牛m1和m5受体内环3区的克隆及主动脉内皮细胞上两种受体mRNA表达的检测

Cloning of the innerloop3 of bovine m1 and m5 receptors and the assay of the mRNA expression of these two receptors in the aortic endothelial cells

目的 :克隆牛m1和m5受体亚型特异性区域内环 (innerloop) 3区 ,并检测牛主动脉内皮细胞上两种受体mRNA的表达。方法 :根据人、小鼠的m1和m5受体序列 ,设计并合成针对两种受体内环 3区的特异性引物。采用RT_PCR法从牛海马组织中扩增牛m1和m5受体内环 3区 ,并通过半定量RT_PCR法检测两种受体mRNA在传代培养前后的牛主动脉内皮细胞中的表达。结果 :序列测定与对比表明克隆得到了牛m1和m5受体内环 3区。半定量RT_PCR结果显示 ,原代牛主动脉内皮细胞表达m1受体mRNA而未检测到m5受体mRNA ,两种受体mRNA在传 10代的培养牛主动脉内皮细胞中均未检测到有表达。结论

Objective:To clone bovine m1 and m5 receptors′ innerloop3 and examine these two receptors mRNA expression in the aortic endothelial cells.Methods: Based on the human and mouse sequences of m1 and m5 receptors, primers specific for the two receptors′ innerloop3 were designed and RT_PCR was used to amplify the two receptors′ innerloop3 from bovine hippocampus. These two receptors mRNA expressed in the bovine aortic endothelium were assayed by semi_quantitative RT_PCR.Results:Cloning of bovine m1 and m5 receptors′ innerloop3 was confirmed by DNA sequencing. Semi_quantitative RT_PCR showed that the m1 receptor mRNA was detected on the primary endothelium, whereas m5 receptor mRNA was not,and neither m1 nor m5 receptor mRNA was detected on the 10th passage_cultured endothelium. Conclusion:This study gives clues for research on the relationship of the vascular endothelial receptor that can mediate acetylcholine_induced relaxation of blood vessels with M receptor.