降低斑马鱼cntnap2表达建立注意缺陷多动障碍遗传模型  

Reduce the expression of cntnap2 in zebrafish to explore a genetic model of attention-deficit/hyperactivity disorder

作　　者：陈维 赵峰 张樱腊 刘东 杨莉[1] Chen Wei;Zhao Feng;Zhang Yingla;Liu Dong;Yang Li(Peking University Sixth Hospital/Institute of Mental Health,National Clinical Research Center for Mental Disorders(Peking University Sixth Hospital),Key Laboratory of Mental Health(Peking University),Beijing 100191,China;Department of Biology,Southern University of Science and Technology,Shenzhen 518055,China;School of Life Sciences,Peking University,Beijing 100871,China)

机构地区：[1]北京大学第六医院北京大学精神卫生研究所国家精神心理疾病临床医学研究中心(北京大学第六医院)卫生部精神卫生学重点实验室(北京大学),100191 [2]南方科技大学生物系,深圳518055 [3]北京大学生命科学学院,100871

出　　处：《中华精神科杂志》2020年第5期431-437,共7页Chinese Journal of Psychiatry

基　　金：国家自然科学基金面上项目(81873803,31571496)。

摘　　要：目的通过降低斑马鱼接触蛋白相关蛋白基因2(contact protein-related protein gene 2,cntnap2)表达,建立注意缺陷多动障碍(attention-deficit/hyperactivity disorder,ADHD)遗传模型。方法以注射剪接抑制型吗啉代寡聚核糖核酸降低cntnap2表达的斑马鱼作为实验组(n=48),注射随机寡核苷酸片段的斑马鱼作为对照组(n=48),通过原位杂交观察斑马鱼神经递质系统相关基因th、dbh、gad1b、glyt2、vglut2表达的变化,使用实时荧光定量PCR检测神经递质系统及神经元发育相关基因表达量变化。使用斑马鱼行为轨迹跟踪系统记录(Noldus行为仪)检测cntnap2表达降低后多动/冲动行为及托莫西汀对多动/冲动行为的改善作用。结果原位杂交实验结果显示实验组较对照组神经递质系统相关基因dbh表达减少,gad1b表达增加;th、glyt2、vglut2表达未见明显差异。实时荧光定量PCR显示实验组较对照组神经递质系统相关基因dbh表达降低(t=5.772,P=0.005),snap25、dat、gad1b表达增高(t=14.310、22.210、22.090,均P<0.01);神经元发育相关基因shha、epha4b、netrin1b、noi及神经递质系统相关基因drd4、th、vglut2、glyt2表达差异均无统计学意义。6 dpf时药物未处理实验组较药物未处理对照组幼鱼游动总距离长[143.12(98.56,212.22)cm与99.03(80.54,133.96)cm,Z=-3.547,P<0.01],平均游动速度快[0.050(0.041,0.067)cm/s与0.033(0.025,0.042)cm/s,Z=-5.495,P<0.01];经托莫西汀处理后,实验组药物处理较药物未处理幼鱼游动总距离减少[106.59(95.28,120.10)cm与143.12(98.56,212.22)cm,Z=-3.591,P<0.01],平均游动速度减慢[0.031(0.028,0.037)cm/s与0.050(0.041,0.067)cm/s,Z=-7.035,P<0.01]。结论cntnap2表达降低斑马鱼可通过改变dbh、snap25、dat、gad1b表达量而产生多动/冲动表型,托莫西汀可有效缓解多动/冲动表型,cntnap2表达降低斑马鱼可作为ADHD遗传模型。Objective By knocking down the expression of contact protein-related protein gene 2(cntnap2)in zebrafish,this current study explored whether the reduced expression of cntnap2 could be used as a genetic model for attention-deficit/hyperactivity disorder(ADHD)in zebrafish.Methods Forty-eight zebrafish were allocated in the experimental group using splicing Morpholino oligonucleotides(MO)to knock down cntnap2 expression while another 48 zebrafish were assigned to the control group by injecting Random MO(RM).In situ hybridization was used to observe the expression changes of neurotransmitter system related genes,such as th,dbh,gad1b at 1-3 dpf,glyt2 at 2-4dpf and vglut2 at 2-3 dpf,after knockdown of cntnap2.Real-time fluorescence quantitative PCR was used to detect the above neurotransmitter system related genes at 3dpf,the expression changes of axon-guided genes shha,epha4b,netrin1b,and noi at 1dpf and neurotransmitter system related genes drd4,snap25,and dat at 3 dpf.The hyperactivity/impulsivity behavior of zebrafish after knockdown of cntnap2 was detected by the Noldus behavioral instrument,which is also used to detect the improvement of hyperactivity/impulsivity by Atomoxetine(5μmol/L),a type of medication to alleviate ADHD.Results After cntnap2 knockdown,the expression area of dbh decreased and the expression area of gad1b increased,which were detected by in situ hybridization at 1-3 dpf.No expression area in the brain changes of th at 1-3 dpf,glyt2 at 2-4 dpf and vglut2 at 2-3 dpf were found.Compared to RM group,real-time quantitative PCR results showed that after knockdown cntnap2,the expression level of shha,epha4b,netrin1b,and noi did not change significantly at 1dpf.The expression level of dbh(t=5.772,P=0.005)was found to be decreased while dat(t=22.21,P<0.01),snap25(t=14.31,P<0.01),and gad1b(t=22.09,P<0.01)increased at 3dpf.Besides,the expression level of drd4,th,vglut2,and glyt2 showed no significant changes at 3dpf.At 6dpf,the total swimming distance(143.12(98.56,212.22)cm vs.99.03(80.54,133.96)cm,Z=-3

关 键 词：注意力缺陷障碍伴多动 斑马鱼 模型 动物 接触蛋白相关蛋白基因2 

分 类 号：R749.94[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]