本文選題：NNK　切入點：V79　出處：《鄭州輕工業(yè)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：4-(N-甲基亞硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)是煙草中主要的致癌物之一,具有促進細胞凋亡的作用。本實驗主要研究NNK在不同濃度和時間下對細胞形態(tài)、細胞增值、細胞凋亡、細胞周期,以及對抗氧化基因GCLC、Nrf2和凋亡基因Bax、Bcl-2表達的影響。在前期動物毒理實驗的基礎(chǔ)上,以中國倉鼠肺細胞(V79)和小鼠肝細胞(NCTC 1469)為研究對象,運用細胞形態(tài)學(xué),MTT法、Hoechst33258染色,流式細胞儀、熒光定量PCR等方法,研究不同濃度(0.1、0.2、0.3、0.4、0.5mg/m L)和不同脅迫時間(12、24、36h)下,NNK對細胞的脅迫作用,主要以細胞及其細胞核形態(tài)的變化,細胞凋亡和細胞周期的變化,并以相關(guān)基因(GCLC、Nrf2、Bax、Bcl-2)在NNK誘導(dǎo)下所產(chǎn)生的調(diào)控變化為檢測指標(biāo),其結(jié)果如下:1、NNK能導(dǎo)致細胞形態(tài)發(fā)生改變。對照組細胞貼壁生長,排列均勻,大小基本一致,輪廓清晰,懸浮細胞少。在實驗組中,隨NNK濃度上升,細胞出現(xiàn)皺縮,細胞數(shù)量及生長密度逐漸減少,細胞顆粒增多。2、MTT結(jié)果顯示:相同脅迫時間下,NNK對兩種細胞增殖的抑制均隨NNK濃度的增加而升高,與陰性對照組比較,差異均有統(tǒng)計學(xué)意義(P0.05),存在明顯的劑量-效應(yīng)關(guān)系。3、Hochest33258染色結(jié)果顯示:NNK脅迫細胞24h后,與正常細胞相比,細胞核均出現(xiàn)不同程度藍色熒光,可見核致密、濃染等典型的凋亡形態(tài)學(xué)特征。4、流式細胞儀檢測結(jié)果顯示:細胞凋亡率隨NNK濃度的升高而增大;且能將細胞周期阻滯在G0/G1期。5、熒光定量PCR結(jié)果顯示:經(jīng)NNK脅迫后,兩種細胞中GCLC、Nrf2、Bax表達均上調(diào),Bcl-2表達明顯下調(diào);其表達變化與這些基因的作用機理相一致。由以上結(jié)果可得出:NNK能明顯抑制V79和NCTC 1469細胞增殖,存在正向的劑量-效應(yīng)關(guān)系;NNK對V79和NCTC 1469有促凋亡作用;NNK可使V79和NCTC 1469的細胞周期阻滯在G0/G1期;NNK可使V79和NCTC 1469的GCLC、Nrf2、Bax表達上調(diào),Bcl-2表達下調(diào)。
[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.
【學(xué)位授予單位】：鄭州輕工業(yè)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R73-3;TS41

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相關(guān)期刊論文 前1條

1 姚二民;張峻松;梁永林;;茶葉對降低吸煙危害的應(yīng)用研究[J];茶葉科學(xué);2009年02期

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本文編號：1608747