AAV9-OPTN对肌萎缩侧索硬化模型小鼠自噬通路的作用机制研究  

Mechanism of AAV9-OPTN on autophagy pathway in atrophic lateral sclerosis model mice

作　　者：温迪[1] 李秋生[2] 李媛媛[1] 王燕燕[1] 刘亚坤[1] Wen Di;Li Qiusheng;Li Yuanyuan;Wang Yanyan;Liu Yakun(Department of Neurology,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学第二医院神经内科,石家庄050000 [2]河北医科大学第二医院肝胆外科,石家庄050000

出　　处：《脑与神经疾病杂志》2025年第2期77-81,共5页Journal of Brain and Nervous Diseases

基　　金：河北省自然科学基金青年科学基金项目(H2021206048);河北省医学科学研究课题计划(20240793)。

摘　　要：目的探索以自我互补双链腺相关病毒9为载体介导的视神经病变诱导反应蛋白(AAV9-OPTN)对肌萎缩侧索硬化(ALS)SOD1-G93A转基因模型小鼠自噬通路的作用。方法采用ALS的转基因SOD1-G93A突变型小鼠动物模型,在其出生后30~40 d龄时采用随机的方法分配到治疗组及对照组,治疗组鞘内注射AAV9-OPTN,对照组鞘内注射AAV9-GFP。在注射40~50 d后,利用蛋白印迹技术检测腰髓中OPTN含量的变化,利用免疫荧光染色观察OPTN的定位情况,同时利用免疫荧光染色观察自噬通路相关蛋白p62、TBK1、LC3的染色情况及荧光含量变化。结果蛋白印迹结果显示鞘内注射AAV9-OPTN后,腰髓中OPTN的蛋白含量显著高于GFP对照组,免疫荧光染色OPTN的荧光强度显著高于溶剂对照组(~均P<0.01)。对自噬通路蛋白的免疫荧光染色结果显示,AAV9-OPTN治疗组中TBK1、LC3的荧光强度显著高于对照组,而p62的荧光强度降低,与对照组差异比较均有统计学意义。结论AAV9-OPTN可以转导至小鼠腰髓中并诱导OPTN蛋白表达,能够激活SOD1-G93A转基因小鼠模型中的自噬通路。Objective To explore the effect of self complementary double stranded adenovirus 9 mediated optineurin(AAV9-OPTN)on the autophagy pathway in a transgenic mouse model of amyotrophic lateral sclerosis(ALS)SOD1-G93A.Methods The animal model of ALS,transgenic SOD1-G93A mutant mice were used.At 30-40 days after birth,they were randomly assigned to the treatment group and control group.The treatment group received intrathecal injection of AAV9-PTN,while the control group received intrathecal injection of AAV9-GFP.After 40-50 days of Oinjection,immunoblotting technology was used to detect changes in the content of OPTN in the lumbar spinal cord.Immunofluorescence staining was used to observe the localization of OPTN,while immunofluorescence staining was used to observe the staining and fluorescence content changes of autophagy pathway related proteins p62,TBK1,LC3.Results The immunoblotting showed that after intrathecal injection of AAV9-OPTN,the protein content of OPTN in the lumbar spinal cord was significantly higher than that in the GFP control group,and the fluorescence intensity of immunofluorescence staining for OPTN was significantly higher than that in the solvent control group(~(all)P<0.01).The immunofluorescence staining results of autophagy pathway proteins showed that the fluorescence intensity of TBK1 and LC3 in the AAV9-OPTN treatment group were significantly higher than that in the control group,while the fluorescence intensity of p62 decreased,showing statistical differences compared to the control group.Conclusion AAV9-OPTN can be transferred into the lumbar spinal cord of mice and induced the expression of OPTN protein,which can activate the autophagy pathway in the SOD 1-G93A transgenic mouse model.

关 键 词：肌萎缩侧索硬化 SOD1-G93A转基因小鼠 腺相关病毒9 视神经病变诱导反应蛋白 自噬通路 

分 类 号：R741[医药卫生—神经病学与精神病学]