5-Aza-CdR促进小鼠海马神经细胞HT22自噬增加通过启动TSC1/mTOR通路  

5-Aza-2′-deoxycytidine induce hypoxia tolerance dependent autophagy in mouse neuronal cells by initiating TSC1/mTOR pathway

作　　者：齐瑞芳[1] 包素雅 邵国[1] QI Rui-fang;BAO Su-ya;SHAO Guo(Department of Key Laboratory of Hypoxic Adaptive Translational Medicine,Institute of Neuroscience,Preclinical and Forensic Medicine of Baotao Medical College,Baotou Inner Mongolia 014060,China;West China clinical medical college,Sichuan university,Chengdu 610041,China)

机构地区：[1]包头医学院低氧适应转化医学重点实验室,基础医学与法医学院,包头医学院神经科学研究所,内蒙古包头014060 [2]四川大学华西临床医学院,四川成都610041

出　　处：《中国药理学通报》2020年第9期1265-1269,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81660307);内蒙古自治区自然科学基金资助项目(No 2019MS03093,2018LH08078);内蒙古自治区高等学校科学研究资助项目(No NJZY20171);包头医学院科学基金资助项目(No BYJJ-DF201602,BYJJ-QM 201644)。

摘　　要：目的研究5-杂氮脱氧胞嘧啶核苷(5-Aza-2′-deoxycytidine,5-Aza-CdR)能否诱导小鼠海马神经细胞HT22产生自噬对以及TSC1/mTOR通路是否参与其中。方法通过体外培养HT22细胞,5-Aza-CdR孵育24 h后,采用MTS检测对细胞活力的影响。采用RT-PCR和Western blot,检测TSC1、mTOR和LC3的mRNA和蛋白表达水平;采用组织免疫荧光的方法检测TSC1在不同组别HT22细胞中的表达;通过转染TSC1质粒,检测TSC1/mTOR途径在自噬中的作用。结果10μmol·L^-15-Aza-CdR可降低HT22细胞的活力,5-Aza-CdR使TSC1 mRNA和蛋白表达水平增加,p-mTOR(Ser2448)蛋白水平降低,LC3-Ⅱ/LC3-Ⅰ比值升高;过表达TSC1后小鼠海马HT22细胞LC3-Ⅱ/LC3-Ⅰ比值升高。结论5-Aza-CdR可以诱导HT22自噬增加,可能是通过启动TSC1/mTOR通路。Aim To investigate whether 5-Aza-CdR can induce autophagy in mouse hippocampal neurons HT22 and whether TSC1/mTOR pathway is involved.Methods HT22 cells were cultured in vitro and 5-Aza-CdR was incubated for 24 hours,then cell viability was detected by MTS.Real time RT-PCR and Western blot were used to detect the mRNA and protein expression levels of TSC1,mTOR,and LC3.Tissue immunofluorescence was used to detect the expression of TSC1 of HT22 cells in different groups.TSC1 plasmid was transfected to HT22 cells and the role of TSC1/mTOR pathway in autophagy was assessed.Results 10μmol·L^-15-Aza-CdR reduced the viability of HT22 cells,5-Aza-CdR increased the expression level of TSC1 mRNA and protein,p-mTOR(Ser2448)protein level,LC3-Ⅱ/LC3-Ⅰratio increased.LC3-Ⅱ/LC3-Ⅰratio increased in mouse hippocampal HT22 cells after over-expression of TSC1.Conclusion 5-Aza-CdR can induce increased autophagy of HT22,possibly by activating TSC1/mTOR pathway.

关 键 词：5-AZA-CDR HT22细胞 自噬 TSC1 MTOR 细胞活力 

分 类 号：R-332[医药卫生] R322.81