新城疫病毒D90杀伤口腔鳞癌细胞系CAL－27机制的研究

摘要

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 stai-ning and flow cytometry; CAL-27 cells were cultured in a 24-well plate and infected with D90 at an MOI of 0.1 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 and 36 h after infection ) ,while the normal control group was set up.The protein content of the lysates was meas-ured 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 fragmenta-tion;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-αprotein 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.%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。