【作者】 刘海波；

【导师】 刘红春；

【作者基本信息】 郑州大学 ， 临床检验诊断学， 2013， 硕士

【摘要】 类风湿性关节炎(rheumatoid arthritis,RA)是临床上常见的疑难病症,是一种慢性、进行性的自身免疫性疾病(autoimmune disease, AID)。该病病因至今不明,可能与遗传、内分泌、地理、职业、营养、细菌和病毒感染以及心理和社会环境的差异等因素有关系,以慢性对称性的多滑膜关节炎和关节外病变为其主要临床表现。临床上该病好发于手、腕、足等一些小关节,反复发作,呈对称性分布,疾病早期表现为关节肿痛,继而软骨破坏、关节间隙变窄,晚期因严重的骨质破坏、吸收而导致关节出现僵直、畸形、功能障碍。该病预后不良,致残率较高。临床上RA活动情况和病变程度的判断对该病的诊疗及预后均有很大意义,’一直以来,关于RA病情严重程度和早期诊断的评价指标都是研究的热点问题。目前临床上评价RA病情活动度需要综合诸多因素来判断,如肿胀关节数、疼痛及压痛关节数、晨僵时间、C-反应蛋白(C-reaction protein, CRP)、血沉(erythrocyte sedimentation rate, ESR)等,目前应用最广泛的评价标准是其中的DAS评分系统。近年来,随着仪器和检验技术的不断进步,临床上越来越重视对炎症指标的测定,希望找到更加敏感和特异的炎症指标,以便更好的为临床服务。Clq作为补体经典激活途径的启动子,在防止组织免疫性损伤和清除体内循环免疫复合物等方面发挥着重要作用。目前,关于Clq的研究主要集中在系统性红斑狼疮(systemic lupus_erythematosus, SLE)患者中,而在RA患者中的研究比较少见,血清淀粉样蛋白A(serum amyloid A protein, SAA)是一种急性时相反应蛋白,属于载脂蛋白家族中的异质类蛋白质,相对分子量约12000。在急性时相反应中,SAA含量急剧升高,研究发现,在患炎性关节炎的案例中,血清淀粉样蛋白A与疾病活动性的关系最为密切。相关研究表明,SAA水平在RA患者血清中亦升高,并且更能反映RA的活动度。但SAA在RA发病机制中的作用目前尚不明确,需要继续探讨。RA是由免疫介导的慢性炎性疾病,其炎症和组织损伤涉及多种炎症因子,各种异常表达的炎症因子在RA的病程进展中均起着重要作用。研究发现,IL-6(lnterleukin-6)主要是由活化的单核细胞产生,是RA滑膜炎症和关节损伤的重要介质之一。相关研究表明, RA患者活动期血清中IL-6水平升高,并与病情活动的某些指标相关。因此本研究拟对不同时期的RA患者血清中C1q、SAA及IL-6的水平进行检测并与正常对照组进行比较,以探讨三者水平变化在RA不同时期患者中的临床诊断意义。材料和方法1.临床资料和方法收集符合美国风湿病学会(ARA)1987年修订的分类标准的RA患者活动期和缓解期各50例及30例健康体检者的血液标本,离心后置于—80℃冰箱中保存备用,以备测定各组血清C1q、SAA及IL-6的水平。采用德国罗氏公司生产的罗氏P-800全自动生化分析仪,上海北加生化试剂有限公司生产的C1q试剂,以免疫透射比浊法(immunoturbidimetricassay, ITA)测定血清C1q。采用德国罗氏公司生产的罗氏cobas8000全自动生化分析和IL-6试剂,以电发学发光(electro chemiluminescence, ECL)法测定血清IL-6。采用上海赛默飞世尔仪器有限公司生产的Multiskan Mk3型酶标仪和美国R&D公司生产的SAA试剂盒,以酶联免疫吸附测定(enzyme-linkedimmunosorbent assay, ELISA)法测定血清SAA含量。以上所有操作均按试剂盒说明书严格操作。2.统计学处理采用SPSS17.0统计软件进行统计学分析,计量资料以均数±标准差(x±s)表示,组间比较采用单因素方差分析,以P<0.05为差异具有统计学意义。结果1.RA活动组患者50例,男性12例,女性38例,平均年龄(46.54±13.16)岁。RA缓解组患者50例,男性10例,女性40例；平均年龄(41.82±14.34)岁。正常对照组30例,男性10例,女性20例；平均年龄(43.03±13.35)岁。RA活动组、RA缓解组及正常对照组三组之间在年龄及性别构成等方面均无统计学差异(P均>0.05)。2.RA活动组患者血清Clq、SAA和IL-6的水平分别为(27.32±8.31)mg/dl、(2953.91±874.17) μg/ml、(78.06±36.22) pg/ml, RA缓解组患者血清C1q, SAA和IL-6的水平分别为(22.41±4.91)mg/dl、(211.91±52.56)μg/ml、(5.47±1.65) pg/ml,正常对照组血清C1q, SAA和IL-6的水平分别为(16.62±4.86) mg/dl、(185.08±61.38)μg/ml、(2.38±0.47) pg/ml,RA患者活动组Clq水平高于缓解组和正常对照组,缓解组Clq水平高于正常对照组,差异具有统计学意义(P<0.05),RA患者活动组SAA和IL-6水平高于缓解组和正常对照组,差异具有统计学意义(P<0.05),RA患者缓解组SAA和IL-6水平与正常对照组相比无统计学意义(P>0.05)。结论1.RA活动期患者血清C1q、SAA及IL-6的水平均有高表达,RA患者体内C1q、SAA及IL-6水平的变化与RA疾病的活动度有关,它们可能成为RA病情监测和疗效判断的临床指标。2. C1q、SAA及IL-6在RA的发生、发展过程中起着重要作用,探讨三者在RA发病机制中的作用,有利于观察RA的治疗效果,从而更好地指导RA的临床治疗。更多还原

【Abstract】 Rheumatoid arthritis(RA) is a common difficult and baffling diseases in clinic, it is a chronic and progressive autoimmune disease(AID). Its etiology is unknown so far,and may be associated with heredity, endocrine, geography, occupation, nutrition, bacteria and virus infection,and psychological and social environment difference factors.The main clinical manifestations of RA include chronic, symmetry and many synovial arthritis and the extra-articular changes. Clinically, the disease occurs in the hand, wrist, feet and other small joints,it attacks recurrently and shows symmetric distribution.In the early stage，joint manifests swelling and pain,then articular cartilage is damaged and joint space narrows down,in the late stage, serious bone destruction and absorption lead to joint