【摘要】 目的探讨新城疫病毒(NDV)D90杀伤口腔鳞癌细胞系CAL-27的机制。方法 CAL-27细胞用稀释10倍的D90病毒作用0、12、24、36 h后用以Annexin V-异硫氰酸荧光素/碘化丙啶(PI)双染色流式细胞术检测NDV D90对口腔鳞癌细胞系CAL-27细胞凋亡情况的影响,接种于24孔板中的CAL-27细胞用稀释10倍的D90病毒作用12 h,4’,6-二脒基-2-苯基吲哚荧光染色检测NDV D90对CAL-27细胞作用前后细胞形态学的变化;用稀释10倍的病毒与细胞分别共同孵育12、24、36 h,同时设置对照组。使用BCA蛋白测定试剂盒的裂解物中的蛋白质含量进行测定。免疫蛋白印迹法检测NDV D90作用CAL-27细胞后对凋亡相关蛋白表达量的影响。结果 NDV D90能引起细胞CAL-27的凋亡,并且凋亡率随着病毒作用的时间增加而升高[0 h,12 h,24 h,36 h的凋亡率分别为(3.68±1.50)%、(19.22±0.99)%、(82.80±0.74)%、(99.69±0.13)%];D90作用于CAL-27后细胞核出现凋亡的相关形态改变,如核固缩以及核碎裂;随着病毒作用时间的增加,细胞色素C蛋白表达水平升高,胱天蛋白酶3前体蛋白(procaspase-3)以及procaspase-9蛋白表达水平降低,procaspase-8蛋白表达水平没有明显变化,同时未检测到肿瘤坏死因子α蛋白的表达。结论 NDV D90毒株能引起人口腔鳞癌细胞系CAL-27的凋亡,且凋亡率呈时间依赖性;NDV D90引起CAL-27细胞凋亡主要是通过线粒体途径,死亡受体途径没有被激活或者作用非常小。更多还原

【Abstract】 Objective To investigate the mechanisms of NDV-D90 killling human oral squamous cell carcinoma CAL-27. Methods CAL-27 cells were infected with D90( MOI = 0. 1) and cells were processed for flow cytometry at different time points( 0,12,24 and 36 h after infection). NDV-D90 strains influence on apoptosis of human oral squamous cell carcinoma CAL-27 cells was detected with Annexin V-FITC / PI staining and flow cytometry; CAL-27 cells were cultured in a 24-well plate and infected with D90 at an MOI of0. 1 for 12 h,the NDV-D90 strains effect on CAL-27 cell morphology were observed with the militiamen DAPI fluorescence; CAL-27 cells were infected with D90 MOI = 0. 1) at different time points( 12,24 and36 h after infection),while the normal control group was set up. The protein content of the lysates was measured using a BCA Protein Assay kit,and the expression of apoptosis-related proteins of cultured CAL-27 cells was detected using Western Blot detection. Results NDV-D90 strain could induce apoptosis of CAL-27,and the percentage of the apoptotic cells increased with increasing of the infection time [the percentage of CAL-27 cells infected by NDV D90 that were apoptotic significantly increased from( 3. 68 ± 1. 50) % at 0 h to( 19. 22 ± 0. 99) %,( 82. 8 ± 0. 74) % and( 99. 69 ± 0. 13) % at 12,24 and 36 h respectively]; D90 induced a typical apoptosis-like morphology,characterized by chromatin condensation and nuclear fragmentation; with the increasing of virus effects,the levels of cytochrome C increased,the levels of procaspase-9 and procaspase-3 decreased,and the level of procaspase-8 was almost unchanged,however,TNF-α protein was not detected. Conclusion NDV-D90 strain can induce apoptosis in human oral squamous cell carcinoma CAL-27,and D90 decreasing the viability of CAL-27 is time-dependent; the apoptosis induced by D90 is mainly dependent on the mitochondrial pathway,while the death receptor pathway is not activated or has very little effect.更多还原