新生大鼠大脑皮质O-2A祖细胞的体外诱导分化

The differentiation of oligodendrocyte / type-2 astrocyte progenitor cells from neonatal rat cerebrum in vitro

目的 在获取高纯度O 2A祖细胞的基础上 ,探讨O 2A祖细胞在体外分化的形态学特点。方法 采用“两次恒温摇床振荡法”和差速贴壁法 ,结合使用神经营养因子 (bFGF、PDGF AA) ,进行O 2A祖细胞体外纯化和扩增培养 ,并观察O 2A祖细胞在有血清和无血清培养条件下的分化情况 ,用免疫细胞化学鉴定分化成熟的 2型星型胶质细胞和少突胶质细胞。结果 培养的O 2A祖细胞能形成克隆球 ,具有增殖能力 ,经免疫细胞化学鉴定细胞纯度达 90 %以上。在含血清的培养基中能定向分化为 2型星形胶质细胞 ,其胞体较大 ,从胞体上伸出细长突起 ,由突起上再发出更为细小的分支 ,GFAP和A2B5抗体标记均为阳性 ;在无血清的化学条件培养基中能定向分化为少突胶质细胞 ,其胞体较小 ,突起短而细 ,相互交织呈“蜘蛛网”样 ,CNPase抗体标记阳性。结论 (1)培养的O 2A祖细胞保持定向干细胞的特性 ,具有增殖和双向分化的潜能 ;(2 )O 2A祖细胞定向分化为 2型星形胶质细胞过程中经历较长的增殖期 ,而分化为少突胶质细胞过程中则增殖期较短 ;(3)分化的 2型星形胶质细胞和少突胶质细胞在形态学和抗原表达上存在差异。

Objective To observe the differentiation of oligodendrocyte / type-2 astrocyte progenitor cells (O-2A progenitors) on the base of gaining high pure O-2A progenitors in vitro. Methods O-2A progenitors were isolated by standard shaking and different adhension methods, combing with growth factors, bFGF and PDGF-AA. With cell culture in vitro, O-2A progenitors differentiated into type-2 astrocytes or oligodendrocytes depending on the culture medium and immunocytochemistry was performed to detect the expression of A2B5(O-2A progenitors), GFAP(type-2 astrocytes) and CNPase(oligodendrocytes). Results The purified O-2A progenitors could form clone ball, and the cell proliferation was confirmed by cell growth curve. Identified by immunocytochemistry of A2B5, the purity of O-2A progenitors was more than 90%. O-2A progenitors developed in vitro into type-2 astrocytes when cultured in fetal calf serum-containing medium, whereas they developed into oligodendrocytes when cultured in serum-free medium. The morphology of type-2 astrocytes were fibrous-like, whereas oligodendrocytes were spider-like. Conclusion (1) O-2A progenitors could be characterized as glial stem cells according to their proliferation and bipotential differentiation features. (2) O-2A progenitors could differentiate into type-2 astrocytes through longer period of proliferation than oligodendrocyte. (3) Type-2 astrocytes and oligodendrocyte were different in morphology and antigenic expression.