Gao Zi-Xun, Song Xi-Ling, Li Shan-Shan, Lai Xiao-Rong, Yang Yu-Lan, Yang Guang, Li Zhi-Jie, Cui Yu-Hong, Pan Hong-Wei
Institute of Ophthalmology School of Medicine, Jinan University, Guangzhou, China 2Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China.
Department of Public Health and Preventive Medicine, Jinan University, Guangzhou, China.
Invest Ophthalmol Vis Sci. 2016 Jun 1;57(7):3093-102. doi: 10.1167/iovs.15-18839.
To assess the genotoxic effect of airborne particulate matter on corneal epithelial cells and investigate the role of reactive oxygen species (ROS) formation in this process.
Immortalized human corneal epithelial cells (HCECs) and primary bovine corneal epithelial cells were exposed to airborne particulate matter collected from Guangzhou for 24 hours. The cell viability and toxicity were measured by the CCK-8 test and lactate dehydrogenase (LDH) release, respectively. The DNA breaks and DNA repair were examined by alkaline comet assay and by immunofluorescence staining of the phosphorylated histone variant H2AX (γH2AX), respectively. Reactive oxygen species production was assessed by the fluorescent probe, CM-H2DCFDA. Cell senescence was evaluated with senescence-associated β-Galactosidase staining, and cell ultrastructure was observed with transmission electron microscopy.
Exposure to PM2.5 at the concentration of 20 μg/mL to 200 μg/mL decreased cell viability and increased LDH release. Remarkably increased DNA double-stand breaks, increased expression of DNA repair-related protein γH2AX, elevated ROS formation, and altered cell ultrastructure were observed in HCECs after treatment with PM2.5. The genotoxic effect of PM2.5 was attenuated by the ROS inhibitor N-acetyl-l-cysteine (NAC).
Particulate matter 2.5 could induce DNA damage and cell senescence in corneal epithelial cells, probably by promoting ROS formation. Thus, whether long-term exposure of PM2.5 might be related to potential risk of abnormality in corneal epithelium renewal and regeneration should be further investigated.
评估空气中颗粒物对角膜上皮细胞的遗传毒性作用，并研究活性氧（ROS）形成在此过程中的作用。
将永生化人角膜上皮细胞（HCECs）和原代牛角膜上皮细胞暴露于从广州采集的空气中颗粒物中24小时。分别通过CCK-8试验和乳酸脱氢酶（LDH）释放来测定细胞活力和毒性。分别通过碱性彗星试验和磷酸化组蛋白变体H2AX（γH2AX）的免疫荧光染色来检测DNA断裂和DNA修复。通过荧光探针CM-H2DCFDA评估活性氧的产生。用衰老相关β-半乳糖苷酶染色评估细胞衰老，并用透射电子显微镜观察细胞超微结构。
暴露于浓度为20μg/mL至200μg/mL的PM2.5会降低细胞活力并增加LDH释放。在用PM2.5处理后的HCECs中观察到DNA双链断裂显著增加、DNA修复相关蛋白γH2AX的表达增加、ROS形成升高以及细胞超微结构改变。ROS抑制剂N-乙酰-L-半胱氨酸（NAC）减弱了PM2.5的遗传毒性作用。
颗粒物2.5可能通过促进ROS形成诱导角膜上皮细胞中的DNA损伤和细胞衰老。因此，PM2.5的长期暴露是否可能与角膜上皮更新和再生异常的潜在风险有关，应进一步研究。
