Rac1蛋白表达与神经前体细胞增殖分化的关系

目的探讨Rac1蛋白表达与神经前体细胞增殖分化的关系。方法取新生大鼠大脑皮层,原代培养获得混合细胞培养体系,在含10%血清的培养基中培养6 d细胞汇合后更换无血清培养基继续培养。实验中设计4个时间点,用倒置相差显微镜观察细胞增殖分化的变化,免疫荧光染色技术鉴定细胞种类以及Rac1蛋白在细胞内的表达,通过Western blot检测Rac1蛋白的表达变化。结果建立了新生SD大鼠大脑皮层原代混合细胞培养体系,此体系底层主要是由Ⅰ型胶质细胞组成的单层细胞,GFAP染色呈阳性;在单层细胞上面有一些小圆形强折光性神经前体细胞生长,βⅢ-Tubulin染色呈阳性。更换无血清培养基后上层神经前体细胞大量增殖,βⅢ-Tubulin、Rac1染色呈阳性。Western blot定量分析结果显示,神经前体细胞增殖过程中Rac1蛋白表达上升,而分化过程中Rac1蛋白的表达下降。结论Rac1蛋白表达与神经前体细胞的增殖分化过程密切相关,可能参与了神经前体细胞的增殖分化过程。

Study on the Relationship Between Rac1 Expression and the Proliferation and Differentiation of Neural Precursor Cells

Objective To investigate the relationship between Racl expression and the proliferation and differentiation of neural precursor cells. Methods Mixed primary cultures were prepared from the cerebral cortex of newborn SD rats. Cells were cultured in DMEM （H） plus 10% FCS, and subsequently in serum-free medium （SF） after confluence. Immunofluorescence staining was used to characterize the mixed cultures. Racl expression was analyzed by immunofluorescence and western blot. Results Cerebral Cortex culture system was established. After cultured for 6 days, glial cells were of the most abundant cell type in the primary mixed neuronal-glial cultures of newborn rat cortex and formed a cell monolayer. Neural precursor cells grew on top of the monolayer. The mixed cell cultures were positive for GFAP and β Ⅲ-Tubulin by immunofluorescence staining. After confluence of astrocytes, change of medium to SF resulting in a considerable proliferation of the neural precursor cells for up to 4 d and the differentiation of these cells afterwards, which was accompanied by varied expression level of Racl detected by immunofluorescence and westem blot. The expression of Racl protein increased in the process of the neural precursor cell proliferation, but decreased when these cells began to differentiate. Conclusion Rael protein expression is closely related to the state of proliferation and differentiation of neural precursor cells.