MAPK、PI3K途径在蛇毒神经生长因子诱导PC12细胞分化的作用.docVIP

MAPK、PI3K途径在蛇毒神经生长因子诱导PC12细胞分化的作用 【关键词】 神经生长因子;,PC12细胞;,细胞分化;,信号传导 　　摘要: 目的 研究 丝裂原激活蛋白激酶(MAPK)和磷脂酰肌醇3激酶(PI3K)/Akt途径在蛇毒神经生长因子(NGF)诱导PC12细胞分化中的作用。 方法 应用 蛋白免疫印迹法 分析 p44/p42MAPK和Akt的磷酸化水平,并利用MAPK抑制剂PD98059和PI3K抑制剂LY294002,观察其对NGF诱导的PC12细胞形态学改变的 影响 。结果 眼镜蛇毒NGF在10 ng/mL即可诱导PC12细胞长出突起,100,1000 ng/mL作用明显,呈剂量效应关系;NGF能有效刺激p44/p42MAPK、Akt的磷酸化;分别用特异抑制剂PD98059抑制MAPK活性,LY294002抑制PI3K活性后,NGF诱导的细胞分化均受抑制。 结论 蛇毒神经生长因子诱导PC12细胞的分化作用与p44/p42MAPK和PI3K/Akt途径相关。 　　关键词: 神经生长因子; PC12细胞; 细胞分化; 信号传导 　　ABSTRACT: ObjectiveTo investigate the role of mitogenactivated protEin kinase(MAPK) and phosphoinositide 3kinase(PI3K)/Akt pathways in differentiation of PC12 cells induced by nerve growth factor(NGF) from venom. Methods The phosphorylation of p44/p42MAPK and Akt were detected by Western blot. The role of MAPK and PI3K in the cellular action of PVC12 cells induced by NGF were assessed by use MAPK and PI3K specific inhibitor PD98059 and LY294002 respectively. Results NGF from cobra venom can stimulate p44/p42MAPK and Akt phosphorylation and induce the differentiation of PC12 cells from 10 ng/mL to 1000 ng/mL. The differentiation of PC12 cells induced by NGF was inhibited when MAPK or PI3K pathway was abolished by the specific inhibitor PD98059 and LY294002. Conclusion The differentiation of PC12 cells by NGF from cobra venom relates to p44/p42MAPK and PI3K/Akt pathway. 　　KEY WORDS: nerve growth factor; PC12 cell; cell differentiation; signal transduction 　　神经生长因子(NGF)自发现以来,其在神经系统中的作用倍受关注。实验表明,NGF能促进大鼠嗜铬瘤PC12细胞系的分化。NGF的分化信号是通过与其高亲和力受体TrkA结合而触发的[1],进而依次激活一系列信号途径。然而,信号途径如何参与其激活增殖及分化调控还未完全清楚。多种细胞的生存、分化、凋亡信号通过受体RasRaf丝裂原激活蛋白激酶(MAPK)又称胞外信号调节蛋白激酶(ERK)和受体磷脂酰肌醇3激酶(PI3K)Akt(又称蛋白激酶B)信号途径进行传导[23]。笔者探讨MAPK、PI3K信号途径与眼镜蛇毒来源的神经生长因子诱导的PC12细胞分化间的关系。 　　1材料和方法 　　1.1 材料 　　1.1.1 细胞株 　　PC12细胞(大鼠嗜铬瘤细胞株),由福建医科大学分子医学研究中心郑志教授赠送。 　　1.1.2 主要试剂 　　蛇毒神经生长因子(NGF)用凝胶通透层析和离子交换层析等方法自蛇毒粗毒冻干粉(广东产眼镜蛇)中提纯。信号途径的特异抑制剂――MAPK抑制剂PD98059、PI3K抑制剂LY294002(美国Promega公司),溶于DMSO配制成储存液。抗pERK、pAkt(ser)、βtubulin抗体,辣根过氧化物酶标记的羊抗兔、羊抗小鼠二抗,ECL检测系统