中华皮肤科杂志 ›› 2019, Vol. 52 ›› Issue (10): 753-758.doi: 10.35541/cjd.20190095

• 论著 • 上一篇    下一篇

城市大气细颗粒物PM2.5对人角质形成细胞屏障相关蛋白表达的影响

姚骐羽1    罗阳2    姚煦2    刘军3   

  1. 1昆明医科大学影像医学系  650500；2中国医学科学院  北京协和医学院  皮肤病医院过敏与风湿免疫科，南京  210042；3南京大学医学院附属鼓楼医院皮肤科  210008
  • 收稿日期:2018-12-07 修回日期:2019-06-20 发布日期:2019-09-30
  • 通讯作者: 刘军 E-mail:drliu_jun@126.com
  • 作者简介:由于毕业保研需要，恳请贵刊考虑文章可以尽快发表
  • 基金资助:
    江苏省自然科学基金（BK20171117）；南京市医学发展项目（YKK17081）

Effect of urban atmospheric fine particulate matter PM2.5 on the expression of skin barrier-associated proteins in human keratinocytes

Yao Qiyu1, Luo Yang2, Yao Xu2, Liu Jun3   

  1. 1Department of Imaging Medicine, Kunming Medical University, Kunming 650500, China; 2Department of Allergy and Rheumatology, Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China; 3Department of Dermatology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China
  • Received:2018-12-07 Revised:2019-06-20 Published:2019-09-30
  • Contact: Liu Jun E-mail:drliu_jun@126.com
  • Supported by:
    Natural Science Foundation of Jiangsu Province of China （BK20171117）; Nanjing Medical Development Project （YKK17081）

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摘要： 【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、200 mg/L PM2.5分别刺激人原代角质形成细胞24 h，CCK-8检测细胞存活率。50 mg/L PM2.5处理原代角质形成细胞0、3、6、9、12、18和24 h，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24 h后细胞内丝聚蛋白、角蛋白14和紧密连接蛋白1 mRNA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24 h后，10 mg/L PM2.5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、200 mg/L PM2.5组细胞存活率均显著低于对照组（均P < 0.05）。50 mg/L PM2.5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，24 h细胞生存率为72.37% ± 3.12%。不同浓度PM2.5刺激角质形成细胞24 h后，10、50 mg/L PM2.5组丝聚蛋白mRNA相对表达量（1.27 ± 0.15、1.32 ± 0.09）和角蛋白14 mRNA相对表达量（1.15 ± 0.13、1.08 ± 0.16）均高于对照组（均P < 0.05）；100、200 mg/L PM2.5组丝聚蛋白mRNA相对表达量（0.84 ± 0.11、0.42 ± 0.12）和角蛋白14 mRNA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、200 mg/L PM2.5组紧密连接蛋白1 mRNA表达均显著低于对照组（均P < 0.05）。Western印迹显示，50、100 mg/L PM2.5组丝聚蛋白表达高于对照组（均P < 0.05），而10、200 mg/L PM2.5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）。10 mg/L PM2.5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、200 mg/L PM2.5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。

关键词: 角蛋白细胞, 颗粒物, 细胞存活, 角蛋白14, 紧密连接蛋白质类, 白细胞介素1α, PM2.5, 屏障蛋白, 丝聚蛋白, 白细胞介素33, 胸腺基质淋巴细胞生成素

Abstract: 【Abstract】 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-1α and IL-33 （all P < 0.01）. Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1α and 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-1α and IL-33, which may lead to impaired skin barrier function.

Key words: Keratinocytes, Particulate matter, Cell survival, Keratin-14, Claudins, Interleukin-1alpha, PM2.5, Barrier-associated proteins, Filaggrin, Interleukin-33, Thymic stromal lymphopoietin

引用本文

姚骐羽 罗阳 姚煦 刘军. 城市大气细颗粒物PM2.5对人角质形成细胞屏障相关蛋白表达的影响[J]. 中华皮肤科杂志, 2019,52(10):753-758. doi:10.35541/cjd.20190095