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.%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(rTH)基因,将rTH基因构建至慢病毒质粒,包装rTH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rTH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-rTH重组慢病毒(Lv-rTH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rTH基因,并将rTH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rTH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-rTH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-rTH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-rTH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-rTH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rTH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.
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