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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
Objective To obtain an engineered cell line (CHO hm1 R) expressing human muscarinic acetylcholine type 1 receptor (hm1 R) and to identify whether the expression receptor has the corresponding functional activity. Methods The hm1 R cDNA was amplified by PCR using the human genomic DNA as template. The pcDNA3. 1 (+) hm1 R expression vector was constructed and transfected into CHO K1 cells to obtain the CHO hm1 R cell line. Different concentrations of acetylcholine Cells with Fura 2 as a fluorescent probe to detect changes in intracellular calcium concentration; the same time, observe the effect of atropine pretreatment on the role of acetylcholine. Results CHO hm1 R cell line was successfully obtained. Acetylcholine 10 0 and 100 μmol·L -1 increased intracellular calcium concentration, which was completely blocked by 50 μmol·L -1 atropine. Conclusion The CHO hm1 R cell line can be used for the detection of hm1 R ligands and provide a reference for establishing a research system of related orphan G protein - coupled receptors