过表达多巴胺脱羧酶、酪氨酸羟化酶和三磷酸鸟苷环化水解酶1重组慢病毒构建及其在帕金森病大鼠模型治疗作用的研究  

Construction of Overexpressed DDC, TH and GCH1 Recombinant Lentivirus Vectors and Its Therapeutic Effect in 6-OHDA PD Rat Model

作　　者：赵慧慧[1] 史明超 金庆文[1,2] ZHAO Hui-hui;SHI Ming-chao;JIN Qing-wen(Department of Neurology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China;Department of Neurology, the Affiliated Sir Run Run Hospital of Nanjing Medical University, Nanjing 211166, China)

机构地区：[1]南京医科大学第一附属医院神经内科,南京210029 [2]南京医科大学附属逸夫医院神经内科,南京211166

出　　处：《中国临床神经科学》2018年第2期133-144,共12页Chinese Journal of Clinical Neurosciences

基　　金：江苏省自然科学基金面上项目(编号:BK20141439);国家自然科学基金面上项目(编号:81671117)

摘　　要：目的构建同时表达3种多巴胺(DA)合成相关酶的重组慢病毒(rLV),验证其在6-羟基多巴胺(6-OHDA)帕金森病(PD)大鼠模型中治疗作用,为PD临床基因治疗提供实验基础。方法构建过表达多巴胺脱羧酶(DDC)、酪氨酸羟化酶(TH)和三磷酸鸟苷环化水解酶1(GCH1)rLV及对照rLV。6-OHDA制备的PD模型大鼠,随机分为生理盐水组(n=6)、阴性病毒组(n=6)和阳性病毒组(n=12),分别经立体定向向大鼠纹状体注射生理盐水、对照rLV和DDC+TH+GCH1-rLV。观察构建病毒对PD大鼠旋转行为的影响,免疫荧光法检测病毒转染情况,同时使用高效液相色谱-串联质谱联用法(HPLC-MS/MS)检测纹状体内DA及其代谢产物改变情况。结果 PD大鼠纹状体注射后,阳性病毒组行为改变与生理盐水组、阴性病毒组比较,差异有显著统计学意义(P<0.01);阳性病毒组呈现显著的旋转行为改善。免疫荧光显示阳性病毒转染表达有TH活性的阳性细胞。阳性病毒组纹状体内DA及其代谢产物与生理盐水组、阴性病毒组比较,显著增高(P<0.01)。结论成功构建过表达DDC+TH+GCH1-rLV,并且在PD模型大鼠中呈现显著的行为改善及DA水平增加,为PD的基因治疗提供实验基础。Aim To construct recombinant lentivirus（rLV） vector transferring system expressing three kinds of dopamine synthesis enzymes simultaneously, test and verify the function of this vector system in 6-hydroxydopamine（6-OHDA） Parkinson＇s disease（PD） rat model in order to provide experimental foundation for PD clinical gene therapy. Methods Overexpressed dopamine decarboxylase（DDC）, tyrosine hydroxylase（TH） and GTP cyclihydrolase 1（GCH1） positive rLV and control rLV were constructed. PD rat models produced by 6-OHDA were divided into three groups randomly： a normal saline group（n=6）, a control rLV group（n=6） and a DDC＋TH＋GCH1-rLV group（n=12）. The striatum of 6-OHDA PD rat models were stereoscopically injected with normal saline, control rLV and DDC＋TH＋GCH1-rLV respectively. The changes of rotation behaviors in animal models were observed and immunofluorescence was used to detect virus transfection. The levels of dopamine and its metabolites in striatum were measured by high performance liquid chromatography combined with tandem mass spectrometry（HPLC-MS/MS） in each group. Results After the striatum injection of PD rat model, DDC＋TH＋GCH1-rLV group showed obvious rotation behavior significance in contrast with the normal saline group and the control rLV group（P0.01）. DDC＋TH＋GCH1-rLV group presented prominent rotation behavior improvement. With TH immunofluorescence staining, TH expressing positive cells can be detected in the striatum of positive rLV transferred group. Compared with the normal saline group and the control rLV group, DA and its metabolites in positive rLV group appeared prominent progress（P〈0.01）. Conclusion DDC, TH and GCH1 rLV were successfully constructed in a single transcription unit. Positive rLV group showed prominent rotation behavior progress and DA level increase, which provided an experimental basis for PD gene therapy.

关 键 词：帕金森病 多巴胺脱羧酶 酪氨酸羟化酶 三磷酸鸟苷环化水解酶1 重组慢病毒 基因治疗 高效液相色谱-串联质谱联用法 

分 类 号：R742.5[医药卫生—神经病学与精神病学] Q189[医药卫生—临床医学]