NNK对V79和NCTC 1469细胞增殖和凋亡的影响

发布时间：2018-03-14 00:12

本文选题：NNK　切入点：V79　出处：《郑州轻工业学院》2015年硕士论文　论文类型：学位论文

【摘要】：4-(N-甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)是烟草中主要的致癌物之一,具有促进细胞凋亡的作用。本实验主要研究NNK在不同浓度和时间下对细胞形态、细胞增值、细胞凋亡、细胞周期,以及对抗氧化基因GCLC、Nrf2和凋亡基因Bax、Bcl-2表达的影响。在前期动物毒理实验的基础上,以中国仓鼠肺细胞(V79)和小鼠肝细胞(NCTC 1469)为研究对象,运用细胞形态学,MTT法、Hoechst33258染色,流式细胞仪、荧光定量PCR等方法,研究不同浓度(0.1、0.2、0.3、0.4、0.5mg/m L)和不同胁迫时间(12、24、36h)下,NNK对细胞的胁迫作用,主要以细胞及其细胞核形态的变化,细胞凋亡和细胞周期的变化,并以相关基因(GCLC、Nrf2、Bax、Bcl-2)在NNK诱导下所产生的调控变化为检测指标,其结果如下:1、NNK能导致细胞形态发生改变。对照组细胞贴壁生长,排列均匀,大小基本一致,轮廓清晰,悬浮细胞少。在实验组中,随NNK浓度上升,细胞出现皱缩,细胞数量及生长密度逐渐减少,细胞颗粒增多。2、MTT结果显示:相同胁迫时间下,NNK对两种细胞增殖的抑制均随NNK浓度的增加而升高,与阴性对照组比较,差异均有统计学意义(P0.05),存在明显的剂量-效应关系。3、Hochest33258染色结果显示:NNK胁迫细胞24h后,与正常细胞相比,细胞核均出现不同程度蓝色荧光,可见核致密、浓染等典型的凋亡形态学特征。4、流式细胞仪检测结果显示:细胞凋亡率随NNK浓度的升高而增大;且能将细胞周期阻滞在G0/G1期。5、荧光定量PCR结果显示:经NNK胁迫后,两种细胞中GCLC、Nrf2、Bax表达均上调,Bcl-2表达明显下调;其表达变化与这些基因的作用机理相一致。由以上结果可得出:NNK能明显抑制V79和NCTC 1469细胞增殖,存在正向的剂量-效应关系;NNK对V79和NCTC 1469有促凋亡作用;NNK可使V79和NCTC 1469的细胞周期阻滞在G0/G1期;NNK可使V79和NCTC 1469的GCLC、Nrf2、Bax表达上调,Bcl-2表达下调。
[Abstract]:NNKK is one of the major carcinogens in tobacco, which can promote cell apoptosis. In this study, we studied the effects of NNK on cell morphology, cell proliferation and cell apoptosis at different concentration and time. Cell cycle, and the expression of anti-oxidation gene GCLCnrf2 and apoptotic gene Baxf2.Based on the previous animal toxicological experiments, Chinese hamster lung cells (V79) and mouse hepatocytes (NCTC 14699) were used to study the expression of anti-oxidation gene GCLCnrf2 and apoptotic gene Baxf2.The cells were stained with Hoechst33258 by MTT assay. Flow cytometry (FCM) and fluorescence quantitative PCR (PCR) were used to study the stress effects of NNK on cells under different concentrations of 0.1mg / mL and 0.4mg / mL and at different stress time (1224g / mL). The changes of cell morphology, cell apoptosis and cell cycle were studied. The regulatory changes induced by NNK were used to detect the regulatory changes induced by NNK. The results were as follows: (1) NNK could cause cell morphological changes. In the control group, the cells grew on the wall, arranged evenly, were basically the same size, and had a clear outline, and the results were as follows: (1) in the control group, the cell growth was uniform, the size was the same, and the outline was clear. In the experimental group, with the increase of NNK concentration, the cells shrank, the number and density of cells gradually decreased. The results showed that the inhibition of the proliferation of the two kinds of cells increased with the increase of NNK concentration at the same stress time, compared with the negative control group. There was a significant dose-effect relationship. The results of Hochest33258 staining showed that after 24 hours of stress, the nuclei of all the cells were blue fluorescence, and the nuclei were dense, compared with the normal cells. The results of flow cytometry showed that the apoptosis rate increased with the increase of NNK concentration, and the cell cycle was blocked at G _ 0 / G _ 1 phase. The results of fluorescence quantitative PCR showed that after NNK stress, the cell cycle was blocked in G _ 0 / G _ 1 phase. The expression of Bcl 2 was down-regulated in both cells, and the changes were consistent with the mechanism of these genes. From the above results, it was concluded that the proliferation of V79 and NCTC 1469 cells could be significantly inhibited by the cell proliferation of V79 and NCTC 1469 cells. There is a positive dose-effect relationship between NNK and V79 and NCTC 1469. NNK can block the cell cycle of V79 and NCTC 1469 at G _ 0 / G _ 1 phase and up-regulate the expression of Bcl-2 in V79 and NCTC 1469.
【学位授予单位】：郑州轻工业学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R73-3;TS41

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