摘要
目的通过建立由H2O2诱导的HEI-OC1细胞氧化应激模型,验证石墨烯对氧化应激后毛细胞的保护作用,并探究石墨烯减轻毛细胞氧化应激损伤的可能机制。方法我们将二维石墨烯材料(2D-G)作为实验组,TC玻片(TCPs)作为对照组。首先用不同浓度的H2O2处理接种于两种材料上的HEI-OC1细胞,分别作用4h和12h后,通过CCK-8法检测两组细胞存活率的差异,与此同时,通过免疫荧光法(通过观察myosin7a抗体,即毛细胞特异性抗体的染色情况)观察两组细胞的存活情况。接下来我们利用DCFH-DA和Mito-sox两种荧光探针分别测定细胞内和线粒体内ROS(活性氧)水平,运用流式分析法和免疫荧光法对氧化应激后的两组细胞内ROS水平的变化进行了直接和间接的检测。随后,我们通过TUNNEL法检测发生氧化应激后两组细胞凋亡水平的差异。结果石墨烯能够显著提高短期/较长时间氧化应激后耳蜗毛细胞系HEI-OC1细胞的存活率;石墨烯能够显著降低氧化应激后HEI-OC1细胞内及线粒体内的ROS水平;石墨烯能够显著降低氧化应激后HEI-OC1细胞的凋亡水平。结论石墨烯能够显著降低氧化应激后耳蜗类毛细胞系HEI-OC1胞质及线粒体内的ROS水平,从而抑制凋亡现象,对氧化应激引起的耳蜗毛细胞损伤具有一定的保护作用。
Objective To verify the protection of the hair cells by 2D-graphene against oxidative stress damage by H2O2 and possible mechanisms.Methods HEI-OC1 cells were treated with 2D-G(study group)or TCPs(control group)and exposed to different concentrations of H2O2.Cell survival was examined by the CCK 8 method and immunofluorescence staining of Myosin7a(a hair-cell-specific protein)after 4 hours and 12 hours of H2O2 exposure and compared between the two groups.Whole ROS level in cells and ROS level in mitochondria were determined by detection of DCFHDA and Mito-sox using fluorescent probes.Changes of ROS levels and levels of apoptosis after oxidative stress were examined by flow analysis and immunofluorescence and by TUNNEL assay,respectively,and compared between the two groups.Results The rate of HEI-OC1 cells survival was greater after oxidative stress,with significantly reduced intracellular and mitochondrial ROS levels and cells apoptosis when treated with graphene.Conclusion Graphene can significantly reduce the level of ROS in cytoplasm and mitochondria in cochlear hair cell line HEI-OC1 cells at risk of oxidative stress damage,thereby inhibiting apoptosis and protecting the cells from oxidative stress.
作者
董健菲
陆玲
林川耀
刘勇
周函
朱成文
潘明洁
唐明亮
高下
DONG Jianfei;LU Ling;LIN Chuanyao;LIU Yong;ZHOU Han;ZHU Chengwen;PAN Mingjie;TANG Mingliang;GAO Xia(Department of Otolaryngology-Head and Neck Surgery,Nanjing Drum Tower Hospital of Nanjing University School of Medicine,Nanjing 210008;Research Institute of Otolaryngology,Nanjing 210008;Central Laboratory of Nanjing Drum Tower Hospital of Nanjing University School of Medicine,Nanjing 210008;College of Life Science,Southeast University,Nanjing,210096)
出处
《中华耳科学杂志》
CSCD
北大核心
2021年第1期101-107,共7页
Chinese Journal of Otology
基金
国家自然科学基金青年基金(81700913)
江苏省科教强卫工程江苏省青年医学人才基金(QNRC2016002)。
关键词
石墨烯
氧化应激
毛细胞损伤
毛细胞保护
Graphene
Oxidative stress
Hair cell injury
Hair cell protection
