【作者】 吕卓；

【导师】 李娟；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2010， 硕士

【摘要】 研究背景类风湿关节炎(Rheumatoid Arthritis, RA)是一种主要以关节关节滑膜炎症和关节外病变为表现的系统性自身免疫性疾病,当累及关节软骨和骨质时,逐渐形成关节畸形和强直,导致关节功能丧失。在我国的患病率约为0.4%。RA的发病机制尚不明确,目前认为RA由多种因素联合介导的自身免疫病,环境和遗传等因素共同发挥作用。与RA相关的白细胞抗原(human leukocyte antigen,HLA)-DR4属HLA-Ⅱ类分子,HLA-Ⅱ类分子在抗原识别、递呈和与TCR结合方面起着重要作用,主要功能是与抗原结合将递呈质T细胞。当受到外界刺激或发生自身改变时,HLA的抗原决定簇发生了改变,借助共同表位和基因剂量效应,使具有HLA成为免疫活化的靶点,产生T细胞激活及一系列免疫介质如细胞因子的释放,导致RA免疫性炎症发生,最终导致滑膜、软骨及骨质的破坏。HLA-DR4阳性的患者更容易发展为侵蚀性病变,预后不好,药物疗效差。RA患者体内存在有多种对瓜氨酸化多肽特异的抗体,瓜氨酸化在RA发病中有很强的特异性,肽基精氨酸脱亚氨酶(peptidylarginine deiminase PAD)所编码的酶具有对mRNA翻译后蛋白进行瓜氨酸化修饰,可以催化抗原蛋白多肽进行瓜氨酸化反应,使蛋白中的精氨酸残基转化为瓜氨酸残基,并通过形成瓜氨酸肽打破机体的免疫耐受而诱发自身免疫反应的发生。2003至今有多项研究显示PADI4基因多态性与RA有很强的相关性。T细胞活化在RA发病中起重要作用,CD4+T细胞受不同抗原的刺激分化为不同效应亚群,此过程受调节性转录因子的调控,转录因子起关键作用。信号转导与转录激活因子4(signal transducer and activator of transcription 4, STAT4)被认为是调节Th1细胞分化的关键的调控因子,亦发现与Th17的分化有关。2006年鉴定出STAT4与RA相关,之后有多项研究结果显示其是RA易感基因。信使核糖核酸(MessengerRNA, mRNA)为由脱氧核糖核酸(DNA)转录合成的带有遗传信息的一类单链核糖核酸(RNA),与遗传性疾病的发生与表达密切相关。探讨RA患者体内易感基因的表达水平对RA发病及疾病进展有重要意义。RA属于祖国传统医学“痹病”范畴,因人体遭受风、寒、湿、热之邪的侵袭,气血为病邪阴闭而引起的疾病。以筋骨、肌肉、关节酸痛、麻木、重着、屈伸不利和关节肿大、灼热等为临床主要表现。其主要病机是气血痹阻不通,筋脉关节失于濡养,以祛风、除湿、舒筋活络为治疗大法。中医药治疗RA历史悠久,疗效确切,且不良反应相对较轻,目前得多越来越关注,以期达到缓解症状和提高整体生存质量的目的。清风藤用于治疗关节炎已经有上千年历史。其主要有效成分为青藤碱,现代药理研究显示,青藤碱具有良好的抗炎和抗风湿的作用。清风痛胶囊(青藤碱制剂)具有祛风化湿、温经散寒、活血通络的功效,既往药理及临床研究显示对急、慢性炎症具有良好的抗炎镇痛效果。研究清风痛胶囊对RA的疗效及对HLA-DR4、PADI4、STAT4基因mRNA表达水平的影响有助于了进一步探讨研究青藤碱制剂对RA的作用和机理。中医学认为体质是个体在先天遗传和后天获得的基础上表现出的形态结构、生理机能和心理状态方面综合的、相对稳定的特质,是制约和影响疾病发生发展变化的基本因素之一,并与疾病的发展过程、预后密切相关。RA发病存在区域差异性,临床表现存在个体差异性,有必要对RA及其相关基因表达水平与中医体质的关系进行深入研究,而目前这方面研究较少。同时定量检测多种基因表达水平依靠传统检测手段无法实现。QGP技术(QuantiGene Plex)结合了b-DNA (Branched DNA)信号放大与Luminex xMAP液相芯片(liquid chip)平台,其优势是可直接从全血样本中检测mRNA表达,无需对进行RNA提纯、反转录、PCR扩增,可以同时对极少量样本中的3至36个基因进行高通量定量,且消除了众多人为因素的干扰。研究目的1.采用QGP技术对RA患者外周血HLADR4、PADI4、STAT4基因表达进行定量,并探讨表达水平与RA病情活动、抗CCP抗体、类风湿因子(RF)的关系。2.评价清风痛胶囊(青藤碱制剂)联合MTX治疗活动性RA12周的疗效、安全性,比较治疗前后HLA-DR4、PADI4、STAT4基因表达水平变化,探讨青藤碱制剂的干预影响。3.分析RA患者中医体质类型分布及常见中医体质类型HLA-DR4、PADI4、STAT4基因表达水平,为进一步研究中医体质分型与RA相关基因表达的关系提供理论依据。方法1研究对象2009年6月至2009年12月在南方医科大学附属南方医院中医风湿科门诊及病房确诊的RA患者38例,男性8例,女性30例,活动期患者24例,其中男性4例,女性20人。缓解期患者14例,男性4例,女性10例。健康对照组为无血缘关系的同期医院门诊健康体检者12人,男性4例,女性8例。2研究方法2.1 RA患者外周血HLA-DR4、PADI4、STAT4基因mRNA表达水平研究。应用QGP技术检测RA患者及健康人外周血HLA-DR4、PADI4、STAT4基因mRNA表达水平,以PPIB、HPRT1为管家基因进行校正。2.2清风痛胶囊(青藤碱制剂)治疗RA的临床评价及对HLA-DR4、PADI4、STAT4基因mRNA表达影响。2.2.118例活动期RA患者予清风痛胶囊联合MTX治疗12周,疗效评价为12周较基线期达到RA疗效评价标准(ACR制定标准)ACR20、ACR50患者的比例、DAS28评分、晨僵持续时间、关节肿胀和关节触痛计数、休息痛、受试者及研究者总体评分,健康评估问卷、血沉(ESR)及C反应蛋白(CRP),抗CCP抗体及RF,定期检查血、尿常规、肝肾功能及心电图等并记录不良事件的发生。2.2.2 QGP方法检测RA患者治疗前后外周血HLA-DR4、PADI4、STAT4基因mRNA的表达水平。2.3通过《中医体质分类与判定》标准对RA患者进行中医体质分类。3统计学处理所有数据用SPSS13.0统计软件分析。计量资料采用X±S表示。两样本计量资料比较采用独立样本t检验,组内前后差异比较用配对t检验或Wilcoxon检验；多组样本均数比较采用one-way ANOVA,相关分析采用Spearman或Person相关分析方法,双侧P<0.05为差异有统计学意义。结果1.QGP方法检测HLA-DR4、PADI4、STAT4基因mRNA表达水平1.1 QGP方法检测HLA-DR4、PADI4、STAT4背景信号荧光强度分别为6.0、9.0、5.5；最低检测限分别为16.4、18.6、15.1,批内CV分别为5.4%、7.7%、6.8%,批间CV分别为7.3%、10.1%、9.8%。1.2 RA患者外周血HLA-DR4、PADI4、STAT4基因mRNA水平较健康对照组上调(P<0.05；P<0.01)。