氨甲蝶呤调控调节性T细胞数量及Foxp3表达在银屑病治疗中的作用  

Effect of methotrexate on regulation for the number of regulatory T cells and expression of Foxp3 in psoriasis

作　　者：匡叶红[1] 张衡 朱武[1] 吴丽莎[1] 陈旺青[1] 鲁艳 覃群师 贾学昆 廖立秋[3] KUANG Yehong;ZHANG Heng;ZHU Wu;WU Lisha;CHEN Wangqing;LU Yan;QIN Qunshi;JIA Xuekun;LIAO Liqiu(Department of Dermatology,Xiangya Hospital,Central South University,Changsha 410008;Department of Dermatology,Central Hospital of Shaoyang,Shaoyang Hunan 422000;Department of Breast Surgery,Xiangya Hospital,Central South University,Changsha 410008,China)

机构地区：[1]中南大学湘雅医院皮肤科,长沙410008 [2]邵阳市中心医院皮肤科,湖南邵阳422000 [3]中南大学湘雅医院乳腺科,长沙410008

出　　处：《中南大学学报（医学版）》2018年第8期835-842,共8页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81101654;81573049)~~

摘　　要：目的:探讨氨甲蝶呤(methotrexate,MTX)对调节性T细胞(regulatory T cells,Treg)细胞数量及关键转录因子Foxp3 m RNA的影响。方法:1)收集初诊的寻常型银屑病患者、健康对照者和经MTX治疗8周后疗效显著的寻常型银屑病患者入组,抽取外周抗凝静脉血并分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC),采用流式细胞术(fl ow cytometry,FCM)检测Treg细胞数量,采用实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)法检测PBMC中Foxp3 m RNA表达水平;2)将咪喹莫特(imiquimod,IMQ)连续外用在BALB/c小鼠背部,观察6 d,每天拍照记录小鼠皮损变化,根据银屑病面积和严重程度指数(psoriasis area and severity index,PASI)进行评分,将小鼠皮损进行HE染色观察其病理改变;3)在IMQ诱导中期(第3天)使用不同剂量MTX(38.5和77.0 nmol/L)进行小鼠腹腔内注射1次,同时设置PBS组和阴性对照组,肉眼观察各组皮损外观并用HE染色方法观察皮损组织病理改变,观察6 d;随后采用FCM检测小鼠脾淋巴细胞中Treg细胞数量,q RT-PCR法检测脾淋巴细胞Foxp3 m RNA表达水平。结果:1)在银屑病患者外周血PBMC中Treg细胞数量及Foxp3 m RNA表达水平比健康对照者明显降低(P<0.05);用MTX治疗8周后疗效显著的寻常型银屑病患者外周血Treg细胞的数量显著高于治疗前水平(P<0.05),Foxp3 m RNA表达水平显著升高(P<0.01);2)由IMQ诱导的BALB/c小鼠表现出银屑病样的皮损外观及组织病理学改变,其脾淋巴细胞中Treg细胞数量下降(P<0.01),Foxp3m RNA表达水平降低(P<0.05);3)使用不同剂量MTX于BALB/c小鼠腹腔内注射可有效缓解其银屑病样皮炎的皮损外观、逆转病理改变,小鼠脾淋巴细胞Treg细胞数量部分逆转,Foxp3 m RNA表达水平增高(P<0.05)。结论:MTX可以通过调控Treg细胞数量及Foxp3 m RNA表达水平来治疗银屑病。Objective- To explore the role of methotrexate （MTX） in regulating the number of regulatory T cells （Treg） and the mRNA expression of transcription factor Foxp3. Methods： 1） We analyzed the number of Treg and the mRNA expression of Foxp3 by flow cytometry （FCM） and quantitative real-time PCR （qRT-PCR） respectively in patients with psoriasis vulgaris, patients with psoriasis vulgaris after the 8-week treatment of MTX, and healthy people. 2） BALB/c female mice were smeared with imiquimod （IMQ） cream for 6 days. We recorded the change of the lesion in mice every day. The morphological changes of lesion in mice were evaluated by the psoriasis area and severity index （PASI） and HE staining. 3） The mouse model was randomly divided into a control group and an MTX group. The MTX group was treated with different doses of MTX （38.5 and 77.0 nmol/L） on the third day of this experiment. The morphological changes of lesion in mice were evaluated by PASI and HE staining. We tested the number of Treg and the expression level ofFoxp3 mRNA in splenic lymphocytes. Results： 1） 3-he number of Treg and the expression level of Foxp3 mRNA were lower in psoriasis vulgaris patients than those in the healthy control group （P〈0.05）. After 8-week treatment of MTX, the number of Treg was increased （P〈0.05） and Foxp3 mRNA level was up-regulated （P〈0.01）. 2） Typical psoriasis-like skin lesions, such as red scaly skin plaque were found after topical application oflMQ：. Both the number of Treg in the splenic lymphocytes of mice and the Foxp3 mRNA level of Trey were reduced by IMQ（P〈0.01 and P〈0.05）. 3） Different doses of MTX for mice showed the ability to improve skin lesion, increase the number of Trey in the spleen of mice and Foxp3 mRNA level in psoriatic dermatitis of mice （P〈 0.05）. Conclusion： MTX is able to regulate the number of Trey and Foxp3 mRNA expression in psoriasis.

关 键 词：银屑病 氨甲喋呤 调节性T细胞 Foxp3 咪喹莫特 

分 类 号：R758.63[医药卫生—皮肤病学与性病学]