目的：探讨mitoKATP通道开放剂二氮嗪对高氧诱导人A549细胞凋亡的保护作用和机制。方法：体外培养人肺泡Ⅱ型上皮细胞（A549），随机分为对照组、高氧组（换液后通入900 mL/L氧气和50 mL/L 二氧化碳高纯混合气，10 min后密闭培养）和二氮嗪组（用终浓度为100 μmol/L的二氮嗪培养液预处理24 h再进行高氧诱导）。3组分别于处理后12、24、48 h收集细胞进行检测。倒置相差显微镜下观察A549细胞形态，流式细胞仪检测48 h的细胞凋亡，免疫组化法检测细胞内Omi/HtrA2的表达。结果：与对照组相比，高氧组细胞受损明显，形态改变，细胞凋亡率增加（P<0.05），胞浆Omi/HtrA2表达增多（P<0.05）。与高氧组相比，二氮嗪组细胞损伤状况明显得到改善，细胞生长状况明显好转，形态明显变好；胞浆Omi/HtrA2表达减少（P<0.05），细胞凋亡率减少（P<0.05）。结论：mitoKATP通道开放剂二氮嗪具有降低Omi/HtrA2表达和A549细胞凋亡，从而减轻高氧诱导的肺损伤的作用。
Abstract
OBJECTIVE: To explore the protective effects of mitochondrial ATP-sensitive potassium channel opener diazoxide on hyperoxia-induced apoptosis of type Ⅱ alveolar epithelial cells (A549 cells) and possible mechanisms. METHODS: A549 cells were cultured in vitro and divided randomly into control, hyperoxia and diazoxide group. The hyperoxia group was exposed to a mixture of O2 (900 mL/L) and CO2 (50 mL/L) for 10 minutes, then cultured in a closed environment. The diazoxide group was pretreated with diazoxide of 100 μmol/L for 24 hrs before hyperxia induction. The cells were collected 12, 24 and 48 hrs after culture. The morphologic changes of A549 cells were observed under an inverted microscope. A549 cell apoptosis was detected by flow cytometry. The expression of Omi/HtrA2 in the endochylema of A549 cells was determined by immunohistochemistry. RESULTS: A549 cells were damaged and the changes in morphology of the cells were serious in the hyperoxia group. The apoptosis rate of A549 cells and the expression of Omi/HtrA2 in the endochylema increased in the hyperoxia group compared with the control group (P<0.05). The growth and the morphology of A549 cells were greatly improved and the cell injuries were obviously alleviated in the diazoxide group. The expression of Omi/HtrA2 in the endochylema and the apoptosis rate of A549 cells were significantly reduced in the diazoxide group compared with the hyperoxia group (P<0.05). CONCLUSIONS: Diazoxide as an opener of mitoKATP channel can reduce the expression of Omi/HtrA2 and the apoptosis rate of A549 cells, thus relieves the injury of A549 cells induced by hyperoxia.
关键词
高氧 /
二氮嗪 /
线粒体ATP敏感钾通道 /
凋亡 /
Omi/HtrA2 /
A549细胞
Key words
Hyperoxia /
Diazoxide /
Mitochondrial ATP-sensitive potassium channel /
Apotosis /
Omi/HtrA2 /
A549 cell
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