活动期与缓解期HLA-DR4mRNA4表达水平无差异(P>0.05)；RA活动期PADI4、STAT4mRNA表达水平高于缓解期(P<0.05)。HLA-DR4mRNA表达与DAS28评分、抗-CCP抗体、RF均无相关性(P>0.05),PADI4与DAS28、抗-CCP抗体水平呈正相关(r=0.521, P=0.01; r=0.558, P=0.000), STAT4mRNA表达水平与DAS28、RF呈正相关((r=0.503,P=0.001；r=0.493, P=0.02)。2.清风痛胶囊(青藤碱制剂)治疗RA的临床评价及对HLA-DR4、PADI4、STAT4基因mRNA表达影响2.1治疗组12周时达到ACR20的患者比例为72.2%,达到ACR50患者比例为22.2%,DAS28评分显著降低(P<0.05),治疗12周后临床症状及实验室指标较基线期均有显著改善(P<0.01,P<0.05),观察过程中未发现严重的不良反应及并发症。2.2治疗后HLA-DR4基因表达表达水平较治疗前无差异(P＞0.05),PADI4、STAT4基因表达水平较治疗前降低(P<0.05)。3.RA中医体质分析及常见体质类型HLA-DR4、PADI4、STAT4表达水平比较3.1本研究RA患者最常见中医体质类型为阳虚质、瘀血质,余见湿热质、痰湿质、气虚质、阴虚质及平和质。3.2阳虚质、瘀血质与其它体质HLA-DR4、STAT4基因mRNA表达水平无差异(P>0.05),瘀血质PADI4基因mRNA表达高于阳虚质及其它体质(P<0.05,P<0.01),阳虚质和其它体质间表达无差异(P>0.05)结论1.QGP方法检测RA患者外周血HLA-DR4、PADI4、STAT4基因mRNA表达有较高的灵敏性,且节省样本用量少,操作简便,可作为同时检测RA多种基因表达水平的方法应用于临床及实验。2.RA患者外周血HLA-DR4、PADI4、STAT4基因mRNA表达水平上调；HLA-DR4表达水平与疾病活动、抗CCP抗体、RF水平无相关性,PADI4、STAT表达水平与病情活动关系密切,并与疾病进展及严重程度有关。检测PADI4、STAT4基因表达水平可能有助于对RA疾病活动的判断。3.清风痛胶囊(青藤碱制剂)联合MTX治疗活动性RA可明显控制RA炎症,减轻RA疾病活动的临床症状和体征,在治疗过程中未发现严重的不良反应及并发症；可降低外周血PADI4、STAT4mRNA表达水平,提示青藤碱制剂和MTX或青藤碱制剂可能直接或间接通过减少PADI4、STAT4基因表达来阻止RA进展。4.阳虚质和瘀血质为本次纳入RA患者最为常见的体质类型,瘀血质PADI4基因表达水平较高,提示RA患者易感基因表达水平可能对RA中医体质分型提供一定参考价值。更多还原

【Abstract】 Rheumatoid arthritis (rheumatoid arthritis, RA) is a systematic autoimmune disease which is characteristic of persistently progressive synovitis and extra-articular lesions. Articular cartilage and bone can be involved, resulting in joint deformity and ankylosis, even loss of joint function.The prevalence is about 0.4% of the population in our county.The pathogenesis of RA is still a dynamic area of researeh. The etiology of RA remains illusive and is most likely multifactorial. Heredity、environment and immunological regulation have palyed significant roles together. HLA-DR4,a member of HLAⅡmolecules, is known to be related to RA. The main function of HLAmolecules is to present antigen to T cells. HLAantigenic determinants will be changed when confronted with external stimuli or mutation. Through shared epitope and dosage effect of gene, HLA molecules become the target of immunological activation and then T cell was activated. A series of immune molecules are released such as cytokines which can induce RA and eventually lead to synovium, cartilage and bone destruction. Patients with HLA-DR4 are most likely to develop erosive lesions and poor prognosis and efficacy. It was considerable observed that a very high proportion of patients with RA have antibodies to citrulline-containing proteins. Peptidylarginine deiminase type4 (PADI4) post-translationally converts peptidylarginine to citrulline, a process called citrullination. As a result, uimmune tolerance is broken and autoimmune reactions occur. Since 2003, numerous studies have shown that PADI4 gene polymorphism has a strong correlation with RA. In addition, activated T cell plays an important role in RA pathogenesis. Transcription factors paly a key rolr in the process of CD4 +T cells differentiation STAT4 is considered as a key regulatory factor for Thl cell