【作者】 梁东风；

【导师】 黄烽；

【作者基本信息】 中国人民解放军军医进修学院 ， 风湿病学， 2009， 博士

【摘要】 目的:1.了解依那西普(ETA)和英夫利西单抗(INF)对体外培养的类风湿关节炎(RA)成纤维细胞样滑膜细胞(FLS)的影响。2.评价单次膝关节腔内注射ETA对脊柱关节病(SpA)和RA导致的X线无中、重度骨破坏表现的膝关节炎的疗效和安全性。比较关节腔注射和皮下注射ETA对SpA膝关节炎的疗效差异。3.了解关节液中下列炎症相关指标与SpA和RA关节炎活动性的关系:TNF-α、IFN-γ、IL-2、IL-4、IL-6、IL-10浓度、Th1/Th2比例以及高敏C反应蛋白(hs-CRP)水平。了解ETA对SpA和RA关节液中上述细胞因子浓度和Th1/Th2平衡性的影响。方法:1.体外培养RA患者FLS,应用WST-8比色法检测ETA和INF对FLS增殖的影响,应用流式细胞术观察ETA和INF是否能诱导FLS凋亡。2.进行单次膝关节腔内注射ETA治疗SpA和RA膝关节炎的随机、单盲、平行、阳性药(复方倍他米松)对照的临床试验。试验对象按2:1比例随机分为2组,分别给予目标膝关节腔注射25mg ETA或2ml复方倍他米松;观察4周;60例完成试验;主要疗效指标为改良HSS(Hospitalfor Special Surgery)膝关节评分。3.对膝关节较多积液的SpA和RA患者给予膝关节腔注射(11例SpA和13例RA)或皮下注射(11例SpA)25mg ETA,同时抽吸出5ml关节液,48h后评价其临床疗效并再次行关节腔穿刺抽吸出关节液;应用酶联免疫吸附法(ELISA)检测其注射前后膝关节液中上述细胞因子的浓度;应用免疫散射比浊法检测其hs-CRP水平。结果:1.不同浓度ETA和INF作用24～72小时对FLS增殖的抑制率分别在1.0～17.9%和6.2～19.8%,10pmol/L ETA和INF未能诱导FLS凋亡。2.关节腔注射ETA或复方倍他米松均可使SpA和RA膝关节炎的多数疗效指标得到改善;ETA组对改良HSS膝关节评分、患者对膝健康状况VAS和医生对膝健康状况VAS的改善率大于复方倍他米松组(P=0.0409～0.0467);不良事件:试验组8例(19.0%),对照组8例(44.4%)。3.膝关节腔注射与皮下注射25mg ETA相比,48小时后对SpA膝关节炎的临床疗效指标、关节液白细胞和hs-CRP的改善率无明显差异。4.SpA膝关节炎改良HSS膝关节评分与关节液中细胞因子TNF-α、IFN-γ、IL-2、IL-4、IL-6、IL-10的浓度(P=0.0079～0.0319,r=0.4585～0.5506)及IFN-γ/IL-4比值(P=0.0678,r=0.3964)呈正线性相关,与关节液中hs-CRP水平呈负线性相关(P=0.0013,r=-0.6528)。对于关节液中上述细胞因子和IFN-γ/IL-4比值原为较高水平的SpA患者而言,ETA对其有降低作用;但对于细胞因子和IFN-γ/IL-4比值原为较低水平的SpA患者而言,ETA对其有升高作用。5.RA膝关节炎改良HSS膝关节评分和关节液细胞因子TNF-α、IFN-γ、IL-2、IL-4、IL-6和IL-10的浓度及IFN-γ/IL-4比值无相关性。ETA降低了多数RA患者膝关节液的TNF-α、IFN-γ、IL-2、IL-4、IL-6和IL-10浓度。结论:1.ETA和INF在体外可以抑制RA患者FLS的增殖。2.单次膝关节腔内注射25mg ETA对于炎性膝关节炎是一种有效、安全的治疗选择,其疗效优于2ml复方倍他米松。膝关节腔注射与皮下注射25mg ETA48小时后对SpA膝关节炎的疗效相似。3.SpA关节炎活动性越强,关节液中TNF-α、IFN-γ、IL-2、IL-4、IL-6和IL-10浓度越低,hs-CRP水平越高。ETA对SpA关节液中上述细胞因子浓度起双向调节作用。4.ETA可降低多数RA患者的关节液TNF-α、IFN-γ、IL-2、IL-4、IL-6和IL-10浓度。更多还原

