摘 要: 目的研究不同剂量UVB辐射对人角质形成细胞HaCaT DNA损伤的影响及机制。方法采用紫外辐照装置照射人角质形成细胞Ha Ca T建立急性紫外辐射细胞模型;MTT法检测不同剂量紫外辐射对细胞存活的影响;免疫荧光和流式细胞术分别检测细胞核内磷酸化γH2AX焦点形成及蛋白表达水平;Annexin V-FITC细胞凋亡检测试剂盒检测不同剂量不同时间照射后早期细胞凋亡百分数;免疫印迹法分别检测Caspase-3活化水平。结果 UVB辐射可抑制HaCaT细胞的存活,有一定的量效关系;相对较大剂量的UVB辐射可观察到明显的焦点形成;UVB照射后各剂量点磷酸化γH2AX的表达均明显高于未照射的对照组;UVB辐射可通过Caspase-3活化诱导细胞发生早期凋亡,照射后20 h,早期凋亡细胞百分数明显增加,特别是在UVB相对较大剂量范围内更明显,并有随照射剂量增高而增加的趋势。结论 UVB辐射可通过诱导DNA双链断裂抑制人HaCaT细胞存活,并通过激活Caspase-3而诱导细胞凋亡,细胞凋亡延迟,可能导致细胞更易于发生突变进而形成皮肤肿瘤。 |
关键词: UVB辐射 DNA损伤 γH2AX 人角质形成细胞HaCaT 皮肤癌 |
中图分类号: B818.03
文献标识码:
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基金项目: |
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Damage effect of UVB radiation on DNA of human keratinocytes HaCaT and its mechanism |
LI Chen,QI Xue song,LI Ning,GOU Qiao,LIU Jian xiang,TIAN Mei
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National Institute for Radiological Protection,Key Laboratory of Radiological Protection and Nuclear
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Abstract: To investigate the DNA damage effect of UVB irradiation on human keratinocytes HaCaT cells and its mechanism. Methods Acute UVB radiation damage models were established by Ultra Violet CL 1000 instrument,cell viability and proliferation were detected by MTT assay,γH2AX foci and its expression were examined by immunofluorescence staining and western blotting respectively,early cell apoptosis were detected by Annexin V FITC kit,the activation level of Caspase 3 was detected by western blotting technique. Results It was showed that the cell viability and proliferation of HaCaT were decreased significantly after UVB irradiation in a dose dependent manner;higher doses of UVB could induce γH2AX foci formation and the expression of γH2AX after UVB irradiation was significantly increased compared with controls,particularly the higher dose group.Besides,UVB irradiation might result in retarding of cell response,which shown by that the early apoptosis cells amount at the 20th hour after UVB irradiation was dramaticlly increased compared with that of the 6 th hour,as well as the Caspase 3 activation also occurred at 6 th hour and reached the top at 24 th hour after UVB irradiation.Conclusion The results suggested that UVB expose might induce DNA double strands break,suppress cell viability of HaCaT,and retard cell apoptosis,which cause cells more prone to mutation and skin cancer. |
Keywords: ultraviolet B(UVB) DNA damage phosphorylated histone H2AX(γH2AX) human keratinocytes HaCaT cells skin cancer |