一种PTEN基因下调的孤独症幼鼠模型构建的改良方法  

An improved method for the establishment of an autistic mouse model with down-regulation of PTEN gene

作　　者：党伟利[1,2] 梁绿圆 曹佳蕾 丁申奥 魏炳琦 邱霞 马丙祥[1,2] Dang Weili;Liang Lvyuan;Cao Jialei;Ding Shenao;Wei Bingqi;Qiu Xia;Ma Bingxiang(Dept of Pediatrics,The First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450099;Pediatric Medical College,Henan University of Chinese Medicine,Zhengzhou 450046)

机构地区：[1]河南中医药大学第一附属医院儿科医院,郑州450099 [2]河南中医药大学儿科医学院,郑州450046

出　　处：《安徽医科大学学报》2025年第3期462-471,共10页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金项目(编号:81973904);河南省特色骨干学科中医学学科建设项目(编号:STG-ZYX06-202144、STG-ZYXKY-2020023);2023年度河南省中医学“双一流”创建科学研究专项课题项目(编号:HSRP-DFCTCM-2023-2-08、HSRP-DFCTCM-2023-3-06、HSRP-DFCTCM-2023-05-08);河南省中医药科学研究专项课题项目(编号:2023ZY2057、2021ZY2111)。

摘　　要：目的构建一种丙戊酸(VPA)和PTEN腺病毒(ADV)联合应用的PTEN基因下调ASD幼鼠模型(VPA-ADV),并与VPA和ADV两种传统模型进行比较,证明此模型在针刺治疗ASD造模的优势。方法将怀孕12.5 d的Wistar大鼠随机分为2组,分别给予VPA(600 mg/kg)和等剂量的生理盐水作为VPA和正常组。记录新生幼鼠体质量、睁眼时间、尾巴畸形情况。正常组幼鼠随机分为3组,每组10只,设置为:正常(NS)组、病毒(ADV)组和病毒阴性对照(ADV-NC)组;VPA组幼鼠20只,随机分为2组,每组10只,设置为丙戊酸(VPA)组、丙戊酸结合病毒干扰(VPA+ADV)组。观察5组幼鼠出生后的体质量、尾长、弯尾、负趋地性反射时间、骨骼畸形情况、21日龄神经行为学表现、脑组织电镜髓鞘结构;通过免疫组化、Western blot、q-PCR方法检测PTEN、PI3K、AKT、GSK3β、MBP表达水平。结果与NS组孕鼠活产幼鼠对比,VPA组孕鼠活产幼鼠畸形率显著增加,体质量增长慢,尾长增长慢,负趋地性反射时间长(P<0.05)。3个模型组较NS组体质量增长慢,尾长增长慢,负趋地性反射时间长(P<0.05),且VPA+ADV组表现最为显著。3组模型旷场试验跨中央格时间、跨边缘格次数、跨边缘格时间与NS组差异均有统计学意义(P<0.05)。自我捋毛实验中VPA+ADV组互动次数最少(P<0.05),且挖掘、自我捋毛次数最多(P<0.05)。三箱社交实验中,VPA+ADV组进入社交箱平均次数以及停留时间最短(P<0.05),其在水迷宫实验中找到平台所用的时间最长,穿越第三象限次数最少(P<0.05)。透射电镜观察胼胝体区髓鞘板层结构显示NS组结构清晰完整。与NS组相比,ADV-NC组髓鞘结构近似,3个模型组髓鞘结构分层、断裂,存在ASD病理改变及髓鞘损伤,其中VPA+ADV组髓鞘增厚、分层明显,严重的可见崩解。免疫组化、Western blot及q-PCR结果均表明VPA+ADV组的PTEN表达下调约50%,最为明显。AKT、MBP均表达增高,GSK3β表达降低(P<0.05)。免疫组化�Objective To establish a PTEN gene-downregulated ASD juvenile rat model(VPA-ADV)through combined application of valproic acid(VPA)and PTEN adenovirus(ADV),then to compare the newly constructed animal model with two traditional autistic animal models of VPA and AD,and ultimately to prove the advantages of this model in animal model establishment of acupuncture treatment for ASD.Methods Wista rats at 12.5 days of gestation were randomly divided into 2 groups.VPA rats were given 600 mg/kg of normal saline(NS)intraperitoneally.Weight,eye opening time and tail deformity were recorded.The newborn mice in NS group were randomly divided into three groups(10 rats in each group):normal(NS)group,virus(ADV)group and virus-negative control(ADV-NC)group;VPA group(20 young rats)was randomly divided into 2 groups(10 rats in each group):valproic acid(VPA)group and valproic acid-binding virus interference(VPA+ADV)group.The body weight,tail length,curved tail,geotaxis text time and skeletal deformity of 5 groups of young rats after birth,the neurobehavioral behavior of 21-day-old rats,and the myelin structure of brain tissue under electron microscope were observed,the expression levels of PTEN,PI3K,AKT,GSK3βand MBP were detected by immunohistochemistry,Western blot and q-PCR.Results Compared with the NS group,the VPA group had significantly increased malformation rate,slow weight gain,slow tail length growth,and long negative geotaxis reflex time(P<0.05).Compared with the NS group,the weight gain,tail length growth and negative geotaxis reflex time of the three model groups were slower(P<0.05),and the performance of VPA+ADV model was the most significant.There were significant differences in the time of crossing the central grid,the number of crossing the edge grid,and the time of crossing the edge grid in the open field test between the three groups and the NS group(P<0.05).In the self-grooming experiment,the number of interactions in VPA-ADV model was the least(P<0.05),and the number of digging and self-grooming was the most(P<0

关 键 词：PTEN 孤独症 动物模型 丙戊酸 

分 类 号：Q95-33[生物学—动物学]