类风湿关节炎患者甲氨蝶呤耐药的相关耐药基因研究

Methotrexate (MTX)，as the recommendation of first-line drug in "treatment for rheumatoid arthritis (RA) ", should be considered to be the first choice, no matter the condition is light, medium or heavy, the course is at early、mid or later stage, the prognosis is good or bad. The latest research shows that resistance to MTX is one of the main reasons bringing about the failure treatment of RA patients. MTX is the competitive inhibitor of DHFR. It enters into the cells through realizing the mechanism of initiative transport mediated by RFC, and accompanishes to outflow from the cells depending on the combination between ATP and ABC to transport super family members, which is the main factors of multidrug resistance to tumor. In these transportors, multi-drug resistance proteins(MRP), multidurg-resistance gene mdr1 and its product P-glycoprotein (P-gp), play an important role in outflowing of MTX from cells. This research puts the monocytes in peripheral blood of patients with RA who are treated by MTX but faliure as the object, plans to start with RFC transportation system that the MTX enters into cells, ABC transport protein that the MTX outflows from cells, and NF-κB signal genes which influence the activity of mdr1 and P-gp,and tries to make an intensive study of the mechanism which influence the curative effect of MTX.This study has significant value in overcoming the resistance to MTX and prospective prediction about improving the curative effect of RA.

甲氨蝶呤（MTX）作为"治疗类风湿关节炎（RA）推荐"的一线用药，无论病情轻、中、重；病程早、中、晚以及预后的好与坏均应首先考虑应用MTX。最新的研究表明，MTX耐药的产生是造成RA患者治疗失败主要原因之一。MTX是二氢叶酸还原酶的竞争性抑制剂，它进入细胞通过还原型叶酸载体(RFC)介导的主动转运机制实现，其流出细胞通过ATP结合盒蛋白（ABC）转运超家族成员完成，该家族是形成肿瘤多药耐药的主要因素，在这些转运体中，多药耐药基因（mdr1）及其产物P-糖蛋白（P-gp）、多药耐药相关蛋白（MRP）在MTX流出细胞中起到重要作用。本研究以MTX治疗失败的RA患者外周血单核细胞为研究对象，拟从MTX进入细胞的RFC转运体系、流出细胞的ABC转运蛋白及影响mdr1、P-gp活性的NF-κB信号基因入手，深入研究影响MTX疗效的机制，对克服MTX耐药和提高RA治疗的前瞻性预测具有重要价值。

甲氨蝶呤（MTX）目前是治疗类风湿关节炎（RA）的一线用药，但最新的研究表明，MTX耐药的产生是造成RA患者治疗失败主要原因之一。本研究以MTX治疗后的RA患者为研究对象，根据其病情活动度分为MTX敏感组、MTX耐药组，以健康体检患者作为正常对照组。ELISA法检测发现MTX敏感组患者红细胞内MTX平均药物浓度明显高于MTX耐药组。流式细胞法检测外周血淋巴细胞显示MTX敏感组及MTX耐药组P-gp表达及功能明显高于正常对照组；MTX耐药组P-gp表达及功能明显高于MTX敏感组高。PCR法检测外周血结果显示MTX耐药组患者MDR1 mRNA表达量高于MTX敏感组；MTX耐药组患者MPR mRNA表达量高于MTX敏感组；MTX耐药组患者RFC mRNA表达量均值低于MTX敏感组；MTX耐药组患者DHFR mRNA表达量均值与MTX敏感组无明显差异。ELISA法检测MTX敏感组及MTX耐药组血清中NF-κB、TNF-α、IL-1β浓度均高于正常对照组；MTX耐药组血清中NF-κB、TNF-α、IL-1β浓度高于MTX敏感组；MTX敏感组及MTX耐药组血清中IκBα浓度低于正常对照组，MTX耐药组血清中IκBα浓度低于MTX敏感组。PCR法检测显示MTX耐药组外周血单个核细胞中mdr1 mRNA、TNF-α mRNA、IL-1βmRNA均高于MTX敏感组。以上研究结果表明RA患者红细胞内MTX药物浓度与RA患者疗效明显相关，RFC的高表达可能是MTX耐药的形成机制，DHRF表达在RA患者MTX耐药形成中并无显著影响。MDR1高表达可能通过上调P-gp、MRP的表达，影响MTX药物的外排能力，从而参与了MTX耐药的形成机制。RA患者中NF-κB信号及下游炎症因子TNF-α、IL-1β的高表达可以激活mdr1，上调其产物P-gp表达与活性，并与MTX耐药相关，提示NF-κB信号通路的激活可能是RA患者MTX耐药形成的机制之一。