腺伴随病毒载体介导多巴胺合成酶的三重基因转移

摘要

Objective To explore triple gene transfer of dopamine synthetic enzymes with separate adeno-associated virus (AAV) vectors.Methods The genes for dopamine synthetic enzymes, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and GTP cyclohydrolase Ⅰ (GCH, an enzyme critical for tetrahydrobiopterin synthesis) were cotransduced into 293 cells with separate AAV vectors. Expressions of TH, AADC and GCH were detected by Western blot analysis. Intracellular dopamine level was assayed by high-performance liquid chromatography.Results TH, AADC and GCH were effectively coexpressed in transduced cells with three separate AAV vectors, AAV-TH, AAV-AADC and AAV-GCH. Furthermore, the coexpression resulted in an effectively spontaneous dopamine production in cotransduced cells.Conclusion The triple transduction of TH, AADC and GCH genes with separate AAV vectors is effective, which might be important to gene therapy for Parkinson's disease.%目的探讨使用腺伴随病毒（AAV）载体介导编码多巴胺合成相关的三种生物酶基因的共转移。方法使用AAV载体将多巴胺生物合成相关的三种生物酶--酪氨酸氢化酶（TH）、芳香族氨基酸脱酶（AADC）、以及三磷酸鸟苷环水解酶Ⅰ（GGH）基因共转导入293细胞，以Western印迹分析检测TH、AADC、GCH的表达，以高效液相色谱法检测细胞内多巴胺水平。结果 TH、AADC、GCH的可在使用三种AAV载体（AAV-TH、AAV-AADC、AAV-GCH）转导的细胞中有效共表达，进一步分析发现，这种共表达可使该细胞有效地自发生成多巴胺。结论使用AAV载体进行TH、AADC、GCH三重基因共转导是有效的，这对帕金森病基因治疗的研究可能具有重要意义。