【Abstract】 Objectives:1.To investigate the effects of etanercept(ETA) and infliximab(INF) on the cultured fibroblast-like synoviocytes(FLS) in patients with rheumatoid arthritis(RA).2.To evaluate the efficacy and safety of single intra-articular ETA injection in patients with RA and spondyloarthropathy(SPA) who had knee arthritis without moderate to severe bone erosion in radiography.To compare the efficacy between intra-articular ETA injection and subcutaneous ETA injection in SpA patients with knee arthritis.3.To investigate the relationships between the disease activities of SpA and RA and the inflammatory parameters including Th1/Th2 ratio,hs-CRP level and TNF-α,IFN-γ,Il-2,IL-4,IL-6 and IL-10 levels in synovial fluid.Toinvestigate the effect of ETA on Th1/Th2 balance and the cytokines mentioned above in the synovial fluid of SpA or RA patients.Methods:1.FLS obtained from RA patients through synovectomy was cultured in vitro. WST-8 colorimetric assay was applied to detect the effects of ETA and INF on FLS proliferation.Flow cytometry was applied to detect ETA and 1NF induced FLS apoptosis.2.A randomized,single-blinded,controlled study was conducted in SpA and RA patients with knee arthritis.Total 60 patients were randomized in 2:1 ratio to receive either single intra-articular 25 mg ETA injection or 2 ml compound betamethasone to the knee joints at baseline.They were followed up four weeks after injection.3.Arthrocenteses were done in SpA and RA patients with knee effusion.5 ml synovial fluid was drawn out at baseline and 48 hours after ETA injection.13 RA patients and 11 SpA patients were treated with 25 mg intra-articular ETA injection.11 SpA patients were treated with subcutaneous ETA injection.The synovial fluid cytokines concentrations before and after ETA injection were detected by Enzyme-linked immunosorbent assay(ELISA) and the synovial fluid hs-CRP levels were detected by Nephelometry.Results:1.1.0 to 17.9%and 6.2 to 19.8%FLS proliferations were suppressed by different concentrations of ETA and IFN respectively at 24 to 72 hours.Both ETA and INF at concentration 10μmol/L could not induce FLS apoptosis.2.Significant improvements in the modified Hospital for Special Surgery(HSS) knee score,patient global assessment and physical global evaluation were observed in ETA treated group when compared with compound betamethasone treated group(p= 0.0409～0.0467).Adverse effects were observed in eight ETA treated patients(19.0%) and eight compound betamethasone treated patients(44.4%).3.When comparing with intra-articular 25 mg ETA injection and subcutaneous 25 mg ETA injection in SpA patients,there was no significant difference in clinical response,synovial fluid white cell count and synovial fluid hs-CRP concentrations after 48 hours upon ETA injection.4.In SpA patients,the synovial fluid IFN-γ/IL-4 ratio(p= 0.0678,r= 0.3964) and cytokines concentrations including TNF-α,IFN-γ,IL-2,IL-4,IL-6 and IL-10 positively correlated with the modified HSS knee scores(p= 0.0079 0.0319,r= 0.4585～0.5506).But the synovial fluid hs-CRP concentrations inversely correlated with the modified HSS knee scores(p= 0.0013,r= -0.6528).In SpA patients who had high synovial fluid IFN-γ/IL-4 ratio and cytokines concentrations before treatment,ETA decreased the synovial fluid IFN-γ/IL-4 ratio and cytokines concentrations.But in SpA patients who had low synovial fluid IFN-γ/IL-4 ratio and cytokines concentrations before treatment,ETA increased their concentrations subsequently.5.In RA patients,the synovial fluid IFN-γ/IL-4 ratio and the cvtokines concentrations including TNF-α,IFN-γ,IL-2,IL-4 IL-6 and IL-10 had no correlation with the modified HSS knee scores.ETA decreased the synovial fluid cytokines concentrations including TNF-α,IFN-γ,IL-2,IL-4,IL-6 and IL-10 in most of the RA patients.Conclusions:1.ETA and INF were capable to suppress FLS proliferation in RA patients.2.Single intra-articular 25 mg ETA injection had a better efficacy than 2 ml compound betamethasone.It was an effective and safe therapeutic option for SpA and RA patients who had knee arthritis.There was no difference in 48 hours efficacy between intra-articular 25 mg ETA injection and subcutaneous 25 mg ETA injection in SpA patients with knee arthritis.3.SpA patients with knee arthritis had a higher synovial fluid hs-CRP level when the synovial fluid cytokines concentrations including TNF-α,IFN-γ, Il-2,IL-4,IL-6 and IL-10 were low.ETA had a bi-directional effect in regulating synovial fluid cytokines concentrations in SpA patients.4.ETA was capable to decrease the synovial fluid cytokines concentrations including TNF-α,IFN-γ,IL-2,IL-4,IL-6 and IL-10 in most of the RA patients.更多还原