塞来昔布增强肿瘤坏死因子相关凋亡诱导配体对甲状腺髓样癌TT细胞凋亡作用的研究

The synergistic effect of celecoxib on TRAIL-reduced apoptosis of medullary thyroid cancer TT cell linenecrosis factor-related apoptosis-inducing ligand; Cell cycle arrest ; Apoptosis; tiff cell line

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 factorrelated 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MTr法检测TRAIL、celecoxib及2者联合应用对TI’细胞的增殖作用；PI单染流式细胞仪检测联合用药对r丌细胞周期的影响；Hoehest33258染色和DNAladder检测联合用药对，ITI’细胞凋亡的作用；Westernblot检测cyclinA、Cdk2、caspase-8、c-FLIP、RIP蛋白表达改变。结果①MTT实验显示：TRAIL1000ng／ml与celecoxib50μM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）；③West—ernblot检测：celecoxib组及联合用药组CyclinA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celecoxib11（20％±1．24％）之和（t检验，F=1．824，P〈0．01），差异具有统计学意义；⑤Westernblot检测发现：联合用药组caspase-8活化裂解程度明显增加，C—FLIP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GO／G1期，并下调c—FLIP及RIP的表达，促进TRAIL对caspase-8的活化，诱导细胞凋亡。

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. tit cell cycle distribution was analyzed by flowcytometry. Hochesi33258 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 inter- vened by the combination of TRAIL and celecoxib was 47.53% ：i： 1.34% , which was much higher than that inter- vened 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 TF cell line, which may due to the cell cycle arrest at G0/G1 ohase, down-regulation of c-FLIP and RIP and subsequent activation of casoase-8.