中华皮肤科杂志 ›› 2005, Vol. 38 ›› Issue (7): 435-438.

• 论著 • 上一篇    下一篇

中波紫外线辐射剂量与HaCaT细胞凋亡时相的相关性研究

王平1, 毕志刚1, 寿伟璋2, 尹琰2   

  1. 1. 南京医科大学第一附属医院皮肤科 210029;
    2. 东南大学医学院分析细胞室 210029
  • 收稿日期:2004-07-30 发布日期:2005-07-15
  • 基金资助:
    江苏省重点学科基金资助项目(135-03)

Correlation Between Dose of Ultraviolet B Irradiation and Apoptotic Phase of HaCaT Cells

WANG Ping1, BI Zhi-gang1, SHOU Wei-zhang2, YIN Yan2   

  1. Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
  • Received:2004-07-30 Published:2005-07-15

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摘要： 目的 探讨中波紫外线(UVB)辐射角质形成细胞后,UVB辐射剂量与角质形成细胞凋亡时相的相关性。方法 以20,40,60,80,100和120mJ/cm2的UVB辐射永生化人角质形成细胞株HaCaT细胞,检测辐射后2,12,24,48和72h的细胞活性和细胞周期的变化,以及代表不同时相的凋亡通路:即应用广谱Caspase抑制剂VAD-FMK与凋亡细胞中活化的Caspase不可逆的结合检测早期Caspase介导的凋亡;应用AnnexinV及PI双染方法检测后发的细胞膜介导的凋亡;以及应用DNA的梯度凝胶电泳检测晚期细胞核介导的凋亡。结果 UVB辐射可使HaCaT细胞的细胞周期受滞于G2/M期,G2/M期的百分比与辐射剂量成正比。细胞活性于24h后与辐射剂量成反比。各时相的凋亡率均与辐射剂量成正比,但凋亡通路有明显的时相性:即早期(48h内)由Caspase和细胞膜介导凋亡为主,晚期(48h后)则由细胞核介导凋亡为主。结论 UVB诱导的角质形成细胞凋亡具有显著的剂量与时相依赖性特征。宜对其进行多指标、多时相的检测。

关键词: 紫外线, 角蛋白细胞, 细胞,培养的, 细胞凋亡

Abstract: Objective To elucidate the correlation between the dose of ultraviolet B (UVB) irradiation and apoptotic phase of keratinocytes (HaCaT cells).Methods Cultured immortalized human keratinocyte cell line,HaCaT cells,was irradiated with ultraviolet B (UVB) in doses of 20,40,60,80,100 and 120 mJ/cm2.Cellular viability,cell cycles and apoptosis were simultaneously detected at 2,12,24,48 and 72 h after irradiation.The caspase-mediated apoptosis was detected by FITC-labelled VAD-FMK,which could irreversibly bind to activated caspases.FITC-Annexin V and PI double staining detected cell membrane-mediated apoptosis.Cell nucleus-mediated apoptosis was detected by DNA ladder electrophoresis.Results Cell cycle analysis revealed that UVB treated HaCaT cells were blocked predominantly in G2/M phase in a dose-dependent manner.Treatment with UVB decreased the viability of HaCaT cells in dose-and timedependent manners.The rates of apoptosis were increased with increasing dose and prolongation of time after UVB irradiation.The apoptosis of UVB-treated HaCaT presented in an obvious time-dependent manner,in which caspase-and cell membrane-mediated apoptosis was predominantly in early phase and cell nucleus-mediated apoptosis predominantly in late phase.Conclusions The apoptosis of UVB treated HaCaT cells appears in dose-and timedependent manners,which warrants further detection with multiple parameters and at different phases.

Key words: Utraviolet rays, Keratinocytes, Cells,cultured, Apoptosis