雷公藤内酯醇诱导皮肤鳞状细胞癌细胞系A431凋亡机制的探讨

目的 探讨雷公藤内酯醇诱导体外培养皮肤鳞状细胞癌细胞系A431凋亡的作用机制.方法 将雷公藤内酯醇按不同浓度、不同作用时间分别培养A431,Annexin V-FITC/PI染色经流式细胞仪检测细胞凋亡,荧光显微镜下观察FITC标记的早期凋亡细胞及PI阳性的中晚期凋亡细胞.通过分光光度计检测凋亡相关基因Caspase-3、Caspase-8、Caspase-9的活性.结果 A431细胞经1、10、100 mg/L雷公藤内酯醇作用后.在12 h 出现早期凋亡,其凋亡率与雷公藤内酯醇的浓度及作用时间有依赖关系;荧光显微镜下12 h出现早期凋亡细胞,24 h出现典型的凋亡细胞.雷公藤内酯醇作用A431细胞后,Caspase-3、Caspase-8、Caspase-9活性在12 h开始增高,Caspase-3、Caspase-9的活性在24 h达到最高,Caspase-3活性三个浓度组的A值分别是0.723,0.850,1.127,Caspase-9活性则为1.072,1.091,1.191,Caspase-8在18 h达到高峰,其A值分别是0.641,0.780,0.910,三项指标的活性均明显高于对照组(P<0.05).结论 雷公藤内酯醇能诱导A431细胞凋亡,其作用存在浓度及时间依赖关系.

Mechanisms of apoptosis of the human squamous cell carcinoma cell line A431 induced by triptolide

Objective To study the mechanisms of apoptosis of the human squamous cell carcinoma cell line A431 induced bv triptolide.Methods Cultured A431 cells were treated with triptolide at difierent concentrations(1,10,100 mg/L)for various durations(12,18,24,48 hours).Various stages of apoptosis of A431 cells were quantified using flow cytometry and dual staining with Annexin V-FITC and propidium iodide (PI).The expressions of Caspase 3,8 and 9 in triptolide-treated or untreated A431 cells were examined by microspectrophotometry.Results As flow cytometry and fluorescence microscopy revealed,early apoptosis of A431 cells was observed 12 hours after treatment with triptolide,and typical apoptosis was noticed at 24 hours.Moreover,the apoptosis rate was positively correlated with the treatment concentration and durations of trilptolide.The activities of Caspase 3,8 and 9 commenced to increase at 12 hours,peaked at 24 hours (Caspase 3 and 9)or 18 hours (Caspase 8)after triptolide treatment,and the top activities expressed as absorbence value were 0.723,0.850,1.127 for Caspase 3,1.072,1.091 and 1.191 for Caspase-9,0.641,0.780 and 0.910 for Caspase 8 after treatment with triptolide of 1,10 and 100 mg/L,respectively,which were significantly higher than those in untreated control cells at corresponding time points (all P<0.05).Conclusion Triptolide could induce the apoptosis of A431 cells,and the induction effect seems to be concentration-and time-dependent.