亚砷酸钠诱导大鼠胰岛素瘤细胞自噬性死亡作用  

Sodium arsenite-induced autophagic cell death in rat insulinoma cells

作　　者：程勇[1] 唐文娟[1] 万亭池 梁冰[1,2] CHENG Yong;TANG Wen-juan;WAN Ting-chi(Pharmacology Department,Guizhou Medical Univesity,Guiyang,Guizhou Province 550025,China)

机构地区：[1]贵州医科大学药理学教研室,贵阳550025 [2]环境污染与疾病监控省部共建教育部重点实验室

出　　处：《中国公共卫生》2019年第12期1637-1640,共4页Chinese Journal of Public Health

基　　金：国家自然科学基金(81760575,81430077);贵阳市科技计划项目(筑科合同[2017]5–31号);贵州省科技创新人才团队项目[黔科合平台人才(2016)5625号]

摘　　要：目的探讨自噬在亚砷酸钠(NaAsO2)诱导大鼠胰岛素瘤细胞(INS-1)死亡过程中的作用及分子机制。方法实验设阴性对照组,NaAsO2(30、15、5μmol/L)组;NaAsO2作用INS-1细胞24 h后,采用CCK8比色法检测INS-1细胞的存活率;透射电子显微镜观察INS-1细胞超微形态;激光扫描共聚焦显微镜观察转染质粒GFP-LC3后INS-1细胞内荧光的表达;蛋白印迹法(WB)法检测LC3、P62、mTOR、PI3K蛋白表达。结果经NaAsO2作用后,INS-1细胞存活率明显下降;NaAsO2组INS-1细胞中可见双层膜自噬小体与自噬空泡明显增多;转染质粒GFP-LC3后NaAsO2组荧光显微镜下可见绿色点状荧光斑点聚集增多。WB结果表明与阴性对照组LC3Ⅱ/LC3Ⅰ[(0.93±0.04)]、P62[(0.57±0.01)]、mTOR[(1.44±0.03)]、PI3K[(1.26±0.07)]比较,30、15、5μmol/L NaAsO2组INS-1细胞中LC3Ⅱ/LC3Ⅰ蛋白表达量[分别为(1.34±0.02、1.16±0.02、1.13±0.04)]升高,P62蛋白表达量[分别为(1.89±0.14、1.08±0.06、0.71±0.03)]升高,mTOR蛋白表达量[分别为(0.46±0.04、0.92±0.06、1.15±0.06)]降低,PI3K蛋白表达量[分别为(0.78±0.04、0.87±0.03、0.91±0.04)]降低,差异具有统计学意义(P<0.05)。结论NaAsO2暴露可诱导INS-1细胞自噬性死亡,其机制可能与调控PI3K/mTOR信号通路有关。Objective To study the role and molecular mechanism of autophagy in death of rat insulinoma cells(INS-1)induced by sodium arsenite(NaAsO2).Methods Three dose groups(administered with NaAsO2 of 30,15,5μmol/L for 24 hours)and one negative control group of rat INS-1 cells were established.The survival rate of INS-1 cells was detected with cholecystokinin-8(CCK8)colorimetry.The ultrastructure of INS-1 cells was observed with transmission electron microscope.The fluorescence expression of INS-1 cells after transfection of plasmid GFP-LC3 was observed with laser scanning confocal microscopy.The expression of light chain 3(LC3),P62,mammalian target of rapamycin(mTOR)and phosphatidylinositol 3-kinase(PI3K)protein was detected with Western blot.Results After NaAsO2 treatment,the survival rate of INS-1 cells decreased significantly.Two-layer membrane autophagosomes and autophagic vacuoles were significantly increased in INS-1 cells of NaAsO2 groups.After transfection of plasmid GFP-LC3,the aggregation of green dot-like fluorescent spots was observed under the fluorescence microscope of NaAsO2 groups.Compared with those in the control cells,significantly increased protein expressions of LC3II/LC3I(1.34±0.02,1.16±0.02,and 1.13±0.04 vs.0.93±0.04)and P62(1.89±0.14,1.08±0.06,and 0.71±0.03 vs.0.57±0.01),but decreased protein expressions of mTOR(0.46±0.04,0.92±0.06,and 1.15±0.06 vs.1.44±0.03)and PI3K(0.78±0.04,0.87±0.03,and 0.91±0.04 vs.1.26±0.07)were detected in the INS-1 cells treated with 30,15,and 5μmol/L NaAsO2(P<0.05 for all).Conclusion NaAsO2 treatment can induce autophagic death in INS-1 cells and the mechanism of the effect may be related to the regulation of PI3K/mTOR signaling pathway.

关 键 词：自噬 大鼠胰岛素瘤细胞 亚碑酸钠 

分 类 号：TQ126.41[化学工程—无机化工]