摘要
[目的]构建斑马鱼可调控突变型APPswe表达的阿尔茨海默症(AD)动物模型,以探究Aβ淀粉样蛋白Aβ沉积对脑血管系统的损伤及动物行为学的影响,为研究AD的治疗构建一个具有时间空间可控性的动物模型。[方法]构建重组质粒pTetlIP-APPswe-EGFP,导入慢病毒细胞并感染人神经母细胞瘤(SH-SY5Y)细胞,最后注入斑马鱼囊胚期的囊胚内;孵化APPswe细胞移植斑马鱼胚胎并培育至成年斑马鱼;评估斑马鱼行为学,如游泳、觅食及躲避风险等,并进行斑马鱼脑神经细胞透射电镜超微结构观察尼氏染色神经元计数。[结果]建立了APPswe斑马鱼体系,经过T型迷宫行为学检测显示,6月龄及12月龄的APPswe细胞移植斑马鱼经过训练后,寻找到营养丰富区(enriched chamber,EC)的时间改变不明显并随月龄增加呈延长趋势,提示6月龄及12月龄转基因斑马鱼出现了游泳、觅食及躲避风险障碍;斑马鱼脑超微结构变化在透射电镜下显示,6月龄APPswe斑马鱼细胞核形态不规则,核膜皱缩;12月龄APPswe斑马鱼的细胞器崩解破损,线粒体肿胀并出现空化,线粒体嵴间腔消失,内质网及高尔基体肿胀,细胞核塌陷皱缩;斑马鱼脑组织常规尼氏染色结果显示,6月龄及12月龄APPswe斑马鱼脑内神经元数量与正常野生型斑马鱼相比,神经元减少;12月龄APPswe斑马鱼脑内神经元数量与6月龄APPswe斑马鱼相比,神经元减少。[结论]APP诱导生成的Aβ蛋白能够造成斑马鱼脑血管系统的损伤及影响动物行为学,斑马鱼作为研究对象可为AD的治疗构建一个具有时间、空间可控性AD动物模型,但是不能培育APPswe细胞移植斑马鱼后代,限制了实验可持续研究。
[Objective]To construct an animal model of Alzheimer’s disease(AD)in which zebrafish can regulate the expression of mutant APPswe in order to investigate the effects of amyloid proteinβ(Aβ)deposition on cerebrovascular system damage and animal behavior.To build an animal model with time and space controllability for studying the treatment of AD.[Methods]Construct a recombinant plasmid pTetlIP-APPswe-EGFP,then introduce it into lentiviral cells and infect human neuroblastoma(SH-SY5 Y)cells,and finally inject them into the blastocysts of the zebrafish blastocyst stage;incubate APPswe cells to transplant zebrafish embryos and cultivate to adult zebrafish;evaluate zebrafish behaviors,such as swimming,foraging,and risk avoidance,and perform transmission electron microscopic observation of zebrafish brain nerve cells and Nissl-stained neuron counts.[Results]The APPswe zebrafish system was successfully established.T-maze behavioral testing showed that after 6 months and 12 months of APPswe cell transplantation,zebrafish were trained to find an enriched chamber(EC).It is obvious and shows a prolonged trend with increasing age,suggesting that genetically modified zebrafish at 6 and 12 months of age have experienced obstacles to swimming,foraging and avoiding risks.Zebrafish brain ultrastructural changes were observed under transmission electron microscopy.6-month-old APPswe zebrafish had irregular nucleus morphology and nuclear membrane shrinkage;12-month-old APPswe zebrafish had collapsed and damaged organelles,mitochondria swollen and cavitated,and mitochondria The lumen disappeared,endoplasmic reticulum and Golgi swelled,and the nucleus collapsed and shrunk.The conventional Nissl staining of the brain tissue of zebrafish showed that the number of neurons in the brain of APPswe zebrafish at 6 months and 12 months was compared with that of normal wild type zebrafish.Neurons decreased;the number of neurons in the brain of APPswe zebrafish at 12 months of age was reduced compared with that of APPswe zebrafish at 6 mont
作者
郭双磊
张悠然
董军成
苑兵舰
李凯歌
刘红林
GUO Shuanglei;ZHANG Youran;DONG Juncheng;YUAN Bingjian;LI Kaige;LIU Honglin(Department of neurosurgery Huaihe Hospital of Henan University,Kaifeng 475000,China;Department of neurology Huaihe Hospital of Henan University,Kaifeng 475000,China)
出处
《河南大学学报（医学版）》
CAS
2020年第2期102-107,共6页
Journal of Henan University:Medical Science
基金
河南省高等学校重点科研项目(16A320035)。
