白藜芦醇对人髓母细胞瘤细胞Daoy增殖的影响及其作用机制

Effect of resveratrol on proliferation of human medulloblastoma Daoy cells and the relevant mechanism

目的探讨白藜芦醇(resveratrol,Res)对人髓母细胞瘤(medulloblastoma,MB)Daoy细胞增殖的影响及其可能的作用机制。方法体外培养Daoy细胞,用不同浓度的Res(10、20、40、80μmol/L)作用不同时间(24、48和72 h),CCK-8法检测Res对Daoy细胞的增殖抑制率;电镜观察Res对细胞内自噬溶酶体的形成情况;Cathepsin D活性检测试剂盒检测Cathepsin D活性;Western blot法检测Daoy细胞中Beclin 1、LC3、p62和溶酶体相关膜蛋白2(lysosomeassociated membrane proteins 2,LAMP2)的表达及3-甲基腺嘌呤(3-MA)和巴弗洛霉素A1(Bafilomycin A1)对Res作用的影响。结果 Res作用后,Daoy细胞的增殖抑制率随Res浓度的升高及作用时间的延长呈升高趋势;随着Res浓度的升高,Daoy细胞内自噬溶酶体形成数量、Cathepsin D酶活性、LC3Ⅱ/LC3Ⅰ的比值、Beclin 1、LAMP2蛋白的表达均呈上升趋势,p62蛋白表达下降。3-MA作用后,LC3Ⅱ/LC3Ⅰ的比值、Beclin 1、LAMP2的蛋白表达水平明显下降(P <0. 05),p62的表达水平明显上调(P <0. 05);Bafilomycin A1处理后,Beclin 1、p62及LAMP2表达明显降低(P <0. 05),LC3Ⅱ/LC3Ⅰ的比值明显增高(P <0. 05)。结论 Res可抑制髓母细胞瘤细胞Daoy的增殖,其机制可能与Res促进自噬形成与成熟过程有关。

Objective To investigate the effect of resveratrol(Res)on proliferation of human medulloblastoma Daoy cells and the possible mechanism.Methods Daoy cells were cultured in vitro and treated with Res at various concentrations(10,20,40,80μmol/L)for various time durations(24,48 and 72 h),then determined for the inhibitory rate of proliferation by CCK-8 assay,and observed for formation of autophagolysosome by electron microscopy.The activity of Cathepsin D was determined by Cathepsin D activity detection kit,while the expressions of Beclin 1,LC3,p62 and lysosome associated membrane protein 3(LAMP2)as well as the influence of 3-methyladenine(MA)and Bafilomycin A1 on the effect of Res were evaluated by Western blot.Results After treatment with Res,the inhibitory rate of Daoy cell proliferation showed an increasing tendency with the increasing Res concentration and time.However,with the increasing Res concentration,the number of autophagolysosome formed in Daoy cells,Cathepsin D activity,the ratio of LC3Ⅱ/LC3Ⅰas well as expression levels of Beclin 1 and LAMP2 showed increasing tendencies,while the expression level of p62 decreased.However,after treatment with 3-MA,the LC3Ⅱ/LC3Ⅰratio as well as expression levels of Beclin 1 and LAMP2 decreased significantly(P<0.05),while the expression level of p62 increased significantly(P<0.05).After treatment with Bafilomycin,the expression levels of p62,LAMP2 and Beclin1 decreased significantly(P<0.05),while the LC3Ⅱ/LC3Ⅰratio increased significantly(P<0.05).Conclusion Res inhibited the proliferation of human medulloblastoma Daoy cells,of which the mechanism might be associated with the formation and maturation of autophagolysosome.