低氧条件下MLE-12细胞自噬及ROS表达变化的研究  

Autophagy and active oxygen species level of MLE-12 cells under hypoxic condition

作　　者：王婷婷[1] 胡天玉[2] 任飞[1] 

机构地区：[1]中国医科大学附属第一医院老年医学科,沈阳110001 [2]中国医科大学附属盛京医院第二肿瘤病房,沈阳110001

出　　处：《中国医学前沿杂志（电子版）》2016年第4期116-120,共5页Chinese Journal of the Frontiers of Medical Science(Electronic Version)

摘　　要：目的观察低氧对小鼠Ⅱ型肺泡上皮细胞(mouse lung epithelial 12,MLE-12)氧化应激损伤及细胞自噬活动的影响。方法将MLE-12细胞进行低氧处理,分别于0、4、6、12小时4个时间点采用四甲基偶氮唑盐比色法(MTT法)检测细胞的存活率;采用二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)探针法检测细胞内活性氧类(reactive oxygen species,ROS)生成量;采用Western-blot法检测自噬标志物LC3Ⅱ及Beclin-1的表达水平。同时在MLE-12细胞低氧暴露前1小时将N-乙酰半胱氨酸(N-acetylcysteine,NAC)加入培养基中进行预处理,通过同样的方法检测细胞存活率及ROS生成量,并与单纯低氧组细胞进行比较。结果与低氧0小时组细胞相比,随着低氧刺激时间的延长,MLE-12细胞的存活率明显下降(P<0.05),ROS的生成量显著升高(P<0.05,P<0.01),同时伴细胞自噬活动增强。此外,加入NAC可明显减轻低氧造成的MLE-12细胞存活率减低,减少细胞内ROS的蓄积。结论低氧可以诱导MLE-12细胞产生氧化应激反应并激活细胞的自噬现象,NAC可以对抗低氧引起的损伤,保护MLE-12细胞,提高细胞存活率。Objective To investigate the influences of hypoxia on oxidative stress injury and autophagy activity of MLE-12 cells.Method MLE-12 cells were treated with hypoxia for different treat time（0,4,6,12hours）.The survival rate of MLE-12 cells was measured by MTT assay;the level of reactive oxygen species（ROS） was analyzed by DCFH-DA probe;the expression levels of LC3 Ⅱ and Beclin-1 were detected by western blot.Added N-acetylcysteine（NAC） into medium of MLE-12 cells 1 hour prior to hypoxia exposure and the cell survival rate and ROS levels were measured by the same way.The results were compared with the simple hypoxic group.Result Compared with control group,the cell survival rate of hypoxia group had significantly decreased（P 〈0.05）.The ROS detection rate had significantly increased while at the same time the autophagy activity was enhanced in MLE-12 cells（P 〈0.05,P 〈0.01）.All these indexes were positive correlated to hypoxia exposure time.After treated with NAC,the inhibition of cell viability and ROS accumulation were significantly attenuated,and ROS expression was significantly inhibited.Conclusion Hypoxia can induce oxidative stress injuries and activate cell autophagy activity on MLE-12 cells.NAC can protect the MLE-12 cells against the damage caused by hypoxia,and improve the survuval rate of cells.

关 键 词：低氧 自噬 N-乙酰半胱氨酸 

分 类 号：R364.4[医药卫生—病理学]