目的 建立神经介素Neuromedin U 2 Receptor（NMU2R）激动剂的高通量筛选模型，从中药中筛选NMU2R受体激动剂。方法 将人NMU2R受体基因质粒（hNMU2R/pCDNA3.1）与报告基因质粒（MRE/CRE/LUC）按1∶5的比例共转染到HEK293细胞，通过G418筛选，建立稳定的hNMU2R受体激动剂筛选细胞株。利用CRE的激活剂Forskolin和NMU2R内源激动剂Neuromedin U探索和优化荧光素酶表达时间、每孔细胞数目、荧光素酶底物浓度等筛选条件，建立可靠的筛选方法，并对部分中药水提液进行筛选。结果 已建立了一个稳定的hNMU2R受体激动剂筛选细胞株和可靠的筛选方法，其Z′-因子为0.75，荧光素酶表达时间为6～8 h，细胞密度为每孔4×104～8×104个。结论 该系统能够有效地应用于NMU2R受体激动剂的高通量筛选，能从中药中筛选出NMU2R受体的激动剂。
Abstract
OBJECTIVE To establish a high throughput screening method for finding agonist for NMU2R receptor METHODS The human NMU2R plasmid (hNMU2R/pCDNA3.1) and reporter gene plasmid (MRE/CRE/LUC) were cotransfected HEK293 to establish a cell line for agonist screening To identify and optimize the assay condition, the effects of some factors on the assay were examined by using forskolin and neuromedin U, such as incubation time, cell number in each well and the concentration of luciferase′s substrate The extracts of some traditional Chinese medicine were screened RESULTS A steady cell line and a reliable method for NMU2R agonist screening were established. The Z′-factor value was 0.75. The agonist incubation time was 6 to 8 hours. The cell number per well was 4×104to 8×104 CONCLUSION This technology was successfully applied to identify agonist for NMU2R receptor from the traditional Chinese medicines.
关键词
NMU2R受体 /
报告基因 /
激动剂 /
高通量药物筛选
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Key words
NMU2R /
gene /
agonist /
high throughput screening (HTS)
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参考文献
［1］Shan LX,Qiao XD,Crona JH,et al. Identification of a novel neuromedin U receptor subtype expressed in the central nervous system［J］.J Bilo Chem,2000,275(50):39482.
［2］Howard AD,Wang RP,Pong SS,et al. Identification of receptors for neuromedin U and its role in feeding［J］.Nature,2000,406:70.
［3］郑旭煦，胡应和，欧阳克清,等. 神经介素U（neuromedin U）研究概况［J］. 生物化学与生物物理进展，2002，29（5）：765.
［4］Broach JR,Thorner J. Highthroughput screening for drug discovery［J］.Nature,1996,384(6604 Suppl):14.
［5］黄家学，胡娟娟，杜冠华. 化合物药物活性的高通量筛选［J］. 中国药理学通报,1999,16(5): 401.
［6］Major J. What is the future of high throughput screening?［J］.J Biomol Screen，1999,4(3):119.
［7］梁乾德，王升启. 药物高通量筛选分析技术［J］. 国外医学·药学分册，2002,29(1):22.
［8］Terstappen GC,Giacometti A,Ballini E,et al. Development of a functional reporter gene HTS assay for the identification of mGluR7 modulators［J］.J Biomol Screen, 2000,5(4):255.
［9］Jiang C,Chen G,Zeng X,et al. Generation of a bioactive neuropeptide in a cellfree system［J］.Anal Biochem,2003,316(1):34.
［10］Zhang JH,Chung TD,Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays［J］.J Biomol Screen,1999,4(2):67.
［11］Rosenberg D,Groussin L,Jullian E,et al. Role of the PKAregulated transcription factor CREB in development and tumorigenesis of endocrine tissues［J］.Ann N Y Acad Sci,2002,968:65.
［12］Dendorfer U,Oettgen P,Libermann TA. Multiple regulatory elements in the interleukin6 gene mediate induction by prostaglandins,cyclic AMP,and lipopolys accharide［J］.Mol Cell Biol,1994,14(7):4443.
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脚注
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基金
重庆市科委攻关项目(渝科发计字[2002]1号)和重庆市计委重点高新技术项目（渝计委计[2001]1192号）
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