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目的:观察原代海马神经元不同时期树突棘的变化以及树突棘蛋白Drebrin和突触囊泡膜蛋白SYN的表达水平。方法:光镜观察原代培养7、14和20 d的海马神经元的形态改变。应用免疫荧光对Drebrin和SYN进行染色,观察树突棘形态和数量的改变。进一步应用Western Blot检测Drebrin和SYN蛋白的表达变化。结果:培养7 d的海马神经元,未见树突棘结构,SYN斑点状阳性产物较少。随着海马神经元培养时间的加长,突起数量逐渐增加,14 d和20 d的树突棘数量和SYN阳性产物均显著增加。Drebrin和SYN蛋白表达水平也随培养时间的加长而显著增加。结论:原代培养的海马神经元在14 d已有树突棘形成,20 d显示出成熟树突棘的形态和数量。Drebrin和SYN的蛋白表达水平反映了树突棘和突触的形成和功能状态。
OBJECTIVE: To observe the changes of dendritic spines and expression of dendritic protein Drebrin and synaptic vesicle protein SYN in primary cultured hippocampal neurons. Methods: The morphological changes of hippocampal neurons were observed under light microscope at 7, 14 and 20 days. Drebrin and SYN were stained with immunofluorescence to observe the morphological and quantitative changes of dendritic spines. Western Blot was used to detect the expression of Drebrin and SYN proteins. Results: There was no dendritic spine structure in hippocampal neurons cultured for 7 days and there were fewer SYN positive products. With the prolongation of hippocampal neuron culture, the number of neurites gradually increased, and the numbers of dendritic spines and SYN positive products increased significantly on the 14th and 20th day. Drebrin and SYN protein expression levels also increased significantly with the incubation time. CONCLUSION: Primary cultured hippocampal neurons have dendritic spines formed on the 14th day and morphology and number of mature dendritic spines on the 20th day. Protein expression levels of Drebrin and SYN reflect the formation and functional status of dendritic spines and synapses.