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Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (3): 276-282    DOI: 10.3785/j.issn.1008-9292.2013.
    
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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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 wordsCyclic AMP      Receptors      histamine H3      Histamine H3 antagonists      Imidazoles      Transfection      Genes      Gene expression      Reverse transcriptase polymerase chain reaction      Immunoblotting     
Received: 20 February 2013      Published: 25 May 2013
CLC:  R 965.1  
Cite this article:

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.

URL:

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


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

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 

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