【作者】 欧阳霞；

【导师】 戴冽；

【作者基本信息】 中山大学 ， 内科学， 2012， 硕士

【摘要】 研究背景和目的类风湿关节炎（Rheumatoid arthritis, RA）是一种常见的以慢性滑膜增生为特点，进而导致关节软骨和骨进行性和不可逆性破坏的慢性炎症性自身免疫性疾病，最终导致关节畸形和功能丧失。滑膜炎和关节破坏是RA发病机制中两个最重要的病理环节。滑膜炎主要表现为衬里层滑膜细胞异常增生及大量炎症细胞浸润，其中成纤维细胞样滑膜细胞是介导RA滑膜炎症和关节破坏的主要效应细胞。关节破坏是RA危害最大的一个病理环节，滑膜血管翳侵入软骨分泌基质金属蛋白酶（Matrix metalloproteinase, MMPs）分解软骨基质，引起软骨及骨的破坏。MMPs的异常表达可促进RA的发生和发展，其中以MMP-3最为重要。在MMPs酶原激活的过程中，MMP-3酶原首先被纤溶酶激活为活性MMP-3，再由它激活其他MMPs酶原。MMP-3是对基质降解起广泛作用的一种蛋白酶，主要由炎症因子IL-1和TNF-α刺激滑膜细胞、成纤维细胞和软骨细胞产生。RA滑膜组织中过度表达MMP-3；关节滑液中MMP-3含量也明显升高，且明显高于血清MMP-3水平；随受损关节数量增多，血清MMP-3水平也升高，说明RA血清中MMP-3主要源自关节。MMP-3的降解产物可促进T细胞活化并诱导多种细胞因子的产生，进一步加重关节的炎症反应。MMP-3是降解关节软骨关键的蛋白酶，它对RA的关节破坏作用主要包括两条途径：首先，软骨与滑膜血管翳连接部位MMP-3的mRNA表达水平明显升高，MMP-3对软骨和骨质有直接降解作用。滑液中的MMP-3也可直接降解无滑膜覆盖的软骨和骨质；其次，血管生成是RA侵蚀性滑膜血管翳的显著特征，滑膜血管生成时，MMP-3分解微血管基底膜和间质成分。MMP-3在RA滑膜炎症和关节破坏中具有重要的作用，因此RA患者血清MMP-3水平是否可作为RA滑膜炎和关节损害的指标值得进一步探讨。有文献报道活动期RA患者血清MMP-3水平较正常人明显升高，但有关活动期RA患者治疗前后血清MMP-3水平的动态随访及血清MMP-3水平与关节破坏的关系的报道较少。本研究旨在通过动态观察RA患者治疗前后血清MMP-3水平，分析MMP-3与病情活动性指标和关节破坏指标的相关性，了解血清MMP-3能否反映RA病情活动程度及预测关节破坏的发生，为血清MMP-3应用于临床提供依据。材料和方法病例组女性RA患者83例，对照组正常女性31例。RA患者包括活动期58例和缓解期25例，对活动期RA患者治疗后的3、6、9和12个月进行了随访，收集各时间点RA患者各项病情活动指标并计算DAS28(3)-CRP评分，评估双手X线片Sharp评分，ELISA法检测血清MMP-3水平，电化学发光法检测骨形成标记物PINP、N-MID.OS和骨吸收标记物β-crosslap。比较各组血清MMP-3水平的差异及RA患者治疗前后血清MMP-3水平的动态变化，分析RA患者血清MMP-3水平与病情活动性指标和关节破坏指标的相关性。结果1、女性RA患者的基线资料活动期RA患者、缓解期RA患者和正常人三组间的年龄无统计学差异（P>0.05）。与缓解期RA患者相比，活动期RA患者的病情活动指如晨僵时间、患者疼痛VAS评分、医生疼痛VAS评分、关节肿胀数（SJC）、关节压痛数（TJC）、患者功能状态分级、HAQ、CRP、ESR、RF和DAS28(3)-CRP均明显升高，差异有统计学意义（P<0.05）。2、基线女性RA患者血清MMP-3水平与病情活动性的关系2.1基线女性活动期RA患者、缓解期RA患者和正常人血清MMP-3水平的比较女性活动期RA患者、缓解期RA患者和正常人的血清MMP-3水平分别为227.35（19.79~913.40） ng/ml、77.34（16.15~308.30）ng/ml和49.00（22.00~73.00）ng/ml，三组血清MMP-3水平有明显统计学差异（χ~2=61.88，P<0.001）。进一步两两比较的结果显示，活动期和缓解期RA患者血清MMP-3水平均较正常人明显升高（P<0.001），活动期RA患者血清MMP-3水平较缓解期RA患者也明显升高（P<0.001）。2.2基线不同活动期女性RA患者血清MMP-3水平的比较按DAS28(3)-CRP评分将活动期RA患者分为低度、中度和高度活动期，缓解期、低度活动期、中度活动期和高度活动期RA患者血清MMP-3水平分别为77.34（16.15~308.30）ng/ml、183.60（77.34~634.90)）ng/ml、204.20（32.03~913.40）ng/ml和244.80（19.79~810.04）ng/ml，比较四组血清MMP-3水平，结果有明显差异（χ~2=17.63，P=0.001）。进一步两两比较的结果显示，低度、中度和高度活动期RA患者血清MMP-3水平较缓解期RA患者均明显升高（P<0.05）。2.3基线女性RA患者血清MMP-3水平与病情活动指标之间的相关性所有83例女性RA患者血清MMP-3水平与患者疼痛VAS评分、医生疼痛VAS评分、SJC、TJC、患者功能状态分级、HAQ、CRP、ESR和DAS28(3)-CRP均呈明显正相关（r=0.35、0.38、0.42、0.32、0.34、0.43、0.58、0.48、0.46，P<0.05）。2.4基线血清MMP-3对诊断女性RA及RA病情活动性的价值2.4.1血清MMP-3对诊断女性RA的敏感性和特异性根据本研究血清MMP-3检测试剂盒说明书提供的女性正常值范围（18~60ng/ml），基线RA患者血清MMP-3水平大于60ng/ml为真阳性，小于60ng/ml为假阴性；正常女性血清MMP-3水平大于60ng/ml为假阳性，小于60ng/ml为真阴性。结果提示，血清MMP-3诊断RA、活动期RA和缓解期RA的敏感性分别为79.52%、91.38%和52.00%，以诊断活动期RA的敏感性最高。2.4.2血清MMP-3对女性RA的诊断价值与正常女性相比，血清MMP-3水平越高，诊断为RA的可能性越大；RA、活动期RA和缓解期RA的AUC均大于0.80；诊断活动期RA的价值最高，AUC=0.97，当约登指数最大为0.88时所对应的血清MMP-3最佳截断值为57.81ng/ml；当RA组的约登指数最大为0.66时所对应的血清MMP-3最佳截断值为59.38ng/ml。2.4.3血清MMP-3对女性RA患者病情活动性的诊断价值与缓解期RA患者相比，血清MMP-3水平越高，诊断为活动期的可能性越大，活动期、低度活动期、中度活动期和高度活动期的AUC均大于0.75；诊断低度活动期RA的价值最高，AUC=0.81，约登指数最大为0.51时所对应的血清MMP-3最佳截断值为128.90ng/ml。3、女性活动期RA患者治疗后血清MMP-3水平的动态变化及其与RA病情活动性的关系3.1女性活动期RA患者治疗后病情活动性的的变化14例女性活动期RA患者治疗后3、6、9、12个月分别进行了随访，各个随访月份分别有14例、11例、8例和6例RA患者。