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目的观察永生化人支气管上皮细胞(BEAS-2B)经香烟烟雾长期处理后FHIT基因甲基化状况及其mRNA表达改变,探索吸烟致肺癌的机制。方法 BEAS-2B以20%烟雾浓度,每次10min作为染烟条件,细胞连续染烟2次作为染烟1代。细胞染烟30代且传代40代后,应用流式细胞仪分析细胞周期及凋亡和软琼脂克隆法检测克隆形成率判断其恶性转化程度,用甲基化特异性PCR(MS-PCR)检测FHIT基因甲基化状况,并通过Q-PCR检测FHIT的mRNA表达量。结果与对照组比较,各染烟组细胞出现了一定的S期阻滞,染烟后传代至40代细胞凋亡率较对照组下降明显(P<0.05)。染烟20代传代至40代细胞(Sm20-40)与染烟30代传代至40代细胞(Sm30-40)FHIT基因启动子区呈高甲基化状态,且Q-PCR结果显示Sm20-40和Sm30-40组细胞FHIT基因mRNA均低于对照组(P<0.05)。结论香烟烟雾可导致BEAS-2B细胞FHIT基因启动子区呈高甲基化状态,引起FHIT基因mRNA表达降低,可能是吸烟致肺癌的机制之一。
Objective To investigate the methylation of FHIT gene and its mRNA expression in human immortalized human bronchial epithelial cells (BEAS-2B) after long-term treatment with cigarette smoke to explore the mechanism of smoking-induced lung cancer. Method BEAS-2B with 20% smoke concentration, each 10min as a dye-smoked conditions, the cells were smoked two consecutive smoked tobacco as a generation of generation. Cells were stained for 30 passages and passaged for 40 passages. The cell cycle and apoptosis were analyzed by flow cytometry and the rate of colony formation was determined by soft agar cloning. The methylation-specific PCR (MS-PCR) FHIT gene methylation status, and FHIT mRNA expression was detected by Q-PCR. Results Compared with the control group, there was a certain S phase retardation in all the groups of infected tobacco. The apoptotic rate of passage 40 to passage 40 was significantly lower than that of the control group (P <0.05). The FHIT gene promoter region was hypermethylated in passage 20 to generation 40 (Sm20-40) and passage 30 to passage 40 (Sm30-40), and Q-PCR showed that Sm20-40 and Sm30 The mRNA level of FHIT in -40 group was lower than that in control group (P <0.05). Conclusions Cigarette smoke can cause the hypermethylation of FHIT gene promoter region in BEAS-2B cells and cause the decrease of FHIT gene mRNA expression, which may be one of the mechanisms of smoking-induced lung cancer.