摘要
目的探讨细颗粒物(PM2.5)对人正常鼻黏膜上皮细胞间闭合蛋白(occludin)表达及分布的影响。方法采用(0、 50、 100、 200、 400、 500、 800、 1000)μg/mL PM2.5处理体外培养的正常人HNEpC鼻黏膜上皮细胞,采用CCK-8法检测细胞活力,确定最佳处理剂量;后续采用(200、 400、 500)μg/mL PM2.5处理HNEpC细胞,用免疫荧光细胞化学染色法检测细胞occludin的表达和分布情况,实时荧光定量PCR检测occludin的mRNA水平, Western blot法检测occludin蛋白水平。结果与阴性对照组(0μg/mL PM2.5)相比,各剂量PM2.5处理鼻黏膜上皮细胞24 h和48 h后,细胞活性均降低,且细胞活性与PM2.5剂量呈负相关。与阴性对照组相比, 200μg/mL PM2.5组,细胞形态及细胞间occludin蛋白分布表达无明显改变; 400μg/mL PM2.5组,细胞形态变为梭形,细胞间隙增宽; 500μg/mL PM2.5组,细胞数量明显减少,细胞形态变为长梭形,细胞间隙增宽明显;除200μg/mL PM2.5处理细胞24 h外,其余各组细胞occludin mRNA及蛋白水平均下调,且occludin蛋白表达水平呈时间依赖性。结论 PM2.5可降低正常人HNEpC鼻黏膜上皮细胞活性、降低occludin的表达和分布。
Objective To investigate the effect of PM2.5 on the distribution and expression of occludin in nasal mucosal epithelial cells. Methods The normal human nasal epithelial cell line HNEpCs were stimulated with PM2.5 in vitro. The incubated cells were treated with 0, 50, 100, 200, 400, 500, 800, 1000 μg/mL of PM2.5. The suitable concentration of PM2.5 was determined according to the cell viability measured by CCK-8 assay. Then the incubated cells were treated with 200, 400, 500 μg/mL of PM2.5. The occludin expression and distribution on the levels of protein and mRNA were detected by immunofluorescence assay, Western blot analysis and real-time fluorescence quantitative PCR. Results Compared with the control group, the cell viability of HNEpCs treated with different concentrations of PM2.5 decreased after 24 and 48 hours. The cell viability was negatively correlated with PM2.5 concentrations. Compared with the control group, there was no obvious change in the cell morphology and occluding expression of the 200 μg/mL PM2.5 group. The cell morphology changed into the spindle shape in the 400 μg/mL group, and a small number of vacuoles appeared in the cell connection. After treated with 500 μg/mL PM2.5, the cell density decreased, normal cell morphology changed into irregular long spindle shape and the intercellular space was enlarged. Occludin mRNA and protein levels were down-regulated in all the groups except for the cells treated with 200 g/mL PM2.5 for 24 hours, and occludin protein expression levels were time-dependent. Conclusion PM2.5 can reduce the viability of HNEpCs in normal human nasal epithelial cell line, and reduce the mRNA and protein expression of the intercellular occludin.
作者
冯彦
蔺慧文
朱蓉娜
雒红霞
皇甫辉
王彤
FENG Yan;LIN Huiwen;ZHU Rongna;LUO Hongxia;HUANGFU Hui;WANG Tong(Department of Otolaryngology, Head & Neck Surgery, First Hospital, Shanxi Medical University, Taiyuan 030001, China;School of Nursing, Shanxi Medical University, Taiyuan 030001, China;Department of Health Statistics, School of Public Health, Shanxi Medical University, Taiyuan 030001, China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2019年第2期152-157,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
中国博士后科学基金(2016M591413)
山西省软科学研究计划(2018041032-5)