随访RA患者治疗后3、6、9、12个月的患者疼痛VAS评分、医生疼痛VAS评分、SJC、HAQ、CRP、ESR和DAS(3)-28CRP均较基线明显降低（P <0.05）；治疗后3、6、9个月的晨僵时间和TJC均较基线明显降低（P <0.05）；治疗后12个月的CRP较治疗后6个月时明显降低（P <0.05）。3.2女性活动期RA患者治疗后血清MMP-3水平的变化随访RA患者基线和治疗后3、6、9、12个月血清MMP-3水平分别为131.00（19.79~759.40）ng/ml、97.40（23.96~560.40）ng/ml、34.38（16.15~658.30）ng/ml、35.16（22.92~77.34）ng/ml和44.92（25.78~77.34）ng/ml。治疗后6、9、12个月的血清MMP-3水平均较基线明显下降（P<0.05），并且以治疗后12个月的血清MMP-3水平降低最明显，12月血清MMP-3降低了的水平为114.31（-5.99~664.32）ng/ml；与治疗后3个月相比，治疗后9个月的血清MMP-3水平也明显减低（P<0.05）。4、女性RA患者血清MMP-3水平与关节破坏的关系4.1基线女性RA患者血清MMP-3水平与血清骨代谢标记物的相关性基线83例女性RA患者中有58例（包括活动期48例和缓解期10例）检测了血清骨形成标志物PINP、N-MID.OS和骨吸收标志物β-crosslap。相关分析结果显示基线女性RA患者、活动期RA患者和缓解期RA患者血清MMP-3水平与骨形成标志物PINP、N-MID.OS和骨吸收标志物β-crosslap均无明显相关性（P>0.05）。4.2女性活动期RA患者治疗后血清MMP-3水平的动态变化与血清骨代谢指标的相关性本研究入组的女性活动期RA患者有7例在治疗后3个月、6个月复查了血清骨代谢标记物。比较这7例患者基线、治疗后3个月和6个月时骨代谢标记物水平，结果显示各随访月份的PINP（χ~2=3.43）、N-MID.OS（χ~2=1.05）、β-crosslap（χ~2=3.30）和MMP-3（χ~2=3.07）均无明显统计学差异（P>0.05）。进一步的相关分析结果显示，治疗后3个月和6个月的血清MMP-3变化水平均与治疗后6个月的骨吸收标志物β-crosslap变化水平呈明显正相关（r=0.83和0.83，P<0.05），与骨形成标志物PINP、N-MID.OS无明显相关（P>0.05）。4.3基线女性RA患者血清MMP-3水平与关节Sharp评分的相关性基线入组的83例女性RA患者有48例（包括活动期38例和缓解期10例）行双手X线片Sharp评分。相关分析结果显示48例女性RA患者血清MMP-3水平与双手X线Sharp狭窄评分、Sharp侵蚀评分和Sharp总分均呈明显正相关（r=0.43、0.30和0.42，P<0.05）；38例活动期RA患者血清MMP-3水平与双手X线Sharp狭窄评分和Sharp总分呈明显正相关（r=0.37和0.33，P<0.05）；10例缓解期RA患者血清MMP-3水平与关节Sharp评分无明显相关（P>0.05）。4.4女性活动期RA患者治疗后血清MMP-3水平的动态变化与关节Sharp评分变化的相关性本研究入组的14例女性活动期RA患者在动态随访过程中，分别于治疗后6个月、12个月复查了双手关节X线片并进行Sharp评分。比较14例女性活动性RA患者基线、治疗后6个月和12个月的关节Sharp评分，结果显示Sharp狭窄评分（χ~2=3.43）、Sharp侵蚀评分（χ~2=1.05）、Sharp总分（χ~2=3.30）和MMP-3（χ~2=3.30）均无明显统计学差异（P≥0.05）。进一步的相关分析结果显示，治疗后3个月血清MMP-3的变化水平与6个月关节Sharp侵蚀评分的变化水平呈明显正相关（r=0.60，P<0.05）；治疗后6个月血清MMP-3的变化水平与6和12个月关节Sharp总分变化呈明显正相关（r=0.75和0.81，P<0.05）。结论1、血清MMP-3水平与女性RA患者病情活动相关，血清MMP-3的水平越高，RA病情活动度越高；治疗后随病情活动度的下降或缓解，血清MMP-3水平也随之下降；血清MMP-3的检测可作为监测治疗前后病情活动动态变化的指标。2、女性RA患者治疗后血清MMP-3降低水平与骨吸收标志物血清β-crosslap降低水平呈正相关，提示血清MMP-3可用于监测RA患者骨吸收的情况。3、女性RA患者血清MMP-3水平能反映活动期RA的关节狭窄和侵蚀情况，可作为监测关节软骨和骨破坏的指标。更多还原

【Abstract】 Backgroud and objectiveRheumatoid arthritis (RA) is a common chronic autoimmune diseasecharacterized by synovial proliferation, which causes progressive and irreversiblearticular cartilage and bone destruction, and ultimately lead to joint deformity anddisability. Two of the most important pathological features in RA are synovitis andjoint destruction. Synovitis mainly manifest as dysplasia of lining layer of synovialcells and infiltration of inflammatory cells. Fibroblast-like synoviocytes (FLS) are keyeffector cells mediating synovium inflammation and joint destruction. Jointdestruction is the most harmful pathological process in RA. Synovial pannus invadecartilage and secrete matrix metalloproteinases (MMPs) to decompose cartilagematrix, and thus causing damage of cartilage and bone.The occurrence and development of RA are promoted by abnormal expression ofMMPs, and MMP-3is the most important one. In the process of activating MMPsproenzymes, MMP-3proenzyme is activated as active MMP-3by fibrinolysin firstly,and then other MMPs proenzymes are actived by active MMP-3. MMP-3plays