多巴胺合成酶相关基因治疗帕金森病的实验研究  

Gene therapy of tyrosine hydroxylase,aromatic L-amino acid decarboxylase,and GTP cyclohydrolase genes in rat model of Parkinson′s disease

作　　者：鲁玲玲[1] 苏月[1] 段春礼[1] 赵春礼[1] 苏玉金[1] 吴均[1] 赵焕英[1] 徐群渊[1] 杨慧[1] 

机构地区：[1]首都医科大学北京神经科学研究所北京市神经再生与修复研究重点实验室,100054

出　　处：《中华医学杂志》2004年第18期1528-1532,共5页National Medical Journal of China

基　　金：北京市自然科学基金资助项目 (70 2 2 0 0 5);北京市教委科技发展基金重点资助项目(KZ2 0 0 3 10 0 2 5 0 0 92 )

摘　　要：目的　探讨重组多巴胺合成酶基因在体内外的表达、生成蛋白的活性及对帕金森病模型大鼠的治疗作用。方法　在腺相关病毒介导下 ,将多巴胺合成酶相关基因酪氨酸羟化酶 (TH)基因、芳香族氨基酸脱羧酶 (AADC)基因与GTP环化水解酶Ⅰ (GCH Ⅰ )基因导入COS7细胞 ,分别通过原位杂交与免疫组化的方法检测目的基因的转录与表达 ,通过高压液相分析检测酶活性。将携带目的基因的运载细胞移植到帕金森病模型大鼠纹状体 ,每周进行诱发旋转行为的检测。 10周后高压液相分析脑内多巴胺含量 ,以进一步评估复合基因的治疗效果。结果　重组多巴胺合成酶基因在体外可以表达并被正确修饰 ,具有联合酶促催化功能。当把表达TH、AADC与GCH Ⅰ基因的细胞共培养时 ,在底物左旋酪氨酸存在的情况下 ,可以生成大量多巴胺。而且 ,把携带治疗基因的细胞移植到大脑后 ,经检测三基因移植组脑内多巴胺的生成量较单基因移植组多 ,且前者与后者相比 ,其行为学改善更为明显。但是三基因移植组与双基因组相比 ,脑内多巴胺生成量差异无显著意义。结论　在帕金森病基因治疗策略中 ,应根据多巴胺能神经元的损伤程度来选择加入基因的组合量。Objective To detect the expression and function of enzyme genes involved in biosynthetic pathway for dopamine in vitro and assess their effect in rat model of Parkinson′s disease. Methods Cos7 cells were transfected with separate adeno associated virus(AAV) expressing tyrosine hydroxylase(TH) gene, aromatic L amino acid decarboxylase(AADC) gene and GTP cyclohydrolase Ⅰ (GCH Ⅰ) gene. The expression and function of the three genes were detected by methods of immunohistochemistry, in situ hybridization and high performance liquid chromatograph and electrochemical detection (HPLC ECD). Gene engineered cells were sequentially transplanted into the striatum of 6 hydroxy dopamine leisioned Parkinsonian rat by stereotaxic instrastriatal injection. The asymmetric rotations of these rats after apomorphine administration were detected every week after transplantation. 10 weeks after grafting, the animals were sacrificed and the dopamine produced in the striatum was detected by HPLC ECD. Results In vitro experiments showed that the three genes were high expressed in Cos7 cells. When Cos7 cells expressing TH, AADC and GCH I were cocultured, they produced large amount of dopamine in the condition of existance of L tyrosine. Furthermore, triple genes therapy resulted in greater dopamine production in the striatum of Parkinsonian rats and improved the rotational behavior of the rats more efficiently than did single gene therapy. However, the production of dopamine in the rats with triple genes therapy is no more than double genes therapy. Conclusion For gene therapy in Parkinson′s disease, the amount of target genes to be used should be determined by the level of doperminergic neurons damaged. In the present study, the efficiency of multiple genes therapy is significantly better than that of single gene therapy.

关 键 词：多巴胺 帕金森病 治疗 脑内 表达 相关基因 模型大鼠 合成酶 目的基因 单基因 

分 类 号：R742.5[医药卫生—神经病学与精神病学] R735.7[医药卫生—临床医学]