新生大鼠尾核神经元的体外原代培养及神经元鉴定

目的建立一种大鼠尾核神经元的原代培养方法,并对其进行形态学观察和免疫组织化学鉴定。方法自新生24 h内的乳鼠大脑中钝性取出尾核,胰酶消化后采用含有B27的Neurobasal A培养液进行体外原代培养。采用神经元特异性烯醇化酶(NSE)免疫组织化学法对神经元进行鉴定,应用酪氨酸羟化酶(TH)免疫组织化学法对多巴胺(DA)能神经元进行鉴定。结果①从新生鼠分离的尾核神经元在本实验条件下生长良好。原代培养7～11 d的尾核神经细胞在形态上趋向成熟。②NSE免疫组织化学染色显示所培养细胞90%以上为神经元细胞。③TH免疫组织化学染色结果表明培养的神经细胞多数呈多巴胺免疫反应阳性,为多巴胺能神经细胞。结论新生鼠的尾核神经元可进行体外原代培养,其中存在大量的多巴胺能神经元。它为研究神经变性疾病的发病及干预机制提供了体外细胞模型。

Primary Culture of Neurons from Caudate Nucleus from New Born Rats in Vitro and the Study on Identification of Neurons

Objective To establish a method for primary cultured neurons from caudate nucleus of rodent for the study on the characteristics of the morphology and identification of neurons. Methods Primaryly cultured neurons from caudate nucleus in neonatal Wistar rats within 24h after birth were processed by using enzyme digestion in the presence of neurobasal A medium containing B27.Neuron-specific enolase(NSE) immunohistochemical staining was used to identify neurons.The dopaminergic neurons was detected by tyrosine hydroxylase(TH) immunohistochemical staining. Results ①Primaryly cultured neurons from caudate nucleus in neonatal rats were in high quality with smooth membrane,elasticity.The cultured neurons from 7d to 11d in vitro tended to mature in cellular morphology.②90% of cultured cells were neurons by NSE immunohistochemical staining.③TH positive neurons and fine dendrites were identified clearly.The stained neurons comprised about 90% of all neurons. Conclusion Pimaryly cultured neurons from caudate nucleus of neonatal rats is established and most of cells are dopaminergic neurons.The cultured cells are useful cellular model for carrying out in vitro studies on the mechanisms of etiology and intervention of neurodegenerative diseases.