特应性皮炎差异表达基因筛选及KLF15对AD小鼠血清中Th1/Th2型细胞因子水平的影响  

Screening of Differentially Expressed Genes and the Effect of KLF15 on Th1/Th2 Cytokines in AD Mouse

作　　者：唐情 汪海珍[2] 汪碧滢 易婷婷 王力 罗菲菲 冉崇军 罗美俊子[2] 严伊宁 黄盼[2] 彭友华[2] 潘意 周蓉 TANG Qing;WANG Haizhen;WANG Biying;YI Tingting;WANG Li;LUO Feifei;RAN Chongjun;LUO Meijunzi;YAN Yining;HUANG Pan;PENG Youhua;PAN Yi;ZHOU Rong(Hunan University of Chinese Medicine,Changsha 410208,China;The Second Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410005,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南中医药大学第二附属医院,湖南长沙410005

出　　处：《中国皮肤性病学杂志》2023年第6期645-651,共7页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金项目(82004381);湖南省卫生计生委科研课题(20201213);湖南省自然科学基金项目(2020JJ5431)。

摘　　要：目的分析特应性皮炎(AD)患者皮损组织中差异表达基因(DEGs)及其功能作用,并探究其中的Krüppel样因子15(KLF15)对AD小鼠血清中Th1/Th2型细胞因子水平的影响。方法基于GSE157194芯片筛选AD患者皮损组织中的差异表达基因,通过Metascape在线分析工具进行基因本体(GO)功能分析和京都基因与基因组百科全书(KEGG)通路富集分析。进一步通过基因芯片验证KLF15在AD皮损组织与正常皮肤组织中的差异表达水平及治疗前后的表达变化。将16只BALB/c小鼠随机分为对照组(n=4),模型组(n=4),OE-NC组(n=4,空载质粒),OE-KLF15组(n=4,KLF15过表达质粒)。利用二硝基氯苯(DNCB)涂抹小鼠左耳构建AD小鼠模型,验证KLF15在AD小鼠组织中的表达水平。构建KLF15过表达载体,并注射至各小鼠皮损处,qPCR和Western blot检测转染效率,酶联免疫吸附试验(ELISA)检测白细胞介素(IL)-4、IL-13、IL-31和干扰素(IFN)-γ的表达水平。结果与正常皮肤组织相比,治疗前AD患者的皮损组织中有74个下调的DEGs及331个上调的DEGs,主要与细菌应答、炎症反应、Th2型免疫激活、皮肤发育、细胞离子代谢、调节类固醇代谢过程以及Wnt通路调控显著相关。与治疗前正常皮肤组织样本(AN)相比,皮损组织样本(AL)中KLF15表达显著下调(|logFC|>1,P<0.05)。使用度普利尤单抗或环孢菌素治疗3个月后,AN与AL中KLF15表达水平差异无统计学意义(|logFC|>1,P>0.05)。对照组小鼠表皮、真皮结构正常,无炎性细胞浸润。模型组小鼠出现棘层肥厚、真皮层水肿并伴有大量炎细胞浸润。与对照组小鼠比较,AD小鼠KLF15 mRNA及蛋白水平显著下调(P<0.05)。与OE-NC组比较,OE-KLF15组小鼠血清中IL-4、IL-13和IL-31的表达水平显著下调(P<0.01),KLF15、IFN-γ的表达水平显著上升(P<0.01)。结论KLF15在AD患者皮损组织中下调,KLF15可能通过改善AD小鼠血清中Th1/Th2平衡,减轻炎症反应,缓解皮肤损伤。Objective To explore the differentially expressed genes(DEGs)and their functional roles in the skin lesions of patients with atopic dermatitis(AD),and explore the effect of Krüppel like factor 15(KLF15)on the levels of Th1/Th2 cytokines in serum of AD mouse.Methods Based on the GSE157194 chip to screen differentially expressed genes in skin lesions of AD patients,Gene Ontology(GO)function analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed by Metascape online analysis tools.The differential expression level of KLF15 in AD skin lesions and normal skin tissues and the expression changes before and after treatment were further verified by gene chip.Sixteen BALB/c mouse were randomly divided into control group(n=4),model group(n=4),OE-NC group(n=4),OE-KLF15 group(n=4).The AD mouse model was established by smearing the left ear of mice with dinitrochlorobenzene(DNCB),and the expression level of KLF15 in AD mouse tissues was verified.The KLF15 overexpression vector was constructed and injected into the skin lesions of each mouse.The transfection efficiency was detected by qPCR and Western blot,and interleukin(IL)-4,IL-13,IL-31 and interferon(IFN)-γ.Results Compared with the normal skin tissue before treatment,there were 74 down-regulated DEGs and 331 up-regulated DEGs in the skin lesions.AD-related DEGs are mainly associated with response to bacterium,inflammatory response,Th2-type immune activation,skin development,response to metal ion,regulation of steroid metabolism,and regulation of Wnt signaling pathway.Compared with normal skin tissue samples(AN)before treatment,the expression of KLF15 in skin lesion tissue samples(AL)was significantly down-regulated(|logFC|>1,P<0.05).After 3 months of treatment with dupilumab or cyclosporine,there was no significant difference in the expression level of KLF15 between AN and AL(|logFC|>1,P>0.05).In the control group,the epidermis and dermis of the mouse were normal,and there was no inflammatory cell infiltration.The mouse in the

关 键 词：特应性皮炎 Krü ppel样因子15(KLF15) TH1/TH2平衡 炎症因子 

分 类 号：R758.2[医药卫生—皮肤病学与性病学]