痹肿消汤干预类风湿关节炎患者外周血单个核细胞蛋白质组学研究

作者	单位
蒋勇前	中南大学湘雅医院中西医结合研究所
梁清华	中南大学湘雅医院中西医结合研究所
熊新贵	中南大学湘雅医院中西医结合研究所
杨波	中南大学湘雅医院中西医结合研究所
萧梅芳	中南大学湘雅医院中西医结合研究所
区建刚	中南大学湘雅医院中西医结合研究所
曾年菊	中南大学湘雅医院中西医结合研究所
陈疆	中南大学湘雅医院中西医结合研究所

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中文摘要:

目的观察痹肿消汤干预前后的早期活动性类风湿关节炎(rheumatoid arthritis,RA)患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)蛋白质的双向凝胶电泳(2-DE)图谱,并分析其差异表达的蛋白质,寻找RA疾病相关蛋白,探讨痹肿消汤治疗类风湿关节炎可能的作用机制。方法 18例早期活动性RA患者随机分为两组,治疗组9例口服痹肿消汤(白花蛇舌草25g肿节风30g丹参15g络石藤20g骨碎补15g薏苡仁30g等15味中药),对照组9例口服甲氨蝶呤(methotrexate,MTX)和普威(nimesulide tablets),疗程均为3个月。9名健康成年人为正常对照组。健康成年人及RA患者治疗前后分别采血10mL,用淋巴细胞分离液分离单个核细胞,抽提外周血单个核细胞中的蛋白,采用固相pH梯度(IPG)2-DE分离正常人及RA患者PBMCs总蛋白质。凝胶经考染显色后,PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质,对差异蛋白质点用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)进行鉴定。同时运用反转录-聚合酶链反应(RT-PCR)方法验证部分差异蛋白质。结果分别获得健康人、RA患者治疗前、后PBMCs总蛋白2-DE图谱,通过RA患者治疗前与健康人比较分析,差异表达蛋白质点数为23,选取18个点进行质谱鉴定,成功鉴定14个蛋白质,其中8个蛋白质点在RA中表达上调,6个表达下调。与对照组治疗后比较,治疗组治疗后有5个蛋白质表达差异更为明显。RT-PCR方法证实,ApoA-I在健康人、RA治疗组、对照组的表达水平与2-DE结果一致。结论 (1)RA患者PBMCs存在一些差异表达的蛋白质,这些差异蛋白质可能是RA发病的内在因素;(2)痹肿消汤可能通过对RA患者PBMCs中某些蛋白质表达的调控达到治疗目的。

英文摘要:

Objective To probe in the possible acting mechanism of Bizhongxiao Decoction(BZXD) for treatment of early active rheumatoid arthritis(RA) by way of observing the two-dimensional gel electrophoresis map of proteins in peripheral blood mononuclear cells(PBMCs) of healthy persons and RA patients(intervened or un-intervened with BZXD),analyzing the differential proteins and seeking out the RA associated proteins.Methods Eighteen patients with early active RA were randomized into the BZXD group and the methotrexate(MTX) group,nine in each group,they were treated with BZXD(contained 15 Chinese herbs,as Herba Hedyotis diffusae,Herba Sarcandrae glabrae,Radix Salviae miltiorrhizae,Caulis Trachelosperi,Rhizoma Drynariae,Semen Coicis,etc.) and MTX combined with nimesulide Tablets respectively,three months as a treatment course,and their blood samples were collected for observation.Besides,blood samples from 9 healthy persons were taken as normal controls.PBMCs were isolated from blood using lymphozytes separation medium,and total protein in the cells was extracted through immobilized pH gradient two-dimensional gel electrophoresis.After Coomassie brilliant blue G250 staining,gel-image analysis was performed using PDQuest software.The differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry(MALDI-TOF-MS).Then partial proteins were validated by reverse transcription polymerase chain reaction(RT-PCR).Results The 2-DE protein profile of PBMCs from healthy persons and RA patients before and 3 months after treatment were obtained,and 23 differential protein spots were found,14 from 18 differential protein spots were successfully identified,of which 8 proteins were up-regulated and 6 proteins were down-regulated in RA patients as compared with control.After 3-month treatment,5 differentially expressed proteins showed more obvious in the BZXD group than in the MTX group.RT-PCR verified that the expression of ApoA-I in all the three groups was consistent with the outcomes of 2-DE.Conclusions Some differentially expressed proteins exist in the PBMCs of RA patients,which may play a potential role in the pathogenesis of RA;BZXD may treat RA by way of regulating the expression of some differential proteins in patients.

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