腺病毒载体介导的人β_2-肾上腺素能受体基因导入心肌细胞的研究

The study of beta2-AR gene transfected to cardiomyocytes by adenovirus vector

目的 观察重组缺陷型腺病毒即腺病毒载体介导的人 β2 肾上腺素能受体 (β2 AR)基因导入大鼠心肌细胞后 β2 AR的表达及其功能。方法 构建含人 β2 AR基因的腺病毒载体 (Adβ2 AR) ,转染大鼠的心肌细胞 ,检测心肌细胞对人 β2 AR的表达及环磷酸腺苷 (cAMP)的含量。结果 Northern印迹杂交显示Adβ2 AR转染的心肌细胞表达人 β2 -ARmRNA ;Western免疫印迹杂交分析表明转染的心肌细胞有人 β2 AR ;放射性配基检测显示转基因心肌细胞的 β AR密度 [(2 34± 6 .4)fmol/mg蛋白 ]是未转染的心肌细胞密度 [(76± 2 .8)fmol/mg蛋白 ]的 3倍 (P <0 .0 1 ) ;经 1 0 μmol/L异丙肾上腺素作用 ,转基因的心肌细胞的cAMP量 [(1 2 4± 6 .8)pmol/ 1 0 6 细胞 ]明显高于对照组 [(58.4± 4 .6)pmol/ 1 0 6 细胞 ] (P <0 .0 1 )。结论 人 β2

Objective To investigate the expression of beta 2 adrenergic receptors (β 2 AR) on cultured neonate rat ventricular myocytes transfected by Adeno β 2AR and their ability to potentiate beta adrenergic signaling. Methods Recombinant adenoviruses encoding the β 2 AR (Adeno β 2AR) was created and the β 2 AR cDNA was transfected in vitro to the neonate rat myocytes.The expression of β 2 AR on cultured neonate rat ventricular myocytes and the ability to potentiate β 2 AR signaling after transfection were assessed. Results Northern blot demonstrated the presence of β 2 AR mRNA and protein immunoblots demonstrated the expression of the Adeno β 2AR transgene. Expression of beta adrenergic receptor in the transfected myocytes was approximately 3 times higher than that in control (non transfected,234±6.4 fmol/mg protein vs 76 ±2.8 fmol/mg protein) according to a ligand binding assay. Transgene significantly increased isoproterenol stimulated cAMP as compared with control (124±6.8 pmol/10 6 cells vs 58.4±4.6 pmol/10 6cells). Conclusion β 2 adrenergic receptor gene transfected to myocytes by Adeno β 2AR potentiate beta adrenergic signaling.