长期砷暴露诱导人膀胱上皮细胞恶性转化的研究
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摘要
目的构建低浓度长期亚砷酸钠(NaAsO_2)诱导人膀胱上皮细胞(SV-HUC-1)恶性转化模型,研究低浓度长期砷暴露对SV-HUC-1恶性转化过程的影响。方法 0和0. 5μmol/L NaAsO_2分别长期处理SV-HUC-1至40周(约80代,10个月),观察暴露组及对照组细胞在培养至10、20、30和40周时形态,四氮唑盐比色(MTT)验证细胞增殖速率;划痕实验、Transwell细胞迁移实验验证细胞迁移能力;软琼脂集落形成实验观察细胞肿瘤形成能力。结果随NaAsO_2处理时间的延长,细胞由连接紧密的扁平多变形状向松散的长梭型转变; 0. 5μmol/L NaAsO_2处理至20周后,细胞活力显著增强,与处理10周时比较差异有统计学意义(P<0. 01);划痕愈合实验显示,砷暴露20周后,划痕愈合面积显著大于10周(P<0. 01); Transwell细胞迁移侵袭能力30周时显著高于10周(P<0. 01);软琼脂集落形成实验显示,砷暴露30周后软琼脂上出现大量明显的锚定生长集落,20、30、40周组集落形成数量分别为10周组的1. 85、9. 79和15. 36倍(P<0. 01),未经砷暴露组各代数有极少集落,且体积小,故忽略不计。结论 0. 5μmol/LNaAsO_2长期处理的SV-HUC-1可逐渐恶转,在砷处理20～30周时已初具恶性表型,40周后细胞恶性转化。
Objective To construct amalignant transformation model of human bladder epithelial cells( SV-HUC-1)induced by low-concentration long-term sodium arsenite( NaAsO_2),studying the effect of arsenic treatment on the malignant transformation process of SV-HUC-1 cells. Methods SV-HUC-1 cells were treated with 0( control) and 0. 5 μmol/L NaAsO_2( exposed),respectively for 40 weeks( about 80 passages,10 months). In the 10 th,20 th,30 th and 40 th weeks after exposure,the cells both treated and control groups were taken for study,such as cell morphology,cell proliferation rate( with tetrazolium salt colorimetric-MTT),scratch test,cell migration ability( with Transwell migration assay),and cell tumor formation ability( with soft agar experiment),etc.. Results It was showed that with the prolongation of NaAsO_2 treatment,the cells changed from a tightly connected,flat and polymorphic to a loose long,spindle type. After 20 weeks treatment with 0. 5 μmol/L NaAsO_2,the viability of cells was significantly enhanced,the difference was statis tical significance( P<0. 01) compared with that of 10 weeks treated group,after 20 weeks of arsenic treatment,the scratch healing area was also significantly larger than that of 10 weeks treated cells( P< 0. 01); after 30 weeks of arsenic treatment,the migration and invasion ability of transwell cells was significantly higher than that of 10 weeks treated cells( P<0. 01) as well. Additionally,soft agar colony formation experiments showed that a large number of obvious anchored colonies appeared on soft agar after 30 weeks of arsenic treatment,the colony formation numbers in 20,30 and 40 weeks groups were 1. 85 times,9. 79 times and 15. 36 times of the 10 weeks group,respectively( P<0. 01),nearly no colony was found in control group. Conclusion The results suggested that under long-term and low dose arsenic treatment( 0. 5 μmol/L NaAsO_2) might make SV-HUC-1 cells gradually metastasize,after 20 to 30 weeks of arsenic treatment,the cell already had initial malignant phenotype and after 40 weeks treatment with arsenic the malignant transformation completed.
Objective To construct amalignant transformation model of human bladder epithelial cells( SV-HUC-1)induced by low-concentration long-term sodium arsenite( NaAsO_2),studying the effect of arsenic treatment on the malignant transformation process of SV-HUC-1 cells. Methods SV-HUC-1 cells were treated with 0( control) and 0. 5 μmol/L NaAsO_2( exposed),respectively for 40 weeks( about 80 passages,10 months). In the 10 th,20 th,30 th and 40 th weeks after exposure,the cells both treated and control groups were taken for study,such as cell morphology,cell proliferation rate( with tetrazolium salt colorimetric-MTT),scratch test,cell migration ability( with Transwell migration assay),and cell tumor formation ability( with soft agar experiment),etc.. Results It was showed that with the prolongation of NaAsO_2 treatment,the cells changed from a tightly connected,flat and polymorphic to a loose long,spindle type. After 20 weeks treatment with 0. 5 μmol/L NaAsO_2,the viability of cells was significantly enhanced,the difference was statis tical significance( P<0. 01) compared with that of 10 weeks treated group,after 20 weeks of arsenic treatment,the scratch healing area was also significantly larger than that of 10 weeks treated cells( P< 0. 01); after 30 weeks of arsenic treatment,the migration and invasion ability of transwell cells was significantly higher than that of 10 weeks treated cells( P<0. 01) as well. Additionally,soft agar colony formation experiments showed that a large number of obvious anchored colonies appeared on soft agar after 30 weeks of arsenic treatment,the colony formation numbers in 20,30 and 40 weeks groups were 1. 85 times,9. 79 times and 15. 36 times of the 10 weeks group,respectively( P<0. 01),nearly no colony was found in control group. Conclusion The results suggested that under long-term and low dose arsenic treatment( 0. 5 μmol/L NaAsO_2) might make SV-HUC-1 cells gradually metastasize,after 20 to 30 weeks of arsenic treatment,the cell already had initial malignant phenotype and after 40 weeks treatment with arsenic the malignant transformation completed.
引文
[1]刘世宜,李昕.低剂量长期砷暴露对Ha Cat细胞增殖及凋亡水平的影响[J].中国地方病防治杂志,2013,23(1):1-3.
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[4]谷仕艳,陈承志,蒋学君,等. Nrf2信号通路在亚砷酸钠诱导支气管上皮细胞恶性转化中的作用[J].卫生研究,2016,45(3):356-361.
[5]邓晗依,王小娟,朱震,等.长期低剂量亚砷酸钠染毒对人支气管上皮细胞和人角质形成细胞增殖的影响[J].卫生研究,2017,46(1):126-131.
[2] Imura J,Hayashi S,Ichikawa K,et al. Malignant transformation ofhyperplastic gastric polyps:An immunohistochemical and pathologicalstudy of the changes of neoplastic phenotype[J]. Oncol Lett,2014,7(5):1459-1463.
[3]王晨,王菲,刘盛男,等.长时间低浓度暴露亚砷酸钠促进人正常膀胱上皮细胞增殖[J].中国工业医学杂志,2015,28(3):195-196.
[4]谷仕艳,陈承志,蒋学君,等. Nrf2信号通路在亚砷酸钠诱导支气管上皮细胞恶性转化中的作用[J].卫生研究,2016,45(3):356-361.
[5]邓晗依,王小娟,朱震,等.长期低剂量亚砷酸钠染毒对人支气管上皮细胞和人角质形成细胞增殖的影响[J].卫生研究,2017,46(1):126-131.