PI3K/Akt信号通路在染尘大鼠肺泡巨噬细胞自噬中作用  

Effects of PI3K / Akt signaling pathway on the autophagy of rat alveolar macrophages exposed to dust

作　　者：王晓艳 宋瑞瑞 张晓雪 沈福海 蒋守芳 李小明 曹燕花 时亚欣 王永斌 金玉兰 

机构地区：[1]华北理工大学公共卫生学院,河北唐山063000

出　　处：《中国职业医学》2016年第3期247-255,共9页China Occupational Medicine

基　　金：国家自然科学基金(81202161)

摘　　要：目的观察大鼠肺泡巨噬细胞株NR8383细胞在染尘处理后的自噬情况,以LY294002阻断磷脂酰肌醇3-激酶(PI3K)途径,探讨PI3K/蛋白激酶B(Akt)信号通路在其自噬中的调控作用。方法 1常规培养NR8383细胞,分为空白组和染尘处理组,分别采用终浓度为0、50 mg/L的矽尘混悬液处理3、6、12、20和24 h后,收集细胞上清液,采用酶联免疫吸附实验(ELISA)法检测肿瘤坏死因子-α(TNF-α)和转化生长因子-β1(TGF-β1)水平,选择最佳染尘时间。2NR8383细胞分为对照组(予等体积不含血清的F-12K培养基处理)、矽尘组(予终浓度为50 mg/L矽尘混悬液处理)和干预组(予终浓度分别为50 mg/L、20μmol/L的矽尘混悬液和PI3K抑制剂LY294002处理),孵育后收集样品。采用ELISA法检测孵育后20 h时间点细胞上清液中TNF-α和TGF-β1水平;采用蛋白免疫印迹方法测定孵育后20 h时间点细胞中Akt和自噬标记蛋白微管相关蛋白1轻链3(LC3)的水平;采用激光扫描共聚焦显微镜(LSCM)观察孵育3、6、12和20 h时间点细胞自噬免疫荧光表达情况,以及加入溶酶体抑制剂磷酸氯喹(CDP)后细胞的自噬表达情况。结果 1离体NR8383细胞染尘模型最佳处理时间为20 h。2矽尘组细胞上清液中TNF-α和TGF-β1水平均高于对照组(P<0.05),干预组细胞上清液中TNF-α和TGF-β1水平均高于对照组和矽尘组(P<0.05)。干预组细胞中Akt蛋白表达水平分别低于对照组和矽尘组;矽尘组细胞中LC3Ⅱ/Ⅰ蛋白水平分别高于对照组和干预组(P<0.05),干预组和对照组细胞中LC3Ⅱ/Ⅰ蛋白水平比较,差异无统计学意义(P>0.05)。LSCM观察结果显示:对照组细胞3和6 h时间点自噬表达分别强于同组12和20 h时间点;矽尘组细胞12 h时间点自噬表达分别强于同组3和6 h,20 h时间点自噬表达稍弱于同组12 h,但仍强于同组3和6 h。与同时间点对照组比较,矽尘组细胞3和6 h时间点的自噬表达略减弱,但12和20 h时间点自噬表达�Objective To determine the regulating role of phosphatidylinositol 3-kinase（ PI3K） / protein kinase B（ Akt）signaling pathway in the autophagy activity of rat NR8383 cells exposed to silicon dioxide（ SiO_2）. LY294002 was used to block PI3 K pathway. Methods i） The normal NR8383 cells were used and divided into blank group and silica exposure group（ final concentrations of SiO_2 suspension were 0 and 50 mg / L respectively）. They were cultured for 3,6,12,20 and24 hours. The enzyme linked immunosorbent assay（ ELISA） was used to assess the amount of tumor necrosis factor-α（ TNF-α） and transforming growth factor-β1（ TGF-β1） in supernatants of cultured cells,and then the optimal time of cells exposed to dust was determined. ii） NR8383 cells were divided into control group（ treated with a same volume of F-12 K medium without serum）,silica group（ treated with SiO_2 suspension,final concentration 50 mg / L） and intervention group（ treated with SiO_2 suspension and PI3 K inhibitor LY294002,final concentration 50 mg / L and 20 μmol / L,respectively）.Cells were harvested following incubation. ELISA was used to detect the levels of TNF-α and TGF-β1 at the time point of20 hours after incubation. To reveal the autophagy status of cells,Western blotting was used to detect Akt and microtubuleassociated proteins 1 light chain 3（ LC3） protein at time point of 20 hours; laser scanning confocal microscope（ LSCM）was used to observe the immunofluorescence expression of autophagy at time points of 3,6,12 and 20 hours. The cells were also treated with the lysosomal inhibitor chloroquine diphosphate（ CDP） at the same time of SiO_2 treatment. Results i） The time point of 20 hours was confirmed to be the best dust exposure time for in vitro cell model of NR8383 cells.ii） The levels of TNF-α and TGF-β1 of supernatant in the silica group were higher than those of the control group（ P〈0. 05）. The levels of TNF-α and TGF-β1 of supernatant in the intervention group were

关 键 词：磷脂酰肌醇3-激酶 蛋白激酶B PI3K/AKT信号通路 自噬 肺泡巨噬细胞 肿瘤坏死因子-α 转化生长因子-β1 微管相关蛋白1轻链3 

分 类 号：R135.2[医药卫生—劳动卫生] R114[医药卫生—公共卫生与预防医学]