iTRAQ技术筛查TNF-α抑制剂治疗RA的疗效预测蛋白及其信号通路研究

Rheumatoid arthritis (RA) is a chronic and disabling disease. TNF-α inhibitors have demonstrated an outstanding performance in relieving joint inflammation and retarding bone erosion involved in RA. However, there is still about one-thirds of RA patients had a poor response to TNF α inhibitors. Currently the personalized biological treatment is the research hotspot. Recent studies focuses on exploring biomarkers predictive of drug response. The research methods such as genomics, transcriptomics, proteomics, metabolomics and immunocytology, have been applied, but they are not successfully integrated. The related studies in China are still at an initial stage, which necessitates an in-depth study in this area. Our preliminary study showed that TNF-α-308 gene polymorphisms existed in Chinese RA patients and PI3K/Akt signal pathway was ativated in proliferated synovial fibroblasts stimulated by TNF-α. Therefore, for the first attempt in China, we intend to screen for differential proteins by using iTRAQ technique in RA patients receiving anti-TNF-α therapy, and then verify the predictive effects of selected differential proteins from the upstream gene polymorphism to the downstream protein expression. We will also investigate the mechanisms of differential proteins involved in TNF-α related signal pathway by using in vitro gene transfer, siRNA interference, and RA animal models. Through our study we hope to discover a prediction protein with a domestic genetic background and finally establish a prediction system with Chinese characteristics.

类风湿关节炎（RA）是慢性致残的炎性关节病，TNF-α抑制剂在减轻关节炎症和抑制骨质破坏方面作用突出，但1/3的患者疗效不佳。如何提高治疗精准性，是个性化医疗的研究热点。此类研究重点在于找到预测疗效的生物标志物，已知研究方法包括药物基因组学、转录组学、蛋白质组学、代谢组学以及免疫细胞等，但各有优劣，且结论互不关联，国内此类研究才刚起步，面对挑战，深入开展研究确有必要。我们发现中国RA患者存在TNF-α-308位点多态性，TNF-α诱导的滑膜成纤维细胞增殖中存在PI3K/Akt信号通路活化，因此我们在国内首次采用iTRAQ技术，在TNF-α抑制剂治疗的患者中筛查出差异蛋白质，从上游基因多态性层面和下游表达层面验证差异蛋白质的作用，并通过细胞转染，siRNA干扰和关节炎动物模型，研究差异蛋白质影响TNF-α诱导信号通路的机制，最终找到具有中国遗传背景的疗效预测蛋白，形成中国特色的疗效预测体系。

背景：临床至少20%的RA患者对于生物制剂的反应不良，因此，研究MTX+LEF+IFX三联治疗RA的蛋白质谱变化，确定预测该三联疗法短期疗效的生物标志物以及机制。主要研究内容：纳入经MTX+LEF治疗一月后DAS28-CRP>5.1的RA患者（n=20），所有患者给予MTX+LEF+IFX疗法。14周末，将患者分类为应答者（R）和无应答者（NR）。生物发光法测量治疗前、后血清TNF-α水平，ELISA法测量治疗第14周末血清中英夫利西单抗浓度和抗IFX药物抗体水平，同量异位标签相对和绝对定量（iTRAQ）技术对R和NR治疗前、后血清蛋白质进行质谱分析，筛选出差异表达蛋白（DEPs）；通过平行反应监测（PRM）技术进行定量验证。重要结果和关键数据：①R组和NR组治疗前、后TNF-α的变化值、14周末血清ITL水平及ATI抗体阳性率组间差异无统计学意义。②iTRAQ技术检测发现，治疗前R组和NR组存在51个DEPs，主要参与急性期反应、免疫反应、炎症调节和脂质代谢过程；其中，APOM、GSN，TSP-1、FGB、S100、S100-A8、SAA、SAA-1、SAA-2、RF、CRP、CFH已被报导为IFX治疗前RA的疗效预测蛋白，新发现治疗前高水平的纤维蛋白原β链，附睾分泌蛋白Li282和睾丸组织蛋白Li70与较好疗效有关。③R组经IFX治疗前、后存在56种DEPs，NR组有62个DEPs，生信分析发现主要集中在p53信号通路、TGF-β信号通路、氧化途径、及吞噬过程，PRM证实转铁蛋白参与了疗效机制。④IL-17通路、TGF-β通路、NF-B/HIF-1通路和PI3K/Akt/HIF-1a通路有望成为高疾病活动度的RA患者的新靶标。科学意义：新的信号通路在RA治疗中的研究值得进一步重视。