【作者】 王静；

【导师】 蒋云生； 陈进伟；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 研究背景类风湿关节炎(Rheumatoid Arthritis, RA)是一种以关节滑膜为主要靶组织的慢性高致残性自身免疫性疾病,以进展性、侵蚀性关节炎为主要表现。RA是造成我国人群丧失劳动力和致残的主要疾病之一。目前类风湿关节炎的治疗主要包括非甾体抗炎药(non-steroidal antiinflammatory drugs, NSAIDs)、改善病情抗风湿药(disease modifying antirheumatic drug, DMARDs)及生物制剂。虽经过正规早期联合多种DMARDs药物治疗,仍有部分RA患者疗效不佳；同时随着治疗的延长,药物疗效逐渐减弱,对治疗药物无反应或低反应,出现耐药现象,导致治疗失败。耐药尤其是多药耐药(Multiple Drug Resistance, MDR)的形成是影响RA治疗效果的重要因素。作为治疗失败的原因之一,MDR在肿瘤和感染性疾病的治疗中已得以足够重视,但在RA的治疗中的研究还较少。跨膜转运蛋白介导的细胞内药物排出增加,导致细胞内药物降低,是MDR的重要机制。P-糖蛋白(P-glycoprotein, P-gp)是迄今为止研究的最多也是最重要的MDR相关的蛋白。它的作用底物广泛,在抗风湿药物中,目前已明确糖皮质激素、氯喹等是P-gp的底物。有研究报道难治性类风湿关节炎P-gp表达和活性增高,P-gp参与类风湿关节炎多药耐药的产生。TNF-α是RA发病机制中居中心地位的促炎症因子,参与RA复杂的细胞网络反应,其水平与疾病活动程度相关,并随疾病的缓解而降低。目前已证实TNF-α可影响多种细胞系P-gp的表达和活性,参与MDR的形成。但在RA中TNF-α是否影响P-gp的表达和活性,参与MDR的形成,国内外尚未见报道。本研究拟通过检测RA初治组、RA治疗有效组、RA难治组患者外周血单个核细胞P-gp表达和功能、TNF-αmRNA,血清TNF-α水平,对P-gp与类风湿关节炎多药耐药进行相关性研究,筛选RA耐药的分子标志,研究RA对DMARDs的耐药特征,分析P-gp与TNF-α及疾病活动的相关性。在此基础上,在体外实验中我们观察TNF-α对RA外周血单个核细胞P-gp的影响,探寻TNF-α对RA外周血单个核细胞P-gp的调控机制,从整体、细胞、胞内信号转导水平深入了解炎性因子TNF-α对RA P-gp的作用及RA多药耐药的影响。这一研究将为我们更深入了解RA多药耐药的特征及形成机制提供理论基础,并进一步探寻预防和逆转耐药的方法和途径,给类风湿关节炎的治疗带来新的突破。第一部分P-糖蛋白与类风湿关节炎多药耐药及TNF-α水平的相关性研究目的：检测正常对照组、RA初发未治组、RA治疗有效组、RA难治组患者外周血单个核细胞P-gp表达和功能、TNF-αmRNA,血清’TNF-α水平,分析P-gp与RA多药耐药及TNF-α相关性,探讨P-gp、TNF-α在RA多药耐药中的作用及其相关性。方法：入选RA初治组、治疗有效组、难治组患者和正常对照组各20例为研究对象。RA初治组未经任何DMARDs及生物制剂治疗。RA治疗有效组和难治组以联合甲氨蝶呤、来氟米特为基础治疗药物,未经糖皮质激素和生物制剂治疗。入组RA患者进行疾病活动度评分(DAS28)。采集入组患者血标本进行血清和外周血单个核细胞的分离。以流式细胞仪检测外周血单个核细胞的P-gp的表达,罗丹明123蓄积试验检测外周血单个核细胞的P-gp的功能,RT-PCR检测外周血单个核细胞TNF-αmRNA水平,ELISA检测血清TNF-α浓度。结果：1.外周血单个核细胞P-gp的表达和功能：在正常对照组有P-gp表达,但表达量和功能低；RA初治组、RA治疗有效组及RA难治组P-gp表达和功能均高于正常对照；RA难治组P-gp的表达和功能较RA初治组、RA治疗有效组明显增高,差异有统计学意义(P<0.01)；RA初治组P-gp的表达和功能高于RA治疗有效组(P<0.05)。2.外周血单个核细胞TNF-αmRNA和血清TNF-α水平：正常对照组TNF-αmRNA表达水平明显低于RA各组(P<0.01)。RA各组TNF-αmRNA表达水平两两比较,差异有统计学意义(P<0.01),其中RA难治组TNF-αmRNA表达水平最高,RA治疗有效组TNF-αmRNA表达水平最低。RA各组血清TNF-α含量较正常对照组高(P<0.01),其中RA难治组明显高于RA初治组和RA治疗有效组(P<0.01),RA初治组高于RA治疗有效组(P<0.01)。3.RA各组疾病活动的比较：RA初治组DAS28评分为5.70士1.46,明显高于RA治疗有效组(3.78±0.79,P<0.01)；RA难治组DAS28评分为7.02±0.93,明显高于其它两组(P<0.01)。4.PBMC P-gp的表达和功能与TNF-αmRNA、血清TNF-α水平和疾病活动度的相关性分析：PBMC P-gp的表达与PBMC TNF-αmRNA水平、血清TNF-α的水平均呈正相关(r=0.29,P<0.01；r=0.758,P<0.01)；罗丹明蓄积细胞内荧光强度与PBMC TNF-αmRNA.血清TNF-α的水平呈负相关(r=-0.843,P<0.01；r=-0.863,P<0.01)。PBMC P-gp的表达与DAS28做相关性分析,显示二者呈正相关(r=0.588,,P<0.01),罗丹明蓄积细胞内荧光强度与DAS28做相关性分析,显示二者呈负相关(r=-0.702,P<0.01)。5.PBMC P-gp的表达和功能与病程和用药时间的相关性分析：P-gp表达和功能与RA的病程和用药时间无明显关联。结论：1.RA难治组的PBMC P-gp表达和功能明显高于与RA初治组和RA治疗有效组,P-gp表达升高功能增强与RA的难治程度有关,P-gp参与难治性RA多药耐药的形成。2.RA治疗有效组P-gp表达和功能较RA初治组下降,且RA各组P-gp表达和功能与疾病活动评分DAS28呈正相关,提示P-gp可作为RA治疗效果的监测指标之一。3.P-gp表达和功能与RA的病程和用药时间无明显关联。4.P-gp表达和功能与PBMC TNF-αmRNA、血清TNF-α呈正相关,TNF-α可能参与P-gp介导的RRA多药耐药的形成。5.P-gp的表达和活性受原发耐药、继发耐药、疾病活动内环境改变等多重因素的影响。疾病活动内环境改变可能在RA P-gp的表达和活性增强中起着重要作用。第二部分TNF-α对RA外周血单个核细胞P-gp表达及活性的影响目的：观察TNF-α对RA外周血单个核细胞后P-gp表达及活性的影响,探讨TNF-α在RA MDR形成中的作用。方法：入选RA初治组、治疗有效组、难治组患者和正常对照组各20例为研究对象。