【摘要】 目的探讨慢病毒介导的新型Tet-On系统大鼠胶质细胞源性神经营养因子(GDNF)基因和酪氨酸羟化酶(TH)双基因直接转移对帕金森病(PD)大鼠的保护作用。方法将携带TH、GDNF基因并含启动子为Palb的四环素应答元件的慢病毒(Lv-TH-GDNF)和四环素反式作用子rtTA2s-M2病毒共同注射到SD大鼠左侧纹状体,用强力霉素(DOX)诱导大鼠目的基因GDNF和TH的表达。1周后在大鼠的同侧纹状体注射6-羟基多巴胺(6-OHDA)损毁纹状体的DA能神经元。通过旋转行为、黑质TH免疫组化染色以及高效液相色谱-电化学方法(HPLC-ECD)检测纹状体DA含量评估其保护效应;通过RT-PCR、免疫印迹观察Lv-TH-GDNF在脑内的表达。实验与健康对照组、磷酸缓冲液(PBS)对照组进行比较。结果在6-OHDA损伤后4周,Lv-TH-GDNF+rt-TA2s-M2+DOX组大鼠阿扑吗啡诱发的旋转效应均明显低于PBS对照组(P<0.01),损毁侧的的黑质区TH阳性细胞表达强度、纹状体DA含量均明显高于PBS对照组(P<0.01),但二者均低于健康对照组(P<0.01)。RT-PCR、免疫印迹结果显示,与PBS对照组比较,Lv-TH-GDNF+rtTA2s-M2+DOX组大鼠纹状体TH、GDNF基因的表达明显增高(P<0.01)。结论慢病毒介导的新型Tet-On系统大鼠GDNF和TH双基因脑内直接转移,在强力霉素诱导下可减缓6-OHDA诱发的大鼠DA能神经元进行性变性,具有保护作用。更多还原

【Abstract】 Aim To study the protective effect of lentiviral-mediated rat glial cell line-derived neurotrophic factor(GDNF)and tyrosine hydroxylase(TH)on the basis of improved Tet-on system gene transfer on dopaminergic neurons in a rat model of Parkinson′s disease(PD).Methods Recombinant lentivirus(Lv-TH-GDNF)carrying rat GDNF and TH genes and tetracycline response element containing mouse albumin gene promoter(Palb) together with tetracycline-controlled transactivator rtTA2s-M2 virus was injected into the left striate bodies SD rats.After that,the expression of GDNF and TH genes was regulated by doxycycline.One week later,ipsilateral intrastriatal injection of 6-hydroxydopamine(6-OHDA)was to injury dopaminergic neurons.The neuroprotective effects of Lv-TH-GDNF were evaluated by apomorphine-induced rotational behavior,immunohistochemistry assay of the tyrosine hydroxylase positive neurons in the substantia nigra,and the measurement of dopamine level in the striatum by high performance liquid chromatography-electric chemical discharge(HPLC-ECD).RT-PCR and western blotting were performed to check the expression of Lv-TH-GDNF in the brain.Tests were compared with the PBS control group and the non-injected control group.Results Tests showed apomorphine-induced contralateral turning effect was significantly reduced in Lv-TH-GDNF+rtTA2s-M2+DOX-pre-treated rats as compared with PBS-pre-treated rats when measured at 4 weeks post lesion by 6-OHDA(P<0.01),the expression of TH-positive cells in the lesioned substantia nigra and DA levels in the lesioned striatum were significantly higher in Lv-TH-GDNF+rtTA2s-M2+DOX-pre-treated rats than that in PBS-pre-treated rats(P<0.01),but both were significantly lower than that of in non-injected rats(P<0.01).RT-PCR and western blotting showed the expression of exogenous GDNF and TH genes was significantly raised in the lesioned striatum in Lv-TH-GDNF+rtTA2s-M2+DOX-pre-treated rats as compared with PBS-pre-treated rats.Conclusion The results suggest that lentiviral-mediated rat GDNF and TH bi-gene on the basis of improved Tet-on system,which is intrastriataly transferred to be induced by doxycycline,can significantly retard progressive degeneration of dopaminergic neurons of nigrostriatal system from 6-OHDA-induced injury,which shows that it has neuroprotective effect on the dopaminergic neurons of PD.更多还原