rigidity, deformity, dysfunction.The disease shows poor prognosis and has a high probability of disability.Clinically,the judgment of pathological changes degree and activities of RA has great significance for the diagnosis and treatment and prognosis.All the time, the evaluation indictor,which evaluates early diagnosis and disease severity of RA,is the research hotspot problem.At present,the disease activity evaluation of RA needs to comprehensive judgment by many factors in clinic,such as the number of joint swelling,pain and tenderness joint number,morning stiffness time, C-reaction protein (CRP), erythrocyte sedimentation rate(ESR) and so on,disease activity score(DAS) rating system is currently the most widely used evaluation standard among the evaluation standards. In recent years, with the continuous improvement of instrument and inspection technique, the measurement of inflammation marker is also more and more be taken seriously,Clinicians hope to find more sensitive and specific inflammation marker, in order to provide better service for clinic.Clq as the promoter of complement classic activation pathway, it plays an important role in preventing tissue autoimmune injury and clearing circulating immune complexes in vivo.At present, the research about Clq is mainly concentrated in patients with systemic lupus erythematosus,but in patients with RA is rarely founded. Serum amyloid A protein(SAA) is an acute phase protein, belongs to heterogeneous class protein of the apolipoprotein family, its relative molecular weight is about12000. In the acute phase reaction, SAA content increased sharply, the research found that serum amyloid A protein is most closely related to disease activity in the case of suffering from inflammatory arthritis. Related studies have shown that elevated serum SAA levels in patients with RA, and better reflect the activity of RA. However, the role of SAA in the pathogenesis of RA is not clear, so it needs to continue to discuss. RA is a chronic immune-mediated inflammatory diseases, its inflammation and tissue injury involves a variety of inflammatory cytokines, abnormal expression of various inflammatory factors play an important role in the progression of RA. The study found that lnterleukin-6(IL-6) is mainly produced by activated monocytes. It is one of the important mediators of synovial inflammation and joint injury of RA. Related studies have shown that IL-6levels increase in serum of patients with with RA in active stage, and is related to some indicators of disease activity. Therefore, this study intends to detect C1q,SAA and IL-6levels of different periods in the serum of patients with RA, and compares their levels with that of the normal control group,in order to investigate clinical significance of changes of the three levels of different periods in patients with RA.Materials and Methods1. Clinical data and methods We collect50blood specimens of patients with RA in active stage and inactive stage respectively, all the RA patients fulfilled the American College of Rheumatology (ARA) revised criteria for classification in1987, and we also collect30healthy human blood specimens, then store them at-80℃refrigerator after centrifugation, our purpose is to detect Clq, SAA and