多壁碳纳米管长期染毒对人胸膜间皮细胞的影响  

Chronic toxicity of Multi-walled carbon nanotubes in human pleural mesothelial cells

作　　者：鞠莉 余珉 朱丽瑾 贾振宇 张敏 陈钧强 Ju Li;Yu Min;Zhu Lijin;Jia Zhenyu;Zhang Min;Chen Junqiang(Institute of Occupational Diseases,Hangzhou Medical College,Hangzhou 310013,China)

机构地区：[1]杭州医学院职业病防治研究所,310013

出　　处：《中华劳动卫生职业病杂志》2021年第3期173-177,共5页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金青年项目(81402718);浙江省医药卫生科技计划项目平台计划(2019ZD026);浙江省医学支撑学科劳动卫生学(11-ZC02)。

摘　　要：目的探讨低剂量多壁碳纳米管(MWCNT)长期慢性染毒对人胸膜间皮细胞的毒性效应及可能机制。方法于2016年,使用10μg/cm^(2) MWCNT对人胸膜间皮细胞MeT-5A反复持续染毒1年作为染毒组,每4周收集1次细胞并观察细胞形态和计数增殖情况。对照组细胞同期培养但不进行染毒处理。染毒后细胞经流式细胞仪测量细胞周期和细胞凋亡的改变;用Transwell小室测量细胞侵袭和迁移能力的改变;用Affymetrix clariom D芯片分析染毒后MeT-5A细胞基因表达谱的改变。部分差异表达基因经实时定量荧光PCR进一步验证其表达变化。结果与对照组比较,染毒1年组细胞增殖能力明显增强,增殖速度约为对照组2~3倍(F=481.32,P<0.05)。MWCNT染毒1年和6个月组MeT-5A细胞均呈现细胞周期阻滞效应,表现为G1期增加,S期、G2期减少(F=14.94,P<0.05)。染毒6个月组细胞凋亡率较对照组明显增加(F=15.12,P<0.05),染毒1年组细胞的早期凋亡率和总凋亡率较对照组细胞均差异无统计学意义(F=3.97,P>0.05)。对染毒1年组细胞进行基因芯片检测,差异倍数2倍、平均信号值>7的差异基因共2878个,其中上调基因986个,下调基因为1892个。表达变化的基因主要参与Wnt信号通路、VEGF信号通路和mTOR信号通路等途径,功能主要涉及细胞增殖、细胞迁移和细胞骨架调节等过程。其中与上述细胞表型改变密切相关的PIK3R3、WNT2B、VANGL2和ANXA1基因的表达改变经RT-PCR验证,结果与基因芯片趋势一致。结论长期反复染毒后MWCNT对MeT-5A细胞可能存在潜在恶性转化能力。Objective To explore the chronic toxicity and its potential mechanism of multi-walled carbon nanotube(MWCNT)in human pleural mesothelial cells.Methods A sustainable exposure of MeT-5A cells to MWCNT at 10μg/cm2 for one year was conducted in 2016.During the exposure,the cell images and cell proliferation was recorded every 4 weeks.The cell apoptosis,cell cycle,cell migration and cell invasion were compared between the control cells and the cells after MWCNT exposure.Finally,the gene expression was screened with Affymetrix clariom D assay,and some of the significantly differential expressed genes was verified by RT-PCR.Results Compared with the control group,the proliferation ability of the cells in the 1-year exposed group was significantly increased,and the rate of proliferation was about 2-3 times as that in the Control Group(F=481.32,P<0.05).MeT-5A cells all showed cell cycle arrest effect,which showed the increase of G1 phase and the decrease of s phase and G2 phase(F=14.94,P<0.05).The apoptosis rate of cells in the treated group was significantly higher than that in the control group after 6 months(F=15.12,P<0.05),but the early apoptosis rate and the total apoptosis rate of cells in the treated group were not significantly different from those in the control group after 1 year(F=3.97,P<0.05).The cell migration and invasion were both promoted by MWCNT.Furthermore,the differentially expressed genes was screened,to find 2,878 genes with more than 2 folds changes.To further verified,RT-PCR was conducted with PIK3R3、WNT2B、VANGL2、ANXA1,and their expression changes were consistent with above.Conclusion MWCNT might have a carcinogenic potential to MeT-5A cells after the long term exposure.

关 键 词：细胞迁移分析 基因 多壁碳纳米管 长期染毒 人胸膜间皮细胞 细胞应答 

分 类 号：R114[医药卫生—卫生毒理学]