中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (9): 759-766.doi: 10.35541/cjd.20210876

• 论著 • 上一篇    下一篇

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

姚玲玲1，2    余增洋2    郭春源3    周静3    崔莲2    于倩2    虞英媛3    周雪2    蔡江鲁伊3    史玉玲1，2，3   

  1. 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
  • 收稿日期:2021-12-02 修回日期:2022-04-28 发布日期:2022-09-02
  • 通讯作者: 史玉玲 E-mail:shiyuling1973@tongji.edu.cn
  • 基金资助:
    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）

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

Yao Lingling1,2, Yu Zengyang2, Guo Chunyuan3, Zhou Jing3, Cui Lian2, Yu Qian2, Yu Yingyuan3, Zhou Xue2, Cai Jiangluyi3, Shi Yuling1,2,3   

  1. 1Department of Dermatology, Shanghai Clinical College of Anhui Medical University, Hefei 230000, China; 2Department of Dermatology, Shanghai Tenth People′s Hospital, Shanghai 200072, China; 3Department of Dermatology, Skin Disease Hospital of Tongji University, Shanghai 200443, China
  • Received:2021-12-02 Revised:2022-04-28 Published:2022-09-02
  • Contact: Shi Yuling E-mail:shiyuling1973@tongji.edu.cn
  • Supported by:
    National Natural Science Foundation of China （81872522, 82073429, 81900612, 82103720）; Innovation Program of Shanghai Municipal Education Commission （2019-01-07-00-07-E00046）; The Program of Science and Technology Commission of Shanghai Municipality （18140901800）;Excellent Subject Leader Program of Shanghai Municipal Commission of Health and Family Planning （2018BR30）; Program of Shanghai Academic Research Leader （20XD1403300）; Clinical Research Program of Shanghai Hospital Development Center （SHDC2020CR1014B, SHDC12018X06） 

PDF

12

摘要： 【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siRNA-NC + TNF-α组（均P < 0.05）。结论 CRY2在银屑病小鼠模型中表达降低，促进了角质形成细胞的增殖以及趋化因子CXCL1、CXCL8和CCL20的表达，且TNF-α可能是下调CRY2表达的上游细胞因子。

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

Abstract: 【Abstract】 Objective 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 group （0.94 ± 0.23） than in the control group （2.30 ± 0.25, t = 3.99, P = 0.016）. Compared with the siRNA-NC group, the siRNA-CRY2 group showed significantly increased proportions of EdU-positive cells （48.13% ± 10.97% vs. 38.23% ± 0.81%, t = 5.00, P = 0.007）, mRNA expression levels of chemokines CXCL1 and CXCL8, as well as significantly increased phosphorylated （p）-ERK1/2 protein expression levels （all P < 0.05）, while there were no significant differences in the CCL20 mRNA expression or ERK1/2 protein expression between the two groups （both P > 0.05）. Immunofluorescence staining showed significantly decreased CRY2 protein expression level in the mouse ear epidermis in the TNF-α group （0.37 ± 0.34） compared with the PBS group （2.04 ± 0.17, t = 4.38, P = 0.012）; the relative mRNA expression levels of chemokines CXCL1, CXCL8, and CCL20 in HaCaT cells were significantly higher in the siRNA-CRY2 + TNF-α group than in the siRNA-NC + TNF-α group （all P < 0.05）. Conclusion CRY2 was markedly underexpressed in psoriasis, which might promote the proliferation of keratinocytes and expression of chemokines CXCL1, CXCL8 and CCL20, and TNF-α might be an upstream cytokine that could downregulate CRY2 expression.

Key words: Psoriasis, Cryptochromes, Circadian rhythm, Models, animal, Keratinocytes, Chemokines, Tumor necrosis factor-alpha, Extracellular signal-regulated kinase-1/2