CAJ | 학술논문

目的探讨塞来昔布(celecoxib)增强肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)诱导甲状腺髓样癌TT细胞凋亡的效应及相关机制.方法 MTT法检测TRAIL、celecoxib及2者联合应用对TT细胞的增殖作用;PI单染流式细胞仪检测联合用药对TT细胞周期的影响;Hochest33258染色和DNA ladder检测联合用药对TT细胞凋亡的作用;Western blot检测cyclin A、Cdk2、caspase-8、c-FLIP、RIP蛋白表达改变.结果 ①MTT实验显示:TRAIL1000 ng/ml与celecoxib 50 μM联合用药的增殖抑制率为(47.53％±1.34％),明显高于单用TRAIL(7.75％±3.84％)及celecoxib (24.49％±1.57％)之和组(t检验,F=5.234,P＜0.01),差异有统计学意义;②PI检测显示:celecoxib组和联合用药组G0/G1期细胞明显高于Control组和TRAIL组(F=242.694,P＜0.01);③Westem blot检测:celecoxib组及联合用药组Cyclin A和Cdk2蛋白表达明显下调,且2者之间未见明显差异;④Hochest33258染色观察细胞核的凋亡形态学改变发现:联合用药组核固缩及核碎裂现象明显增多,其细胞凋亡率为(24.23％±2.91％),明显高于单用TRAIL(5.86％±1.41％)及celecoxib 11 (20％±1.24％)之和(t检验,F=1.824,P＜0.01),差异具有统计学意义;⑤Western blot检测发现:联合用药组caspase-8活化裂解程度明显增加,c-FHP及RIP蛋白表达亦明显下调.结论 celecoxib能增强TRAIL对甲状腺髓样癌TT细胞增殖的抑制作用,其机制是通过celecoxib将TT细胞阻滞于G0/G1期,并下调c-FHP及RIP的表达,促进TRAIL对caspase-8的活化,诱导细胞凋亡.
목적 탐토새래석포(celecoxib)증강종류배사인자상관조망유도배체(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)유도갑상선수양암TT세포조망적효응급상관궤제.방법 MTT법검측TRAIL、celecoxib급2자연합응용대TT세포적증식작용;PI단염류식세포의검측연합용약대TT세포주기적영향;Hochest33258염색화DNA ladder검측연합용약대TT세포조망적작용;Western blot검측cyclin A、Cdk2、caspase-8、c-FLIP、RIP단백표체개변.결과 ①MTT실험현시:TRAIL1000 ng/ml여celecoxib 50 μM연합용약적증식억제솔위(47.53％±1.34％),명현고우단용TRAIL(7.75％±3.84％)급celecoxib (24.49％±1.57％)지화조(t검험,F=5.234,P＜0.01),차이유통계학의의;②PI검측현시:celecoxib조화연합용약조G0/G1기세포명현고우Control조화TRAIL조(F=242.694,P＜0.01);③Westem blot검측:celecoxib조급연합용약조Cyclin A화Cdk2단백표체명현하조,차2자지간미견명현차이;④Hochest33258염색관찰세포핵적조망형태학개변발현:연합용약조핵고축급핵쇄렬현상명현증다,기세포조망솔위(24.23％±2.91％),명현고우단용TRAIL(5.86％±1.41％)급celecoxib 11 (20％±1.24％)지화(t검험,F=1.824,P＜0.01),차이구유통계학의의;⑤Western blot검측발현:연합용약조caspase-8활화렬해정도명현증가,c-FHP급RIP단백표체역명현하조.결론 celecoxib능증강TRAIL대갑상선수양암TT세포증식적억제작용,기궤제시통과celecoxib장TT세포조체우G0/G1기,병하조c-FHP급RIP적표체,촉진TRAIL대caspase-8적활화,유도세포조망.
Objective To study the effect and related mechanism of celecoxib on tumor necrosis factorrelated apoptosis-inducing ligand(TRAIL) induced apoptosis of medullary thyroid cancer TT cell line.Methods MTT assay was used to measure the growth inhibition induced by TRAIL and celecoxib alone and their combination.TT cell cycle distribution was analyzed by flowcytometry.Hochest33258 staining and DNA ladder was used to detect the apoptosis of drug combination on TT cells.Western blot was used detect the protein change of cyclin A,Cdk2,caspase-8,c-FLIP,and RIP.Results ①MTT showed the growth inhibition ratio of TT cell intervened by the combination of TRAIL and celecoxib was 47.53％ ± 1.34％,which was much higher than that intervened by TRAIL(7.75 ％ ± 3.84％)and celecoxib alone.The differences had statistical significance (t test,F =5.234,P ＜0.01);②PI detection found the cells' number in G0/G1 phase in celecoxib group and combination group were increased compared to that in control group and TRAIL group(F =242.694,P ＜ 0.01);③Western blot indicated the expression of Cyclin A and Cdk2 were down regulated,there was no statistic significance;④ The apoptosis morph in nuclus was detected by Hochest33258 staining and showed the karyopycnosis and muclear fragmentation were increased in combination group with the apoptosis rate 24.23％ ± 2.91％,which was much higher than that in TRAIL(5.86％ ± 1.41％) and celecoxib(20％ ± 1.24％) (t test,F =1.824,P ＜0.01),the difference has statistic significance;⑤Western blot illustrated the active schizolysis of casplase-8 was higher and the expression of c-FLIP and RIP was down regulated in combination group.Conclusion celecoxib plays a positive effect on TRAIL-reduced apoptosis of medullary thyroid cancer TT cell line,which may due to the cell cycle arrest at G0/G1 phase,down-regulation of c-FLIP and RIP and subsequent activation of caspase-8.