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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。
Objective To investigate the relationship between Rac1 protein expression and proliferation and differentiation of neural precursor cells. Methods Neonatal rat cerebral cortex was obtained. Primary culture was performed to obtain a mixed cell culture system. After cultured for 6 days in medium containing 10% serum, the cells were replaced with serum-free medium and cultured. The experimental design of four time points, inverted phase contrast microscope observation of cell proliferation and differentiation, immunofluorescence staining to identify cell types and Rac1 protein expression in the cell by Western blot Rac1 protein expression changes. Results The primary mixed culture system of neonatal SD rats was established. The bottom of this system consisted primarily of monolayers of type I glial cells with positive staining for GFAP. In the monolayer cells, there were some small round strong refraction Proliferation of neural precursor cells, β Ⅲ-Tubulin staining was positive. After replacing serum-free medium, the upper neural precursor cells proliferated in a large number, positive for βⅢ-Tubulin and Rac1 staining. Western blot analysis showed that the expression of Rac1 protein increased during the proliferation of neural precursor cells, while the expression of Rac1 protein decreased during differentiation. Conclusion The expression of Rac1 protein is closely related to the proliferation and differentiation of neural precursor cells and may be involved in the process of proliferation and differentiation of neural precursor cells.