小鼠胚胎神经干细胞培养方法的改良、优化及鉴定

目的通过培养神经球来培养神经干细胞的方法是最常用的扩增神经干细胞方法。但是该方法的缺陷是神经干细胞在冻存和复苏的过程中死亡率高,不能达到高效地扩增神经干细胞的目的。本文章的实验目的是建立不通过培养神经球而直接扩增神经干细胞的方法。方法通过从小鼠大脑胚层组织中获取神经干细胞、如何传代以及冻存的步骤进行了优化比较并对复苏后的神经干细胞进行干性鉴定。结果通过比较使用胰蛋白酶、胰蛋白酶和核酸酶、木瓜酶在消化小鼠大脑皮层组织并获得神经干细胞的效率,发现使用胰蛋白酶获得的神经干细胞的数量最多,在质量上和其它两种方法没有明显的差异。在传代过程中,通过比较使用化学消化液和胰蛋白酶消化液,发现胰蛋白酶消化后的神经干细胞存活率比化学消化液高。结论使用收集的神经干细胞培养液和10%DMSO作为冻存液,神经干细胞的复苏存活率达到90%以上。复苏后的神经干细胞在传代两次后仍然高表达干性基因Sox2、Nestin和Pax6。

Improvement,optimization and identification of the methods for deriving the mouse embryonic neural progenitor cells

Objective The method most commonly used for amplification of neural stem cells is through deriving neurospheres cultured neural stem cells,but the defect of the method is the high mortality of the neural stem cells in the process of cryopreservation and recovery,which leads to people can not amplify neural stem cells efficeiently.The experimental purposes of this article is to establish new methods by direct amplification of neural stem cells not through cultured neurospheres.Methods Optimization and comparison the conditions of how to obtain the neural stem cells from the mesodermal tissues in mouse brain,how to passage and cryopreservation,and do the dry identification for the recoveried neural stem cells.Results By comparing the efficiency of digesting the mouse cerebral cortex to obtain the neural stem cells by using trypsin,trypsin and ribonuclease,papain completely,we found that using trypsin can obtain the largest number of neural stem cells,and the quality had not significant differences compared to other two methods.In the process of passage,we found the neural stem cells survival rate by trypsin digestion was higher than the chemical digestion by comparing using different digestive juice.Conclusions The recovery survival rate of neural stem cells was more than 90% by using the collected neural cell culture fluid and 10% DMSO as the freezing medium.The recoveried neural stem cells still high express dry genes Sox 2,Nestin and Pax 6 after passaged twice.