环境浓度下铜锰复合暴露对人角膜上皮细胞的毒性效应  

Toxic effects of copper and manganese on human corneal epithelial cells at environmental concentrations

作　　者：张振宁 毕珏 杨丹蕾 崔烨 刘海 郑钦象 杨极 向萍 ZHANG Zhenning;BI Jue;YANG Danlei;CUI Ye;LIU Hai;ZHENG Qinxiang;YANG Ji;XIANG Ping(Institute of Environmental Remediation and Human Health,School of Ecology and Environment,Southwest Forestry University,Kunming 650224;Affiliated Hospital of Yunnan University,Eye Hospital of Yunnan Province,Kunming 650224;Eye Hospital of Wenzhou Medical University,Wenzhou 325027)

机构地区：[1]西南林业大学生态与环境学院,环境修复与健康研究院,昆明650224 [2]云南大学附属医院,云南省眼科医院,昆明650224 [3]温州医科大学附属眼视光医院,温州325027

出　　处：《环境科学学报》2022年第2期458-467,共10页Acta Scientiae Circumstantiae

基　　金：国家自然科学基金(No.21906134);云南省基础研究计划面上项目(No.2019FB014);云南省创新团队项目(No.202005AE160017);云南省高层次人才引进计划项目(No.YNQR-QNRC-2018-049);国家大学生创新训练项目(No.202010677004)。

摘　　要：人正常角膜上皮细胞(HCE)经室内环境浓度下的铜(10μmol·L^(-1))、锰(20、40μmol·L^(-1))单独及复合暴露后,采用CCK-8法对细胞活力进行检测,使用Annexin V-FITC/PI试剂盒测定了细胞凋亡率,并通过实时荧光定量PCR法从分子水平比较了不同暴露组炎症(NLRP3,IL^(-1)β)和凋亡调控关键基因(Caspase-1,Caspase-3)表达差异.结果显示:低浓度铜或锰单独暴露时,未明显降低细胞活力,而复合暴露后,细胞活力降低至20%~77%,大量细胞变圆并形成空泡,细胞凋亡率增加至69%;铜、锰单独及复合暴露后均会引起炎症基因(IL^(-1)β和NLRP3)的差异表达,铜暴露诱发了Caspase-3的表达,而锰则促进了Caspase-1的转录,铜锰复合暴露后Cu10+Mn20组的Caspase-3在mRNA表达水平上增加了1.8倍,Cu10+Mn40组促炎因子NLRP3表达量明显上升,为对照组的7.32倍(p<0.0001).综上,比起单独暴露,室内环境浓度铜锰复合暴露显著降低了人角膜上皮细胞活力,促进了细胞凋亡.可见,在室内环境健康风险评价或毒性效应研究中应该综合考虑复合污染.Normal human corneal epithelial cells were employed to analyze the effects of single and combined exposure to Cu(10μmol·L^(-1))and Mn(20,40μmol·L^(-1)).Cell viability was measured by a commercial CCK-8 assay,and cell apoptosis rate of HCE was detected by Annexin V-FITC/PI kit.The expression of inflammation mediators(NLRP3,IL^(-1)β)and the key apoptotic genes(Caspase-1,Caspase-3)in different exposure groups were detected at the mRNA level by Real-Time quantitative PCR.The results showed that cell viability was not affected when HCE were exposed to Cu or Mn at low concentrations individually,while reducing to 20%~77%after Cu and Mn combined exposure.Many cells with round shapes and formed vacuoles after exposure were observed,and cell apoptosis rate increased to 69%after being treated with Cu(10μmol·L^(-1))and Mn(40μmol·L^(-1)).Single or combined exposure to Cu and Mn triggered differential expression patterns of inflammatory genes(IL^(-1)βand NLRP3).Cu exposure induced the expression of Caspase-3,while Mn promoted the transcription of Caspase-1,and the mRNA expression level of Caspase-3 was increased by 1.8 times after combined exposure in the Cu10+Mn20 group.Besides,the gene expression of NLRP3 in the Cu10+Mn40 group increased sharply,which was increased by 7.32 times(p<0.0001).In conclusion,combined exposure to indoor environmental concentrations of Cu and Mn apparently reduced the viability of HCE and increased cell apoptosis.Therefore,complex pollution should be comprehensively considered in the research of indoor environmental health risks or toxic effects.

关 键 词：室内灰尘 重金属 复合暴露 人角膜上皮细胞(HCE) 细胞凋亡 炎症 

分 类 号：X171.5[环境科学与工程—环境科学]