摘要
目的检测过表达婆罗双树样基因2(Sall2)对人超氧化物歧化酶1(hSOD1)-G93A突变型肌萎缩侧索硬化症(ALS)转基因小鼠疾病进展的影响,为ALS基因治疗提供潜在靶点。方法通过生物信息学分析筛选出差异基因Sall2。选取48只ALS转基因小鼠,通过构建具有神经元特异性启动子人突触蛋白Ⅰ(hSyn)的AAV-PHP.eB-Sall2腺相关病毒,尾静脉输入6周龄小鼠体内,以回输AAV-PHP.eB-GFP腺相关病毒到同窝ALS小鼠作为对照组。免疫荧光双标染色检测小鼠脊髓和大脑皮层Sall2与神经元核抗原(NeuN)/GFAP染色情况,期间检测小鼠生存期、体重、运动能力和腓肠肌肌电变化;尼氏染色检测小鼠脊髓前角神经元形态学变化;Western blotting检测过表达Sall2基因对细胞周期蛋白E1(cyclin E1)表达的影响。结果生物信息学分析筛选到Sall2在ALS小鼠脊髓中异常低表达;相较于对照组ALS小鼠,过表达Sall2基因组ALS小鼠脊髓和大脑皮层中Sall2蛋白表达均增多,Sall2^(+)/NeuN+双阳性细胞中Sall2积分吸光度值均升高。过表达Sall2基因组ALS小鼠生存时间延长,体重下降速度变缓,转棒时间和倒置时间延长,腓肠肌肌电正锐波和纤颤电位减少。过表达Sall2基因组ALS小鼠脊髓前角尼氏体标记的神经元数量增加,神经元受损情况得到改善。过表达Sall2基因降低ALS小鼠脊髓和大脑皮层中cyclin E1的表达。结论过表达Sall2基因可延缓ALS转基因小鼠疾病进展,改善运动能力,影响cyclin E1的表达,对ALS转基因小鼠有一定的治疗效果。
Objective To investigate the effect of sal⁃like gene 2(Sall2)gene overexpression on the progression of disease in human superoxide dismutase 1(hSOD1)⁃G93A mutant amyotrophic lateral sclerosis(ALS)transgenic mice,with the aim of identifying potential therapeutic targets for ALS gene therapy.Methods Differential Sall2 gene were screened through bioinformatics analysis.Forty⁃eight ALS transgenic mice were selected for this study.AAV⁃PHP.eB⁃Sall2 adeno⁃associated virus with a neuron⁃specific promoter,human synapsin I(hSyn),was constructed and administered via tail vein injection to six⁃week⁃old mice.In parallel,the same litter of ALS mice received an injection of AAV⁃PHP.eB⁃GFP.The staining of Sall2 and neuron⁃specific nuclear protein(NeuN)/GFAP in the spinal cord and cerebral cortex of mice were detected through immunofluorescent double⁃label staining technology.The survival period,weight changes,exercise ability,and electromyographic changes of the gastrocnemius muscle were detected.The morphological changes in the spinal cord anterior horn neurons were detected through Nissl staining.The effect of Sall2 gene overexpression on the expression of the cell cycle protein E1(cyclin E1)was investigated through Western blotting.Results Bioinformatics analysis showed out that Sall2 was differentially expressed in ALS mice.Compared with ALS mice in the control group,the Sall2 protein expression of ALS mice in the overexpressing Sall2 gene group increased in both the spinal cord and cerebral cortex,and the Sall2 integral absorbance values of Sall2^(+)/NeuN^(+)double⁃positive cells were higher.The survival time of ALS mice in the Sall2 gene overexpressing group was prolonged,the rate of weight loss was slowed down,the performance in the rotarod and inverted grid tests was improved with longer times,and the positive sharp waves and fibrillation potentials in the gastrocnemius electromyography were reduced.The number of Nissl bodies labeled neurons increased in the spinal cord anterior horn of the Sall2 gene overexpressing mice,and the condition of neuronal damage was improved.Overexpression of the Sall2 gene also reduced the expression of cyclin E1 in both the spinal cord and cerebral cortex of ALS transgenic mice.Conclusion Overexpression of the Sall2 gene can delay disease progression and improve motor performance in ALS transgenic mice,affecting the expression of cyclin E1,thus exerting a therapeutic effect on these mice.
作者
张雪
王晨晨
高学帅
白雪
王雪枚
刘金梦
管英俊
陈燕春
ZHANG Xue;WANG Chen-chen;GAO Xue-shuai;BAI Xue;WANG Xue-mei;LIU Jin-meng;GUAN Ying-jun;CHEN Yan-chun(Histology and Embryology Department,Shandong Second Medical University,Shandong Weifang 261053,China;Neurologic Disorders and Regenerative Repair Lab of Shandong Higher Education,Shandong Weifang 261053,China)
出处
《解剖学报》
2025年第2期127-135,共9页
Acta Anatomica Sinica
基金
国家自然科学基金(82271483)
山东省自然科学基金(ZR2020MH150)
山东省高等学校青创科技支持计划项目(2019KJK004)。
