胎鼠神经干细胞原代培养及传代条件优化  

Primary culture and optimization of subculture conditions of neural stem cells from fetal rats

作　　者：朱文豪 刘天一[1] 何川[1] 张晓宇 辛强 王海峰[1] ZHU Wenhao;LIU Tianyi;HE Chuan;ZHANG Xiaoyu;XIN Qiang;WANG Haifeng(Department of Neurotrauma Surgery,First Hospital,Jilin University,Changchun 130021,China)

机构地区：[1]吉林大学第一医院神经创伤外科,吉林长春130021

出　　处：《吉林大学学报（医学版）》2023年第6期1642-1648,共7页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金面上项目(81871555);吉林省财政厅卫生人才专项项目(JLSWSRCZX2021-028)。

摘　　要：目的:探讨胎鼠神经干细胞(NSCs)原代培养方法和活性评价体系,确定NSCs的最佳传代条件。方法:选取孕11~14 d SD大鼠,提取胎鼠原代NSCs,采用巢蛋白免疫荧光染色鉴定NSCs。将NSCs以1.0×10^(4)、2.0×10^(4)、6.0×10^(4)、1.0×10^(5)、1.6×10^(5)和2.0×10^(5) mL^(-1)的细胞密度进行传代培养48 h后观察神经球的形态表现,测定神经球直径。活死细胞染色法检测不同直径神经球中NSCs存活率。结果:NSCs呈巢蛋白阳性,培养的细胞为神经干细胞。NSCs呈聚集生长和牢固细胞间黏附聚集模式,形成神经球,平均直径为(152.72±47.52)μm,球与球之间少有单独的细胞散在。与2.0×10^(4) mL^(-1)传代密度比较,6.0×10^(4) mL^(-1)和1.0×10^(5) mL^(-1)传代密度的神经球直径增大(P<0.05)。1.0×10^(5) mL^(-1)、1.6×10^(5) mL^(-1)和2.0×10^(5) mL^(-1)传代密度的神经球直径比较差异无统计学意义(P>0.05)。与0~40μm、40~60μm、60~80μm和80~100μm直径神经球比较,100~200μm直径神经球中NSCs存活率降低(P<0.05)。结论:以1.0×10^(5) mL^(-1)的密度进行传代时神经球直径为80~100μm,可有效提高NSCs传代培养效率和活性。Objective:To discuss the method of primary culture and activity evaluation system of the neural stem cells(NSCs)of the fetal rats,and to confirm the optimal subculture conditions of the NSCs.Methods:The SD rats with 11-14 d gestation were chosen,and the primary NSCs of the fetal rats were extracted.Nestin immunofluorescence staining was used to identify the NSCs.The NSCs were subcultured at the cell densities of 1.0×10^(4),2.0×10^(4),6.0×10^(4),1.0×105,1.6×10^(5 )and 2.0×10^(5 )mL^(-1),then the morpholgy of the neurospheres was observed 48 h after passage and the diameter of the neurospheres was calculated.The survival rates of NSCs in the neurospheres with different diameters were detected by live-dead cell staining.Results:The expression of nestin in the NSCs was positive,indicating that the cultured cells were the NSCs.The NSCs showed aggregation growth and strong cell-cell adhesion pattern forming neurospheres with an average diameter of(152.72±47.52)μm,and there were few single cells scattered between the neurospheres.Compared with 2.0×10^(4) mL^(-1) subculture density,the diameters of the neurospheres at the subculture densities of differences 6.0×10^(4) mL^(-1) and 1.0×10^(5 )mL^(-1) were increased(P<0.05).There were no significant differences in the diameters of the neurospheres at the subculture densities of 1.0×10^(5 )mL^(-1),1.6×10^(5 )mL^(-1),and 2.0×10^(5 )mL^(-1)(P>0.05).Compared with neurospheres with diameters of 0—40μm,40—60μm,60—80μm,and 80—100μm,the survival rate of NSCs in the neurospheres with the diameters of 100—200μm was decreased(P<0.05).Conclusion:The neurospheres with the diameters of 80—100μm when subcultured at the density of 1.0×10^(5 )mL^(-1) can effectively improve the efficiency and activity of subculture of the NSCs.

关 键 词：神经干细胞 胎鼠 原代培养 传代条件 神经球 

分 类 号：Q254[生物学—细胞生物学]