anextensive role in degrade matrix. MMP-3is mainly produced by synovial cells,fibroblasts and chondrocytes which are stimulated by IL-1and TNF-α. MMP-3isoverexpressed in RA synovial tissue; MMP-3levels are increase dramatically insynovial fluid, MMP-3levels in synovial fluid are significantly higher than in serum;serum MMP-3levels elevate with increased number of damaged joints. The above indicates that serum MMP-3is mainly derived from joints. The degradated productsof MMP-3can promote activation of T cell and induce the generation of multiplecytokines, resulting in aggravation of inflammation in joints. MMP-3is a criticalprotease for degrading cartilage; the role of MMP-3in RA joint destruction includestwo main parts: Firstly, MMP-3mRNA is overexpressed in the connection betweensynovial pannus and cartilage, MMP-3can directly degrade cartilage and bone.Synovial fluid MMP-3also directly degrades cartilage and bone where withoutsynovial membrane covering. Secondly, angiogenesis is a distinguishing feature ofRA aggressive synovial pannus. MMP-3digests microvascular basement membraneand stroma in the process of synovial angiogenesis.MMP-3plays an important role in RA synovial inflammation and jointdestruction. So, further exploration is warranted to analyze whether serum MMP-3inRA patients can be used as indicator of synovitis and joint destruction. It had beenreported that serum MMP-3levels in active RA patients was significantly higher thanin normal control. But there were few reports about close dynamic follow-up of serumMMP-3levels before and after treatment and the relationship between serum MMP-3levels and joint destruction in active RA patients.This study was designed to observechanges of serum MMP-3levels in RA patients before and after treatment, andanalyzes the correlation between serum MMP-3levels and disease activity indicatorsand or joint destruction indicators. The aim of this study is to evaluate whether serumMMP-3can reflect disease activity and predict the occurrence of joint destruction inRA patients, and to provide theory evidence for the clinical application of serumMMP-3.Materials and methodsWe recruited eighty-three female RA patients and thirty-one normal women ascontrol group. RA patients included fifty-eight active patients and twenty-fiveremissive patients，active RA patients were followed-up at the3th,6th,9th, and12thmonths after treatment. At different followed-up times, parameters of disease activityindicators were collected and DAS28(3)-CRP scores were counted by rheumatologist,hands X-ray Sharp scores were assessed by a radiologist. Serum MMP-3levels weremeasured by ELISA; serum bone formation markers such as N-MID.OS and PINPand bone resorption marker such as β-crosslap were measured by electrochemilumin- escence. We compared the difference of serum MMP-3levels among active RApatients, remissive RA patients and normal control, and observed the changes ofserum MMP-3levels before and after treatment, and analyzed the correlationsbetween serum MMP-3levels and