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目的:探讨1-甲基-4-苯基-四氢吡啶离子(MPP+)对小鼠胚胎中脑原代培养多巴胺能神经元的毒性作用及其机制。在体外建立帕金森病(PD)实验研究的细胞模型。方法:采用小鼠胚胎(孕14d)中脑进行原代细胞培养,实验分为对照组和不同浓度(0.1、1、10和15μmol.L-1)MPP+药物实验组,应用酪氨酸羟化酶(TH)免疫化学细胞染色方法和Hoechst33342荧光染色对多巴胺能神经元的损伤及凋亡进行测定。结果:对照组中多巴胺能神经元的数量为(875±23)个/孔。在0.1、1、10和15μmol.L-1MPP+实验组,多巴胺能神经元的数量分别为(612±25)、(586±32)、(459±16)和(435±19)个/孔,与对照组比较,MPP+实验组多巴胺能神经元数量均明显降低(P<0.05),多巴胺能神经元突起的数目和长度明显减少。Hoechst33342染色发现,对照组中凋亡细胞数占总细胞数的(5.45±0.29)%,10μmol.L-1MPP+药物治疗组细胞凋亡率为(26.97±1.36)%,显著高于对照组水平(P<0.05)。结论:0.1、1、10和15μmol.L-1MPP+均引起多巴胺能神经元的数量显著减少,随着药物浓度的增加,多巴胺能神经元减少的程度越显著。MPP+引起的多巴胺能神经元的变性死亡可能通过凋亡途径所诱导。
AIM: To investigate the toxic effects of 1-methyl-4-phenyl-tetrahydropyridine ion (MPP +) on primary cultured dopaminergic neurons in midbrain of mouse embryos and its mechanism. Cell models of Parkinson’s disease (PD) experimental studies were established in vitro. Methods: Primary cultured cells were obtained from the midbrain of mouse embryos (pregnant 14d). The experiment was divided into control group and MPP + groups with different concentrations (0.1, 1, 10 and 15μmol.L-1) Enzyme (TH) immunocytochemical staining and Hoechst33342 staining were used to detect the damage and apoptosis of dopaminergic neurons. Results: The number of dopaminergic neurons in the control group was (875 ± 23) per well. The numbers of dopaminergic neurons were (612 ± 25), (586 ± 32), (459 ± 16) and (435 ± 19) per well in 0.1, 1, 10 and 15 μmol.L- Compared with the control group, the number of dopaminergic neurons in MPP + experimental group was significantly decreased (P <0.05), and the number and length of dopaminergic neuron protrusions were significantly reduced. Hoechst33342 staining showed that the number of apoptotic cells in the control group was (5.45 ± 0.29)% of the total cell number, and the apoptosis rate was (26.97 ± 1.36)% in the 10μmol.L-1MPP + drug treatment group, which was significantly higher than that in the control group P <0.05). CONCLUSION: The numbers of dopaminergic neurons in 0.1, 1, 10 and 15μmol.L-1MPP + all significantly decrease, and the more significant the decrease of dopaminergic neurons with the increase of drug concentration. Degeneration of dopaminergic neurons caused by MPP + may be induced by the apoptotic pathway.