Notch信号通路体外和细胞的药物筛选模型的建立和研究.docVIP

Notch信号通路体外和细胞的药物筛选模型的建立和研究 钮 琦, 吴 方* (上海交通大学系统生物医学研究院, 上海 200240 摘要: Notch信号通路与生物体的发育及多种癌症的发生密切相关, 目前针对Notch信号通路的药物筛选模 型均以鼠源Notch蛋白为底物, 未见以人源Notch为底物的报道。本文克隆并表达提纯了人源Notch1截短形式 N100, 以之为底物首次构建了Notch信号通路体外和细胞的药物筛选模型。同时, 用已知Notch通路抑制剂对此 模型进行了验证, 得到了相应的IC50值。本研究构建的模型能有效应用于人源Notch信号通路的调控剂筛选, 为 高效发现靶向Notch通路的调控剂奠定了基础。 关键词: Notch信号通路; 癌症; 调控剂; 药物筛选模型 中图分类号: R965.1 文献标识码:A 文章编号: 0513-4870 (2014 06-0837-06 Establishment of in vitro and cell-based drug screening model for Notch signaling pathway NIU Qi, WU Fang* (Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University, Shanghai 200240, China Abstract: Notch signal pathway is closely related to the organism’s development and a variety of cancers. Current models available for screening modulators of Notch signal pathway all use mouse Notch protein as substrates and those models which use human Notch protein have not been reported. To make the screen results much more reliable, the authors cloned a truncated form of human Notch1 called N100, and built the screening models for the use of it instead of mouse Notch protein. The models included an in vitro screening model based on the purified γ-secretase enzyme and a cell model using luciferase reporter system. The screening models then have been verified by the known modulators of Notch signal pathway and the IC50 values have been obtained. The verified models can be used to screen modulators of human Notch signaling pathway effectively and it can lay the foundation for finding new modulators of this kind effectively. Key words: Notch signaling pathway; cancer; modulator; drug screening model Notch信号通路是一类高度保守、不依赖第二信使的信号通路。它由Notch配体蛋白、Notch受体蛋白及下游的转录抑制蛋白CSL构成。当与相邻细胞上的配体结合后, 细胞膜上的Notch受体蛋白发生构象变化, 从而引起之后的两次水解。其中, Notch受体首先被金属蛋白酶 (TACE 在位于胞外区的S2位点剪切, 产生的C端片段再被γ分泌酶在跨膜区的S3 收稿日期: 2014-01-29; 修回日期: 2014-03-18. 基金项目: 国家自然科学基金面上基金资助项目 *通讯作者 Tel / Fax: 86-21 E-mail: fang.wu@sjtu.edu.cn 位点剪切, 得到的胞内片段进入细胞核, 与CSL结合, 参与下游靶基因表达的激活[1]。 Notch信号通路对癌症的影响具有双重性, 在T 淋