断藤益母汤对类风湿关节炎患者树突状细胞趋化因子分泌和受体表达的影响

发布时间：2018-04-05 00:22

本文选题：类风湿关节炎　切入点：树突状细胞　出处：《广州中医药大学》2017年硕士论文

【摘要】：目的:通过体外实验,研究断藤益母汤对类风湿关节炎(rheumatoid arthritis,RA)患者外周血树突状细胞(dendriticcell,DC)迁移功能相关的趋化因子和受体表达的影响,从DC迁移功能的角度来探讨断藤益母汤的免疫抑制和治疗RA的机制。方法:密度梯度离心法分离RA患者外周血单个核细胞(PBMCs),采用贴壁法分离出单核细胞,并以粒细胞巨噬细胞集落刺激因子(GM-CSF)及白介素-4(IL-4)刺激7天以使单核细胞分化成树突状细胞,分别加入甲氨蝶呤(阳性对照)、断藤益母汤冻干粉(试验组)进行干预,随后以酶联免疫吸附测定(ELISA)测定上清液中细胞因子CXCL9和CXCL10的含量,并收集细胞,利用流式细胞技术检测DC表面CCR5和CCR7的表达水平,并与空白组进行对比。结果:1.在GM-CSF和IL-4的共同刺激下,外周血单核细胞成功分化成为DC,在光镜下可观察到DC的典型树突状形态。2.根据CCK8的细胞毒理-增殖实验结果,比较相同时间点下,不同断藤益母汤浓度组与正常组的A值,得出断藤益母汤冻干粉的最佳给药质量体积比为l00mg/ml。3.经ELISA法检测后,结果显示,与空白组对比,经断藤益母汤冻干粉及甲氨蝶呤干预后DC培养的上清液中CXCL9、CXCL10浓度均明显下降(P0.05),差异具有统计学意义;而断藤益母汤冻干粉干预组与甲氨蝶呤组对比,CXCL9、CXCL10浓度下降不明显(P0.05),差异没有统计学意义。4.流式细胞结果显示,经断藤益母汤冻干粉及甲氨蝶呤干预后的DC表面CCR5和CCR7的表达均明显下调(P0.05),差异具有统计学意义;而断藤益母汤冻干粉干预组与甲氨蝶呤组对比,CCR5和CCR7的表达略有下调(P0.05),但差异没有统计学意义。结论:断藤益母汤可以下调RA患者外周血DC表面CCR5和CCR7的表达,减少趋化因子CXCL9和CXCL10的分泌,从而通过抑制DC往次级淋巴器官的迁移,减少其与T细胞的特异性结合,从而起到治疗RA的作用。
[Abstract]:Objective: to study the effect of Shuteng Yimu decoction on the expression of chemokines and receptors related to the migration function of peripheral blood dendritic cells (DC) in patients with rheumatoid arthritis (RA).From the point of view of DC migration function, the mechanism of immunosuppression and treatment of RA was studied.Methods: peripheral blood mononuclear cells from patients with RA were isolated by density gradient centrifugation. Monocytes were isolated by adherent method and stimulated with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4IL-4 (IL-4) for 7 days to differentiate monocytes into dendritic cells.Methotrexate (positive control group, Shutentenyimu decoction freeze-dried powder) was added to the experiment group, then the levels of cytokines CXCL9 and CXCL10 in the supernatant were measured by enzyme-linked immunosorbent assay (Elisa), and the cells were collected.Flow cytometry was used to detect the expression of CCR5 and CCR7 on DC surface and compared with the blank group.The result is 1: 1.Under the co-stimulation of GM-CSF and IL-4, peripheral blood mononuclear cells were successfully differentiated into DC.The typical dendritic morphology of DC was observed under light microscope.According to the results of cell toxicology and proliferation test of CCK8, the A value of different concentration groups and normal group were compared at the same time point, and the optimum volume ratio of the lyophilized powder of Shuteng Yimu decoction was 1 00mg / ml 路ml. 3.The results of ELISA test showed that the concentration of CXCL9 and CXCL10 in the supernatant of DC cultured after the intervention of Shuteng Yimu decoction and methotrexate was significantly lower than that of the blank group, and the difference was statistically significant.The concentration of CXCL9 and CXCL10 in the intervention group and methotrexate group were not significantly decreased (P 0.05), and there was no significant difference between them.The results of flow cytometry showed that the expression of CCR5 and CCR7 on the DC surface was significantly down-regulated after the intervention of Shutenyimu decoction freeze-dried powder and methotrexate, and the difference was statistically significant.Compared with methotrexate group, the expression of CCR5 and CCR7 was slightly down-regulated, but there was no significant difference in the expression of CCR5 and CCR7 between the intervention group and the methotrexate group.Conclusion: TYD can down-regulate the expression of CCR5 and CCR7 on peripheral blood DC of RA patients, reduce the secretion of chemokine CXCL9 and CXCL10, and reduce the specific binding of DC to T cells by inhibiting the migration of DC to secondary lymphoid organs.So as to play a role in the treatment of RA.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R259

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