星形胶质细胞重编程多巴胺能神经元对帕金森病模型大鼠运动功能障碍的修复效应  

Reprogramming astrocytes into dopaminergic neurons to restore motor dysfunction in Parkinson’s disease model rats

作　　者：刘淳博 应梦娇 王澳 刘宇萌 陈颖 叶文豪 文河保 马彩云 刘长青 郭俣 LIU Chunbo;YING Mengjiao;WANG Ao;LIU Yumeng;CHEN Ying;YE Wenhao;WEN Hebao;MA Caiyun;LIU Changqing;GUO Yu(School of Life Sciences,Bengbu Medical University,Bengbu Anhui 233030;School of Laboratory Medicine,Bengbu Medical University,Bengbu Anhui 233030;Department of Sports and Art,Bengbu Medical University,Bengbu Anhui 233030,China)

机构地区：[1]蚌埠医科大学生命科学学院,安徽蚌埠233030 [2]蚌埠医科大学检验医学院,安徽蚌埠233030 [3]蚌埠医科大学体育艺术部,安徽蚌埠233030

出　　处：《中南大学学报(医学版)》2024年第9期1377-1389,共13页Journal of Central South University :Medical Science

基　　金：国家自然科学基金(82371382,81771381);国家级大学生创新训练项目(202210367019,202310367005,202310367061);安徽省重点研究与开发计划项目(2022e07020030,2022e07020032);安徽省高校自然科学基金重点项目(KJ2021ZD0085,KJ2021A0784,2022AH051434,2024AH051296);安徽省大学生创新训练项目(S202210367043);蚌埠医学院重大科技项目计划(2021byfy002,2023byfy007)。

摘　　要：目的:帕金森病(Parkinson’s disease,PD)是一种神经退行性变性疾病,其根本原因是患者脑内多巴胺能神经元(dopaminergic neuron,DA)的死亡。由于DA不可再生,传统治疗手段只能缓解PD的症状。本研究旨在通过将易于获得的星形胶质细胞(astrocyte,AS)重编程为DA后再移植入脑内,重建受损的神经通路,以达到治疗PD的目的。方法:从新生乳鼠脑组织中分离得到AS。构建携带转录因子核受体相关因子1(nuclear receptor-related factor 1,NURR1)和achaete-scute家族bHLH转录因子1(achaete-scute family bHLH transcription factor 1,ASCL1)的慢病毒载体LV-NURR1-ASCL1,用LV-NURR1-ASCL1感染体外培养的大鼠AS。采用免疫荧光技术、蛋白质印迹法、反转录聚合酶链反应(reverse transcription PCR,RT-PCR)检测并比较慢病毒感染后的AS(LV组)和未添加LV-NURR1-ASCL1的完全培养基中培养的AS(Con组)NURR1、ASCL1的表达水平。将慢病毒感染后的AS细胞置于神经元诱导培养基中培养18 d后,采用免疫荧光技术检测其是否表达DA标志物酪氨酸羟化酶(tyrosine hydroxylase,TH)、叉头框蛋白A2(forkhead boxA2,FOXA2)及神经元标志物β-微管蛋白III型(class III beta-tubulin,TUJ1)。予大鼠右脑内侧前脑束(medial forebrain bundle,MFB)区域2个位点注射6-羟基多巴胺(6-hydroxy-dopamine,6-OHDA),以建立PD大鼠模型。将转分化后的细胞(AS-iDA)定量后通过立体定位仪移植入PD模型大鼠右脑MFB区域。移植后4周通过免疫荧光技术检测AS-iDA在PD大鼠脑内的存活、分化、迁移情况,以及PD大鼠大脑组织中TH、TUJ1、FOXA2的表达情况;移植8周后,通过阿扑吗啡(apomorphine,APO)诱导旋转实验、转棒疲劳实验和旷场实验检测移植后PD大鼠运动功能的恢复情况。结果:免疫荧光结果显示慢病毒感染后的AS中NURR1、ASCL1表达阳性。RT-PCR结果显示LV组AS中NURR1和ASCL1的mRNA表达量相较于Con组分别上升了(7.483±0.706)倍和(10.830±1.940)倍。蛋�Objective:Parkinson’s disease(PD)is a neurodegenerative disorder primarily caused by the loss of dopaminergic neurons(DA)in the brain.Since DA neurons are non-renewable,conventional therapies only alleviate symptoms without addressing the root cause.This study aims to reprogram astrocyte(AS)into DA neurons for transplantation into the brain to reconstruct damaged neural circuits and treat PD.Methods:Astrocytes were isolated from neonatal rat brain tissues.A lentiviral vector carrying the transcription factors nuclear receptor-related factor 1(UNRR1)and achaete-scute family bHLH transcription factor 1(ASCL1),named LV-NURR1-ASCL1,was constructed and used to infect cultured rat AS in vitro.Immunofluorescence,Western blotting,and reverse transcription polymerase chain reaction(RT-PCR)were employed to detect and compare the expression levels of NURR1 and ASCL1 in lentivirus-infected AS(LV group)and AS cultured in complete medium without LV-NURR1-ASCL1(Con group).The virus-infected AS was then cultured in neuronal induction medium for 18 days.Immunofluorescence was used to detect the expression of DA markers,including tyrosine hydroxylase(TH)and forkhead box A2(FOXA2),as well as the neuronal marker class III beta tubulin(TUJ1).To establish the PD rat model,6-hydroxydopamine(6-OHDA)was injected into 2 sites in the medial forebrain bundle(MFB)region of the right brain in rats.The reprogrammed cells(AS-iDA)were quantified and transplanted into the right MFB region of the PD model rats using a stereotaxic instrument.Four weeks after transplantation,immunofluorescence was used to assess the survival,differentiation,and migration of AS-iDA in the brain and the expression of TH,TUJ1,and FOXA2 in the brain tissue of PD rats.Eight weeks post-transplantation,the recovery of motor function in PD rats was evaluated using the apomorphine(APO)-induced rotation test,rotarod fatigue test,and open-field test.Results:Immunofluorescence analysis showed positive expression of NURR1 and ASCL1 in AS after lentiviral infection.RT-PCR resul

关 键 词：星形胶质细胞 转录因子 多巴胺能神经元 帕金森病 细胞转分化 

分 类 号：R-332[医药卫生] R742.5