采集入组患者血标本并进行外周血单个核细胞的分离,以终浓度0.5ng/ml的TNF-α作用于外周血单个核细胞,置于37℃C02培养箱中饱和湿度下培养,分别在TNF-α干预前和干预后2h、6h、12h、24h后,收集细胞,流式检测PBMC P-gp的表达,罗丹明123蓄积试验检测PBMC P-gp功能。结果：在TNF-α干预2h后,各组P-gp的表达与干预前无明显变化,但Rh123蓄积试验荧光强度下降,即P-gp的功能增强。干预12h各组的P-gp的表达和功能都能达到最大值并维持在高水平的平台上。干预12h与干预24h P-gp的表达和功能差异无统计学意义。在6h和12h的P-gp的检测中发现,各组在起效时间和达峰时间上不一致。在正常对照组干预6h,P-gp的表达较干预前升高(P<0.05),干预12h P-gp的表达达峰。在RA初治组,在干预6h,P-gp的表达较干预前升高(P<0.05),干预12h P-gp的表达达峰。在RA治疗有效组,干预12h,P-gp的表达才较干预前升高(P<0.01)并达到峰值。在RA难治组,干预6h P-gp的表达较干预前明显升高(P<0.01)并达峰。干预12h P-gp的表达和功能达峰,比较各组P-gp的表达和功能的情况,RA各组P-gp表达和功能高于正常对照组(P<0.01)；RA各组中难治组P-gp表达最高、功能最强,治疗有效组P-gp表达最低、功能最弱。结论：1.TNF-α可增强RA患者的PBMC P-gp的表达和功能,介导RA的多药耐药,参与难治性类风湿关节炎的产生。2.RA各组PBMC对相同浓度的TNF-α刺激上调P-gp的速度和效率不同,难治组起效和达峰所需时间短,治疗有效组起效和达峰所需时间长,这种差异性可能与细胞自身对TNF-α的内在反应性有关。3. TNF-α对各组PBMC P-gp的表达和功能的影响,在作用12h均可达到最大值,此后维持在高水平的平台上4.RA P-gp表达和活性增高的机制复杂,TNF-α的作用仅为影响P-gp的表达和活性增高的机制之一。第三部分TNF-α对RA外周血单个核细胞P-gp调控机制的研究目的：研究TNF-α信号转导途径中的MAPKs(ERK1/2、JNK和p38)及NF-κB信号转导途径在TNF-α增强RA PBMC P-gp表达和活性中的作用,探讨TNF-α对RA PBMC P-gp的调控机制。方法：入选RA初治组患者20例,采集入组患者血标本并进行外周血单个核细胞的分离。每份标本设6组：A组、B组、C组、D组、E组和F组。A组为空白对照组。在C、D、E、F组分别加入NF-κB、JNK、ERK1/2、p38的抑制剂PDTC、SP600125、U0126、SB202190进行预处理。处理30min后,B、C、D、E、F组均加入TNF-α,调整TNF-α终浓度为0.5ng/ml,置于37℃C02培养箱中饱和湿度下培养,12h后收集细胞,流式检测PBMC P-gp的表达,罗丹明蓄积试验检测PBMC P-gp功能。结果：与空白对照组相比,TNF-α组P-gp表达升高(P<0.01),Rh123荧光强度降低(P<0.01)即P-gp功能增强。用PDTC预处理抑制NF-κB活性或P600125预处理抑制JNK活性,与未预处理的TNF-α组相比,预处理组P-gp表达降低、功能减弱。用U0126预处理抑制ERK1/2活性或SB202190预处理抑制p38活性,与未预处理的TNF-α组相比,两组P-gp表达和功能差异无统计学意义结论：1. NF-κB的抑制剂PDTC和JNK抑制剂SP600125均可下调TNF-α诱导的RA PBMC P-gp的表达增加和功能增强。NF-κB和JNK信号转导通路介导TNF-a对RA PBMC P-gp的表达和功能的调控。2.ERK1/2抑制剂U0126和p38-MAPK抑制剂SB202190对TNF-α诱导的RA PBMC P-gp的表达增加和功能增强无影响。ERK1/2通路和p38-MAPK信号通路不直接参与TNF-α对RA PBMC P-gp的表达和功能的调控。3.TNF-α通过NF-κB和JNK信号转导通路的激活对RA PBMCP-gp的表达和功能进行调控。更多还原

【Abstract】 Background:Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease with high rate of disability.The main manifestation is progressive and erosive arthritis. Progression of RA results in severe disability and loss of function with severe pain. The main treatments currently of RA include non-steroidal anti-inflammatory drug (NSAIDs), disease modifying antirheumatic drug (DMARDs) and biological agents. Although combination of two or three DMARDs was administered to the patients with RA in the earlier stage of the disease, some patients displayed Poor efficacy to the treatment. Moreover, as the extension of the treatment, drug efficacy gradually reduced, show low response or even no response and drug resistance occurred. Emergence of multiple drug resistance (MDR) plays an important part in therapeutic efficacy of RA. As one of the cause of treatment failure, MDR was take account in the domain of carcinoma and infective disease. The research of MDR in RA was less.Molecular mechanisms underlying drug resistance include the action of transmembrane transporters, which mediate the cellular extrusion of a large variety of therapeutic drugs. P-glycoprotein (P-gp) is the most important and classic transmembrane transporter up to now. Overexpressi -on of P-gp results in reduction of intracellular concentrations of substrates involving xenobiotics and various drugs such as vinca alkaloids, anthracyclines, verapamil, some DMARDs (eg, hydroxyl chloroquine, D-penicillamine, colchicines) and corticosteroids. It was reported that the expression and activity of P-gp increased in refractory RA and play a part in MDR of RA. Tumor necrosis factor-alpha (TNF-α) is proinflammatory factor which play a core role in the pathogenesis of RA. Many studies confirmed that TNF-αregulates P-gp in different cell lines and involved in MDR. It is unknown that whether TNF-αregulate P-gp and involved in MDR in RA. The main target of DMARDs is the lymphocyte, so we choose peripheral blood mononuclear cells (PBMC) as research object. In this study, we investigated the level of the expression and activity of P-gp, TNF-αand analyzed their correlation. On this basis, we also investigated the effect of TNF-αon the expression and activity of P-gp of PBMC in vitro and explored the possible mechanisms. Our study is helpful to elucidate the character of MDR in RA and provides a basis of the molecular mechanisms of MDR in RA, which can offer a new idea and strategy for the treatment of RA especially refractory RA. Objective:To investigate the expression and function of P-gp, TNF-αmRNA of PBMC and the level of TNF-αin serum in normal and RA patients (refractory, effective to treatment or untreated). To analyses correlations among multidrug resistance, TNF-αand expression and function of P-gp.Methods:20 normal control and 60 RA patients (20 untreated,20 ffective to treatment,20 refractory for each group) were selected as object of this study. The clinical activity of RA was assessed by the Disease Activity Score (DAS)28 in the selected RA patients. PBMC from the selected RA patients and normal control was analyzed by flow cytometry for P-gp expression, rhodamine123 accumulation test for P-gp function and RT-PC R for TNF-αmRNA. The serum was analyzed by ELISA for TNF-α.Results:1. The expression and function of P-gp on PBMC was lower in normal control group compared with that in RA groups. Among the groups of RA, the expression and function of P-gp in RA refractory group was higher than that in RA untreated group and RA effective to treatment group(P<0.01). The expression and function of P-gp in RA untreated group was higher than that in RA effective to treatment group(P<0.05)2. The level of PBMC TNF-αmRNA and serum TNF-αin RA groups were significantly higher than that of normal control group(P< 0.01). Among the groups of RA, the level of PBMC TNF-αmRNA and serum TNF-αin RA refractory group was highest, and that in RA effective to treatment group was lowest.3. The score of DAS28 in RA untreated group(5.70±1.46) was higher than that of RA effective to