贴壁培养法鉴定海马神经干/祖细胞的增殖与分化能力

摘要

BACKGROUND: In vitro isolation and culture of neural stem/progenitor cells will provide a good cell model for the study of neurodevelopment, neurological diseases, and neural transplantation. OBJECTIVE: To study the highly effective method for isolation and expansion of hippocampal neural stem/progenitor cells from newborn mice, and to identify the proliferation and differentiation of hippocampal neural stem/progenitor cells using improved adherent culture method. METHODS: Neural stem/progenitor cells were isolated from the hippocampus of newborn C57BL/6 mice and were expanded for several passages. Combination of polylysine and laminin were used for adherent culture to promote cell attachment. Morphological observation and immunofluorescence cytochemical staining were conducted to detect the expression of neural progenitor-specific marker protein Nestin and proliferation index Ki-67. After 7 days of induction and differentiation, the expression of GFAP, DCX, Tuj1 and S100β was detected by immunofluorescence. RESULTS AND CONCLUSION: About 82％ of the cultured neural stem/progenitor cells expressed Nestin, and about 49％ expressed Ki-67. A small number of cells were DCX-positive neurons after induction and differentiation, while most of the cells were positive for GFAP. The ratio of neurons to astrocytes was 1:1.7 identified by Tuj1 and S100β double staining. The neural stem/progenitor cells derived from the hippocampus were efficiently isolated and cultured. The cell proliferation and differentiation abilities were effectively identified after adherent culture, which can provide sufficient cell sources for further experimental research.%背景:神经干/祖细胞广泛应用于神经发育、神经相关性疾病和细胞移植等研究,体外分离培养的神经干/祖细胞能够为相关研究提供细胞模型.目的:探讨分离新生小鼠海马组织并高效培养出神经干/祖细胞的方法,并使用贴壁培养法对其增殖以及分化能力进行鉴定.方法:分离新生C57BL/6 小鼠海马组织,培养原代神经干/祖细胞并扩增,进行形态学观察;联合使用多聚赖氨酸和层粘连蛋白促进细胞贴附,进行贴壁培养;免疫荧光法检测特异性标志蛋白Nestin 表达和增殖指标Ki-67的表达;使用神经干/祖细胞分化培养基诱导分化培养7 d 后,免疫荧光法检测GFAP、DCX、Tuj1 和S100β的表达.结果与结论:①培养的神经干/祖细胞约82％表达Nestin,约49％表达Ki-67;②诱导分化后,少数细胞为DCX阳性,大多数细胞为GFAP 阳性.荧光双色染色后显示Tuj1 和S100β 表达的神经元和星形胶质细胞比例为1∶1.7;③结果显示,从新生C57BL/6 小鼠海马组织中成功分离并高效培养出神经干/祖细胞,贴壁培养后有效地对其增殖和分化能力进行了鉴定,能够为进一步实验研究提供足够的细胞来源.