【作者】 张婷；

【导师】 唐福林； 尤欣；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 【目的】深入探讨p53在滑膜成纤维细胞(FLS)炎症与增生中的作用,包括p53与NF-κB、p38、JNK及其下游促炎症性细胞因子和基质金属蛋白酶(MMP)之间的关系,以及p53对FLS增生相关信号通路的影响,揭示p53在类风湿关节炎(RA)发病机制中的重要作用。【材料与方法】取人膝关节滑膜组织体外培养FLS。(1)分别与0.1 ng/ml、1 ng/ml、10ng/ml IL-1β或TNF-α共同孵育24小时,收集培养上清行ELISA检测细胞因子IL-6、IL-1β以及MMP-1和MMP-13的水平;同时裂解细胞收获蛋白,利用Western Blot方法检测p53和p21等蛋白的表达;(2)用电转法将p53 siRNA(small interfering RNA)转入FLS细胞并设立对照。3日后,或在这些细胞中重复步骤(1),或与IL-1β(2ng/ml)共同孵育15分钟后裂解细胞收集蛋白,用Western Blot方法检测信号通路NF-κB、p38、JNK和ERK激活情况。【结果】(1)TNF-α和IL-1β可以抑制FLS p53表达,并刺激FLS分泌IL-6、MMP-1显著增加(P＜0.001);(2)p53 siRNA可以有效抑制FLS p53表达。在加或不加IL-1β刺激时,p53表达显著下降的FLS分泌IL-6和MMP-1均较对照组显著增加(P值均＜0.05);在IL-1β刺激下,p53 si RNA抑制p53引起P-IκBα、P-JNK和P-p38表达增高;(3)抑制p53引起p21的表达随之而下降;在IL-1β刺激下,p53低表达的FLS P-ERK较对照组增高。【结论】(1)FLS中p53缺陷对滑膜炎症可以产生重要影响:抑制p53表达可以使NF-κB、p38、JNK活性显著增高,进而促进FLS分泌促炎症性细胞因子和MMPs,并且FLS p53缺陷可能对以上分子各自发挥功能存在易化作用;而促炎症性细胞因子增高可进一步抑制p53表达。由此构成的反馈循环可能是RA进展和维持的关键因素。(2)FLS中p53缺陷还可能对滑膜增生可以产生重要影响:除了通过影响细胞因子分泌以调控FLS增生,p53还可能通过影响细胞周期途径的重要分子p21和ERK调控FLS增生。更多还原

【Abstract】 Objective.To investigate the role of p53 on inflammation and proliferation of fibroblast-like synoviocytes(FLS),including the relationship between p53 and NF-κB, p38,JNK and their downstream pro-inflammatory cytokines and matrix metalloproteinases(MMPs),as well as the influence of p53 on synovial proliferation and related signal transduction pathway.Materials and Methods.Synovial tissues were obtained from patients at joint replacement surgery,and FLS were cultured in vitro.(1) FLS was co-incubated with TNF-αor IL-1βat the concentration of 0.1 ng/ml,1 ng/ml,10ng/ml respectively for 24 hours.The supernatant was then collected for ELISA assay to detect cytokines and MMPs,while the cells were lysed for Western blot to evaluate the expression of p53 and p21.(2) p53 small interfering RNA(siRNA) or scrambled siRNA(sc siRNA) were electrotransfected into FLS.Three days later,step(1) was repeated,or FLS was co-incubated with IL-1β2ng/ml for 15 minutes and then lyzed for Western Blot to identify the activation of signal transduction pathways like NF-κB,p38,JNK and ERK.Results.(1) TNF-αand IL-1βcan inhibit expression of p53,and they both can stimulate FLS overexpressing IL-6 and MMP-1(P＜0.001)(2) p53 siRNA can effectively knockdown p53,leading to significant increase of IL-6 and MMP-1 secretion (P＜0.05).When IL-1βwas introduced,suppression of p53 by p53 siRNA provokes activation of P-JNK,P-p38 and P-IκBα,compared with control group.(3) Transfection of p53 siRNA into FLS induces p21 decline along with the suppression of p53.When coincubated with IL-1β,suppression of p53 results in elevated P-ERK in FLS.Conclusions.(1) p53 defect in FLS may play an essential role in synovitis.Inhibition of p53 leads to activation of signal transduction pathways like NF-κB,p38 and JNK, results in subsequent elevation of downstream expression of pro-inflammatory cytokines as well as MMPs.Meanwhile,cytokines further downregulate expression of p53.This feedback loop involving p53 may well explain the development and propagation of synovitis.(2) In addition to promote FLS proliferation by inducing elevated cytokines, suppression of p53 also contributes to FLS proliferation via p21 and ERK in the pathway of cell cycle.更多还原