自噬在肿瘤坏死因子相关凋亡诱导配体诱导甲状腺髓样癌TT细胞凋亡中的作用

目的 观察在肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand,TRAIL）作用下甲状腺髓样癌TT细胞自噬发生情况,明确自噬在TRAIL诱导甲状腺癌TT细胞凋亡中的作用.方法 采用MTT法检测细胞增殖抑制率;MDC染色检测自噬的发生情况;流式细胞仪检测细胞凋亡;Western blot检测相关蛋白的表达.结果 ①MTT实验显示：TRAIL对TT细胞增殖抑制作用弱,在浓度为250、500、1000、2000 ng/ml的TRAIL作用48 h后对TT细胞增殖抑制率分别为（3.02±1.82）％、（4.87±1.45）％、（7.51±1.57）％及（12.76±3.23）％;②TRAIL作用48 h后,胞内代表自噬小体的绿色荧光亮点明显增多,且随TRAIL浓度的增加而增加;③3-MA预处理TT细胞4h,TRAIL 500 ng/ml及1000 ng/ml的凋亡率分别为（17.83 ±1.54）％及（27.81±1.79）％,明显高于单用TRAIL（3.70±0.34、6.55 ±0.59）％与3-MA （7.71±0.64）％之和,差异有统计学意义（t=3.282,P＜0.05;t=7.830,P＜0.01）.④各实验组可见明显的caspase-8的活化裂解片段,自噬相关蛋白Beclin 1的表达明显减低.结论 甲状腺髓样癌TT细胞对TRAIL不敏感,TRAIL能诱导TT细胞内自噬发生,抑制自噬可明显改善TT细胞对TRAIL的敏感性.

The role of autophagy in TRAIL-induced apoptosis of medullary thyroid cancer TT cells

Objective To observe the level of autophagy induced by TRAIL in TT cell line and identify the role of autophagy in TRAIL-inducing apoptosis of TT cell line.Methods The growth inhibition of TT cells was measured by MTT assay.MDC staining was used to identify the happening of autophagy.Annexin V/PI double staining was used to analyze the apoptosis rate of TT cells by flowcytometry.The protein expression of caspase-8 and Beclin1 was detected by Western blot.Results ① The growth inhibition ratio of TT cells induced by TRAIL at the concentration of 250,500,1000 and 2000 ng/ml was （3.02 ± 1.82）％,（4.87 ± 1.45）％,（7.51 ± 1.57） ％,（12.76 ± 3.23） ％ respectively,which suggested significant resistance of TT cells to TRAIL.② MDC-labeled green light vesicles was significantly increased after the treatment of TRAIL for 48 h.③ The apoptosis rate of TT cells induced by TRAIL at the concentration of 500 ng/ml and 1000 ng/ml after the pretreat ment of 3-MA for 4 h was（17.83 ± 1.54） ％ and（27.81 ± 1.79） ％ respectively,which was significantly higher than the apoptosis rate induced by TRAIL（3.70 ± 0.34） ％,（6.55 ± 0.59） ％ alone and that induced by 3-MA（7.71 ± 0.64） ％ （t =3.282,P ＜ 0.05 ; t =7.830,P ＜ 0.01）.④ The combination treatment of TRAIL and 3-MA increased the cleavage of caspase-8 and down-regulated the expression of Beclin 1.Conclusion Autophagy induced by TRAIL may contributes to the resistance of TT cells to TRAIL,which can be reversed by the inhibition of autophagy.