摘要
研究4-(甲基亚硝氨基)-1-(3-吡啶基)-1-丁酮(NNK)对小鼠肝细胞NCTC 1469细胞的毒性作用。利用MTT比色法检测5个不同NNK浓度(0.1、0.2、0.3、0.4、0.5 mg/m L)对细胞的毒性作用;倒置相差显微镜观察细胞的形态变化;流式细胞仪检测细胞凋亡率;实时荧光定量PCR检测凋亡基因Bcl-2和Bax的表达。MTT显示,NNK能降低NCTC 1469细胞存活率,并呈剂量和时间依赖性。镜检结果可见细胞皱缩,密度降低;流式细胞仪检测显示,0.1、0.2、0.3、0.4、0.5 mg/m L的NNK作用24 h后凋亡率分别为(16.79±2.01)%、(26.87±1.67)%、(41.78±3.19)%、(50.89±3.94)%和(65.86±4.54)%,显著高于对照组(7.46±1.58)%;荧光定量PCR结果显示,随着NNK浓度的增加,Bcl-2 m RNA的表达减少,Bax m RNA的表达逐渐上升。NNK能够降低NCTC 1469细胞存活率并诱导其凋亡,作用呈剂量和时间依赖性。
This work is to investigate the cytotoxicity in NCTC 1469 cells induced by 4-（methyInitrosamino）-1-（3-pyridyl）-1-butanone （NNK）. MTT assay was used to analyze the toxic effects on NCTC 1469 cells under various concentrations（0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL） of NNK at different times. Morphologic changes of treated NCTC 1469 cells with NNK were observed by the inverted microscopy. Flow cytometry （FCM）and real-time quantitative PCR were applied to measure the apoptosis rate and the expression of apoptotic gene Bcl-2 and Bax mRNA. NNK depressed the viability of NCTC 1469 cells with a pattern of the dose-dependent and time-dependent. Typical morphological changes of cells shrinkage and the decrease of density were observed by the inverted microscopy. FCW revealed that inhibited cell proliferation and induced death were of dose-dependent, i.e., apoptotic rates were（16.79 ± 2.01）%,（26.87 ± 1.67）%,（41.78 ± 3.19）%,（50.89 ± 3.94）%and （65.86 ± 4.54）%respectively at 24 h after the cells treated with the concentrations（0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL）of NNK, showing much higher than the control of（7.46 ± 1.58）%. Real-time quantitative PCR showed that with the raising of NNH concentration, the expression of gene Bcl-2 decreased and the expression of gene Bax gradually increased. Conclusively, NNK could obviously abate the viability of NCTC 1469 cells and induce the apoptosis of NCTC 1469 cells in a pattern of dose-dependent and time-dependent.
出处
《生物技术通报》
CAS
CSCD
北大核心
2015年第9期218-223,共6页
Biotechnology Bulletin
基金
郑州市科技局科技攻关项目(121PPTGG362-16)
河南省教育厅青年骨干教师项目(2012GG-117)
