牛膝多肽可能通过ERK1/2途径诱导PC12细胞向神经元分化

Neuronal differentiation of PC12 cells through ERK1/2 pathways

目的:研究牛膝多肽(ABPP)诱导PC12细胞向神经元分化的作用,初步探讨ABPP作用于PC12细胞的信号转导途径。方法:以体外培养的PC12细胞为研究模型,观察在低血清的情况下不同浓度ABPP(0.25、0.50、1.00μg/ml)诱导PC12细胞发生的形态学改变。采用免疫荧光细胞化学法,观察神经丝蛋白(NF-H)在ABPP诱导分化的PC12细胞中的表达;采用Western blot法观察ABPP(1.0μg/ml)加药后不同时间段(0、6、12h和1、2、3、7d)和不同浓度ABPP(0.25、0.50、1.00μg/ml)加药2 d后对PC12细胞ERK1/2活性的影响,同时应用ERK1/2特异性拮抗剂PD98059与ABPP共培养PC12细胞,分析ABPP对PC12细胞的作用与ERK1/2通路的关系。结果:ABPP处理3 d后,部分PC12细胞开始出现神经元样的形态。随着加药时间延长具有神经元样的细胞逐渐增多,到7 d时,可以见到神经生长因子(NGF)和ABPP处理组PC12细胞的突起都显著增多,能形成网络;14 d时,这种现象愈发显著。加药后7 d和14 d,ABPP各浓度组的细胞分化率以及细胞突起长度均明显提高,且存在明显的剂量反应关系。加药第7天和第14天,ABPP高剂量组与NGF组的PC12细胞均出现NF-H标记阳性的分化细胞。ABPP对ERK1/2的激活作用存在明显的量效关系,以1.0μg/ml作用2 d为最大。当用PD98059抑制ERK1/2的活化时,ABPP对ERK1/2的激活作用被部分阻断。结论:ABPP具有诱导PC12细胞神经元性分化的作用,此作用可能是通过ERK1/2信号转导途径实现的。

Objective：To study the potential ability of ABPP on the neuronal differentiation of PC12 cells and to preliminarily investigate the activation of ERK1/2 cascade involved. Methods：By cell culture in the low-concentration of serum,the morphological changes of PC12 cells treated with ABPP at different concentration（0.25,0.50,1.00 μg/ml） were detected. Fluorescent immunocytochemistry was performed to examine the expression of NF-H in differentiated PC12 cells induced by 1.0 μg/ml ABPP. Western blot was performed to detect the acitvities of ERK1/2 in PC12 cells activated by 1.0 μg / ml ABPP for different periods（0h,6h,12h,1d,2d,3d and 7d） and by different concentrations（0.25,0.50,1.00 μg / ml） of ABPP for 2 days co-cultured with activated（diphosphorylated ERK1 / 2） antibody and mitogen activated protein kinase（MAP Kinase,MAPK,ERK-1 ERK-2）antibody. Results：After 3 days treated with ABPP（0.25,0.50,1.00 μg / ml）,PC12 cells started to present neuron morphology characteristics. As time went on,the number of the differentiated PC12 cells increased. At the 7th day the differentiated PC12 cells produced the neurite network and at the 14th day the phenomena became more obvious. It showed that ABPP promoted the differentiation of PC12 by in a dose-dependent manner. ABPP activated ERK1 / 2 in PC12 cells in a dose-dependent manner and a time-dependent manner, with the best concentration of 1.0 μg / ml for 2 days. PD98059 significantly inhibited ABPP-induced phosphorylation of ERK1 / 2. Conclusion：ABPP could induce the neuronal differentiation of PC12 cells through ERK1/2 pathways