。RT-PCR结果显示bFGF诱导体外PRG-B表达上调。此外，我们检测了RA软骨临床样本中PRG-B衍生蛋白的表达。我们的结果也表明，rPRP-B在体外下调VEGF的表达。在关节炎关节中，关节软骨受到滑膜产生的促血管生成和炎性细胞因子（如bFGF和IL-1β）的刺激。我们在51^<st> ORS会议上报道了rPRP-B在体外FLS中下调VEGF表达。因此，我们的研究结果表明，PRG-B在关节软骨中表达，并对关节炎关节的滑膜细胞和软骨细胞具有自分泌和旁分泌的抗血管生成作用。因此，PRG-B可能是关节炎疾病中抗血管生成靶向基因的候选基因。少