摘要
目的:探讨3-磷酸甘油醛脱氢酶(GAP-DH)在肿瘤坏死因子相关凋亡诱导配体(TRAIL)促人甲状腺癌细胞凋亡中的作用机制。方法:利用微小干扰RNA(siRNA)技术,设计沉默GAPDH基因表达序列,分别按经N-硝基-L-精氨酸甲酯(L-NAME)预处理前、后(si-GAPDH、siGAPDH+L-NAME)组及siRNA对照进行分组,并设空白对照组同步进行甲状腺未分化癌细胞系(FRO)转染实验。蛋白质印迹法检测细胞中GAPDH蛋白表达,台盼蓝法检测细胞生存率,DNA裂解分析检测细胞凋亡,Caspases-3酶分析测定活性,一氧化氮(NO)产物测定法检测各组细胞亚硝酸盐和硝酸盐水平。结果:siGAPDH下调FRO细胞中GAPDH蛋白表达,细胞核中GAPDH几乎完全被封闭;与siRNA对照组细胞生存率(37%)相比,siGAP-DH组(60%)和siGAPDH+L-NAME组细胞生存率(67%)显著增加(χ2=4.88,P=0.027;χ2=7.98,P=0.026);Caspase-3活性显著下降(t=9.59,P=0.001;t=11.86,P=0.000),但两组间差异无统计学意义,t=1.65,P=0.172;siGAP-DH组和siRNA对照组NO产物较siGAPDH+L-NAME组显著增加(t=36.95,P=0.000;t=52.1,P=0.000),但两组间差异无统计学意义,t=2.563,P=0.062。结论:GAPDH作为NO产物作用的下游因子参与TRAIL促人甲状腺癌细胞凋亡作用。
OBJECTIVE： To investigate the mechanism by which glyceraldehydes-3-phosphate dehydrogenase （GAP- DH） affects tumor necrosis factor related apoptosis-inducing ligand （TRAIL）-mediated apoptosis in thyroid cancer cells. METHODS： Western blotting analysis was used for GAPDH protein analyses in total cell lysates and nuclear fractions. Trypan blue staining, nuclear DNA fragmentation, and Caspase 3 activity assay were used to detect apoptotic cells. Nitrite/nitrate concentration were measured to detect cellular production of nitric oxide（NO）. RESULTS： siGAPDH treatment significantly decreased the level of GAPDH protein in total cell lysates, and almost blocked nuclear transloeation of GAPDH; compared with scramble siRNA-treated group （37%）, cell death in siGAPDH （60%） and siGAPDH＋ L-NAME treated groups （67%） significantly increased （x^2 ： 4.88, P = 0.027; Z2 = 7.98, P = 0.026）; Activity of Caspase 3 decreased significantly （t = 9.59, P 0. 001; t = 11.86, P 0. 000）, whereas there was no significant difference between siGAPDH and siGAPDH ＋ L-NAME treated groups（t = 1.65, P = 0. 172） ; siGAPDH ＋ L-NAME treated group showed a higher production of NO than siGAPDH and scramble siRNA treated groups（t= 36.95, P= 0.000;t= 52.1, P = 0. 000）, without significant difference between si- GAPDH and scramble siRNA treated groups（t = 2. 563, P= 0. 062）. CONCLUSION： GAPDH may be involved in TRAIL-induced thyroid cancer cell apoptosis as a downstream factor of NO production.
出处
《中华肿瘤防治杂志》
CAS
2009年第9期641-644,共4页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(30740086)
辽宁省科技攻关项目(2007225021)
关键词
甲状腺肿瘤
细胞凋亡
受体
肿瘤坏死因子
thyroid neoplasms
apoptosis
receptors, tumor necrosis factor
