CHO-hm_1R工程细胞株的建立及乙酰胆碱对其细胞内钙离子浓度的影响  

Establishment of an engineered CHO-hm_1R cells and the effect of acetylcholine on intracellular calcium concentration inthe engineered cells

作　　者：余少平[1] 熊永炎[1] 王琼[2] 贺艳丽[3] 伍仕敏[3] 高月[2] 高沛永[2] 刘永学[2] 

机构地区：[1]武汉大学中南医院病理科,湖北武汉430071 [2]北京放射医学研究所药理毒理研究室,北京100850 [3]武汉大学中南医院检验科,湖北武汉430071

出　　处：《中国药理学与毒理学杂志》2004年第1期17-21,共5页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金资助项目 (3 0 1710 96);全军医药卫生"十五"面上资助项目 (0 1MB0 56)~~

摘　　要：目的　获得表达人毒蕈碱样乙酰胆碱Ⅰ型受体(hm1 R)的工程细胞株 (CHO hm1 R)并鉴定表达受体是否具有相应的功能活性。方法　以健康人基因组DNA为模板 ,PCR扩增hm1 R之cDNA ,并构建pcDNA3 .1 (+) hm1 R表达载体 ,转染CHO K1 细胞获得CHO hm1 R工程细胞株 ,将不同浓度的乙酰胆碱作用于细胞 ,以Fura 2为荧光探针检测细胞内钙离子浓度变化 ;同时 ,观察阿托品预处理对乙酰胆碱作用的影响。结果　成功得到CHO hm1 R工程细胞株 ;乙酰胆碱 1 0 ,1 0 0 μmol·L-1 均可增高细胞内钙离子浓度 ,此反应可被阿托品 50 0 μmol·L-1 完全阻断。结论　CHO hm1 R工程细胞株可以用于hm1 R配基的检测 。AIM To obtain the genetic modified cell line CHO hm 1R transfected with a recombinant vector hm 1R pcDNA3.1(+), and evaluate the feasibility of the activation of the expressing human muscarinic receptor 1(hm 1R) in CHO hm 1R with the corresponding cognate agonist. METHODS The cDNA of hm 1R had been amplified with PCR from the healthy volunteer′s genomic DNA, and the vector pcDNA3.1(+) hm 1R carrying the cDNA of hm 1R had been recombined. CHO K 1 cells had been transfected with the recombinant pcDNA3.1(+) hm 1R, and then the cells had been cultured in DMEM with G 418 (600 μg·L -1 ) for about 2 weeks to select the CHO hm 1R cells. Fura 2/AM, a fluorescence probe, had been used in assaying the [Ca 2+ ] i changes induced by different concentration of acetylcholine (at 10 and 100 μmol·L -1 ) with or without pretreatment with atropine (at 500 μmol·L -1 ) in the CHO hm 1R cells. RESULTS CHO hm 1R cells expressing hm 1R was obtained successfully. Acetylcholine significantly increased [Ca 2+ ] i of CHO hm 1R cells. However, this effect of acetylcholine was blocked by atropine, an antagonist of hm 1R. CONCLUSION CHO hm 1R cells are feasible for analyzing the ligands of hm 1R and can provide useful clues for investigating the related members of orphan G protein coupled receptors.

关 键 词：受体 毒草碱样 乙酰胆碱 阿托品 钙离子浓度 细胞 CHO—K1 

分 类 号：R96[医药卫生—药理学]