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

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。

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.