含不同启动子的腺病毒载体介导的人β_2肾上腺素能受体基因在心肌细胞的表达

The study of beta-2 adrenergic receptors gene transferred into cardiomyocytes by adenovirus vectors contain different promoter

目的 观察不同的启动子cmv、mlc 2v对腺病毒载体介导的人 β2 肾上腺素能受体 (β2 AR)基因在大鼠心肌细胞中表达。方法 分别构建含cmv、mlc 2v启动子并携带人 β2 AR基因的重组腺病毒Adcmvβ2 AR、Admlcβ2 AR ,用以感染心肌细胞 ,并检测心肌细胞对人 β2 AR基因的表达及cAMP的含量。结果 经RT PCR、Western免疫印迹分析证实感染的心肌细胞均表达人 β2 AR ;放射性配基检测显示含mlc 2v启动子Admlcβ2 AR感染的心肌细胞 β2 AR密度低于含cmv启动子的Adcmvβ2 AR感染组 (P <0 0 1) ;经 10 μmol/L异丙肾上腺素作用 ,感染组的心肌细胞内cAMP含量明显高于对照组 ,感染组间存在明显差异 (P <0 0 1)。结论 含不同启动子的腺病毒载体感染心肌细胞后均可表达具有生物活性的人 β2 AR ,但两者存在差异。

Objective To observe human β 2 adrenergic receptors (β 2 AR) gene expression in cardiomyocytes transfected by adenoviral vector contain cmv or mlc 2v promoter. Methods Two adenoviral vectors (Adcmv β 2 AR, Admlc β 2 AR) were constructed, in which β 2 AR gene is under control of either the ventricle specific myosin light chain 2 (mlc 2v) or the cmv promoter. The cultured neonate rat ventricular myocytes were infected by two adenoviral vectors, and the β 2 AR expression of on cultured neonate rat ventricular myocytes and their ability to potentiate β adrenergic signaling were assessed. Results The presence of β 2 AR mRNA was detected by RT PCR, and the expression of the β 2 AR gene was demonstrated by protein immunoblots. According to a ligand binding assay, the density of β AR in the cardiomyocytes infected by Admlc β 2 AR and Adcmv β 2 AR was greater than that in the control. Moreover, the latter was higher than the former(234±6.4 fmol/mg protein vs 153± 5.2 fmol/mg protein)( P <0.01). It was demonstrated that β 2 AR gene significantly increased isoproterenol stimulated cAMP in cardiomyocytes. Conclusion The results above demonstrated that human β 2 AR gene was expressed in the cardiomyocytes infected by recombinant adenovirus contain cmv or mlc 2v promoter, but both is different.