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本文旨在研究鼠双微体基因2(murine double minute 2,MDM2)在啮齿动物脑内的分布及表达模式。应用免疫组化、免疫荧光双标及免疫印迹等技术检测MDM2蛋白在出生后不同阶段昆明小鼠大脑皮层和海马内的表达,及其在成年SD大鼠脑内和原代培养神经元中的分布,观察MDM2与神经元轴突起始段(axon initial segment,AIS)标志蛋白在分布上的相关性。进而采用MDM2抑制剂Nutlin-3在SD大鼠海马内注射,检测MDM2的分布及Nav1.6分布变化。结果显示,昆明小鼠出生后不同时程MDM2在皮层和海马的表达呈现不同的变化模式,但都显示随发育的进展,MDM2逐渐向AIS富集,这一现象在出生7天后逐渐显著。在成年SD大鼠不同脑区的神经元和原代培养神经元中也观察到MDM2富集于AIS的现象,并且其与AIS标志蛋白AnkG、Nav1.6共定位。脑内注射Nutlin-3不仅抑制MDM2在海马神经元AIS的分布,还导致Nav1.6在AIS的分布减少,并且树突标志物MAP2在神经元中的极性分布也发生改变。结果提示MDM2蛋白可在正常成年啮齿类动物脑神经元AIS富集表达,对维持AIS特性和神经元极性起调节作用。
This article aims to investigate the distribution and expression pattern of murine double minute 2 (MDM2) in rodent brain. The expression of MDM2 protein in the cerebral cortex and hippocampus of Kunming mice at various stages after birth was detected by immunohistochemistry, immunofluorescence double-labeled and Western blotting, and its expression in brain and primary cultured neurons of adult SD rats Distribution, to observe the MDM2 neuronal axonal initial segment (axon initial segment, AIS) marker protein in the distribution of the correlation. Then MDM2 inhibitor Nutlin-3 was injected into the hippocampus of SD rats to detect the distribution of MDM2 and the distribution of Nav1.6. The results showed that the expressions of MDM2 in cortex and hippocampus at different time points after birth in Kunming mice showed different patterns of change. However, MDM2 was gradually enriched in AIS as the development progressed. This phenomenon became obvious after 7 days of birth. The phenomenon of MDM2 enrichment in AIS was also observed in neurons and primary cultured neurons in different brain regions of adult SD rats and co-localized with AIS marker protein AnkG, Navl.6. Intranasal injection of Nutlin-3 not only inhibited the distribution of MDM2 in hippocampal neurons AIS, but also reduced the distribution of Nav1.6 in AIS and the distribution of dendritic marker MAP2 in neurons. The results suggest that MDM2 protein can be expressed in AIS of normal adult rodent neurons and play an important role in maintaining the AIS characteristics and neuronal polarity.