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目的:探讨谷氨酸兴奋性损伤诱导小鼠胚胎中脑原代培养多巴胺能神经元损伤的时程效应,阐明谷氨酸兴奋性毒性在帕金森病(PD)模型中对多巴胺能神经元损伤的作用。方法:取孕14d小鼠胚胎的中脑组织制备成细胞悬液,进行原代培养,分为对照组和谷氨酸用药组。细胞在体外培养第10天,加入500μmol·L-1谷氨酸并作用15min,分别于用药后2、4、6、24和48h进行酪氨酸羟化酶(TH)免疫细胞化学染色。在显微镜下计数TH染色阳性神经元数量及每个神经元突起数量。结果:对照组中每个培养孔多巴胺能神经元的数量平均为(778.0±18.6)个,谷氨酸用药组2、4、6、24和48h多巴胺能神经元数量平均为(306.0±8.6)、(314.0±15.4)、(298.0±19.1)、(307.0±18.5)及(323.0±23.8)个。谷氨酸用药各组中多巴胺能神经元数量较对照组明显减少(P<0.05),但谷氨酸用药组2、4、6、24和48h多巴胺能神经元数量比较差异无显著性(P>0.05)。随机选取每组100个多巴胺能神经元的突起进行计数,对照组多巴胺能神经元的突起数量平均为(3.440±0.106)个,谷氨酸用药组2、4、6、24和48h时多巴胺能神经元突起数量分别为(2.240±0.105)、(2.390±0.103)、(3.070±0.105)、(3.420±0.987)和(3.420±0.923)个。谷氨酸用药后2和4h多巴胺能神经元的突起数量较其他各组均明显减少(P<0.05)。结论:谷氨酸可在体外诱导小鼠胚胎中脑原代培养中多巴胺能神经元的损伤和死亡,是一个相对急性、不可逆的损伤过程,但随着损伤后恢复时间的延长,损伤的多巴胺能神经元形态可恢复。
Aims: To investigate the time-course effect of glutamate excitotoxicity injury on primary cultured dopaminergic neurons in midbrain of mice and to elucidate the effect of glutamate excitotoxicity on dopaminergic neuron damage in Parkinson’s disease (PD) model Role. Methods: The midbrain tissue of embryonic day 14 embryos was prepared into cell suspension and cultured in primary culture. The cells were divided into control group and glutamate group. Cells cultured in vitro for 10 days, 500μmol·L-1 glutamate was added and treated for 15min. Immunocytochemical staining of tyrosine hydroxylase (TH) was performed at 2,4,6,24 and 48h after treatment. The number of TH-stained positive neurons and the number of neurites per neuron were counted under a microscope. Results: The average number of dopaminergic neurons per culture hole in the control group was (778.0 ± 18.6). The mean numbers of dopaminergic neurons in the glutamate group at 2,4,6,24 and 48h were (306.0 ± 8.6) , (314.0 ± 15.4), (298.0 ± 19.1), (307.0 ± 18.5) and (323.0 ± 23.8), respectively. Compared with the control group, the number of dopaminergic neurons in the glutamate group was significantly decreased (P <0.05), but there was no significant difference in the number of dopaminergic neurons at 2,4,6,24 and 48h (P > 0.05). The number of protrusions of 100 dopaminergic neurons in each group were randomly selected. The average numbers of neurites in the control group were (3.440 ± 0.106). In the glutamate group, the dopaminergic neurons at 2, 4, 6, 24 and 48 h The number of neurons was (2.240 ± 0.105), (2.390 ± 0.103), (3.070 ± 0.105), (3.420 ± 0.987) and (3.420 ± 0.923) respectively. The numbers of neurites in dopaminergic neurons decreased significantly at 2 and 4 h after glutamate treatment (P <0.05). CONCLUSION: Glutamate induces the injury and death of dopaminergic neurons in the primary cultures of mouse embryonic midbrain in vitro, which is a relatively acute and irreversible injury process. However, with the recovery time after injury, the damage of dopamine Can be neuronal recovery.