甲氨蝶呤通过诱导自噬促进类风湿关节炎滑膜成纤维细胞凋亡机制的研究

The study on the resistance of autophagy of fibroblast-like synovial cells to apoptosis induced by methotrexate in rheumatoid arthritis

目的明确甲氨蝶呤对RA成纤维样滑膜细胞(RA-FLS)的作用机制,为RA的药物治疗提供新的理论依据。方法分离并培养RA-FLS,用四甲基偶氮唑蓝(MTT)法和流式细胞术检测甲氨蝶呤作用下的细胞活力及细胞凋亡;不用浓度或不同时间点,甲氨蝶呤作用于RA-FLS,蛋白质印迹法及免疫荧光检测RA-FLS自噬的诱导情况;微RNA干扰(siRNA)Beclin1基因,流式细胞仪及PARP p85检测甲氨蝶呤作用下RA-FLS的凋亡情况;实验数据采用独立样本的t检验,GraphPad Prism 5.0软件绘制统计图。结果甲氨蝶呤可诱导RA-FLS细胞凋亡增加,增强RA-FLS自噬,诱导自噬体形成。在RA-FLS中,转染Beclin1基因的siRNA抑制自噬并增加甲氨蝶呤诱导的细胞凋亡。结论RA-FLS通过自噬抵抗甲氨蝶呤的促凋亡作用,甲氨蝶呤联合自噬抑制剂可增加RA的治疗效果。

Objective To clarify the mechanisms that the response of fibroblast-like synovial (FLS) cells to methotrexate (MTX) in rheumatoid arthritis (RA) and to provide theory basis for the drug treatment of RA. MethodsSynovial fibroblasts were isolated from synovial tissue specimens obtained from patients with RA and exposed to MTX. Cell viability was measured using a MTT assay and cell apoptosis was valued by flow cytometry. Western blotting analysis of LC3 and immunocytochemistry were used to analyze the induction of autophagy in RA-FLS after treating with MTX. Transfection of siRNA was used to interfere the expression of Beclin1 to down-regulate the autophagy, cell apoptosis was valued by flow cytometry and western blot analysis was used to test the PARPp85 with or without the presence of MTX. Statistical product and service solutions (SPSS) 18.0 statistical software was used for statistical analysis of all experimental data. Independent sample t test was used according to data distribution status, homogeneity of variance, and normal distribution. GraphPad Prism 5.0 was used to draw statistical graphs. ResultsMTX induced apoptosis was increased in RA-FLS. MTX stimulated the autophagy response in RA-FLS by inducing autophagosome formation. In RA-FLS, transfection with Beclin1 siRNA inhibited autophagy and increased the susceptibility to MTX, which induced cell death. ConclusionAutophagy of RA-FLS contributes to the resistance to apoptosis induced by methotrexate.