巴豆醛诱导人支气管上皮细胞(BEAS-2B)自噬及相关通路的时间效应变化  

Time-effect on alterations of autophagy and related pathways in human bronchial epithelial cells BEAS-2B exposed to crotonaldehyde

作　　者：王利蒙 李翔[3] 杨陟华[4] 朱茂祥[4] 谢剑平[3] WANG Limeng LI Xiang YANG Zhihua ZHU Maoxiang XIE Jianping.(Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning, China University of Chinese Academy of Sciences, Beijing 100049, China Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001, China Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China)

机构地区：[1]中国科学院大连化学物理研究所,辽宁省大连市中山路457号116023 [2]中国科学院大学,北京市石景山区玉泉路19号100049 [3]中国烟草总公司郑州烟草研究院 [4]军事医学科学院放射与辐射医学研究所,北京市太平路27号100850

出　　处：《烟草科技》2017年第10期35-41,共7页Tobacco Science & Technology

摘　　要：为评估巴豆醛对人支气管上皮细胞(BEAS-2B)的毒性效应,使用细胞计数试剂盒(CCK-8)检测BEAS-2B细胞存活率,免疫荧光法检测自噬小体的聚集和定位,Western Blot实验检测自噬标志物微管相关蛋白1轻链3β(LC3B-Ⅱ)及两个自噬相关通路磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶标(mTOR)和肝脏激酶B1(LKB1)/单磷酸腺苷活化蛋白激酶(AMPK)/Unc-51-类激酶(ULK1)的变化。结果表明:(1)巴豆醛暴露可显著降低BEAS-2B细胞存活率。(2)巴豆醛处理可引起自噬小体的聚集,改变LC3B-Ⅱ蛋白表达量;巴豆醛诱导细胞自噬具有明显的时间效应关系,BEAS-2B细胞自噬强度随巴豆醛暴露时间延长先升高后降低。(3)PI3K/Akt/m TOR通路中磷酸化的PI3K、Akt和4E结合蛋白1(4E-BP1)呈下降趋势,磷酸化的m TOR和核糖体蛋白S6激酶(p70 S6K)先下降后升高;LKB1/AMPK/ULK1通路中LKB1、AMPK和ULK1活性均呈升高趋势。因此,两个通路均参与调控巴豆醛诱导的BEAS-2B细胞自噬水平的升高。To assess the toxic effects of crotonaldehyde on human bronchial epithelial cells BEAS-2 B,cell counting kit-8（CCK-8）assay was adopted to detect the viability of BEAS-2 B cells; immunofluorescence assay to the accumulation and location of autophagosomes; and Western Blot assay to autophagy-marker microtubuleassociated protein 1 light chain 3 beta（LC3 B-Ⅱ）, the variations of two pathways of phosphatidylinositol 3-kinase（PI3 K）/protein kinase B（Akt）/mammalian target of rapamycin（m TOR）and liver kinase B1（LKB1）/adenosine monophosphate-activated protein kinase（AMPK）/Unc-51-like kinase 1（ULK1）. The results indicated that：1） The viability of BEAS-2 B cells decreased significantly after exposure to crotonaldehyde. 2）Crotonaldehyde significantly promoted the accumulation of autophagosomes and changed the expression level of LC3 B-Ⅱ. Induced autophagy had apparent time-effect relationship. The level of autophagy in BEAS-2 B cells was up-regulated prior to down-regulation with the increase of exposure time. 3）The relative expression of phospho-（P-） PI3 K,P-Akt and P-4 E-binding protein1（4 E-BP1） in PI3 K/Akt/m TOR pathway decreased,while that of P-m TOR and P-ribosomal protein S6 kinase（p70 S6 K）decreased first and then increased. The activities of LKB1,AMPK,and ULK1 in LKB1/AMPK/ULK1 pathway increased. Collectively,the two pathways mediated the up-regulation of autophagy in BEAS-2 B cells exposed to crotonaldehyde.

关 键 词：巴豆醛 人支气管上皮细胞 自噬 PI3K AMPK 

分 类 号：TS416[农业科学—烟草工业]