NGF诱导PC12细胞神经元样细胞分化

PC12 Cells Neuron Differentiation Induced by NGF

目的:建立PC12细胞的神经元样细胞分化模型,并探讨ERK蛋白在PC12细胞神经元样细胞分化中的可能机制.方法:以10、20、50 g/L的NGF(NGF溶于PBS,培养基中PBS的终浓度不超过2%)培养PC12细胞,应用倒置相差显微镜、显微镜测微尺及流式细胞仪鉴定PC12细胞的分化,以确定NGF使用剂量.运用免疫印迹检测不同浓度、不同作用时间时ERK蛋白在PC12细胞中的表达,并进行统计学分析.结果:随NGF剂量的升高,PC12细胞的体积、最长突起长度和突起数目均会增大或增多。统计学分析证明50 g/L的NFG作用48 h足以诱导PC12细胞的交感神经样改变。尽管ERK总蛋白水平在NFG作用前后无明显改变,但在NFG作用5 min后磷酸化的ERK蛋白水平即显著升高,达到峰值,并持续约1 h.50 g/L的NFG作用于PC12细胞48 h可使细胞发生明显的G1期阻滞.结论:PC12细胞可在NGF作用下出现交感神经样改变,并且细胞的分化程度依赖于NGF的使用剂量和作用时间.在NGF诱导的PC12细胞神经元样分化中ERK蛋白的磷酸化对NGF呈现剂量和时间依赖性,提示ERK蛋白在分化的早期发挥重要作用.

Objective： Establishing the NGF-induced PC12 cell neuron differentiation model, and observing the effect of NGF on protein expression of ERK and pp-ERK, exploring the possible mechanism of ERK on PC12 cell neuron differentiation. Methods： PC12 cells were treated with NGF in different concentration （10, 20, 50 lag/L）. Phase-contrast microscope, microscopy planometer and the flow cytometer were used to observe cell differentiation. Using immunoblot to investigate the expression of ERK in PC 12 cell with different concentration and different action time of NGF. Results： Along with the increase of NGF concentration, the diameter, the length and the number of axon-like processes of PC 12 cells all added or extended obviously. Statistical analysis demonstrate that the effect of 50 μg/L NGF will induce neuron differentiation of PC12 cells. There were not distinct difference of ERK protein between the normal cells and the treated cells. However, NGF maximally increased the pp-ERK protein within 5 min, and the activation ofpp-ERK remained forat least 1 h. 50 μg/L NGF could induce the G1 cycle block in PC12 cells. Conclusion： The neuron differentiation of PC 12 cells will happen at the effect of NGF, and it depends on NGF dosage and action time. In the PC12 cell neuron differentiation induced by NGF, the phosphorylation of ERK protein depends on NGF dosage and action time, prompt that the ERK has important function on the early stage of differentiation.