中国全科医学2018，Vol.21Issue(8)：933-939,7.DOI:10.3969/j.issn.1007-9572.2017.00.244
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
摘要
Abstract
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 significantly slower than in the Lv-NC group (P<0.05). The proportions of TH-positive neurons in the substantia nigra in Lv-NC and Lv-rTH groups were both lower than in the control group.The Lv-rTH group had a higher proportion of TH-positive neurons than the Lv-NC group (P<0.05).Conclusion Parkinson's disease symptoms in rats are improved by stereotactic injection of rTH-carrying recombinant lentiviral vectors into the striatum and substantia nigra.关键词
帕金森病/酪氨酸单氧化酶/慢病毒属/基因治疗Key words
Parkinson disease/Tyrosine 3-monooxygenase/Lentivirus/Gene therapy分类
医药卫生引用本文复制引用
董小林,战丽萍,邬刚,李青芸,魏欢,李建辉,李妍平..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[J].中国全科医学,2018,21(8):933-939,7.基金项目
国家卫生计生委医药卫生科技发展中心项目资助(W2014HB183) （W2014HB183）
云南省科技厅-昆医联合专项资助(2011FB236) （2011FB236）
