咖啡因促受照射肝癌细胞株MHCC97H凋亡的实验研究

Caffeine enhanced radiation-induced apoptosis in heptocellular carcinoma cell line

目的观察咖啡因促受照射MHCC97H细胞株凋亡的作用及机制。方法通过流式细胞仪分析不同条件下细胞凋亡比例和周期分布,应用Western Blotting动态观察磷酸化CDC2 Tyr15的表达量,利用细胞克隆集落试验证明咖啡因的放疗增敏效果。结果MHCC97H细胞照射后48小时,0、8、16、24 Gy组的凋亡率分别为5.7%、12.0%、19.4%、21.7%;咖啡因促进凋亡,2.5 mmol/L咖啡因组的凋亡比例在照射16 Gy 48小时左右由对照组的19.4%升至32.3%,并且随着时间的推移凋亡比例逐渐增加。辐射引起细胞G2期阻滞,0、8、16、24 Gy组的G2期比例分别为13.9%、25.6%、41.4%、51.6%,G2期阻滞的程度与剂量呈正相关;而咖啡因可去除由辐射引起的G2期阻滞,2.5 mmol/L咖啡因组的G2期细胞比例在照射12小时左右由对照组的37.6%降为29.6%。MHCC97H细胞照射后,G2期阻滞程度与磷酸化CDC2 Tyr15的表达量一致;咖啡因则抑制磷酸化CDC2 Tyr15的表达量。细胞克隆集落试验显示,咖啡因可降低放射后MHCC97H细胞的存活分数,放疗增敏比(SER)为1.28。结论咖啡因通过增加CDC2 Tyr15的脱磷酸化,去除由辐射引起的G2期阻滞,从而促进凋亡,达到放疗增敏的效果。

Objective To study caffeine enhanced radiation-induced apoptosis and it＇s mechanism in heptocellular carcinoma cell line MHCC97H. Methods Apoptosis and the cell cycle ratio were observed by the flow cytometry. Western Blotting was used to detect the expression of the phosphorylated CDC2 Tyr15. The cell clonogenic assay was used to confirm the radiosensitization of caffeine. Results The apoptosis rates were 5. 7%, 12. 0%, 19. 4%, 21. 7% at 0, 8, 16, 24 Gy, respectively at the 48th hour after completion of radiation. Caffeine could enhance radiation-induced apoptosis, the rate increased from 19.4% in 0 Gy to 32.3% at the concentration of 2.5 mmol/L caffeine at the 48th hour after completion of radiation. Irradiation could induce G2 arrest in a dose-dependent manner,the proportion of G2 phase increased from 13.9% in 0 Gy to 25.6% in 8 Gy,41.4% in 16 Gy,and 51.6% in 24 Gy. Caffeine could abolish G2 delay. The proportion of G2 phase decreased from 37. 6% in 0 Gy to 29. 6% at the concentration of 2. 5 mmol/L after incubation 12 hours and delivered 16 Gy. Radiation induced phosphorylation of CDC2 Tyr15. However, caffeine could inhibit the expression of phosphorylated CDC2 Tyr15. The survival fraction became smaller in the caffeine group, and the SER （sensitization enhancement ratio） was 1.28 in the cell clonogenic assay. Conclusion Radiosensitization from caffeine in MHCC97H line is associated with abolish of the irradiation-induced G2 delay through the suppression of the phosphorylation of CDC2 Tyr15.