酪氨酸羟化酶和左旋芳香族氨基酸脱羧酶基因的细胞表达及活性检测

由于在帕金森病中合成多巴胺所需的酪氨酸羟化酶(tyrosine hydroxylase,TH)和左旋芳香族氨基酸脱羧酶(aromatic L-amino acid decarboxylase,AADC)活性明显降低,所以补充多巴胺合成酶成为基因治疗帕金森病研究的主要手段。我们分别构建了重组逆转录病毒载体pLHCX／TH及pLNCX2／AADC,通过脂质体介导将带有目的基因的载体分别转到包装细胞PA317中,经筛选得到产病毒的细胞PA317／TH和PA317／AADC,采用免疫组化、原位杂交方法检测目的基因表达;细胞经裂解后进行的酶促反应产物多巴胺以高压液相电化学方法检测证明所克隆的T‘H及AADC基因具有功能活性;这两种基因工程改造细胞可以完成酶促动力学的功能,使L-dopa及多巴胺产生明显增加。本研究为用TH和AADC双基因对帕金森病进行基因治疗提供了一定的依据。

Expression and assessment of double genes of tyrosine hydroxylase gene and aromatic L-amino acid decarboxylase gene in vitro

The characteristic pathological changes of Parkinson's disease (PD) include a severe loss ofdopamine neurons in the substantia nigra and a severe decrease in dopamine in the striatum. Since the ex-pression of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC ) in the biosynthet-ic pathway for dopamine are low , a promising approach to the gene therapy of PD is to augment the gene ex-pression of the enzymes in the biosynthetic pathway for dopamine. In the present study, human TH andAADC genes were reconstructed into retrovirous vectors pLHCX and pLNCX_2 respectively. Then pLHCX/TH and pLNCX_2/AADC were transfected into packaging cell line PA317 with liposome. PA317/TH andPA317/AADC were selected by different antibiotics. Gene expression was examined by methods of immuno-histochemistry and in situ hybridization. The catalytic activity of two cloned gene enzymes was assessed invitro by HPLC-EC. Immunocytochemical staining showed that TH and AADC were expressed efficiently invitro. Both TH and AADC mRNA were transcripted in PA317 cell lines by using in situ hybridazation.HPLC-EC experiments revealed that the transfected cells produced a significantly higher level of dopamineand L-dopa than the untransfected cells. The two genetically modified cells could improve the production ofL-dopa and dopamine in response to suitable substrate. The present results suggest that not only recombinant TH and AADC genes are successfully expressed in vitro, but also the enzymes have respective functional ac-tivities. These results have set up a way for in vivo gene therapy of PD with TH and AADC genes.