disease activity indicators or joint damageindicators.Results1Baseline data in female RA patientsThere was no statistically difference of age among active RA patients, remissiveRA patients and normal control. Compared with remission RA patients, diseaseactivity indicators such as duration of morning stiffness, pain VAS scores of patient,pain VAS scores of doctor, TJC, SJC, functional status score of patient, HAQ, CRP,ESR, RF and DAS28(3)-CRP were increased obviously in active RA patients(P<0.05).2The relationship between serum MMP-3levels and disease activity in female RApatients at baseline2.1The comparison of serum MMP-3levels among female active RA patients,remission RA patients and normal control at baseline.Serum MMP-3levels of active RA patients, remission RA patients and normalcontrol were227.35(19.79~913.40) ng/ml,77.34(16.15~308.30) ng/ml and49.00(22.00~73.00) ng/ml respectively. There was statistically difference of serum MMP-3levels among active RA patients, remission RA patients and normal control (χ~2=61.88,P<0.001). Further pairwise comparisons showed serum MMP-3levels of active orremissive RA patients were higher than normal control (P<0.001). Serum MMP-3levels of active RA patients were higher than remissive RA patients, too (P<0.001).2.2The comparison of serum MMP-3levels among different grades of activity RApatients at baselineAccording to DAS28(3)-CRP, active RA patients were divided into low, mediumand high activity patients. Serum MMP-3levels of remission, low activity, mediumactivity and high activity RA patients were227.35(19.79~913.40) ng/ml,77.34(16.15~308.30) ng/ml and49.00(22.00~73.00) ng/ml respectively. Further pairwise compar- esons showed serum MMP-3levels of RA patients were higher in high, medium andlow activity groups than in remission group (P<0.05).2.3The correlation between serum MMP-3levels and disease activity indicators infemale RA patients at baseline.Serum MMP-3levels of all RA patients were positive correlated with pain VASscores of patients, pain VAS scores of doctors, functional status score of patients, SJC,TJC, HAQ, CRP, ESR and DAS28(3)-CRP (r=0.35,0.38,0.42,0.32,0.34,0.43,0.58,0.48,0.46; P<0.05).2.4The value of serum MMP-3levels for diagnosing female RA and disease activityat baseline2.4.1The sensitivity and specificity of serum MMP-3levels for diagnosing RApatientsAccording to serum MMP-3detective kit instruction, the female normal range is18~60ng/ml. At baseline, serum MMP-3levels of all RA patients, active RA patientsand remission RA patients greater than60ng/ml were true positive, less than60ng/mlwere false negative. Serum MMP-3levels of normal female greater than60ng/mlwere false positive, less than60ng/ml were true negative. The results suggested thatsensitivities of serum MMP-3for diagnosis of RA patients, active RA patients andremission RA patients were79.52%,91.38%and52.00%; the highest sensitivity wasfor diagnosing active RA.2.4.2The value of serum MMP-3levels for diagnosing female RA patientsCompared with normal control, the higher