两种冻存液中海马神经元细胞复苏后生物学特性比较  

A biological study on two kinds of cryopresevations to the hippocampal neurons after resuscitation in vitro

作　　者：王洪美[1,2] 胡琴[3] 肖娟[1] 姜晓荣[1] 赵艳廷[1] 曹敬丽[1] 任玉水[1] 田国忠[1] 黄红云[1,2] 

机构地区：[1]北京市虹天济神经科学研究院,北京100144 [2]北京康复中心神经外科,北京100144 [3]北京大学医学部人体解剖学与组织胚胎学系,北京100191

出　　处：《解剖学杂志》2010年第3期345-348,共4页Chinese Journal of Anatomy

摘　　要：目的：建立海马神经元适宜的冻存方法。方法：将新生24h以内的大鼠海马消化成单个细胞后分别直接冻存于血清浓度不同的冻存液中，复苏后进行台盼蓝染色观察细胞活性；培养后观察其生长状态并在第7天固定细胞，分别进行Hoechst，微管蛋白（Tubulin）和神经胶质纤维酸性蛋白（GFAP）显色后，荧光显微镜下计数海马神经元、胶质细胞的百分率，从而建立最佳冻存方法。结果：冻存于高浓度血清冻存液中的神经元百分率为87．5％，冻存于低浓度血清冻存液中的神经元百分率为73．8％，高浓度血清冻存液中的细胞复苏后的状态及神经元存活率明显优于保存于低浓度血清冻存液中的；而冻存于高低浓度血清冻存液中的胶质细胞百分率分别为9．5％和16．5％，胶质细胞在高浓度血清的相对含量明显低于低血清组。结论：高浓度血清冻存液更利于海马神经细胞冻存后的复苏和生长。Objective： To establish feasible cryopreservation method for primary cultured hippocampal neurons. Methods： The cells isolated from the hippocampus of neonatal rats were quickly frozen in two different serum concentrations of cryopreservation mlution for one day after digestion and centrifugal separation. After being resuscitated, the cells were stained with trypan blue to calculate the survival rates of cells. The cells were stained with Hoechst, tubulin and GFAP on the 7^th day to calculate of hippocampal neurons and glia cells. Results： The percentages of the hippocampal neurons in a high serum concentration and a low serum concentration were 87.5% and 73.8%, and those of gtial cells were 9. 5% and 16.5% respectively. Cells growth status in high serum concentration was much better than that in a low serum concentration. Conclusion： High serum concentration is more conducive to hippoeampal neurons in later resuscitation and culture after eryopreservation.

关 键 词：海马神经元 冻存 复苏 存活率 微管蛋白 神经胶质纤维酸性蛋白 

分 类 号：R827.172[医药卫生—临床医学]