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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。复合基因治疗的效果明显好于单基因
Objective To investigate the expression of recombinant dopamine synthetase gene in vitro and in vivo, the activity of protein production and the therapeutic effect on rat model of Parkinson’s disease. Methods The tyrosine hydroxylase (TH) gene, the aromatic amino acid decarboxylase (AADC) gene and the GTP Ⅰ (GCH Ⅰ) gene of dopamine synthase gene were introduced into COS7 cells mediated by adeno-associated virus The transcription and expression of the target gene were detected by in situ hybridization and immunohistochemistry respectively. The enzyme activity was detected by HPLC. The carrying cells carrying the gene of interest were transplanted to the striatum of rat model of Parkinson’s disease and the detection of induced rotational behavior was performed weekly. High-pressure liquid phase analysis of brain dopamine levels was performed 10 weeks later to further evaluate the therapeutic effect of the composite gene. Results The recombinant dopamine synthetase gene could be expressed in vitro and correctly modified, which combined with enzymatic catalysis. When cells expressing TH, AADC and GCH I genes are co-cultured, a large amount of dopamine can be generated in the presence of substrate tyrosine. Moreover, after transplanting the cells carrying the therapeutic gene into the brain, the generation of dopamine in the brain of the three-gene transplantation group was more than that in the single-gene transplantation group, and the former was more improved in behavioral improvement than the latter. However, there was no significant difference in brain dopamine production between the three gene-transplantation groups and the double genome. Conclusion In the strategy of gene therapy for Parkinson’s disease, the combined amount of genes should be selected according to the degree of damage of dopaminergic neurons. Compound gene therapy is significantly better than single gene