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

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 ,为建立相关孤儿G蛋白偶联受体的研究体系提供借鉴

Establishment of an engineered CHO-hm1R cells and the effect of acetylcholine on intracellular calcium concentration in the engineered cells

AIM To obtain the genetic-modified cell line CHO-hm₁R transfected with a recombinant vector hm₁R-pcDNA3.1(+), and evaluate the feasibility of the activation of the expressing human muscarinic receptor 1(hm₁R) in CHO-hm₁R with the corresponding cognate agonist. METHODS The cDNA of hm₁R had been amplified with PCR from the healthy volunteer′s genomic DNA, and the vector pcDNA3.1(+)-hm₁R carrying the cDNA of hm₁R had been recombined. CHO-K₁ cells had been transfected with the recombinant pcDNA3.1(+)-hm₁R, and then the cells had been cultured in DMEM with G418 (600 μg•L^-1) for about 2 weeks to select the CHO-hm₁R cells. Fura-2/AM, a fluorescence probe, had been used in assaying the ［Ca²⁺］_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-hm₁R cells. RESULTS CHO-hm₁R cells expressing hm₁R was obtained successfully. Acetylcholine significantly increased ［Ca²⁺］_i of CHO-hm₁R cells. However, this effect of acetylcholine was blocked by atropine, an antagonist of hm₁R. CONCLUSION CHO-hm₁R cells are feasible for analyzing the ligands of hm₁R and can provide useful clues for investigating the related members of orphan G protein-coupled receptors.