姜黄素诱导人髓母细胞瘤Daoy细胞自噬的实验研究

Experiment research of curcumin induced autophagy in human medulloblastoma Daoy cell

目的:观察姜黄素(Curcumin)诱导人髓母细胞瘤Daoy细胞自噬的作用,并探讨其可能的作用机制。方法:体外培养人髓母细胞瘤Daoy细胞,不同浓度姜黄素作用24、48、72 h,MTT法检测细胞增殖;荧光染色、RT-PCR和Western blot检测自噬相关蛋白Beclin1和LC3的表达;RT-PCR和Western blot检测PI3K/Akt/mTOR信号通路中PI3K、p-Akt和p-mTOR的表达。PI3K激动剂insulin处理细胞后,Western blot检测PI3K/Akt/mTOR信号通路相关蛋白的表达变化。结果:姜黄素明显抑制Daoy细胞的生长,呈时间-剂量依赖性。免疫荧光显示Beclin1和LC3在姜黄素处理组中的表达明显增强。姜黄素作用不同时间,Daoy细胞中Beclin1和LC3-Ⅱ/LC3-Ⅰ的mRNA和蛋白水平均增高(mRNA:F=1 360.962、42.366,P=0.000、0.000;蛋白:F=32.723、11.847,P=0.001、0.008),而PI3K、p-Akt和p-mTOR的表达受到明显抑制(F=329.662、80.638、183.536,P=0.000、0.000、0.000)。PI3K激动剂insulin不能有效阻断姜黄素引起的PI3K、p-Akt和p-mTOR的降低。结论:姜黄素诱导自噬抑制髓母细胞瘤Daoy细胞生长,其机制可能与上调自噬相关基因表达,抑制自噬负调控PI3k/Akt/mTOR信号通路有关。

Objective ：To investigate the mechanism of curcumin-induced autophagy in human medulloblastoma Daoy cells. Methods： Cell viability was tested by MTT assay;expression of autophagic markers Beclinl and LC3 was assessed by fluorescence confocal microscopy, RT-PCR and Western blot;expression of PI3K,Akt,p-Akt,mTOR and p-mTOR in PI3K/Akt/mTOR signal pathway was checked by RT-PCR and Western blot. Results： Curcumin inhibited the proliferation of Daoy cells in a concentration and time dependent manner. Immunofluorescence showed that the intensity of Beclinl and LC3 increased after curcumin treatment. The mRNA and protein level of Beclinl and LC3 was increased significantly in curcumin treated group than in control group（mRNA：F=1360.962,42.366, P=0.000,0.000; protein ： F=32.723,11.847, P=-0.001,0.008）. While the expression of PI3K, p-Akt and p-roTOR in curcumin treated group were lower than that in control group（F=329.662,80.638,183.536, P=-0.000,0.000,0.000）. PI3K/Akt/mTOR pathway agonist insulin could not reverse the reduction of PI3K, p-Akt and p-roTOR induced by curcumin. Conclusion ： Curcumin could inhibit the growth of human medulloblastoma Daoy cells via autophagy.