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浙江大学学报(医学版)  2013, Vol. 42 Issue (3): 276-282    DOI: 10.3785/j.issn.1008-9292.2013.
专题报道     
稳定转染H3受体基因的HEK293细胞株的鉴定及新型非咪唑类H3受体拮抗剂的筛选
何萍1,2,谭丽1,2,胡薇薇2,戴海斌1,2,胡永洲3,陈忠2
1.浙江大学医学院附属第二医院药剂科,浙江 杭州 310009;
2.浙江大学药学院药理教研室,浙江 杭州 310058;
3.浙江大学药学院药物研究所,浙江 杭州 310058;
Identification of a HEK-293 cell line containing stably-transfected H3R gene and screening for novel non-imidazole histamine H3 receptor antagonists
 HE Ping1,2, TAN Li1,2, HU Wei-Wei2, DAI Hai-Bin1, HU Yong-Zhou3, CHEN Zhong2
1.Department of Pharmacy,the Second Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310009,China;2.Department of Pharmacology,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058,China; 3.ZJU-ENS Joint Laboratory of Medicinal Chemistry,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058,China
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摘要:

目的:鉴定转染大鼠组胺H3受体基因的HEK293细胞株受体表达情况,筛选具有H3受体拮抗活性的新型非咪唑类化合物。方法:利用RT-PCR和免疫印迹的方法检测转染细胞株组胺H3受体的表达,以阳性刺激物forskolin诱导细胞内cAMP含量升高为指标,初步筛选具有拮抗作用的化合物。结果:细胞鉴定结果显示,稳定转染H3受体基因的HEK293细胞株高表达大鼠H3受体。H3受体选择性激动剂(R)-α-甲基组胺可浓度依赖性抑制阳性刺激物forskolin (10 μmol/L)诱导的cAMP含量升高(P<0.05),当(R)-α-甲基组胺浓度达到30 nmol/L时,抑制率接近平台值(P>0.05)。而H3受体拮抗剂噻普酰胺和新合成的非咪唑类化合物XHA23及XHA25则能够浓度依赖性阻断(R)-α-甲基组胺对forskolin诱导的cAMP含量升高的抑制作用(P<0.05),IC50分别为3.62 μmol/L、0.49 μmol/L、0.14 μmol/L。结论:高表达H3受体的HEK293细胞株可用于有H3受体拮抗活性化合物的筛选,而非咪唑类化合物XHA23和XHA25具有一定的H3受体拮抗剂样活性。

关键词: 环AMP受体组胺H3组胺H3拮抗剂咪唑类转染基因基因表达逆转录聚合酶链反应免疫印迹法    
Abstract:

Objective: To identify a HEK293 cell line containing stably-transfected H3R gene,and to screen the novel non-imidazole compounds with H3R antagonist activity. Methods: The expression of rat H3 receptor in cell line was detected by RT-PCR and Western blot.An elevation of intercellular cAMP concentration induced by forskolin was measured as the index for screening compounds with H3R antagonist activity. Results: The H3R-transfected HEK-293 cells stably expressed high level of rat H3 receptor mRNA and protein.Forskolin significantly increased intercellular cAMP concentration in the H3R-transfected HEK-293 cells.H3R agonist (R)-α-methyhistamine inhibited the forskolin-induced production of intercellular cAMP.H3R antagonist thioperamide and newly synthesized non-imidazole compounds XHA23 and XHA25 blocked (R)-α- methyhistamine reversal of forskolin-induced cAMP formation in a concentration-dependent manner,and the IC50 values were 3.62 μmol/L,0.49 μmol/L,0.14 μmol/L,respectively. Conclusions: The H3R-transfected HEK293 cells stably express high level of rat H3 receptor,and can be used for screening compounds with H3R antagonist activity.The non-imidazole compounds XHA23 and XHA25 may have H3R antagonist activity.

Key words: Cyclic AMP    Receptors    histamine H3    Histamine H3 antagonists    Imidazoles    Transfection    Genes    Gene expression    Reverse transcriptase polymerase chain reaction    Immunoblotting
收稿日期: 2013-02-20 出版日期: 2013-05-25
CLC:  R 965.1  
基金资助:

浙江省卫生高层次创新人才培养工程项目(419000-710908);浙江省重点科技创新团队计划资助(2011R50014);浙江省教育厅科研项目(Y201122427).

通讯作者: 通讯作者:陈忠(1968-),男,博士,教授,博导,从事神经药理学研究;     E-mail: chenzhong@zju.edu.cn
作者简介: 何萍(1984-),女,硕士,药师.
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引用本文:

何萍, 谭丽, 胡薇薇, 戴海斌, 胡永洲, 陈忠. 稳定转染H3受体基因的HEK293细胞株的鉴定及新型非咪唑类H3受体拮抗剂的筛选[J]. 浙江大学学报(医学版), 2013, 42(3): 276-282.

HE Ping, TAN Li, HU Wei-Wei, DAI Hai-Bin, HU Yong-Zhou, CHEN Zhong. Identification of a HEK-293 cell line containing stably-transfected H3R gene and screening for novel non-imidazole histamine H3 receptor antagonists. Journal of ZheJiang University(Medical Science), 2013, 42(3): 276-282.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2013.        http://www.zjujournals.com/med/CN/Y2013/V42/I3/276

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