新生大鼠神经干细胞的体外培养与冷冻复苏

Cryopreservation and Culture of Neural Stem Cells Isolated from Postnatal Rat

摘要: 目的：分离培养新生SD大鼠神经干细胞(NSCs)，观察应用不同浓度的二甲基亚砜(DMSO)作为冷冻保护剂冷冻保存不同时间的神经干细胞复苏后，其活力及生物学特性的鉴定。方法：在含细胞因子表皮生长因子及碱性成纤维细胞生长因子的无血清培养液中培养新生大鼠海马神经干细胞。经传代扩增后，培养于含有0、5%、10%、15%、20%不同浓度的二甲基亚砜和胎牛血清的神经干细胞冻存液中，在–80℃冰箱中分别冻存1周、2周及一个月后，复苏培养并鉴定，然后对其复苏率进行检测。结果：新生大鼠海马神经干细胞经冷冻复苏后，大多数细胞生长良好并形成新的克隆球。台盼蓝染色检测冻存1周、2周及一个月后的复苏率，含有10%二甲基亚砜的复苏率最高且具有显著性差异(P < 0.05)，不同冻存时间的复苏率分别为54.00 ± 1.73，59.00 ± 1.16，58.00 ± 2.08。结论：神经干细胞能在体外适宜的条件下进行培养及冻存、复苏，并且复苏后不影响其原有的生物学特性。

Abstract: Objective: to isolate and culture neural stem cells from postnatal rat and to observe the viability and biological property of these cells after cryopreservation. Methods: the neural stem cells isolated from hippocampus of the postnatal rat were cultured in culture solution without blood serum. After amplification, the cells were cultured in different concentration of freeze-stored liquid which contained 0、5%、10%、15%、20%DMSO and fetal calf serum, then stored in refrigerator of 80 centigrade below zero one week, two weeks and one month respectively. Through resuscitation training and testing, then we detected the recovery rate of the NSCs. Results: after cryopreservation and culture of neural stem cells isolated from postnatal rat, most of them were growth well and formed into the new cell clones, then the recovery rate of the cells were de-tected with Trypan Blue. There was significant difference in 10% DMSO and it has the highest recovery rate，which is 54.00 ± 1.73, 59.00 ± 1.16, 58.00 ± 2.08. Conclusion: the neural stem cells derived from postnatal rat were able to culture, cryopreservation and resuscitation, which did not change their biological properties.