SD大鼠海马神经元原代培养及形态学观察

目的：掌握SD大鼠海马神经元体外培养方法,观察神经元形态学特点,测定细胞生长曲线。方法：新生SD乳鼠,剥离海马,胰酶消化,接种,1 d换为Neurobasal培养基;小鼠抗大鼠微管相关蛋白-2、兔抗鼠神经元特异性烯醇化酶为一抗,免疫细胞化学法计数阳性细胞百分率并显微摄影;1%甲苯胺蓝、苏木精伊红染色。结果：原代培养神经元6~8 h内开始贴壁,培养5~7 d突起增粗增长,连接成网,以多极神经元为主,胞体呈三角形、梭形、椭圆形;MAP-2、NSE染色为棕褐色,经鉴定神经元纯度达90%左右;细胞经历生长潜伏期,对数生长期,平台期;细胞核大而圆,HE染色胞核深蓝色,胞浆红色,胞体中可见尼氏颗粒。结论：相差显微镜下神经元形态多样,MAP-2、NSE染色阳性细胞,HE染色细胞核呈深蓝色,胞浆为红色,尼氏体位于细胞质内,神经突起内少见。

Primary Cultivation and Observation of SD Rat Hippocampal Neuron

Objective： To explore the techniques of primary cultivation of SD rat hippocampal neuron and the morphological characteristic of the neurons, and to determine the cell growth curve. Methods ： The hippocampus of new born SD rat was dissected and digested with trypsin, then was cultivated. Neurobasal medium was added after a day. Immunocytochemical method was used to count the percentage of positive cells. And the cells were stained with 1% toluidine blue and HE. Results ： Primary cultivated neurons adhered in 6 - 8 hours. Neurites elongated, thicked and connected in 5 - 7 days. Most neurons were muhipolar, and cell bodies were triangle, fusiformis and ellipse shape. Cells were brown in MAP -2 and NSE staining. The purity degree of neurons was about 90 %. Cell growth underwent latency, exponential phase, and plateau period. Nucleus was blue by HE staining and Nissl bodied were in the cytoplasm. Conclusion： Neurons are diversified, and are positive in MAP- 2, as well as NSE staining. For HE staining, nucleus and cytoplasm are blue and red, respectively. Nissle bodies are in cytoplasm and seldom in Neurite.