重组慢病毒携带酪氨酸羟化酶对大鼠帕金森病模型的干预研究  

Effect of a Tyrosine Hydroxylase-carrying Recombinant Lentivirus in a Rat Model of Parkinson's Disease

作　　者：董小林 战丽萍 邬刚 李青芸 魏欢 李建辉 李妍平 DONG Xiao-lin;ZHAN Li-ping;WU Gang;LI Qing-yun;WEI Huan;LI Jian-hui;LI Yan-ping(Yan'an Hospital of Kunming City,Kunming 650051,China)

机构地区：[1]云南省昆明市延安医院,650051

出　　处：《中国全科医学》2018年第8期933-939,共7页Chinese General Practice

基　　金：国家卫生计生委医药卫生科技发展中心项目资助(W2014HB183);云南省科技厅-昆医联合专项资助(2011FB236)

摘　　要：目的利用携带大鼠酪氨酸羟化酶(TH)基因的重组慢病毒,对大鼠帕金森病模型进行基因治疗,评估基因疗法对帕金森病症状缓解和黑质神经元恢复的影响。方法 2014年11月—2015年12月,分离、培养和鉴定胎鼠原代神经元,抽提胎鼠原代神经元RNA、反转录聚合酶链式反应(RT-PCR)克隆大鼠TH(r TH)基因,将r TH基因构建至慢病毒质粒,包装r TH重组慢病毒。采用实时荧光定量RT-PCR、免疫荧光实验和Western blotting法体外检测大鼠成纤维细胞REF中r TH基因表达。通过脑内立体定位注射6羟多巴胺(6-OHDA)建立大鼠帕金森病模型,给予Lv-r TH重组慢病毒(Lv-r TH治疗组)、未携带基因的慢病毒Lv-NC(Lv-NC治疗组)及0.9%氯化钠溶液(对照组)注射大鼠纹状体和黑质。通过阿扑吗啡诱发旋转实验进行行为学评分,免疫组化进行神经元恢复评分。结果成功分离、培养了胎鼠原代神经元,成功克隆了r TH基因,并将r TH基因构建至慢病毒质粒,实时荧光定量RT-PCR、免疫荧光实验和Western blotting法检测大鼠成纤维细胞REF中均表达r TH基因。3组大鼠治疗前1周自发旋转速率比较,差异无统计学意义(P>0.05);3组大鼠治疗0、1、2、3、4、5、6、7、8、9、10周自发旋转速率比较,差异均有统计学意义(P<0.05);其中治疗0、1、2、3、4、5、6、7、8、9、10周,Lv-NC治疗组和Lv-r TH治疗组大鼠自发旋转速率较对照组增快(P<0.05);治疗2、3、5、6、8、9、10周,Lv-r TH治疗组大鼠自发旋转速率较Lv-NC治疗组减慢(P<0.05)。Lv-NC治疗组和Lv-r TH治疗组大鼠中脑黑质TH阳性神经元所占比例较对照组降低,Lv-r TH治疗组大鼠中脑黑质TH阳性神经元所占比例较Lv-NC治疗组升高(P<0.05)。结论通过脑内立体定位注射,将表达r TH基因的重组慢病毒递送至帕金森病大鼠纹状体和黑质能缓解帕金森病症状并恢复TH阳性神经元。Objective Rat tyrosine hydroxylase(TH)-carrying recombinant lentivirus was used to perform gene therapy in a rat model of Parkinson's disease.Effects of the therapy on the alleviation of disease symptoms and recovery of the function of substantia nigra neurons were evaluated.Methods The experiment was conducted between November 2014 and December 2015.Primary fetal rat neurons were isolated,cultured and identified.RNA was extracted from the cultured primary neurons,and the rTH gene was cloned by reverse transcription-polymerase chain reaction(RT-PCR).rTH-carrying recombinant lentiviral vector was constructed by incorporating and packaging the rTH gene into lentiviral plasmids.Real-time quantitative RT-PCR(RT-qPCR),immunofluorescence and Western blotting were used to detect the expression of the rTH gene in rat fibroblasts REF.A rat model of Parkinson's disease was established by stereotactic injection of 6-hydroxydopamine(6-OHDA).Lv-rTH recombinant lentivirus(Lv-rTH treatment group),non-rTH-carrying lentivirus Lv-NC(Lv-NC treatment group)or 0.9%sodium chloride solution(control group)were injected into the striatum and substantia nigra of the rats.Behavioral scoring was performed by the apomorphine-induced rotation test,and neuronal recovery was assessed by immunohistochemistry.Results Primary fetal rat neurons were isolated and cultured,the rTH gene was cloned and the rTH-recombinant lentivirus vector was constructed successfully.rTH was confirmed to be expressed in REF by RT-qPCR,immunofluorescence and Western blotting.There were no significant differences in the rate of spontaneous rotation among the three groups 1 week before treatment(P>0.05).Spontaneous rotation among the three groups was tested every week(0-10)and differed at each time point(P<0.05).Specifically,the spontaneous rotation rate in the Lv-NC group and Lv-rTH group was significantly faster than in the control group at each time point(P<0.05).After 2,3,5,6,8,9 and 10 weeks of treatment,the spontaneous rotation rate in the Lv-rTH group was signific

关 键 词：帕金森病 酪氨酸单氧化酶 慢病毒属 基因治疗 

分 类 号：R745.1[医药卫生—神经病学与精神病学]