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目的探讨正常人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)对雷公藤内酯醇(trip-tolide,TP)的耐药特征和机制。方法采用递增TP剂量的浓度梯度诱导法使PBMC产生耐药。MTT比色法测定细胞对药物的敏感性及耐药指数(resistance index,RI),逆转录-聚合酶链反应(RT-PCR)法分别检测正常对照组和耐药组PBMC中多药耐药相关基因(multidrug resistance gene,MDR1)、多药耐药相关蛋白(multidrugresistance-associated protein,MRP)和肺耐药蛋白(lung resistance-related protein,LRP)的表达。酶联免疫吸附试验(ELISA)检测上清液中IL-1β的量。结果和正常细胞组相比,耐药组细胞的群体倍增时间延长2.7 h,RI是1.99,并且MDR1 mRNA、MRP mRNA、LRP mRNA表达量明显增加(P<0.01)。TP能显著抑制正常组PB-MC分泌IL-1β(P<0.05),但对耐药组的抑制作用不明显(P>0.05)。结论TP体外持续诱导可使正常人PB-MC产生耐药,其耐药性的产生涉及多个相关基因的表达,这可能是导致类风湿关节炎(RA)病人临床服用雷公藤一段时间后产生耐受的原因之一。
Objective To investigate the characteristics and mechanisms of resistance to triptolide (TP) in peripheral blood mononuclear cells (PBMCs) of normal people. Methods PBMCs were resistant to drug by concentration gradient induction with increasing TP dose. MTT colorimetric assay was used to determine the cell sensitivity to drugs and the resistance index (RI). Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect multidrug resistance in PBMCs of normal and resistant groups. Multidrug resistance gene (MDR1), multidrug resistance-associated protein (MRP) and lung resistance-related protein (LRP) expression. Enzyme-linked immunosorbent assay (ELISA) was used to determine the amount of IL-1β in the supernatant. Results Compared with the normal cell group, the population doubling time of the drug-resistant group was prolonged by 2.7 h, the RI was 1.99, and the expression levels of MDR1 mRNA, MRP mRNA, and LRP mRNA were significantly increased (P<0.01). TP could significantly inhibit IL-1β secretion in PB-MC of the normal group (P<0.05), but the inhibitory effect on the drug resistance group was not significant (P>0.05). Conclusion The sustained induction of TP in vitro can make normal human PB-MC resistant, and the development of drug resistance involves the expression of multiple related genes, which may be caused by the clinical use of tripterygium in patients with rheumatoid arthritis (RA). One of the reasons for tolerance.