PI3KC3/Beclin1复合物对染矽尘NR8383细胞自噬的调控作用  

Regulation of PI3KC3/Beclin1 complex on autophagy of NR8383 macrophages exposed to silica dust

作　　者：王路遥 李宁 宋瑞瑞 张晓雪 万斌斌 李小明 李清钊 沈福海 蒋守芳 金玉兰 WANG Lu-yao;LINing;SONG Rui-rui;ZHANG Xiao-xue;WAN Bin-bin;LI Xiao-ming;LI Qing-zhao;SHEN Fu-hai;JIANG Shou-fang;JIN Yu-lan(School of Public Health,North China University of Science and Technology,Tangshan,Hebei 063000,China)

机构地区：[1]华北理工大学公共卫生学院,河北唐山063000

出　　处：《环境与职业医学》2018年第9期801-808,共8页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(编号:81202161)

摘　　要：[目的]探讨PI3KC3/Beclin1复合物在染矽尘大鼠肺泡巨噬细胞(NR8383)自噬中的调控作用。[方法]常规培养NR8383细胞,随机分为4组:对照组,矽尘组,3-MA组和3-MA干预组,分别孵育1、3、6、12、20 h后收集样品。采用蛋白免疫印迹方法检测自噬标记蛋白微管相关蛋白1轻链3(LC3)、自噬底物蛋白p62以及自噬相关蛋白Beclin1、PI3KC3;采用激光扫描共聚焦显微镜观察孵育不同时间的自噬免疫荧光表达情况;采用免疫共沉淀检测Beclin1复合物表达。[结果]免疫荧光结果显示,矽尘组LC3点状聚集绿色荧光亮斑随时间先增强后减弱,在6 h时荧光最强,且各时间点较对照组和3-MA干预组荧光亮斑增强;3-MA干预组LC3荧光标记绿色亮斑先增强后减弱,弱于矽尘组,但强于对照组。加入溶酶体抑制剂二磷酸氯喹后,各组LC3绿色荧光信号随时间均呈现增强的趋势。Western blot结果显示,矽尘组LC3-Ⅱ/LC3-Ⅰ值呈先增加后减少趋势,且在各个时间点均高于对照组(P <0.05);在LC3-Ⅱ/LC3-Ⅰ值达最高的6 h时间点,矽尘组的值是对照组的248%,是3-MA组的372%。3-MA干预组LC3-Ⅱ/LC3-Ⅰ值也呈先增加后减少趋势;除1 h时间点外,余各时间点均低于矽尘组,但高于对照组(P <0.05);在LC3-Ⅱ/LC3-Ⅰ值达最高的6 h时间点,3-MA干预组的值是矽尘组的90%,是对照组的223%。Beclin1、PI3KC3蛋白表达趋势与LC3-Ⅱ/LC3-Ⅰ值一致,p62蛋白表达趋势与LC3-Ⅱ/LC3-Ⅰ值相反。免疫共沉淀显示,矽尘组Beclin1与PI3K结合增加,3-MA干预后两者结合减少。[结论]矽尘诱导NR8383细胞的自噬活动随时间发生不同程度的改变,3-MA可下调矽尘诱导的NR8383细胞自噬活动,推测PI3KC3/Beclin1信号通路参与矽尘诱导肺泡巨噬细胞的自噬过程。[Objective] To observe the regulation of PI3KC3/Beclin 1 on silica dust-induced autophagy in NR8383 cells. [ Methods ] NR8383 cells were randomly divided into four groups： control group, silica dust group, 3-MA group, and 3-MA intervention group. The samples were collected at 1h, 3 h, 6 h, 12 h, and 20 h after incubation. Western blot was used to detect microtube associated protein light chain 3 （LC3）, autophagy substrate p62, as well as autophagy related proteins Bec｜inl and PI3KC3. Laser scanning confocal microscopy was used to observe the green fluorescence emitted by autophagy activity after varied incubation durations. Co-immunoprecipitation was used to detect the expression of Beclin 1 complex. [ Results ] The results of immunofluorescence showed that the green fluorescence spot of the silica dust group was enhanced first and then decreased, the fluorescence intensity was highest at 6 h, and the fluorescence intensity of the silica dust group was elevated at all selected time points compared with the control group and the 3-MA intervention group. In the 3-MA intervention group, the LC3 fluorescent marker green spot was first enhanced and weakened, and was weaker than the silica dust group, but strongerthan the control group. After adding chloroquine diphosphate （CDP）, the LC3 green fluorescence signal of each group showed an increasing trend with time. Western blot results showed that the value of Lc3qI/LC3-1 in the silica group increased first and then decreased, and was higher than the control group at each time point （P〈O.05）. The value of LC3-II/LC3-I was highest at 6h, when the value of the silica dust group was 248% of the control group and 372% of the 3-MA group. The value of LC3-II/LC3-I in the 3-MA intervention group increased first and then decreased, and was lower than the silica dust group at each time point, but was higher than the control group （P〈0.05）. At 6h when LC3-1t/LC3-I value was highest, the value of the 3-MA intervention group was 90% of the silica dust group

关 键 词：自噬 NR8383细胞 Beelin1 Ⅲ型P13K P62 

分 类 号：R135.2[医药卫生—劳动卫生]