用于靶向于NFAT的免疫抑制类小分子化合物筛选的细胞模型的建立  

Establishment of a Cell Model Targeted to NFAT Signal Transduction Pathway for Preliminary Screening of FK506-like Immunosuppressants

作　　者：肖鹤[1] 钱露[1] 秦卫松[1] 李松[2] 沈倍奋[1] 黎燕[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]军事医学科学院毒物药物研究所,北京100850

出　　处：《生物工程学报》2005年第5期759-765,共7页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展规划项目(973)(No.2003CB515508)资助。~~

摘　　要：建立IL2启动子以及NFATAP1增强子调控报告基因包括d2EGFP或Luciferase在Jurkat细胞中瞬时表达的细胞模型。首先,利用三步连接将IL2promoter-255～+285处的序列插入到pNFκBd2EGFP表达载体启动子上游,形成3个拷贝的前后串联的增强子序列;然后从人外周血钓取两条IL2promoter序列,包括-326～+46以及-89～+46两段基因组序列,分别替代上述重组表达载体中的TKminimalpromoter,构建所需的IL2promoter以及NFATAP1enhancer调控的报告基因表达质粒:3×NFATAP1IL2Pd2EGFP和3×NFATAP1TATAd2EGFP;最后,分别将3×NFATAP1IL2P以及3×NFATAP1TATA融合基因克隆到pGL3basic载体中,构建相应的Luciferase报告基因表达质粒。利用电转染方法将重组的报告基因表达载体瞬时转入Jurkat细胞后发现,未刺激以及PMA、离子霉素(Ionomycin)单刺激均不能激活下游报告基因的表达,只有PMA和离子霉素联合刺激才能启动d2EGFP以及Luciferase的转录表达,并且5μgmLFK506预先作用1h能几乎完全阻断刺激剂诱导的无论是IL2promoter还是NFATAP1enhancer调控的报告基因的表达。实验结果提示,所构建的Jurkat细胞瞬时表达模型可用于靶向于NFAT信号通路的FK506类免疫抑制小分子化合物的初步筛选。To screen NFAT antagonistic drugs and research signal transduction pathway related to NFAT. Four recombinant vectors were constructed. Each consists of three tandem copies of the human IL-2 distal NFAT-AP1 binding site in the context of the minimal IL-2 enhancer, either the sequence from - 326 ～ ＋ 46 or the sequence from - 89 ～ ＋ 46（containing only the TATA box）, driving a luciferase reporter gene or a destabilized enhanced green fluorescence protein（d2EGFP） reporter gene, respectively. Transient transfection of Jurkat cells was achieved by electroporation with 5～ 10μg of the above plasmid and one pulse at 200V, 65ms. Plasmid pEFBos-mNFAT1 constitutively expressing murine full length NFAT1 protein was used for transient cotransfection. The results showed that neither of non-stimulation nor PMA or ionomycin stimulation alone could activate the reporter gene except PMA plus ionomycin costimulation. Furthermore, overexpressed murine NFAT1 augmented the activation of either IL-2 promoter or NFAT-API enhancer drived reporter gene compared to the endogenous did. However, the reporter gene expression was nearly completely inhibited by pretreatment for 1 h with FK506 at 5μg/mL and then stimulation for 6-12h with PMA plus ionomycin in the presence of FK506. These findings indicated that such a transient Jurkat cell model offered a potential platform for preliminary screening of FK506 or CsA-like immunosuppressive agents.

关 键 词：IL-2启动子 NFAT-AP1增强子 报告基因 联合刺激 FK506阻断 初步筛选 

分 类 号：R392[医药卫生—免疫学]