卷烟烟气凝集物诱发永生化人支气管上皮细胞恶性转化的实验研究

发布时间：2018-12-15 13:48

【摘要】： 目的：检测卷烟烟气凝集物(cigarette smoking condensates，CSC)，对体外培养的永生化人支气管上皮细胞(immortalized human bronchial epithelial cells，BEP2D)的恶性转化能力，欲建立单因素吸烟致肺癌的细胞模型，并检测不同转化时期差异表达的基因，为深入研究吸烟致肺癌发生多阶段过程中的细胞和分子机理提供实验依据。方法：首先用四唑盐(MTT)比色实验，检测CSC对BEP2D慢性毒性效应，，依据细胞相对存活率，筛选慢性转化剂量，从细胞生长动力学、形态学、血清抗性、锚着独立性及裸鼠成瘤等方面对其转化特性进行鉴定，再利用人类全基因组寡核苷酸芯片，筛选对照及不同染毒代龄细胞间差异表达基因。同时，利用二氯荧光黄双乙酸盐(DCFH-DA)和氢化乙锭(HE)活性氧标记技术，初步探测了CSC对BEP2D及转化细胞的氧化损伤作用。结果：CSC对BEP2D以细胞毒性致死效应为主，随着CSC剂量增加，细胞存活比率下降，据此，我们选定细胞相对存活率在60％-80％下0.001支烟／ml(LHC-8)为转化剂量，其对应的酒精浓度0.0005 ml／ml(酒精／LHC-8)作为溶剂对照，取CSC处理后P10代、P20代、P30代、P40代细胞，对其转化特性进行鉴定，发现第P30代和P40代细胞出现了明显的生长速度加快，倍增时间缩短，对血清抗性及锚着独立性增加。差异表达基因比较结果显示，大多数差异表达基因为下调，极少数基因为上调，其中位于启动子区域表达下调的基因，与肺癌关键基因的甲基化有关。另对CSC致BEP2D细胞的氧化损伤初步探索发现，CSC能够引起BEP2D细胞内产生大量活性氧(ROS)，并有剂量效应；而慢性转化细胞则逐渐获得了一些抗氧化诱导能力，其机制有待进一步探讨。结论：单因素CSC可能对BEP2D细胞有慢性转化效应，有待裸鼠实验进一步证实；转化机制可能与细胞获得抗氧化能力有关。本结果为今后深入研究吸烟致肺癌发病机理奠定基础。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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