【作者】 王文静；

【导师】 陈进伟；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 研究背景类风湿关节炎(rheumatoid arthritis, RA)是一种以慢性侵蚀性关节炎为特征的自身免疫性疾病。改善病情抗风湿药(disease-modifying anti-rheumatic drugs, DMARDs)是RA治疗的首选,经数十年的临床检验被证实对RA疗效肯定,副作用较小且价格相对低廉,容易被我国RA患者接受。但在临床中,一部分患者在治疗过程中逐渐出现DMARDs疗效下降,这时即使加大药物剂量,往往也不能有效控制病情,即患者对DMARDs产生耐药。DMARDs耐药现象在临床中十分普遍,很大程度上限制了药物的使用,影响RA的治疗效果。目前,引起DMARDs耐药的机制尚不清楚,但近年来的研究表明,多药耐药蛋白(multi-drug resistance transporter, MDR)通过药物外排作用,降低靶细胞内的药物浓度是引起DMARDs耐药的重要原因。乳腺癌耐药蛋白(breast cancer resistance protein, BCRP)属于MDR蛋白中的一种,已被证明在多种类型的肿瘤细胞中高表达,可特异性地外排抗肿瘤药物,是导致肿瘤耐药的重要原因。值得注意的是,甲氨蝶呤、来氟米特、柳氮磺吡啶这几种重要的DMARDs均是BCRP的底物。多项研究已证实BCRP表达在RA滑膜巨噬细胞和外周血单核细胞,且与DMARDs治疗效果不佳相关。体外实验表明,柳氮磺吡啶长期作用还可诱导T细胞表面BCRP表达上调,导致细胞内药物浓度降低。这些结果均提示BCRP可能参与了DMARDs耐药。单核苷酸多态性(single nucleotide polymorphisms, SNP)是指由单个核苷酸变异引起的基因组水平上的DNA序列多态性。单核苷酸多态性作为内因不但影响疾病的发生发展,而且造成不同种群、个体对病原体、药物反应的差异性。MDR蛋白的单核苷酸多态性不但影响MDR蛋白的表达及功能,而且广泛参与人体内药物的药代动力学过程,决定了临床常见的群体／个体药物反应差异性。BCRP由BCRP基因(ABCG2)编码,在肿瘤耐药的研究中发现,BCRP基因C421A和G34A两个位点的单核苷酸多态性(SNPs)可影响肿瘤细胞BCRP的表达水平和功能,还影响化疗药物在体内的药代动力学过程,导致患者对化疗的耐药性产生明显不同。目前尚无BCRP基因C421A和G34A位点的单核苷酸多态性对RA患者DMARDs耐药性影响的相关研究。目的：探讨我国湖南汉族人群BCRP基因(ABCG2)C421A和G34A位点的单核苷酸多态性(SNPs)与类风湿关节炎(RA)改善病情抗风湿药(DMARDs)耐药的相关性。方法：研究对象308例,其中RA患者204例,均为2011年3月～2012年2月在中南大学湘雅二医院风湿免疫科就诊的患者,根据患者对(DMARDs)治疗的反应不同,分为治疗有效组112例和难治组92例,另纳入正常对照104例。采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)方法检测BCRP基因在C421A和G34A位点的基因型,比较三组间基因型和等位基因的分布频率。结果：各组研究对象基因型分布符合哈迪-温伯格平衡。C421A位点AC和AA基因型在治疗有效组的出现频率高于正常对照组,但差异无统计学意义(P值分别为0.223,0.420)。AC基因型在治疗有效组的出现频率明显高于难治组(P=0.031, OR=0.521,95%CI:0.287-0.945), A型等位基因在治疗有效组的出现频率也高于难治组(P=0.027, OR=0.625,95%CI:0.412-0.949)。BCRP-G34A位点的各基因型和等位基因在三组间的分布频率无显著性差异(P均>0.05)。结论：在湖南汉族人群中,BCRP基因C421A和G34A位点的单核苷酸多态性与RA易感性无关,但C421A位点突变可能与RA患者对DMARDs的治疗反应相关,AC基因型和A型等位基因可能是DMARDs耐药的保护性因素。BCRP-G34A位点突变与DMARDs耐药无关。更多还原

【Abstract】 BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic erosive arthritis. Disease-modifying anti-rheumatic drugs (DMARDs) are recommended as the first-line treatment in clinical practice for decades, with less adverse effect and low price。However, physicians find that part of the RA patients seems to be less efficient with DMARDs, even increasing dosages cannot redress it. DAMRDs Resistance is a common problem in clinic, which undoubtedly affect the prognosis of RA. So far the mechanism leading to the resistance is not clear. Recently, studies showed that multi-drug resistance transporter (MDR) may play an important role in this process through the exocytosis effect to drugs.Breast cancer resistance protein (BCRP) is a member of MDR which has been proved to play an important role in the resistance to chemotherapeutics through the exocytosis effect to chemotherapeutic agents. It is noted that some DMARDs, including methotrexate, leflunomide and salazosulfadimidine are all substrates of BCRP. Previous studies have showed that BCRP expressed on the macrophages of synovial tissue and related to a poor response to DMARDs. Long-term use of salazosulfadimidine can up-regulate the expression of BCRP on T cells and reduce intracellular drug concentration. BCRP is coded by BCRP gene (ABCG2). Single nucleotide polymorphisms (SNPs) of C421A and G34A sites in BCRP gene can affect the expression of BCRP on cancer cells, resulting in pharmacokinetics change of chemotherapeutic agents and resistance to chemotherapeutics. By far there is no report on the relationship between SNPs of these two sites and resistance of DMARDs in RA patients. Objective:To investigate the relationship of single nucleotide polymorphisms (SNPs) of C421A and G34A sites with the drug-resistance of RA in Hunan Han population.Methods:308subjects of Chinese Han origin were genotyped with polymerase chain reaction-restricted fragment length polymorphisms (PCR-RFLP), with204RA patients and104healthy controls. The RA treated were divided into two groups according the response to disease-modifying antirheumatic drugs (DMARDs). There were108patients in the effective group and115patients in the in-effective group. Genotype distribution and allele frequencies were analyzed between the three groups.Results:No deviations from the Hardy-Weinberge quilibrium(HWE) were observed in all study groups. Compared to non-responders, the responders carried more AC genotype (P=0.031, OR=0.521,95%CI:0.287-0.945), and more A allele (P=0.027, OR=0.625,95%CI:0.412-0.949) in C421A site, but with no statistically differences in genotype nor allele frequency between RA and healthy controls. There were significant differences in the genotype frequency and allele frequency in G34A site among three groups (P>0.5).Conclusion:The results from our study suggest that the C421A BCRP gene polymorphism may not be related with the RA susceptibility, but may influence the efficacy of RA therapy with DMARDs. There is no such relationship found in G34A site.更多还原