新生SD大鼠原代海马组织中神经干细胞的培养和鉴定  

Culture and identification of neural stem cells in hippocampus tissue of newborn SD rats

作　　者：张玲娣 赵亮[2] 由涌 许倩[4] 杨振军[1] ZHANG Lingdi;ZHAO Liang;YONG Yong;XU Qian;YANG Zhenjun(Department of Human Anatomy,Chengde Medical University,Chengde 067000,China;Department of Pharmacology,Chengde Medical University,Chengde 067000,China;Department of Immunology,Chengde Medical University,Chengde 067000,China;Institute of Basic Medicine,Chengde Medical University,Chengde 067000,China)

机构地区：[1]承德医学院人体解剖教研室,河北承德067000 [2]承德医学院药理学教研室,河北承德067000 [3]承德医学院免疫学教研室,河北承德067000 [4]承德医学院基础医学研究所,河北承德067000

出　　处：《吉林大学学报（医学版）》2023年第3期795-801,共7页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(81700310);河北省科技厅重点研发计划项目(22377746D)。

摘　　要：目的:观察原代海马组织中神经干细胞(NSCs)的形态表现和生长规律,为NSCs提取和培养提供依据。方法:提取新生SD大鼠海马组织中NSCs,观察海马组织中NSCs形态表现,流式细胞术检测海马组织中NSCs纯度,免疫荧光法检测NSCs中Nestin和EdU蛋白表达,细胞计数法和CCK-8法检测NSCs增殖活性。结果:培养第0天海马组织中NSCs以单细胞形式悬浮于培养基中;培养第2天海马组织中NSCs开始聚集成大小不等和形态不规则的细胞团块,悬浮生长;培养第8天神经球大小不一,呈圆形或椭圆形,边界清晰,无明显突起。培养第8天NSCs纯度为76.50%~85.40%。NSCs细胞质中Nestin阳性表达呈绿色,细胞核中EdU阳性表达呈红色,神经球为Nestin与EdU共染细胞构成。培养5~11 d,NSCs处于对数生长期,细胞增殖活性较高。结论:NSCs增殖能力强,培养至第8天时纯度较高,体外培养5~11 d时细胞增殖活性较高。Objective:To observe the morphology and growth pattern of the neural stem cells neural stem cells(NSCs)in hippocampus tissue,and to provide the basis for the extraction and culture of the NSCs.Methods:The NSCs were isolated from hippocampus tissue of the newborn SD rats.The morphology of the NSCs in hippocampus tissue was observed;flow cytometry was used to detect the purities of the NSCs in hippocampus tissue;immunofluorescence method was used to detect the expressions of Nestin and EdU proteins in the NSCs;the proliferative activities of the NSCs were analyzed by cell counting and CCK-8 methods.Results:On the 0 day of culture,the NSCs in hippocampus tissue were suspended in the culture medium with the form of single cells;on the 2nd day of culture,the NSCs in hippocampus tissue suspended in the culture medium and began to gather into the cell clumps with different sizes and irregular shapes;On the 8th day of culture,the neurospheres showed round or oval neurosphere with different sizes,and showed clear boundary without synapse.The purity of the NSCs in hippocampus tissue was 76.50%-85.40%On the 8th day of culture,the positive expression of Nestin in the cytoplasm of the NSCs was green,and the positive expression of EdU in the nucleus was red.The neurospheres were composed of the Nestin and EdU co-staining cells.the NSCs were at logarithmic growth period,and the proliferation abilities were high when cultured for 5-11 d.Conclusion:The proliferation ability of the NSCs is high;the purity of the NSCs is high on the 8th day of culture;the proliferation ability of the NSCs is high when cultured for 5-11 d.

关 键 词：神经干细胞 海马组织 增殖活性 生长曲线 免疫荧光 

分 类 号：Q813.1[生物学—生物工程]