levels of serum MMP-3, the greaterpossibility for diagnosing RA; the AUC of RA, active RA and remission RA were allgreater than0.8; the value of diagnosis was best for active RA (AUC=0.97), theoptimal cutoff value of serum MMP-3level was57.81ng/ml when the highest Youdenindex is0.88; the optimal cutoff value of serum MMP-3level in all RA patients was57.81ng/ml when the highest Youden index was0.66.2.4.3The value of serum MMP-3levels for diagnosing disease activity in female RApatientsCompared with remission RA, the higher levels of serum MMP-3, the greater possibility of diagnosing the activity of RA; the AUC of active RA, low, medium andhigh activity RA groups were all greater than0.75, the value of diagnosis was best inlow active RA (AUC=0.81), the optimal cutoff value of serum MMP-3level was128.90ng/ml when the highest Youden index was0.51.3Changes of serum MMP-3levels in dynamic follow-up and its relationship withchanges of disease activity in female active RA patients3.1Changes of disease activity after treatment in female active RA patientsFourteen cases of female active RA patients were followed up at the3th,6th,9th,12th months after treatment. There were fourteen cases, eleven cases, eight cases andsix cases of RA patients at each follow-up month.Parameters of active RA patients at the3th,6th,9th, and12th months weresignificantly lower than at the baseline (P<0.05), these parameters includes pain VASscores of patient, pain VAS scores of doctor, SJC, HAQ, CRP, ESR and DAS28(3)-CRP; morning stiffness time and TJC at the3th,6th and9th months weresignificantly decreased than at the baseline (P<0.05). CRP at the12th month issignificantly lower than at the6th month (P<0.05).3.2Serum MMP-3levels in female active RA patients after treatmentSerum MMP-3levels of active RA patients at baseline, the3th, the6th, the9th,the12th months were131.00(19.79~759.40) ng/ml、97.40(23.96~560.40) ng/ml、34.38(16.15~658.30) ng/ml、35.16(22.92~77.34) ng/ml and44.92(25.78~77.34)ng/ml respectively. Serum MMP-3levels of active RA patients at the6th,9th,12thmonths were decreased significantly than at baseline (P<0.05), and the most obviousreduction of serum MMP-3levels was at the12th month. Serum MMP-3levels at the6th month were also decreased significantly than at the9th month (P<0.05).4The relationship between serum MMP-3levels and joint destruction in RA patients4.1The correlation between serum MMP-3levels in female RA patients and bonemetabolism markers at baselineAt baseline, fifty-eight out of eighty-three cases of recruited female RA patients(including forty-eight active RA patients and ten remissive RA patients) weremeasured peripheral serum bone metabolism markers. Correlation analysis showed that there was no significant correlation between serum MMP-3levels and boneformation markers such as PINP and N-MID.OS or bone resorption markers such asβ-crosslap in RApatients, active RApatients or remission RApatients (P>0.05).4.2Changes of serum MMP-3levels in dynamic follow-up and its relationship withchanges