MPP^+诱导小鼠胚胎中脑组织原代培养多巴胺能神经元的损伤与死亡

Degeneration of dopaminergic neurons induced by MPP^+ in mouse mesencephalic dissociated culture

目的:探讨1-甲基-4-苯基-四氢吡啶离子(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPP+)诱导小鼠胚胎中脑原代培养多巴胺能神经元损伤。方法:采用OF1/SPF小鼠胚胎(孕14d)中脑进行原代细胞培养,将培养细胞分为对照组和MPP+给药组。于体外培养第10天,在MPP+给药组分别加入含有0·1、1·0、10·0和15·0μmol·L-14个不同浓度的MPP+的培养液,持续作用48h后,经酪氨酸羟化酶(TH)免疫组化染色,显微镜下观察、计数多巴胺能神经元。于体外培养10d,在MPP+给药组的培养液中加入终浓度为10μmol·L-1MPP+,分别于给药后2、4、8、24、48、72和96h进行TH免疫组化染色,显微镜下计数TH染色阳性神经元,明确MPP+引起多巴胺能神经元损伤和死亡的时程变化。结果:0·1、1·0、10·0和15·0μmol·L-1浓度的MPP+给药组中平均每培养孔中的TH染色阳性神经元的数量分别为对照组的(70·30±6·38)%、(67·39±4·92)%、(51·68±2·95)%和(50·91±5·60)%。MPP+给药组中,多巴胺能神经元的损伤表现为两种不同形态:绝大多数多巴胺能神经元表现为神经突起的数目及长度明显减少,极少数为胞体空虚、丢失,仅存神经突起。10μmol·L-1MPP+分别作用24、48、72和96h的MPP+给药组中,TH染色阳性神经元的数量每培养孔分别为(677·2±6·1)、(411·5±26·6)、(229·0±20·3)和(191·3±15·2)个,与对照组[(839·8±15·5)个/培养孔]相比明显减少(P<0·05)。结论:0·1、1·0、10·0和15·0μmol·L-1浓度的MPP+均可诱导的多巴胺能神经元的损伤和死亡,其损伤程度与MPP+药物浓度和持续作用的时间呈依赖关系;MPP+诱导多巴胺能神经元的损伤和死亡可能存在2种不同的机制。

Objective To investigate the degeneration of dopaminergic neurons induced by MPP^＋ in mouse mesencephalic dissociated culture. Methods OF1/SPF mouse embroys of 14 d were used to make mecsencephalic dissociated culture, and the culture was divided into control and MPP^＋-treated group. On the lOth day in vitro, different concentrations of MPP^＋ （final concentrations of 0. 1, 1, 10 and 15 μmol·L^-1） were added into the culture and incubated for 48 h. Dopaminergic neurons were identified and counted by using tyrosine hydroxylase （TH） immunochemistry under microscope. In order to investigate the time course effect of MPP^＋ on the degeneration of doapminergic neurons, 10μmol·L^-1 MPP^＋ was used and incubated for different time （2, 4, 8,24, 48, 72and 96h） on the lOth day in vitro. Results The numbers of dopaminergic neurons per well in the 0. 1,1, 10 and 15 μmol·L^-1 MPP^＋ groups were （70.30±6.38）% （67.39±4.92）% （51.68±2.95）% and （50.91±5.60） % of control level, respectively. The cell death of dopaminergic neurons induced by 10μmol·L^-1 MPP^＋ presented in two different ways. Loss of fibers and terminals of dopaminergic neurons was common way of damage, only few degenerated dopaminergic neurons presented as the loss of cell body. The number of dopaminergic neurons in the control group was （839.8±15.5） per well; meanwhile （677.2±6.1）, （411.5±26.6）, （229.0±20.3）, and （191.3±15.2） were found in MPP^＋-treated groups 24, 48, 72 and 96 h after administration, respectively, which were lower significantly than that in control group （P〈0. 05）. Conclusion MPP^＋ with different concentrations （0.1, 1, 10 and 15 μmol·L^-1） could induce the damage and dead of dopaminergicneurons. The degeneration and death are both dose-and time-dependency. And two different mechanism may exist in the damage and death of dopaminergic neurons induced by MPP^＋.