节律基因隐花色素2在银屑病小鼠模型及HaCaT细胞中的表达变化及机制研究  

Changes in circadian gene cryptochrome 2 expression in mouse models of psoriasis and HaCaT cells and their underlying mechanisms

作　　者：姚玲玲[1,2] 余增洋 郭春源 周静 崔莲[2] 于倩 虞英媛 周雪 蔡江鲁伊 史玉玲 Yao Lingling;Yu Zengyang;Guo Chunyuan;Zhou Jing;Cui Lian;Yu Qian;Yu Yingyuan;Zhou Xue;Cai Jiangluyi;Shi Yuling(Department of Dermatology,Shanghai Clinical College of Anhui Medical University,Hefei 230000,China;Department of Dermatology,Shanghai Tenth People's Hospital,Shanghai 200072,China;Department of Dermatology,Skin Disease Hospital of Tongji University,Shanghai 200443,China)

机构地区：[1]安徽医科大学上海临床学院,合肥230000 [2]上海市第十人民医院皮肤科,上海200072 [3]同济大学附属皮肤病医院皮肤科,上海200443

出　　处：《中华皮肤科杂志》2022年第9期759-766,共8页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81872522、82073429、81900612、82103720);上海市教委“科研创新计划”自然科学重大项目(2019-01-07-00-07-E00046);上海市科委“科技创新行动计划”项目(18140901800);上海市卫计委优秀学科带头人计划项目(2018BR30);上海市优秀学术带头人(20XD1403300);上海市促进市级医院临床技能与临床创新三年行动计划重大临床研究项目(SHDC2020CR1014B、SHDC12018X06)。

摘　　要：目的探讨节律基因隐花色素2(CRY2)在银屑病小鼠模型及HaCaT细胞中的表达变化及其机制。方法咪喹莫特诱导小鼠模型实验:12只C57BL/6雌鼠随机均分为咪喹莫特组(连续外用咪喹莫特乳膏5 d诱导构建银屑病小鼠模型,6只)和对照组(不予任何处理,6只),第6天处死小鼠,取其背部皮肤组织,免疫荧光染色检测表皮中CRY2的表达。HaCaT细胞转染实验:使用小干扰RNA(siRNA)技术在HaCaT细胞中敲减CRY2的表达(siRNA-CRY2组),以siRNA-NC组作为对照,5-乙炔基-2'脱氧尿嘧啶核苷(EdU)染色检测HaCaT细胞增殖活力,实时荧光定量PCR(qPCR)检测HaCaT细胞中趋化因子mRNA表达水平,Western印迹检测细胞外信号调节激酶1/2(ERK1/2)蛋白的磷酸化水平。肿瘤坏死因子α(TNF-α)刺激动物和细胞实验:12只C57BL/6雌鼠随机均分为TNF-α组(小鼠耳部皮下连续注射TNF-α溶液6 d,6只)和磷酸盐缓冲液(PBS)组(注射等量的PBS,6只),第7天处死小鼠,取其耳部皮肤组织,免疫荧光染色检测表皮中CRY2的表达;用50 ng/ml TNF-α刺激CRY2基因敲减的HaCaT细胞12 h(siRNA-CRY2+TNF-α组),以siRNA-NC+TNF-α组作为对照,qPCR检测各组细胞中趋化因子mRNA的表达。统计分析采用两独立样本t检验。结果免疫荧光染色显示,咪喹莫特组小鼠背部表皮层中CRY2蛋白的表达(0.94±0.23)显著低于对照组(2.30±0.25,t=3.99,P=0.016)。HaCaT细胞转染实验:siRNA-CRY2组EdU阳性细胞比例(48.13%±10.97%)显著高于siRNA-NC组(38.23%±0.81%,t=5.00,P=0.007),且siRNA-CRY2组趋化因子CXCL1、CXCL8 mRNA相对表达量及p-ERK1/2蛋白相对表达量均显著高于siRNA-NC组(均P<0.05),但两组间CCL20 mRNA表达量及ERK1/2蛋白表达量差异无统计学意义(均P>0.05)。TNF-α刺激实验:免疫荧光染色显示,TNF-α组鼠耳表皮组织中CRY2蛋白表达水平(0.37±0.34)显著低于PBS组(2.04±0.17,t=4.38,P=0.012);siRNA-CRY2+TNF-α组HaCaT细胞趋化因子CXCL1、CXCL8、CCL20 mRNA相对表达量均显著高于siRObjective To investigate changes in circadian gene cryptochrome 2(CRY2)expression in mouse models of psoriasis and HaCaT cells,and to explore underlying mechanisms.Methods Imiquimod-induced mouse model experiment:12 C57BL/6 female mice were randomly and equally divided into imiquimod group receiving topical imiquimod treatment for 5 consecutive days and control group receiving no treatment;these mice were sacrificed on day 6,skin tissues were resected from the back of mice,and immunofluorescence staining was performed to determine the CRY2 expression in the epidermis.HaCaT cell transfection experiment:HaCaT cells with small interfering RNA(siRNA)-mediated knockdown of CRY2 served as siRNA-CRY2 group,and siRNA-NC group as control group;5-ethynyl-2'-deoxyuridine(EdU)staining was performed to evaluate the proliferative activity of the HaCaT cells,real-time fluorescence-based quantitative PCR(qPCR)to determine the mRNA expression of chemokines in the HaCaT cells,and Western blot analysis to determine phosphorylation levels of extracellular signal-regulated kinase 1/2(ERK1/2).Tumor necrosis factor-α(TNF-α)-stimulated animal and cell experiments:12 C57BL/6 female mice were randomly and equally divided into TNF-α group subcutaneously injected with TNF-α solution in the ear for 6 days,and phosphate buffered saline(PBS)group subcutaneously injected with the same amount of PBS;the mice were sacrificed on day 7,skin tissues were resected from the ear of mice,and immunofluorescence staining was conducted to determine the CRY2 expression in the epidermis;CRY2-knockdown HaCaT cells stimulated with 50 ng/ml TNF-α for 12 hours served as siRNA-CRY2+TNF-α group,and siRNA-NC+TNF-α group as control group;qPCR was performed to determine the mRNA expression of chemokines in HaCaT cells in the above groups.Statistical analysis was carried out by using two-independent-sample t test.Results Immunofluorescence staining showed that the CRY2 protein expression was significantly lower in the mouse dorsal epidermis in the imiquimod gro

关 键 词：银屑病 隐花色素类 昼夜节律 模型 动物 角蛋白细胞 炎症趋化因子类 肿瘤坏死因子α 细胞外信号调节激酶1/2 

分 类 号：R758.63[医药卫生—皮肤病学与性病学] R-332[医药卫生—临床医学]