發(fā)布時(shí)間：2018-12-15 13:48

【摘要】： 目的：檢測卷煙煙氣凝集物(cigarette smoking condensates，CSC)，對體外培養(yǎng)的永生化人支氣管上皮細(xì)胞(immortalized human bronchial epithelial cells，BEP2D)的惡性轉(zhuǎn)化能力，欲建立單因素吸煙致肺癌的細(xì)胞模型，并檢測不同轉(zhuǎn)化時(shí)期差異表達(dá)的基因，為深入研究吸煙致肺癌發(fā)生多階段過程中的細(xì)胞和分子機(jī)理提供實(shí)驗(yàn)依據(jù)。方法：首先用四唑鹽(MTT)比色實(shí)驗(yàn)，檢測CSC對BEP2D慢性毒性效應(yīng)，，依據(jù)細(xì)胞相對存活率，篩選慢性轉(zhuǎn)化劑量，從細(xì)胞生長動力學(xué)、形態(tài)學(xué)、血清抗性、錨著獨(dú)立性及裸鼠成瘤等方面對其轉(zhuǎn)化特性進(jìn)行鑒定，再利用人類全基因組寡核苷酸芯片，篩選對照及不同染毒代齡細(xì)胞間差異表達(dá)基因。同時(shí)，利用二氯熒光黃雙乙酸鹽(DCFH-DA)和氫化乙錠(HE)活性氧標(biāo)記技術(shù)，初步探測了CSC對BEP2D及轉(zhuǎn)化細(xì)胞的氧化損傷作用。結(jié)果：CSC對BEP2D以細(xì)胞毒性致死效應(yīng)為主，隨著CSC劑量增加，細(xì)胞存活比率下降，據(jù)此，我們選定細(xì)胞相對存活率在60％-80％下0.001支煙／ml(LHC-8)為轉(zhuǎn)化劑量，其對應(yīng)的酒精濃度0.0005 ml／ml(酒精／LHC-8)作為溶劑對照，取CSC處理后P10代、P20代、P30代、P40代細(xì)胞，對其轉(zhuǎn)化特性進(jìn)行鑒定，發(fā)現(xiàn)第P30代和P40代細(xì)胞出現(xiàn)了明顯的生長速度加快，倍增時(shí)間縮短，對血清抗性及錨著獨(dú)立性增加。差異表達(dá)基因比較結(jié)果顯示，大多數(shù)差異表達(dá)基因?yàn)橄抡{(diào)，極少數(shù)基因?yàn)樯险{(diào)，其中位于啟動子區(qū)域表達(dá)下調(diào)的基因，與肺癌關(guān)鍵基因的甲基化有關(guān)。另對CSC致BEP2D細(xì)胞的氧化損傷初步探索發(fā)現(xiàn)，CSC能夠引起B(yǎng)EP2D細(xì)胞內(nèi)產(chǎn)生大量活性氧(ROS)，并有劑量效應(yīng)；而慢性轉(zhuǎn)化細(xì)胞則逐漸獲得了一些抗氧化誘導(dǎo)能力，其機(jī)制有待進(jìn)一步探討。結(jié)論：單因素CSC可能對BEP2D細(xì)胞有慢性轉(zhuǎn)化效應(yīng)，有待裸鼠實(shí)驗(yàn)進(jìn)一步證實(shí)；轉(zhuǎn)化機(jī)制可能與細(xì)胞獲得抗氧化能力有關(guān)。本結(jié)果為今后深入研究吸煙致肺癌發(fā)病機(jī)理奠定基礎(chǔ)。
[Abstract]:Objective: to detect the malignant transformation ability of cigarette smoke agglutinate (cigarette smoking condensates,CSC) on immortalized human bronchial epithelial cells (immortalized human bronchial epithelial cells,BEP2D) in vitro and to establish a cell model of lung cancer induced by single factor smoking. The differentially expressed genes in different transformation stages were detected to provide experimental evidence for the further study of cellular and molecular mechanisms in the multistage process of lung cancer induced by smoking. Methods: the chronic toxicity of CSC to BEP2D was detected by (MTT) colorimetric assay. According to the relative survival rate of cells, the dose of chronic transformation was screened, and the cell growth kinetics, morphology and serum resistance were analyzed. Anchorage independence and tumorigenesis of nude mice were identified. The differentially expressed genes were screened by using human genome oligonucleotide microarray. At the same time, the oxidative damage of CSC to BEP2D and transformed cells was investigated by using dichlorofluorescein yellow diacetate (DCFH-DA) and hydrogenated ethidium (HE) reactive oxygen species labeling technique. Results: the cytotoxic lethal effect of CSC on BEP2D was predominant. With the increase of CSC dose, the cell survival ratio decreased. According to this, we selected 0.001 cigarettes / ml (LHC-8) as the conversion dose under 60% -80% relative survival rate. The corresponding alcohol concentration of 0.0005 ml/ml (alcohol / LHC-8) was used as a solvent control. After CSC treatment, the cells of P10, P20, P30 and P40 were used to identify the transformation characteristics of P10, P20, P30 and P40 cells. It was found that the growth rate of P30 and P40 cells was accelerated, the doubling time was shortened, the resistance to serum and the independence of anchors were increased. The comparison of differentially expressed genes showed that most of the differentially expressed genes were down-regulated and a few of them were up-regulated. The down-regulated genes located in the promoter region were related to the methylation of the key genes in lung cancer. In addition, the oxidative damage of BEP2D cells induced by CSC was investigated. It was found that CSC could induce a large amount of reactive oxygen species (Ros) (ROS), in BEP2D cells in a dose-dependent manner. However, the chronic transformed cells gradually acquired some antioxidant induction ability, and its mechanism needs further study. Conclusion: univariate CSC may have a chronic transformation effect on BEP2D cells, which needs further confirmation in nude mice, and the mechanism of transformation may be related to the ability of cells to obtain antioxidation. The results lay a foundation for further study on the pathogenesis of lung cancer caused by smoking.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R363

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