differentiation, also partly relevant to Th17 differentiation. A number of studies have demonstrated that STAT4 is one of RA susceptible genes since 2006. It is important to study the expression of these susceptible genes for understanding the pathogenisis and progression of RAIn Traditional Chinese medieine(TCM). the condition that is congruent with arthritis is called "Bi syndrome." Bi syndrome manifests as pain, soreness, or numbness of muscles, tendons and joints which is the result of the body being "invaded" by the external environmental factors such as Wind, Cold, Heat, and/or Dampness. The individual symptoms depend on which external pathogenic factor is strongest. Long-term Bi syndrome can lead to the formation of Phlegn and the deformation of bones and joints. In TCM pain and reduced range of motion indicates the presence of blockage; condition such as RA, therefore are always treated by means of removing Wind-Dampness and smoothing meridian. Sinomenine is an alkaloid extracted from the Chinese medicinal plant,Sinomenine acutum, which has been utilized to treat RA in China for over 2000 years. Sinomenine has been shown to mediate a wide range of pharmacological actions which include anti-inflammatory and anti-rheumatic effects. Qingfengtong capsules(Sinomenine preparation) has an effect of removing wind and dispersing dampness, clearing meridians and collaterals, expelling pathogenic cold from channel.Previous Pharmacological and clinical study on acute and chronic inflammation has shown a good anti-inflammatory and analgesic effects of Qingfengtong capsules. Study of Qingfengtong Capsules for the treatment of rheumatoid arthritis and its effect on the expression of HLA-DR4、STAT、PADI4 genes is helpful for the further study of the role and mechanism of sinomenine.Chinese medical constitution is individually morphological structure, physiological function and psychological state of an integrated, relatively stable characteristics, It is constrained and influenced by changes in the process of disease and is one of the basic factors with close correlation to the development and prognosis of disease.Because there are regional differences in RA morbidity and individual differences in clinical manifestations, it is necessary to make further research on the relationship between RA related gene expression and constitution despite of few research in this respect.Quantitative detection of the expression of genes at the same time can not be achieved by traditional methods. QGP (QuantiGene Plex) technology combines b-DNA (Branched DNA) signal amplification and the Luminex xMAP liquid-chip platform.The advantage is available directly from whole blood samples to detect mRNA expression, no RNA purification, no reverse transcription, no PCR amplification, and it can also high-throughput quantitatively detect 3 to 36 genes from less samples.Objectives1. To detect the expression level of HLA-DR4、PADI4、STAT4 in peripheral blood of RA patients and to explore the relationship