空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响

doi:10.3760/cma.j.issn.0412-4030.2018.03.010

摘要/Abstract

摘要： 目的探讨空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响。方法收集北京市2015—2016年采暖季雾霾天气PM2.5，制备成混悬液。将HaCaT细胞分为空白组（仅用细胞培养基）、对照组（不加PM2.5，其他处理同实验组）和实验组［100 ~ 400 mg/L（细胞形态学观察和增殖实验用50 ~ 800 mg/L）PM2.5混悬液处理］处理24 h后，倒置显微镜下观察细胞形态变化；CCK8法检测细胞存活率；流式细胞仪检测细胞周期分布及细胞凋亡；Western 印迹法检测细胞周期蛋白A2（cyclin A2）及细胞周期蛋白依赖性激酶1（CDK1）的表达水平。结果随着PM2.5浓度升高，HaCaT细胞形态逐渐发生改变，细胞数目逐渐减少。与对照组（100 ± 4.95）%相比，50 mg/L PM2.5组HaCaT细胞存活率无明显变化（P＞0.05），100、200、400、800 mg/L PM2.5组细胞存活率［分别为（91.77 ± 2.04）%、（80.01 ± 1.57）%、（57.80 ± 1.56）%、（21.98 ± 0.86）%］均显著下降（P＜0.05）。流式细胞仪检测显示，与对照组相比，PM2.5组（100、200、400 mg/L）S期细胞比例逐渐增高，G2/M期细胞比例逐渐降低，差异均有统计学意义（P＜0.05）。Western印迹法显示，100、200、400 mg/L PM2.5组cyclin A2、CDK1蛋白表达均较对照组有所降低，尤其以200 mg/L组降低最明显，差异均有统计学意义（P＜0.05）。100、200、400 mg/L PM2.5组细胞总凋亡率分别为（9.98 ± 0.21）%、（12.56 ± 0.74）%、（16.74 ± 1.48）%，与对照组（6.24 ± 0.17）%相比，差异均有统计学意义（P＜0.05）。结论 PM2.5可能通过下调cyclin A2、CDK1表达水平诱导HaCaT细胞发生S期阻滞，从而抑制细胞增殖，促进HaCaT细胞凋亡。

关键词: 细胞增殖, 细胞凋亡, 颗粒物, 空气, HaCaT细胞, PM2.5

Abstract: Xue Chenhong, Zhang Hongwei, Peng Fen, He Congfen, Wang Qiao′e, Chen Zhou, Zhang Jianzhong Department of Dermatology, Peking University People′s Hospital, Beijing 100044, China （Xue CH ［the current affiliation: Department of Dermatology, Henan Provincial People′s Hospital, Zhengzhou 450003, China］, Peng F, Chen Z, Zhang JZ）; Department of Toxicology, National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing 102206, China （Zhang HW）; Beijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, Beijing 102488, China （He CF, Wang QE） Corresponding authors: Chen Zhou, Email: chenzhou@medmail.com.cn; Zhang Jianzhong, Email: rmzjz@126.com 【Abstract】 Objective To evaluate effects of fine particulate matter PM2.5 in ambient air on the proliferation, cell cycle and apoptosis of a human keratinocyte cell line HaCaT. Methods PM2.5 in haze-fog episodes during the heating season was collected in Beijing from 2015 to 2016, and processed into PM2.5 suspensions. HaCaT cells were divided into several groups to be treated with culture medium alone （control group）, PM2.5 suspensions at different concentrations of 100 - 400 mg/L （experiment groups, 50 - 800 mg/L for observation of cellular morphology and analysis of cell proliferation） for 24 hours, or cell culture medium without cells or PM2.5 suspensions （blank group）. Cellular morphological changes were observed under an inverted microscope. Cell counting kit-8（CCK-8） assay was performed to determine cell survival rate, flow cytometry to determine the cell cycle distribution and detect cell apoptosis, and Western blot analysis to determine the protein of cyclin A2 and cyclin-dependent kinase1（CDK1）. Results Along with the increase of PM2.5 concentration, HaCaT cells lost their normal shape gradually, and the number of viable cells gradually decreased. Compared with the control group （100% ± 4.95%）, the 50-mg/L PM2.5 group showed no changes in cell survival rates （P > 0.05）, while the 100-, 200-, 400- and 800-mg/L PM2.5 group showed significantly lower survival rates （91.77% ± 2.04%, 80.01% ± 1.57%, 57.80% ± 1.56%, 21.98% ± 0.86%, respectively, all P < 0.05）. Flow cytometry revealed that the 100-, 200- and 400-mg/L PM2.5 groups showed gradually increased proportion of cells at S phase, but gradually decreased proportion of cells at G2/M phase compared with the control group （all P < 0.05）. As Western blot analysis showed, the protein of cyclin A2 and CDK1 significantly decreased in the 100-, 200- and 400-mg/L PM2.5 groups compared with the control group, which was lowest in the 200-mg/L PM2.5 group（all P < 0.05）. In addition, the 100-, 200- and 400-mg/L PM2.5 groups showed significantly higher total apoptosis rates （9.98% ± 0.21%, 12.56% ± 0.74%, 16.74% ± 1.48%, respectively） compared with the control group （6.24% ± 0.17%, all P < 0.05）. Conclusion PM2.5 can inhibit cell proliferation and promote apoptosis of HaCaT cells, likely by downregulating the of cyclin A2 and CDK1 and arresting HaCaT cells at S phase.

Key words: Cell proliferation, Apoptosis, Particulate matter, Air, HaCaT cells, PM2.5

薛晨红张宏伟彭芬何聪芬王巧娥陈周张建中. 空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响[J]. 中华皮肤科杂志, 2018, 51(3): 204-208.

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