CAJ | 학술논문

目的研究多巴胺(DA)与α-突触核蛋白(α-synuclein)在神经母细胞瘤细胞中的相互作用机制,探讨二者在帕金森病(PD)发病中的作用. 方法 3-(4,5二甲基噻唑-2)-2,5二苯基四氨唑溴盐(MTT)法确定外源添加DA的浓度,检测外源多巴胺对SH-SY5Y细胞α-synuclein、caspase-3活性片段表达及丙二醛含量的影响；野生型α-synuclein重组质粒转染SH-SY5Y细胞,观察细胞内多巴胺浓度、丙二醛含量、caspase-3活性片段表达水平的变化及多巴胺转运体抑制剂GBR12935对上述变化的影响. 结果 (1)外源添加300 μmol/L多巴胺作用24 h后SH-SY5Y细胞内多巴胺浓度较空白对照组升高了接近16倍(t=7.32,P＜0.01),α-synuclein、caspase-3活性片段表达水平及丙二醛含量均升高(t=4.92,P＜0.01；t=17.14,P＜0.01; t=6.55,P＜0.01)；(2)α-synuclein重组质粒转染SH-SY5Y后细胞内多巴胺浓度(F=32.97,P＜0.01)、丙二醛含量(F=107.80,P＜0.01)、caspase-3活性片段表达水平(F=55.54,P＜0.01)均较空载体转染组升高,但这种上升均被GBR12935部分遏制. 结论多巴胺促进SH-SY5Y细胞α-synuclein表达,而过表达α-synuclein又导致SH-SY5Y细胞内多巴胺浓度升高,二者形成产生细胞毒性的恶性循环.
목적 연구다파알(DA)여α-돌촉핵단백(α-synuclein)재신경모세포류세포중적상호작용궤제,탐토이자재파금삼병(PD)발병중적작용. 방법 3-(4,5이갑기새서-2)-2,5이분기사안서추염(MTT)법학정외원첨가DA적농도,검측외원다파알대SH-SY5Y세포α-synuclein、caspase-3활성편단표체급병이철함량적영향；야생형α-synuclein중조질립전염SH-SY5Y세포,관찰세포내다파알농도、병이철함량、caspase-3활성편단표체수평적변화급다파알전운체억제제GBR12935대상술변화적영향. 결과 (1)외원첨가300 μmol/L다파알작용24 h후SH-SY5Y세포내다파알농도교공백대조조승고료접근16배(t=7.32,P＜0.01),α-synuclein、caspase-3활성편단표체수평급병이철함량균승고(t=4.92,P＜0.01；t=17.14,P＜0.01; t=6.55,P＜0.01)；(2)α-synuclein중조질립전염SH-SY5Y후세포내다파알농도(F=32.97,P＜0.01)、병이철함량(F=107.80,P＜0.01)、caspase-3활성편단표체수평(F=55.54,P＜0.01)균교공재체전염조승고,단저충상승균피GBR12935부분알제. 결론 다파알촉진SH-SY5Y세포α-synuclein표체,이과표체α-synuclein우도치SH-SY5Y세포내다파알농도승고,이자형성산생세포독성적악성순배.
Objective To investigate the interactive mechanism of dopamine and α-synuclein in SH-SY5Y cells,and to explore their effects on Parkinson's disease (PD).Methods The concentration of exogenous dopamine was definited by MTT method.The effect of exogenous dopamine on the expressions of α-synuclein and caspase-3 active fragments and malondialdehyde (MDA) content were detected by Western blot and commercial kit.Recombinant plasmid of wild-type α-synuclein was transfected into SH-SY5Y cells.Level changes in dopamine concentration,MDA content and expression of caspase-3 active fragment were observed.Furthermore,the effect of GBR12935,an inhibitor of dopamine transporter,on these outcomes were detected.Results Intracellular dopamine concentration was increased by 16 folds in SH-SY5Y cells which were added 300 umol/L exogenous dopamine for 24 hours as compared with blank controls (t=7.32,P＜0.01).Expressions of α-synuclein and caspase-3 active fragments and MDA content were increased in exogenous dopamine treated group as compared with blank controls (t=4.92,17.14,6.55,all P＜0.01).Intracellular dopamine concentration,MDA content and expression of caspase-3 active fragment were increased in SH-SY5Y cells transfected with recombinant plasmid of wild-type α-synuclein as compared with SH-SY5Y cells transfected with empty plasmid (F =32.97.107.80,55.54,all P＜0.01),whlie these increases could be partially inhibited by GBR12935.Conclusions Dopamine promotes the expression of α-synuclein,while over-expressed α-synuclein increases intracellular dopamine concentration,which forms a vicious cycle of cytotoxicity in SH-SY5Y cells.