两种神经球制片方法的比较研究

Comparison of two methods in neurosphere slide preparation

目的探讨两种神经球制片方法一贴片法和切片法的差异，并初探神经球内部的细胞结构。方法分离培养新生小鼠端脑神经干细胞，收集原代或传代培养7d的神经球用于制片：贴片法是将神经球整体贴于载玻片上，切片法是将神经球用OCT包埋，冷冻切片机切片；通过比较两种制片方法的巢蛋白（nestin）免疫荧光染色的差异说明两种制片方法的差异；用HE染色方法将神经球切片染色，进一步观察其内部结构。结果成功分离培养的新生小鼠端脑神经干细胞在体外培养中形成神经球；贴片法制片神经球表面细胞染色良好，内部细胞不能着色，而且细胞形态显示不清晰．切片法制片神经球表面和内部细胞均能着色，细胞形态显示清晰；HE染色见神经球细胞之间借助突起相互连接，形成错综复杂的细胞网络。结论切片法制片较贴片法制片更有利于神经球的形态学研究：神经球是一个复杂的立体的细胞生长模式。

Objective To explore the differences of 2 slice techniques （paste-up method and section method） in the neurosphere slide preparation and the inside structure of the neurosphere. Methods Neural stem ceils were isolated from the neonatal mouse telencephalon and the neurospheres on the 7^th d of primary culture or subculture were collected. Pasting the intact neurosphere to the slide was the paste-up method. Embedding the spheres with OCT followed by cutting into sections with a frozen section machine was the section method. Immunofluorescence was employed to observe the differences of nestin so as to illustrate the differences of the 2 neurosphere slide preparation methods. Inside structures of the neuroshperes were further studied by HE staining. Results Neural stem cells were successfully isolated and neurospheres formed during in vitro cultures. The superficial cells of the spheres were well stained, but the internal cells could not be stained when paste-up method was adopt; furthermore, the morphology of the cells could not be shown clearly. Both the superficial and internal cells could be well stained with clear morphology when the section method was chosed; as indicated by HE staining, the sphere cells connected to each other and formed complicated cell nets. Conclusion Section method is＇superior to paste-up method in morphology study of neurosphere and neurosphere enjoys a complicated three-dimension cell growth style.