摘要
目的明确大气颗粒物PM2.5对人角质形成细胞中皮肤屏障相关蛋白和前炎症细胞因子表达的影响。方法取5例包皮环切术后的包皮分离培养人原代角质形成细胞。0(对照组)、10、50、100、200mg/LPM2.5分别刺激人原代角质形成细胞24h,CCK-8检测细胞存活率。50mg/LPM2.5处理原代角质形成细胞0、3、6、9、12、18和24h,以只加培养基的孔作为对照组,CCK-8检测细胞存活率。荧光定量PCR、Western印迹法分别检测不同浓度PM2.5刺激角质形成细胞24h后细胞内丝聚蛋白、角蛋白14和紧密连接蛋白1mRNA及蛋白表达,ELISA法检测各组角质形成细胞培养上清液中白细胞介素1α(IL-1α)、胸腺基质淋巴细胞生成素(TSLP)、IL-33水平。采用SPSS13.0统计软件进行单因素方差分析、LSD-t检验及Pearson相关性分析。结果不同浓度PM2.5处理24h后,10mg/LPM2.5组角质形成细胞存活率与对照组相比差异无统计学意义(P>0.05),而50、100、200mg/LPM2.5组细胞存活率均显著低于对照组(均P<0.05)。50mg/LPM2.5处理角质形成细胞不同时间,随PM2.5作用时间的延长,细胞生存率逐渐降低后稳定在一定水平,24h细胞生存率为72.37%±3.12%。不同浓度PM2.5刺激角质形成细胞24h后,10、50mg/LPM2.5组丝聚蛋白mRNA相对表达量(1.27±0.15、1.32±0.09)和角蛋白14mRNA相对表达量(1.15±0.13、1.08±0.16)均高于对照组(均P<0.05);100、200mg/LPM2.5组丝聚蛋白mRNA相对表达量(0.84±0.11、0.42±0.12)和角蛋白14mRNA相对表达量(0.67±0.09、0.74±0.11)均低于对照组(均P<0.05);而10、50、100、200mg/LPM2.5组紧密连接蛋白1mRNA表达均显著低于对照组(均P<0.05)。Western印迹显示,50、100mg/LPM2.5组丝聚蛋白表达高于对照组(均P<0.05),而10、200mg/LPM2.5组与对照组差异无统计学意义(均P>0.05)。各PM2.5组角蛋白14的表达均高于对照组(均P<0.05)。10mg/LPM2.5组紧密连接蛋白1表达与对照组相比差异无统计学意义(P=0.87),50、100、200mg/LPM2.5组均显著高于对照组(均P<0.05)。不同浓度PM2.5刺激角质形成细胞均引起TSLP、IL-1α、IL-33表达升高(均P<0.01)。Pearson相关分析显示,角质形成细胞前炎症因子TSLP、IL-1α、IL-33的分泌量与PM2.5浓度呈正相关(r=0.57、0.67、0.91,均P<0.05)。结论暴露于PM2.5处理可致角质形成细胞生存率下降,丝聚蛋白、角蛋白14和紧密连接蛋白1表达发生异常改变,同时伴有前炎症细胞因子IL-1α、TSLP、IL-33表达升高,可能导致皮肤屏障功能受损。
Objective To evaluate the effect of atmospheric fine particulate matter(PM2.5)on the expression of skin barrier-associated proteins and proinflammatory cytokines in human keratinocytes.Methods Atmospheric PM2.5 samples were given by Professor Yufeng Zhou in Children′s Hospital of Fudan University.Human primary keratinocytes were isolated from circumcised foreskins of 5 males,and subjected to culture.These human primary keratinocytes were divided into several groups to be stimulated with PM2.5 at different concentrations of 0(control group),10,50,100,200 mg/L for 24 hours,and cell counting kit(CCK)-8 assay was performed to determine the survival rates of keratinocytes.Fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of filaggrin,keratin-14 and claudin-1 in these keratinocytes respectively,and enzyme-linked immunosorbent assay(ELISA)was performed to detect levels of interleukin(IL)-1α,thymic stromal lymphopoietin(TSLP)and IL-33 in the culture supernatant of these keratinocytes.Statistical analysis was carried out with SPSS13.0 software by using one-way analysis of variance,least significant difference(LSD)-t test and Pearson correlation analysis.Results After 24-hour treatment with different concentrations of PM2.5,there was no significant difference in the survival rate of keratinocytes between the 10-mg/L PM2.5 group and control group(P>0.05),while the survival rates of keratinocytes were significantly lower in the 50-,100-,200-mg/L PM2.5 groups than in the control group(all P<0.05).After the treatment with 50 mg/L PM2.5,the cell survival rate gradually decreased and then remained stable along with the increase of PM2.5 treatment duration,and the cell survival rate at 24 hours was 72.37%±3.12%.After 24-hour treatment with PM2.5,the mRNA expression of filaggrin and keratin-14 was significantly higher in both the 10-and 50-mg/L PM2.5 groups(filaggrin:1.27±0.15,1.32±0.09 respectively;keratin-14:1.15±0.13,1.08±0.16 respectively)than in the control group(all P<0.05),but lower in both the 100-and 200-mg/L PM2.5 groups(filaggrin:0.84±0.11,0.42±0.12 respectively;keratin-14:0.67±0.09,0.74±0.11 respectively)than in the control group(all P<0.05).The 10-,50-,100-and 200-mg/L PM2.5 groups showed significantly decreased mRNA expression of claudin-1 compared with the control group(all P<0.05).Western blot analysis showed that filaggrin expression was significantly higher in the 50-and 100-mg/L PM2.5 groups than in the control group(both P<0.05),while no significant difference was observed between the 10-,200-mg/L PM2.5 groups and the control group(both P>0.05).The 10-,50-,100-and 200-mg/L PM2.5 groups showed significantly increased keratin-14 expression compared with the control group(all P<0.05).The claudin-1 expression did not differ between the 10-mg/L PM2.5 group and control group(P=0.87),but significantly higher in the 50-,100-and 200-mg/L PM2.5 groups than in the control group(all P<0.05).The stimulation with PM2.5 at 10,50,100 and 200 mg/L could induce an increase in the supernatant levels of TSLP,IL-1αand IL-33(all P<0.01).Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP,IL-1αand IL-33 were positively correlated with the concentration of PM2.5(r=0.57,0.67,0.91 respectively,all P<0.05).Conclusion The exposure to PM2.5 can induce decreased survival rate of keratinocytes,aberrant expression of filaggrin,keratin-14 and claudin-1,and elevated secretion of the proinflammatory cytokines TSLP,IL-1αand IL-33,which may lead to impaired skin barrier function.
作者
姚骐羽
罗阳
姚煦
刘军
Yao Qiyu;Luo Yang;Yao Xu;Liu Jun(Department of Imaging Medicine,Kunming Medical University,Kunming 650500,China;Department of Allergy and Rheumatology,Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing 210042,China;Department of Dermatology,Nanjing Drum Tower Hospital,The Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,China)
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2019年第10期753-758,共6页
Chinese Journal of Dermatology
基金
江苏省自然科学基金(BK20171117)
南京市医学发展项目(YKK17081).