IL-6levels in serum of the three groups.We use Roche P-800automatic biochemical analyzer produced by Germany Roche and Clq reagent produced by Shanghai Beijia biochemical reagent Co, Ltd to detect serum C1q levels by immunoturbidimetric assay(ITA).We use Roche cobas8000automatic biochemical analyzer and IL-6reagent produced by Germany Roche to detect serum IL-6levels by electro chemiluminescence(ECL) method. We use Multiskan Mk3type microplate reader produced by Shanghai Thermo Fisher Scientific Insrument Co, Ltd and SAA kit produced by the United States R&D company to detect serum SAA levels by enzyme-linked immunosorbent assay(ELISA) method. All operations are strictly operated according to the kit instructions.2. Statistical analysisThe SPSS17.0statistical software package was used for statistical analysis.The measurement data were expressed by mean±standard deviation(x±s),the groups were compared by one-way ANOVA analysis, P value<0.05was considered significant.Results1In the group of50patients with RA in active stage,12subjects were male,38subjects were female,the mean age was (46.54±13.16) years old. In the group of50patients with RA in inactive stage,10subjects were male,40subjects were female, the mean age was (41.82±14.34) years old. In the group of30normal controls,10subjects were male,20subjects were female, the mean age was (43.03±13.35) years old. There were no statistical differences among the three groups in age and gender (P>0.05).2The serum levels of C1q,SAA and IL-6in the group of patients with RA in active stage were (27.32±8.31) mg/dl、(2953.91±874.17) μg/ml、(78.06±36.22) pg/ml, the serum levels of C1q,SAA and IL-6in the group of patients with RA in inactive stage were (22.41±4.91) mg/dl、(211.91±52.56)μg/dl、(5.47±1.65) pg/ml, the serum levels of C1q,SAA and IL-6in the group of normal controls were (16.62±4.86) mg/dl、(185.08±61.38)μg/ml、(2.38±0.47) pg/ml. The serum Clq levels in the group of patients with RA in active stage was higher than that in the group of patients with RA in inactive stage and the normal control group, the serum Clq levels in the group of patients with RA in inactive stage was higher than that in the normal control group, and it has the statistical Signifcance (P<0.05), the serum SAA and IL-6levels in the group of patients with RA in active stage were higher than those in the group of patients with RA in inactive stage and the normal control group, and it has the statistical Signifcance (P<0.05), there were no statistical differences between the group of patients with RA in inactive stage and the normal control group in the serum SAA and IL-6levels(P>0.05).Conclusions1The serum levels of Clq, SAA and IL-6are highly expressed in the serum of patients with RA in active stage. The changes of serum C1q、SAA and IL-6levels in patients with RA have relations with the disease activity of RA, and they may become the clinical indicators of condition monitoring and curative effect assessment of patients with RA.2Clq, SAA and IL-6play an important role in the occurrence and development process of RA.Discussing the role of the three in the pathogenesis of RA may be beneficial for observing the therapeutic effect of RA,so as to better guide the clinical treatment opf RA更多还原