海马神经元原代培养方法

目的：掌握海马神经元的有效培养方法和细胞保存培养方法,利于细胞模型的建立。方法：取出出生1 d的SD大鼠海马,联合机械吹打法和胰酶消化法分离海马细胞,加入DMEM/F12＋10%FBS制备细胞悬液,接种在玻璃培养瓶内1～2 d,收集上清细胞液、离心、重悬细胞;再接种在培养板上,培养1 d后,全液更换为Neurobasal＋2%B27,以后每3 d半量换液。利用倒置相差显微镜观察细胞形态变化;利用NF-200、Neu-N抗体免疫荧光、免疫组化技术鉴定海马神经元;利用96孔板培养细胞,计数每个孔海马细胞比例;利用MTT法测定细胞活性。结果：接种在培养板上第1 d细胞贴壁,第3～4 d细胞长出突触,第7～8d细胞突触生长迅速,第11～13 d细胞交织成网状,第15～20 d细胞生长旺盛,20 d后细胞形态开始改变。免疫荧光技术鉴定细胞突触呈红色,细胞核呈绿色;免疫组化技术鉴定突触和胞核都呈棕黄色。细胞纯度95～97.5%,细胞活性以15～17 d最高（93.94～95.13%）,与其他时间点比较,P〈0.01。结论：该方法简便可行,节约取材及时间,可获得纯度高、活性好的海马细胞,为后续研究提供实验基础。

Preparation, cryopreservation and in vitro culture of spermatogonial stem cells of new born calves

The experiment was designed to study the preparation, cryopreservation and culture of calf spermatogonial stem cells in order to provide some data for theoretical research and practical use of calf spermatogonial stem cells. As for enzymolysis of testis tissue, two digestive method A and B. The cryopreservation cooling procedure was that spermatogonial stem cells was equilibrated at 4℃ for lh, at -20℃ for lh, at -40℃ for 2h, at -80℃ overnight and then stored in liquid nitrogen. The different concentration of three eryoprotectants were compared. The results indicated that both method A and method B achieved the same high motility rates of cells with method B needing time longer, calf spermatogonial stem cells could be well cryopreserved by the stage cooling procedure. Satisfactory cryopreservative results were gotten by adding 10% DMSO or 10% EG to cryopreservative medium, the former was better and adding sucrose could improved cryopreservative effect. The culture behaviors of spermatogonial stem cells kept the same before and after cryopreservation. It was concluded that an effective method was verified for preparation, cryopreservation and culture of new calf spermatogonial stem cells.