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预防医学  2022, Vol. 34 Issue (6): 547-554    DOI: 10.19485/j.cnki.issn2096-5087.2022.06.002
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温石棉暴露诱发核糖体DNA拷贝数变异及DNA损伤反应研究
刘佳琪, 冯玲芳, 陈俊斐, 夏海玲, 蒋兆强, 吴帆, 龚晓雪, 楼建林
杭州医学院公共卫生学院,浙江 杭州 310013
Effect of exposure to chrysotile on ribosomal DNA copy number variation and DNA damage response
LIU Jiaqi, FENG Lingfang, CHEN Junfei, XIA Hailing, JIANG Zhaoqiang, WU Fan, GONG Xiaoxue, LOU Jianlin
School of Public Health, Hangzhou Medical College, Hangzhou, Zhejiang 310013, China
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摘要 目的 探索温石棉暴露对核糖体DNA(rDNA)拷贝数的影响及其与DNA损伤反应的关系,为石棉致癌机制研究提供依据。方法 分别用1.25、2.5、5 μg/cm2(低、中、高浓度)的温石棉悬液处理MeT-5A人胸膜间皮细胞,磷酸盐缓冲液处理作为对照。分别收集处理6、24、48和72 h的细胞,采用实时荧光定量PCR检测rDNA拷贝数及核仁蛋白基因BIRC5HRASGINS4RRM2 mRNA表达;使用Muse细胞分析仪及相应试剂盒检测细胞凋亡和DNA损伤。比较不同处理时间不同温石棉浓度组细胞rDNA拷贝数、DNA损伤反应和核仁蛋白基因mRNA表达水平。结果 温石棉悬液处理6、48、72 h时,低、中、高浓度组和对照组45S rDNA拷贝数差异均有统计学意义(P<0.05);处理6 h时,各浓度组45S rDNA拷贝数均低于对照组(P<0.05);处理48、72 h时,高浓度组45S rDNA拷贝数高于低、中浓度组(P<0.05)。处理24、48、72 h时,各组5S rDNA拷贝数比较,差异均有统计学意义(P<0.05);处理24、48 h时,中、高浓度组5S rDNA拷贝数均低于对照组(P<0.05);处理24、72 h时,中、高浓度组5S rDNA拷贝数均低于低浓度组(P<0.05)。不同处理时间点各组总细胞凋亡率比较,差异均有统计学意义(P<0.05);中、高浓度组总细胞凋亡率均高于对照组(P<0.05),以晚期凋亡为主。处理72 h时,各组ATM激活率和DNA双链断裂率比较,差异均有统计学意义(P<0.05);中、高浓度组细胞ATM激活率和DNA双链断裂率均高于对照组(P<0.05)。处理24、48 h时,各组BIRC5HRASGINS4RRM2 mRNA相对表达量比较,差异均有统计学意义(P<0.05);中、高浓度组BIRC5HRASGINS4RRM2 mRNA相对表达量均低于对照组(P<0.05)。结论 温石棉暴露可诱发人胸膜间皮细胞rDNA拷贝数变异,引起相关核仁蛋白表达改变,可能参与DNA损伤反应过程的调控。
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刘佳琪
冯玲芳
陈俊斐
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蒋兆强
吴帆
龚晓雪
楼建林
关键词 温石棉核糖体DNA拷贝数变异DNA损伤反应核仁蛋白    
AbstractObjective To investigate the effect of chrysotile exposure on ribosomal DNA (rDNA) copy number and DNA damage response, so as to provide insights into the mechanism of asbestos-induced carcinogenesis. Methods Human pleural mesothelial MeT-5A cells were treated with chrysotile suspensions at doses of 1.25, 2.5 and 5 μg/cm2 (low-, medium-, high-dose group), while PBS served as controls. MeT-5A cells were harvested 6, 24, 48 and 72 h post-treatment, and the rDNA copy numbers and the BIRC5, HRAS, GINS4 and RRM2 mRNA expression were determined using a quantitative real-time PCR (qPCR) assay. The apoptosis of MeT-5A cells and DNA damage were detected using Muse cell analyzer. The rDNA copy numbers, DNA damage responses and BIRC5, HRAS, GINS4 and RRM2 mRNA expression were compared in MeT-5A cells treated with different doses of chrysotile suspensions. Results There were significant differences in 45S rDNA copy numbers among low-, medium-, high-dose groups and the control groups 6, 48 and 72 h post-treatment with chrysotile suspensions, and significantly lower 45S rDNA copy numbers were measured in low-, medium- and high-dose groups than in the control group 6 h post-treatment, while significantly higher 45S rDNA copy numbers were found in the high-dose group than in low- and medium-dose groups 48 and 72 h post-treatment (all P<0.05). There were significant differences in 5S rDNA copy numbers among low-, medium-, high-dose groups and the control groups 24, 48 and 72 h post-treatment with chrysotile suspensions, and significantly lower 5S rDNA copy numbers were measured in medium- and high-dose groups than in the control group 24 and 48 h post-treatment, while significantly lower 5S rDNA copy numbers were found in medium- and high-dose groups than in the low-dose group 24, 72 h post-treatment (all P<0.05). There were significant differences in the overall apoptotic rate of MeT-5A cells among groups at different time points, and the overall apoptotic rate of MeT-5A cells were significantly higher in medium- and high-dose groups than in the control group (all P<0.05), with late-stage apoptosis predominantly detected. There were significant differences in the rates of ATM activation and DNA double-strand break in MeT-5A cells among groups 72 h post-treatment, and higher rates of ATM activation and DNA double-strand break were measured in medium- and high-dose groups than in the control group (all P<0.05). In addition, there were significant differences in the relative mRNA expression of BIRC5, HRAS, GINS4 and RRM2 genes among groups 24 and 48 h post-treatment, and significantly lower BIRC5, HRAS, GINS4 and RRM2 mRNA expression was quantified in medium- and high-dose groups than in the control group (all P<0.05). Conclusion Exposure to chrysotile may induce rDNA copy number variations and altered expression of nucleolar proteins in human pleural mesothelial cells, which may be involved in the regulation of DNA damage responses.
Key wordschrysotile    ribosomal DNA    copy number variation    DNA damage response    nucleolar protein
收稿日期: 2022-02-21      修回日期: 2022-03-30      出版日期: 2022-06-10
中图分类号:  R135.99  
基金资助:浙江省自然科学基金项目(LY21H260002); 浙江省医药卫生科技计划项目(2020KY100); 杭州医学院基本科研业务费基础科研项目(KYYB202113)
作者简介: 刘佳琪,硕士研究生在读
通信作者: 楼建林,E-mail:jianlinlou@163.com   
引用本文:   
刘佳琪, 冯玲芳, 陈俊斐, 夏海玲, 蒋兆强, 吴帆, 龚晓雪, 楼建林. 温石棉暴露诱发核糖体DNA拷贝数变异及DNA损伤反应研究[J]. 预防医学, 2022, 34(6): 547-554.
LIU Jiaqi, FENG Lingfang, CHEN Junfei, XIA Hailing, JIANG Zhaoqiang, WU Fan, GONG Xiaoxue, LOU Jianlin. Effect of exposure to chrysotile on ribosomal DNA copy number variation and DNA damage response. Preventive Medicine, 2022, 34(6): 547-554.
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http://www.zjyfyxzz.com/CN/10.19485/j.cnki.issn2096-5087.2022.06.002      或      http://www.zjyfyxzz.com/CN/Y2022/V34/I6/547
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