between the expression of genes and disease activity.2. To evaluate the efficacy and safety of Qingfengtong Capsules in active rheumatoid arthritis (RA) patients at 12 weeks,and to study expression changes of HLA-DR4、PADI4、STAT4 genes after the treatment.3. To analyse distribution of Chinese Constitution in RA patients and HLA-DR4、PADI4、STAT4 gene expression of common types.Methods1. Research subjects38 RA patients were obtained from department of Traditional Chinese Rheumatology of Nan-fang hospital from July 2009 to December 2009., including 8 males and 30 females; for RA patients cohorts, including active 24 RA cases,4 males and 20 females; including remissive 14 RA cases,4 males and 10 females. Healthy controls include 4 males and 8 females, none of cases with other autoimmune diseases or severe heart, kidney and liver diseases.2. Research methods2.1 Study of the expression of HLA-DR4、PADI4、STAT4 genes in the peripheral blood of RA patients. The QGP method was used to measure the expression level of HLA-DR4、PADI4、STAT4 and PPIB, HPRT1 genes2.2 Clinical Evaluation of Qingfengtong Capsules(Sinomenine preparation) combined with MTX for treatment of RA and the effect on expression levels of HLA-DR4、STAT4、PADI4mRNA2.2.1 18 active cases were treated with Qingfengtong capsules united MTX for 12 weeks. The primary endpoints includethe proportion of patients achieving ACR20, ACR50, DAS28 score,duration of morning stiffness, joint swelling and joint tenderness count, VAS score, health assessment Questionnaire, the level of ESR,CRP.,Anti-CCP and RF. Regularly examine blood, urine routine test, liver and kidney function and record the occurrence of adverse events2.2.2 The QGP method was used to measure the expression level of HLA-DR4、PADI4. STAT4 mRNA before and after treatment.2.3 Classification of RA patients by using the standard of "Chinese Constitutions classification and determine "3 Statistical MethodsAll data were analysed using statistical software SPSS 13.0. Measurement data were expressed as means±SD. Independent sample t test was used for difference between two groups and paired test or Wilcoxon test for difference before and after treatment. Differences between different dose groups were analyzed by one-Way ANOVA followed by LSD test or Dunnetttest. Correlation analysis using Spearman or Person method. A two-sided P-value less than 0.05 was considered statistically significant.Results1. The QGP method was used to detect the expression level of HLA-DR4、PADI4、STAT4 mRNA1.1 The QGP detect the expression of HLA-DR4、PADI4、STAT4 mRNA,that AVG median fluorescence intensity of assay background (AVG MFI background) were respectively 6.0,9.0,5.5; the limit of detect (LOD) were 16.4,18.6,15.1; the intraplate assay coefficient of variation(CV) were5.4%,7.7%,6.8%; interplate assay coefficient of variation(CV) were 7.3%,10.1%,9.8%.1.2 The expressions of HLA-DR4, PADI4,STAT4 in RA patients were significantly higher than those of healthy controls (P<0.05 or P<0.01). The level