小鼠胚胎脊髓神经干细胞提取与传代培养:3种常用消化酶优劣的比较与分析  

Extraction and subculture of neural stem cells from mouse embryonic spinal cord:comparison and analysis on advantages and disadvantages of three commonly used digestive enzymes

作　　者：罗丹 葛志林 侯永辉 王万舜 詹吉恒 侯宇 林定坤 陈树东 Luo Dan;Ge Zhilin;Hou Yonghui;Wang Wanshun;Zhan Jiheng;Hou Yu;Lin Dingkun;Chen Shudong(Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510000,Guangdong Province,China;Guangdong Academy of Traditional Chinese Medicine Science,Guangzhou 510000,Guangdong Province,China;Guangzhou University of Chinese Medicine,Guangzhou 510000,Guangdong Province,China)

机构地区：[1]广州中医药大学第二附属医院,广东省广州市510000 [2]广东省中医药科学院,广东省广州市510000 [3]广州中医药大学,广东省广州市510000

出　　处：《中国组织工程研究》2025年第31期6609-6615,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81704095),项目负责人:陈树东;广州市基础研究计划基础与应用基础研究项目(202102020542),项目负责人:陈树东;国家自然科学基金项目(82074451),项目负责人:林定坤。

摘　　要：背景:在神经干细胞的研究及应用过程中,细胞的培养与传代是关键环节,直接影响到细胞的质量和实验结果。寻找一种最适宜的消化酶类,对维持胚胎小鼠脊髓神经干细胞的生物学特性并增强其传代效率,具有重要意义。目的:探讨胚胎小鼠脊髓神经干细胞传代最适合的消化酶。方法:显微解剖分离提取孕14 d C57BL/6胎鼠脊髓组织,用含有表皮生长因子、碱性成纤维生长因子及B27的DMEM/F12无血清培养基培养,待成球后进行Nestin与Sox2免疫荧光鉴定。神经干细胞传代培养过程中分别采用胰蛋白酶、木瓜蛋白酶以及TrypLE^(TM)Express酶消化结合吹打法制成单细胞悬液,观察细胞分散效果及成球情况,取第3代细胞进行碘化丙啶染色观察细胞死亡情况,通过计数细胞总数观察细胞增殖情况,通过免疫荧光染色、Western blot和RT-PCR检测Olig2、Tuj1、GFAP、NeuN蛋白和mRNA表达鉴定细胞分化情况。结果与结论:脊髓组织细胞培养72 h后可形成悬浮生长的神经球,5-7 d后即可传代。与胰蛋白酶、木瓜蛋白酶相比,TrypLE^(TM)Express酶结合吹打法进行传代的细胞分散率高,活性较好,分化为NeuN与Tuj1的阳性神经元细胞较多。此研究优化了神经干细胞的培养和传代过程,为进一步开展脊髓疾病的干细胞移植治疗研究奠定了基础。BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLE^(TM)Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLE^(TM)Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

关 键 词：神经干细胞 C57BL/6小鼠 胚胎 脊髓 胰蛋白酶 木瓜蛋白酶 TrypLE^(TM)Express酶 工程化干细胞 

分 类 号：R459.9[医药卫生—治疗学] R338.1[医药卫生—临床医学] R744