of bone metabolism markers in female active RA patientsSeven out of all the baseline female active RA patients were followed-up, bonemetabolism markers were measured at the3th month, the6th month. There was nostatistically difference of PINP (χ~2=3.43), N-MID.OS (χ~2=1.05),β-crosslap (χ~2=3.30),and MMP-3(χ~2=3.07)among the followed-up months (P>0.05). Further pairwisecomparison results showed changes of serum MMP-3levels within three or sixmonths were positive correlated with changes of β-crosslap within six months(r=0.83,0.83;P<0.05). There were no correlation between changes of serum MMP-3levels and changes of bone formation markers (P>0.05).4.3The correlation between serum MMP-3levels and hands X-ray Sharp scores infemale RA patients at baselineAt baseline, forty-eight out of eighty-three cases of recruited female RA patients(including thrity-eight active RA patients and ten remissive RA patients) wereassessed hands X-ray sharp scores. Correlation analysis showed that serum MMP-3levels was positive correlated with hands X-ray Sharp narrow scores, Sharp erosionscores and Sharp total scoresin RA patients (r=0.43,0.42; P<0.05). The serum MMP-3levels was also positive correlated with hands X-ray Sharp narrow scores and Sharptotal scores in active RA patients (r=0.37,0.33; P<0.05). There was no significantcorrelation between serum MMP-3levels and hands X-ray Sharp scores in remissionRA patients.4.4Changes of serum MMP-3levels in dynamic follow-up and its relationship withchanges of hands X-ray Sharp scores in female active RA patientsForteen out of all the baseline female active RA patients were followed-up andassessed hands X-ray sharp scores. Hands X-ray sharp scores were measured at the6th month, the12th month after treatment. There was no statistically difference ofSharp narrow scores (χ~2=3.43), Sharp erosion scores (χ~2=1.05), Sharp total scores(χ~2=3.30) and serum MMP-3level(χ~2=3.30) among the follow-up months (P≥0.05). Further pairwise comparisons showed change of serum MMP-3levels within threemonths was positive correlated with change of Sharp erosion scores within six months(r=0.60, P<0.05). The change of serum MMP-3levels within six months was positivecorrelated with changes of Sharp total scores within six and twelve months (r=0.75,0.81; P<0.05).Conclusions1Serum MMP-3levels were positive correlate with disease activity indicators infemale RA patients, the higher levels of serum MMP-3, the severer disease of activity;the serum MMP-3levels reduce with disease activity decrease or remission aftertreatment; MMP-3may be used to monitor disease activity change before and aftertreatment.2After treatment, the decreased levels of serum MMP-3in female RA patientswere positively correlated with decrease levels of β-crosslap, indicating that serumMMP-3may be used to evalute bone resorption.3The serum MMP-3level in female active RA patients may reflect the narrowand erosion of joints, indicating that serum MMP-3may be used as indicators tomonitor joint cartilage and bone destruction.更多还原