微囊藻毒素-LR对人正常食管上皮细胞凋亡及对Caspase-3和Caspase-9蛋白表达的影响

目的：研究微囊藻毒素-LR对人正常食管上皮细胞活力及凋亡相关蛋白Caspase-3和Caspase-9表达的影响，为探讨其诱导凋亡的途径提供依据。方法：分别用不同浓度（1、3.75、7.5、15、30、50 μg/mL）微囊藻毒素-LR处理人正常食管上皮HEEC细胞24 h，用四甲基偶氮唑蓝（MTT）法检测细胞活力，计算半数抑制浓度（IC₅₀）以确定后续实验的MC-LR浓度。进一步用0、6、12、24 μg/mL微囊藻毒素-LR处理细胞，采用PI-Annexin V双染法检测细胞凋亡，Western blot检测Caspase-3和Caspase-9蛋白的表达水平。结果：与对照组比较，3.75~50 μg/mL的微囊藻毒素-LR均可使人正常食管上皮细胞活力降低（P均 < 0.01），测定得出IC₅₀为24 μg/mL。因此，选择6、12和24 μg/mL的MC-LR用于后续实验。用6~24 μg/mL微囊藻毒素-LR处理细胞24 h后，细胞凋亡率升高（P < 0.05或P < 0.01），Caspase-3和Caspase-9蛋白表达增加（P均 < 0.01）。结论：微囊藻毒素-LR可以诱导人正常食管上皮细胞凋亡，且可能与细胞凋亡相关的信号分子Caspase-3和Caspase-9的激活有关。

Effects of microcystin-LR on apoptosis,and Caspase-3 and Caspase-9 expression in human normal esophageal epithelial cells

OBJECTIVE:To investigate the effects of microcystin-LR on viability and apoptosis of human normal esophageal epithelial cells,and on expression of Caspase-3 and Caspase-9.METHODS:Human normal esophageal epithelial cells were treated with microcystin-LR at different concentrations (1,3.75,7.5,15,30,50 μg/mL) for 24 h,and cell viability was detected by the MTT assay.The data were used to calculate the half inhibitory concentration (IC50) which was used to determine the MC-LR concentrations for subsequent experiments.Accordingly,cells were treated with 6,12 and 24 μg/mL MC-LR,and apoptosis was detected by PI-Annexin V double staining.Expression levels of Caspase-3 and Caspase-9 were detected by Western blot.RESULTS:Compared with the control group,microcystin-LR of 3.75-50 μg/mL decreased the viability of normal esophageal epithelial cells (P＜0.01);the 6-24 μg/mL concentrations increased the apoptosis rate (P＜O.05 or P＜O.01),and the expression of Caspase-3 and Caspase-9 (P＜0.01).CONCLUSION:Induction of apoptosis in human normal esophageal epithelial cells by microcystin-LR may be related to its activation of Caspase-3 and Caspase-9.