CAJ | 학술논문

目的探讨α-突触核蛋白对多巴胺代谢的调节机制.方法以人基因组DNA为模板,PCR法扩增酪氨酸羟化酶(Tyrosine Hydroxylase,TH)基因区的部分DNA片段(-495～+25),将其插入质粒pGL3-Basic,构建荧光素酶报告基因重组质粒pGL3-THprom,转染多巴胺能神经元细胞系MES23.5和MES23.5/hα-Syn+.结果双荧光素酶活性分析表明,pGL3-Basic、pGL3-THprom和pGL3-Control在MES23.5细胞中表达的荧光素酶活性分别为5.60±0.67,26.80±4.11和32.90±4.75,而pGL3-THprom在MES23.5和MES23.5/hα-Syn+中表达的荧光素酶活性分别为26.80±4.11和14.40±0.61,差异极显著(P＜0.01).结论这些结果提示扩增的DNA片段具有启动子活性,而α-Syn作为反式作用因子通过影响TH基因启动子的功能发挥对多巴胺代谢的负性调控作用.
목적 탐토α-돌촉핵단백대다파알대사적조절궤제.방법 이인기인조DNA위모판,PCR법확증락안산간화매(Tyrosine Hydroxylase,TH)기인구적부분DNA편단(-495～+25),장기삽입질립pGL3-Basic,구건형광소매보고기인중조질립pGL3-THprom,전염다파알능신경원세포계MES23.5화MES23.5/hα-Syn+.결과 쌍형광소매활성분석표명,pGL3-Basic、pGL3-THprom화pGL3-Control재MES23.5세포중표체적형광소매활성분별위5.60±0.67,26.80±4.11화32.90±4.75,이pGL3-THprom재MES23.5화MES23.5/hα-Syn+중표체적형광소매활성분별위26.80±4.11화14.40±0.61,차이겁현저(P＜0.01).결론 저사결과제시확증적DNA편단구유계동자활성,이α-Syn작위반식작용인자통과영향TH기인계동자적공능발휘대다파알대사적부성조공작용.
Objective To approach the associated mechanism by which α-synuclein (α-Syn) might regulate the metabolism of dopamine. Methods A DNA fragment, located at -495 to +25 of the human tyrosine hydroxylase (TH) gene, was amplified by PCR and inserted into the pGL3-Basic luciferase reporter vector. The recombinant plasmid pGL3-THprom was transfected into a dopaminergic cell line MES23.5 or a α-Syn over-expressed MES23.5 (named MES23.5/hα-Syn+). The promoter activity was detected by the Dual Luciferase Assay System. Results The luciferase activities in the MES23.5 cells transfected with pGL3-Basic, pGL3-THprom, and pGL3-Control vectors were 5.60±0.67, 26.80±4.11, and 32.90±4.75,respectively. On the other hand, the luciferase activity of pGL3-THprom in the MES23.5 (26.80±4.11) was significantly higher than that in the MES23.5/hα-Syn+ (14.40±0.61) (P＜0.01). Conclusion These results indicate that the -495 to +25 region in the TH gene possesses promoter activity for controlling the gene expression, and that α-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.