成年大鼠侧脑室下区星形胶质细胞的分离培养及纯化研究

成年哺乳动物侧脑室下区星形胶质细胞(AST)的神经干细胞特性研究是当前神经科学领域的热点,为了进一步了解AST生物学特性,体外培养是探讨AST细胞学特性的有力手段,而怎样通过体外培养得到高纯度的成年AST是亟待解决的问题。本研究以2.5月的成年大鼠侧脑室下区为实验材料进行AST培养,并对传统的培养方法进行改良。首先在AST培养基中增加B27添加剂,结合成年AST体外生长特性,采用低浓度血清(2.5%～5%)和血清浓度调整法对培养的AST进行纯化。然后采用GFAP免疫细胞化学染色方法对培养至14d的细胞进行鉴定及纯度检测,采用流式细胞仪检测B27添加剂对AST细胞周期的影响。结果显示:B27添加剂能有效促进体外培养的成年AST的生长和增殖,采用此方法培养的成年AST纯度达90%以上;B27添加剂与低浓度血清的组合以及根据细胞特性及时调整血清浓度是一种有效的纯化成年AST方法。本方法的建立可望为AST的神经干细胞特性研究提供理想的细胞模型。

A study on the isolation,cultivation,purification and morphologic characterization of adult rat subventricular zone astrocytes in vitro

Recent studies have revealed an unexpected new role for astrocytes in subventricular zone (SVZ) that of stem cells/precursors in the adult brain. The present study is aimed to develop a novel cultural method to acquire uniformly enriched population of astrocytes from adult rat SVZ for related studies. Primarily cultured AST were dissociated from SVZ of 2.5-month-old rats and the cultural procedures were optimized as follows: firstly,culture medium was modified by adding B27 supplement into basal medium and AST from SVZ were cultured and further purified by adjusting serum at low concentration (2.5%-5%) combined with limited serum methods; secondly, the purity of cultured astrocytes was identified with glial fibrillary acidic protein (GFAP) immunocytochemical staining and the effect of B27 supplement on growth and proliferation of adult SVZ AST was detected with flow cytometric analysis. Our results showed that the modified medium with B27 supplement could promote the survival as well as the proliferation of AST; the AST purity reached more than 90% by this method, indicating that the optimized medium combined with limited serum methods is effective in preparation of highly purified adult SVZ AST, which would be useful subsequently for in vitro studies on the characteristics of neural stem cells.