摘要
目的观察竹节香附素A(RA)对人结肠癌HT29细胞凋亡及周期的影响,并探讨其相关机制。方法 1、2、4、8、16μmol/L的RA分别作用细胞12、24、48 h,四甲基偶氮唑蓝(MTT)实验检测RA对HT29细胞的增殖抑制作用,计算半数抑制浓度IC50;荧光显微镜下观察Hoechst33258染色,RA对细胞凋亡形态学的影响;流式细胞术(FCM)检测RA对HT29细胞凋亡及细胞周期的影响;蛋白质印迹法(Westernblot,WB)检测Cleaved-caspase-3、Cleaved-PARP、Cyclin B、CDK1等蛋白的表达情况。结果竹节香附素A能够抑制HT29细胞的增殖,并与浓度、时间呈正相关(P<0.05),12、24、48 h IC50分别为为(9.31±0.63)μmol/L,(4.88±1.02)μmol/L,(3.60±0.89)μmol/L。Hochest33258染色发现细胞出现核固缩、核碎裂;流式细胞术显示:随着给药浓度的增大凋亡率逐渐增加(P<0.05);WB结果显示Cleaved-caspase-3、Cleaved-PARP蛋白表达增加。竹节香附素A作用于HT29细胞后,与对照组相比,G2/M期细胞比例逐渐增高;且Cyclin B、CDK1蛋白表达随着给药浓度的增大而减少。信号通路PI3K/AKT中PI3K、AKT蛋白表达上调,p-PI3K、p-AKT表达下调。结论 RA能够抑制肠癌HT29细胞的增殖,其机制可能是通过调控PI3K/AKT信号通路诱导细胞凋亡和阻滞细胞周期实现的。
Objective To investigate the effects of Raddeanin A （RA） on apoptosis and cell cycle of hu- man colon cancer HT29 cells, and discuss the possible mechanism. Methods Different concentration of RA 1,2 4,8,16 μmol/L disposed HT29 cells 12,24,48 h, 3- （4,5-Dimethylthiazol-2-yl） -2,5-diphenyltetrazolium bromide （MTT） assay was used for the cell proliferation ; Hoechst33258 staining was performed to observe the effects of RA on apoptosis morphology ; Flow cytometry （ FCM ） was devoted to calculate apoptosis rate and detect cell cycle ; The expression of Cleaved-caspase-3, Cleaved-PARP, CyclinB, CDK1 proteins and other proteins were tested by West- ern blot （WB）. Results RA could inhibit the proliferation of HT29 cells, and associated with concentration and time（P 〈 0.05） ;The IC50 of 12,24,48 h were（ 9.31 ±0.63） μmol/L, （4.88 ± 1.02） tool/L, （3.60± 0.89） μmol/L. The nucleus pycnosis, fracture were detected by Hochest33258 staining, RA induced apoptosis in HT29 cells;West- ern blot （WB）assay showed that the expression of Cleaved-caspase-3, Cleaved-PARP rised.HT29 cells were arrest- ed at the period of G2/M, and the CyclinB, CDKI proteins down-regulated.WB result showed that PI3K, AKT pro- teins up-regulated, while p-PI3K, p-AKT protein down-regulated. Conclusion RA can inhibit the proliferation of colon cancer HT29 cells and its mechanism is probably by regulating the PI3K/AKT signaling pathways which through inducing apoptosis and blocking the cell cycle.
出处
《实用医学杂志》
CAS
北大核心
2017年第23期3858-3863,共6页
The Journal of Practical Medicine
基金
国家中医临床研究基地业务建设科研专项课题(编号:JDZX2012087)
江苏省中医药领军人才项目(编号:LJ200908)
江苏省卫生计生委课题(编号:BJ14013)
江苏省普通高校研究生科研创新计划项目(编号:KYLX16-1145)
