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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 effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP +) on primary cultured dopaminergic neurons in midbrain of mouse embryos Yuan injury. Methods: Primary cultured cells of OF1 / SPF mouse embryos (14th gestational age) were divided into control group and MPP + administration group. On day 10 of in vitro culture, MPP + -administered medium containing 0.1,1.0,10.0 and 15 · 0μmol·L-14 different concentrations of MPP + were respectively added to the MPP + administration group for 48 hours, Immunohistochemical staining of TH-hydroxylase (TH), under the microscope, counted dopaminergic neurons. The cells were cultured in vitro for 10 days. The final concentration of 10 μmol·L-1 MPP + was added to the culture medium of MPP + administration group, TH immunohistochemistry was performed at 2,4,8,24,48,72 and 96h after administration, Down-count TH-positive neurons, clear MPP + -induced dopaminergic neuron damage and death of the course changes. Results: The average numbers of TH-positive neurons per culture hole in the MPP + administration group at 0 · 1, 1 · 0, 10 · 0 and 15 · 0 μmol·L-1 were (70 · 30 ± 6 · 38)%, (67 · 39 ± 4.92)%, (51 · 68 ± 2 · 95)% and (50 · 91 ± 5 · 60)%, respectively. In the MPP + administration group, dopaminergic neurons showed two different morphological changes: the vast majority of dopaminergic neurons showed a marked decrease in the number and length of neurites, and the few were vacuolar, missing, and only neurite outgrowths. The number of TH-stained neurons in each group was (677 · 2 ± 6 · 1), (411 · 5 ± 26 · 6), (229.0 ± 20.3) and (191.3 ± 15.2), respectively, compared with the control group [(839.8 ± 15.5) cells / culture well] (P <0 · 05). CONCLUSION: MPP + at concentrations of 0 · 1, 1 · 0, 10 · 0 and 15 · 0μmol·L-1 all induced the damage and death of dopaminergic neurons. The degree of injury and the duration of MPP + Dependent relationship; There are two possible mechanisms for MPP + -induced dopaminergic neuron injury and death.