of HLA-DR4 mRNA was no difference between active RA and Remissive RA. The expressions of PADI4 and STAT4 of active RA were significantly higher than remissive group (P<0.05).There were no correlation between the expression of HLA-DR4 of RA patients and DAS28, the level of anti-CCP antibody and RF (P>0.05). The level of PADI4 mRNA of patients was positively associated with DAS28 and the level of Anti-CCP antibody (r=0.521,P=0.01; r=0.558, P=0.000), while the expression of STAT4 of RA patients was positively associated with DAS28 and the level of RF(r=0.503,P=0.001; r=0.493,P=0.02).2. Clinical Evaluation of Qingfengtong Capsules(Sinomenine preparation) combined with MTX for Treatment of RA and its effect on expression levels of HLA-DR4、STAT4、PADI4mRNA2.1 At 12 weeks ACR20 was 72.2%, ACR50 was 22.2%;other indicators shows a similar level and trend of improvement (P<0.05). After 12 weeks of treatment clinical symptoms and laboratory parameters were significantly improved compared with baseline (P<0.05 or P<0.01)2.2 The expression level of HLA-DR4 mRNA was no difference before and after treatment (P＞0.05), while the expression levels of PADI4 and STAT4 after treatment were significantly lower than before treatment (P<0.05).3 Analysis of Chinese medical constitution in RA patients and HLA-DR4, PADI4, STAT4 expression in different types of Chinese medical constitution3.1 The most common type of Chinese medical constitutions were yang deficiency and blood stasis, and Chinese medical constitutions of damp heat, phlegm dampnes, yin deficiency and mild were followed.3.2 The level of HLA-DR4, STAT4 mRNA was no difference in various Chinese medical constitutions groups in RA patients (P＞0.05).The level of PADI4 mRNA of blood stasis constitution was higher than other constitutions (P＜0.05)Conclusion1. The advantages of QGP method to detect peripheral blood expression of HLA-DR4, PADI4, STAT4mRNA in RA patients are simple, reproducible, sensitive, saving samples.2. HLA-DR、PADI4 and STAT4 are overexpressed in peripheral blood of RA patients. The expressions of PADI4 andSTAT4 were closely related to disease activity,while HLA-DR4 expression was not The expressions of PADI4、STAT4 were related with disease progression and severity.The detection of PADI4 and STAT4 genes expression should be helpful for the judgement on disease activity of RA.3.Inflammation of active RA can significantly be relieved by Qingfengtong Capsules(Sinomenine preparation) combined with MTX therapeutics. After 12 weeks of treatment there were no serious adverse events and complications. The expression of PADI4 and STAT4 mRNA were decreased after treatment. That suggests Zhengqingfengtong capsules combined MTX can downregulate the expression of PADI4, STAT4 genes.4.The most common Chinese medical constitution in RA patients was yang deficiency and blood stasis. Morever, the level of PADI4 mRNA in this group was higher, prompting the expression of PADI4 may correlate with Chinese medical constitution types.更多还原