treatment group(3.78±0.79,P< 0.01). The score of DAS28 in RA refractory group(7.02±0.93) was higher than that of RA effective to treatment group and RA untreated group(P< 0.01).4. The expression of P-gp was positively correlated with the level of PBMC TNF-αmRNA and serum TNF-α(r=0.29, P<0.01; r=0.758, P <0.01); Intracellular fluorescence intensity of rhodamine123 was negatively correlated with the level of PBMC TNF-αmRNA and serum TNF-α(r=-0.843, P<0.01; r=-0.863, P<0.01). The expression of P-gp was positively correlated with the scores of DAS28 (r=0.588, P< 0.01). Intracellular fluorescence intensity of rhodamine123 was negatively correlated with the scores of DAS28(r=-0.702, P<0.01).5. No correlation was found among the expression and function of P-gp, duration of disease and time of therapy. Conclusions:1. The expression and function of P-gp on PBMC was highest in RA refractory group and lowest in RA effective to treatment group. The increase of expression and function of P-gp was associated with refractoriness of RA. P-gp played a role in MDR of RA.2. The expression and function of P-gp on PBMC were correlated with disease activity of RA. No correlation was found among the expressi-on and function of P-gp, duration of disease and time of therapy.3. The expression and function of P-gp were positively correlated with the level of PBMC TNF-αmRNA and serum TNF-α.4. We speculated that primary drug resistance, secondary drug resistance, the change of internal environment in disease activities involve in regulation of P-gp and development of MDR in RA. The change of internal environment in disease activities plays an important part in in regulation of P-gp. Objective:To investigate the influence of TNF-αon expression and function of P-gp on PBMC. To study the effect of TNF-αon the development of MDR in RA.Methods:20 normal control and 60 RA patients (20 untreated,20 effective to treatment,20 refractory for each group) were selected as object of this study. PBMC was isolated and treated with TNF-a (0.5ng/ml terminal concentration) in 24-well-plate at 37℃,5% CO2, and 95% relative humidity. PBMC was collected 0,2,6,12, or 24 h after TNF treatment, the 0 time being the untreated control. PBMC analyzed by flow cytometry for P-gp expression, rhodamine123 accumulation test for P-gp function.Results:Results from the experiment showed that 2h after TNF-αtreatment, the function of P-gp enhanced and the expression of P-gp did not change significantly.12h after TNF-αtreatment, the expression and function of P-gp reached it its maximum and then maintain a high level. No significant difference of the expression and function of P-gp were found between 12h and 24h after TNF-αtreatment. At the time of 6h and 12h after TNF-αtreatment, onset time and peak time of expression of P-gp were different in different groups. In normal control group and RA untreated group, onset time was 2h and peak