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目的观察CSC急性染毒对人支气管上皮细胞的毒性特征及Rap2B基因表达的影响,推测Rap2B基因在急性染毒阶段的作用,为后续研究提供实验依据。方法 16HBE细胞暴露香烟烟雾冷凝物(CSC)24 h,于倒置显微镜下观察细胞形态变化,CCK-8法确定染毒剂量,荧光定量PCR检测该基因mRNA表达水平,Western Blot检测其蛋白表达水平。结果 CSC对16HBE细胞有毒性作用,半数抑制浓度(IC50)为(0.08±0.006)mg/ml;CSC染毒16HBE细胞24 h后,细胞形态发生一定改变,高剂量组细胞出现明显形态学改变;与对照组比较,1/8 IC50剂量组mRNA和蛋白表达水平上调较高(P<0.05),随剂量组浓度增高mRNA和蛋白表达下调至最低(IC50组,P<0.05)。结论 Rap2B基因的表达在一定范围随着染毒浓度的改变呈现先升高后下降的趋势,提示Rap2B基因可能作为CSC靶基因在其急性染毒过程中发挥一定的作用。
Objective To observe the effect of acute exposure to CSC on the toxicity of human bronchial epithelial cells and the expression of Rap2B gene, and to infer the role of Rap2B gene in the acute phase of exposure to provide the experimental evidence for the follow-up study. Methods 16HBE cells were exposed to cigarette smoke condensate (CSC) for 24 hours. The morphological changes of the cells were observed under an inverted microscope. The dose of CCK-8 was determined by fluorescence quantitative PCR. The protein expression was detected by Western Blot. Results CSC had a toxic effect on 16HBE cells with an IC50 of (0.08 ± 0.006) mg / ml. After 16 hours of CSC exposure, the morphological changes of 16HBE cells were observed. The morphological changes of cells were observed in high dose group. Compared with the control group, mRNA and protein expressions were upregulated in 1/8 IC50 group (P <0.05). The mRNA and protein expression were down-regulated in the IC50 group (P <0.05). Conclusion The expression of Rap2B gene increases first and then decreases with the change of exposure concentration in a certain range, suggesting that Rap2B gene may play a role in the acute exposure to CSC as a target gene of CSC.