γ-干扰素对TRAIL诱导人甲状腺癌细胞凋亡影响的研究

Effect of IFN-γ on TRAIL-induced apoptosis of human thyroid cancer cells

目的:探讨γ-干扰素(IFN-γ)对TRAIL诱导人甲状腺癌细胞凋亡作用的影响。方法:流式细胞术、蛋白质印迹法、蛋白质羰基测定法和共聚焦显微镜分别用于检测细胞凋亡、GAPDH蛋白表达、氧化损伤程度和细胞核内GAPDH免疫染色表达。运用微小RNA干扰技术下调GAP-DH表达。结果:与耐药细胞系相比,TRAIL敏感的FRO和KTC2细胞核提取液中可见不同程度的GAPDH蛋白表达、细胞内羰基蛋白显著集聚。IFN-γ可增加耐药ARO细胞核中GAPDH水平。IFN-γ或TRAIL单药组细胞凋亡率均<5%,而IFN-γ/TRAIL联合组细胞凋亡率达28.5%。siGAPDH组与siRNA对照组间细胞凋亡率差异有统计学意义(t=4.909,P=0.08),使对照组细胞凋亡率下降11%。结论:IFN-γ加强TRAIL诱导人甲状腺癌细胞凋亡可能是通过上调细胞核内GAP-DH表达实现的。

OBJECTIVE：To determine the effect of IFN-γ on TRAIL-induced apoptosis of human thyroid cancer cells. METHODS： Flow cytometry （FCM） and Western blot analysis were respectively employed for apoptotic analysis and GAPDH protein expression. Protein carbonyl assay and confocal microscopy were respectively used for evaluation of oxidative damage and immunohistochemical expression of GAPDH in nuclei. GAPDH gene expression was downregulated by using small interfering RNA （siRNA） technique. RESULTS： GAPDH was observed in the nuclear fraction in TRAIL-sensitive FRO and KTC2 cells, but not ARO, KTC1, and KTC3 cells. Carbonyls were detected in FRO and KTC2 cells, but not in the other cell lines. IFN-γ increased the expression level of GAPDH in nuclear fraction of TRAIL-resistance ARO cells. The apoptosis rate of the IFN-γ or TRAIL single drug group was less than 5%, but that of the IFN-γ/TRAIL group was 28.5%. The difference of apoptosis rates between the siGAPDH group and siRNA control group（11%） was statistical significant（t=4.909,P=0.08）. CONCLUSION： IFN-γ enhancing TRAIL-induced apoptosis of human thyroid cancer cells may be through the upregulation of nucleic expression of GAPDH.