time was 12h. In RA effective to treatment group, onset time and peak time was 12h. In refractory group onset time and peak time was 6h. We compared the expression and function of P-gp 12h after TNF-αtreatment in each group. The expression and function of P-gp in RA groups were significantly higher than that of normal control group. Among the groups of RA, the expression and function of P-gp in RA refractory group was highest, and that in RA effective to treatment group was lowest.Conclusions:1.TNF-αcan up regulate the expression and function of P-gp on PBMC in RA, and play a part in development of refractory RA and MDR in RA.2. The onset time and peak time of induction of P-gp expression by TNF-αwas short in in RA refractory group and was long in RA effective to treatment group. RA refractory group was more sensitive to TNF-αcompared with RA effective to treatment group.3. The expression and function of P-gp reached it its maximum and then maintain a high level 12h after TNF-αtreatment.4. The mechanisms of the expression and function of P-gp elevated in RA and RRA were complicated and the effect of TNF-αon P-gp was one of the mechanisms. Objective:To study the contribution of two signal transduction pathways of TNF-α-MAPKs(ERK1/2, JNK and p38) and NF-κB in regulation of the expression and function of P-gp on PBMC by TNF-αin RA.Methods:20 RA untreated patients were selected as object of this study. PBMC was isolated one PBMC sample was divided into 6 groups:group A, group B, group C, group D, group E and group F. Group A is blank control. Group C, D, E, F were pretreated with NF-κB, JNK, ERK1/2 and p38 inhibitor (PDTC, SP600125, U0126 and SB202190) for 30min. Then Group B, C, D, E, F were treated with TNF-α(0.5ng/ml terminal concentration) in 24-well-plate at 37℃,5% CO2, and 95% relative humidity for 12h. PBMC was collected and analyzed by flow cytometry for P-gp expression, rhodamine123 accumulation test for P-gp function.Results:The expression and function of P-gp in group B were significantly higher than that in group A. Compared with group B, the expression and function of P-gp decreased in group C and D. No significant differences of the expression and function of P-gp were found among group B, group E and group F.Conclusions:1. Inhibition of NF-κB and JNK can suppress the increased expression and function of P-gp induced by TNF-αsignal transduction pathways of NF-κB and JNK play a role in regulation of the expression and function of P-gp on PBMC by TNF-αin RA.2. Inhibition of ERK1/2 and p38-MAPK had no influence on the increased expression and function of P-gp induced by TNF-α. Signal transduction pathways of ERK1/2 and p38 probably did not play a role in regulation of the expression and function of P-gp on PBMC by TNF-αin RA directly.3. TNF-αregulated the expression and function of P-gp on PBMC through activation of NF-κB and JNK signal transduction pathways.更多还原