以NF-κB为靶的药物筛选方法的建立与应用研究
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国家高技术研究发展计划(863)新药筛选及关键技术研究(2002AA2Z343C)和中法先进性研究计划资助课题(PRA-B-02-01).


Establishment and Application of a Screening Assay Based on NF-κB Signal Transduction
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This work was supported by grants from Hi-Tech Research and Development Programme of China(2002AA2Z343C) and Programme de Recherches Avancees(PRA) (PRA-B-02-01).

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    摘要:

    核转录因子-κB (NF-κB) 通过调节其靶基因的表达,在机体免疫应答、炎症与肿瘤的发生发展过程中发挥着重要功能,因此也被认为是预防和治疗肿瘤与炎症的理想靶点. pNiFty-SEAP质粒被成功地转入HEK293细胞,因此该转染细胞含有5×NF-κB基序的增强子、SEAP报告基因和Zeocin抗生素抗性筛选基因. 通过检测报告基因表达水平可反映NF-κB的活化状态. 在以pNiFty-SEAP/HEK293细胞为基础建立的筛选体系中,TNFα和PMA等已知的NF-κB激活剂能剂量与时间依赖性地促进NF-κB的活化,而PDTC和NAC等NF-κB抑制剂的作用则相反. 该筛选体系不仅具有稳定、特异、高通量等特点,而且可同时检测NF-κB的激活剂与抑制剂,将为发现作用于NF-κB信号传导通路的抗肿瘤与免疫调节药物提供有效的实验方法.

    Abstract:

    NF-κB has been reported to play an important role in immunity, inflammation and carcinogenesis by regulating expression of its target genes. Thus, NF-κB was recognized as a promising target for the prevention and treatment drugs against cancer and inflammation. NF-κB sequence, reporter gene SEAP and selection gene for Zeocine resistance in plasmid pNiFty-SEAP were transfected into HEK293 cells. Therefore, NF-κB activity was able to be evaluated by SEAP activity in the transfected cells. Based on pNiFtySEAP/HEK293 cells, a screening assay was established, by which known NF-κB stimulators TNFα and PMA were demonstrated to activate the NF-κB in dose and time dependent manners. In contrast, NF-κB inhibitors PDTC and NAC showed inhibitory effects on the NF-κB activity in indicated doses and times. Except several characteristics such as stability, sensitivity, sepecifity, the assay was capable to screen both agents inducing and inhibiting NF-κB activity at the same experiment, which will be useful for the discovery of compounds targeting NF-κB signal pathway.

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马艳霞,徐 波,崔景荣,王 鹂,吴 军,李 敏.以NF-κB为靶的药物筛选方法的建立与应用研究[J].生物化学与生物物理进展,2006,33(2):149-154

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