scAAV9-IGF-1转导至G93A-SOD1小鼠神经元及其作用机制  

scAAV9-IGF-1 transduction of G93A-SOD1 transgenic mouse neurons and its mechanism

作　　者：温迪[1,2] 吉盈肖 刘亚坤[1] 李秋生[4] 段伟松[1] 陈相[1] 于秀军[1] Wen Di;Ji Yingxiao;Liu Yakun;Li Qiusheng;Duan Weisong;Chen Xiang;Yu Xiujun(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临床神经病学教育部重点实验室,石家庄050000 [3]河北省人民医院神经内科 [4]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》2023年第12期766-771,共6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)。

摘　　要：目的探索自我互补腺相关病毒载体介导的人胰岛素样生长因子-1(scAAV9-IGF-1)转导至G93A-SOD1转基因小鼠神经元及其对线粒体的作用。方法采用肌萎缩侧索硬化(ALS)的转基因G93A-SOD1及野生型(wild type-SOD1,WT-SOD1)小鼠为动物模型,在60 d龄时,同窝阳性G93A-SOD1转基因雌性小鼠采用随机的方法分配到治疗组肌肉注射scAAV9-IGF-1,载体对照组肌肉注射AAV9-GFP,及同年龄WT-SOD1作为阴性对照。在肌肉注射后约40~50 d时,通过免疫组化染色观察IGF-1在腰髓神经元中的表达,利用免疫荧光染色观测腰髓组织绿色荧光蛋白(GFP)的表达特点;用透射电镜观察腰髓前角运动神经元中线粒体的形态学变化;提取小鼠脊髓细胞进行流式细胞学分析线粒体膜电位变化;腓肠肌进行免疫组化染色观察线粒体变化。结果免疫荧光染色观测腰髓组织GFP的表达主要在神经元,免疫组化染色观察IGF-1表达于腰髓神经元中。透射电镜观察GFP组线粒体存在嵴消失、空泡化、胞浆水肿、核染色质疏松,而IGF-1组线粒体形态与WT组相似,为长椭圆形,未见嵴消失、空泡化等现象。流式细胞学分析IGF-1组降低了线粒体膜电位的除极。在GFP对照组中腓肠肌的苏木精-伊红染色中观察到严重萎缩的肌纤维,而在IGF-1处理的小鼠中肌肉萎缩减少。Gomori染色显示IGF-1处理后,肌肉细胞的线粒体染色增多,同WT组相当。结论scAAV9-IGF-1可以保护G93A-SOD1转基因小鼠模型中的线粒体,其通过减少线粒体膜电位除极,改善线粒体形态,减少腓肠肌肌肉萎缩,从而产生保护线粒体的作用。Objective To explore self-complementary adeno-associated virus human insulin-like growth factor-1(scAAV9-IGF-1)transduction to the neurons of G93A-SOD1 transgenic mouse and its effect on mitochondria.Methods We used amyotrophic lateral sclerosis(ALS)transgenic G93A-SOD1 and wild-type(WT-SODI)mice as animal models.At 60 days of age,littermate-positive G93A-SOD1 transgenic female mice were randomly assigned to the treatment group by intramuscular injection scAAV9-IGF-1,the vector control group was intramuscularly injected with AAV9-GFP,and the same age WT-SOD1 was used as a negative control.About 40-50 days after intramuscular injection,the expression of IGF-1 in the lumbar spinal cord neurons was observed by immunohistochemical staining,and the expression characteristics of GFP in the lumbar spinal cord tissue were observed by immunofluorescence staining;transmission electron microscopy was used to observe the motor nerve of the anterior horn of the lumbar spinal cord.The morphological changes of mitochondria in cells were extracted;the mitochondrial membrane potential changes were analyzed by flow cytometry in mouse spinal cord cells;the mitochondrial changes were observed by immunohistochemical staining of gastrocnemius muscle.Results The expression of GFP in the lumbar spinal cord was observed by immunofluorescence staining,and the expression of IGF-1 was observed in the neurons of the lumbar spinal cord by immunohistochemical staining.The mitochondria in the GFP group had disappearance of cristae,vacuolization,cytoplasmic edema,and loose nuclear chromatin observed by transmission electron microscopy,while the morphology of the mitochondria in the ICF-1 group was similar to that of the WT group,with an oblong shape,without the disappearance of cristae and vacuolization.Flow cytometric analysis showed that the depolarization of mitochondrial membrane potential was reduced in the IGF-1 group.Severely atrophic myofibers were observed in HE staining of gastrocnemius muscle in the GFP control group,whereas muscle at

关 键 词：肌萎缩侧索硬化 转基因小鼠 腺相关病毒 人胰岛素样生长因子-1 线粒体膜电位 

分 类 号：R741[医药卫生—神经病学与精神病学]