β2-肾上腺素能受体下调的细胞模型构建。
Construction of a cell model of β2-adrenoceptor downregulation.
机构信息
Department of Respiratory Medicine, Affiliated Hospital of Nantong University, Nantong 226001, P. R. China.
出版信息
J Thorac Dis. 2013 Aug;5(4):492-7. doi: 10.3978/j.issn.2072-1439.2013.08.32.
OBJECTIVE
To establish a cell model of β2-adrenergic receptor (β2AR) downregulation of murine airway smooth muscle induced by salbutamol to elucidate the molecular and biological mechanisms of β2AR downregulation.
METHODS
Airway smooth muscle cells (ASMCs) derived from Balb/c mice were primary cultured. Passage 2-5 cells were characterized by cell morphology and indirect immunofluorescence. More than 95% pure cells at passage 3 or 4 were randomly divided into two groups: control and salbutamol-treated groups. β2AR mRNA and protein expression levels were then detected by RT-PCR and western blot analyses.
RESULTS
Primary cultured cells demonstrated a typical "peak and valley"-like growth characteristic. Smooth muscle α-actin filaments paralleled the cell longitudinal axis in the cytoplasm. The origin of the ASMCs was validated and consistent with their morphology and biological characteristics. β2AR mRNA expression in the salbutamol-treated group was lower than that in the control group (P<0.05), and β2AR protein expression was also markedly lower than that in the control group (P<0.05).
CONCLUSIONS
We successfully established a cell model of β2AR downregulation in ASMCs, which may provide the foundation for further study of the mechanism of β2AR downregulation in asthmatic patients.
目的
建立沙丁胺醇诱导的β2-肾上腺素能受体（β2AR）下调的小鼠气道平滑肌细胞模型，阐明β2AR下调的分子和生物学机制。
方法
原代培养 Balb/c 小鼠气道平滑肌细胞（ASMCs）。通过细胞形态和间接免疫荧光鉴定第 2-5 代细胞。第 3 或 4 代纯度>95%的细胞随机分为对照组和沙丁胺醇处理组。通过 RT-PCR 和 Western blot 分析检测β2AR mRNA 和蛋白表达水平。
结果
原代培养细胞表现出典型的“峰谷”样生长特征。细胞胞质中的平滑肌α-肌动蛋白丝与细胞长轴平行。ASMCs 的来源得到了验证，其形态和生物学特性与其一致。沙丁胺醇处理组β2AR mRNA 表达低于对照组（P<0.05），β2AR 蛋白表达也明显低于对照组（P<0.05）。
结论
我们成功建立了 ASMCs 中β2AR 下调的细胞模型，为进一步研究哮喘患者中β2AR 下调的机制提供了基础。
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