中波紫外线辐射剂量与HaCaT细胞凋亡时相的相关性研究

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。

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

Objective To elucidate the correlation between the dose of ultraviolet B (UVB) irradiation and apoptotic phase of keratinocytes (HaCaT cells). Methods Cultured immortalized human ker-atinocyte 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 time-dependent 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 time-dependent manners, which warrants further detection with multiple parameters and at different phases.