腺病毒相关病毒载体介导人β_2肾上腺素能受体、增强型绿色荧光蛋白基因导入心肌细胞的表达  

Study on the expression of β_2-adrenergic receptor gene and enhanced green fluorescent protein gene in cardiomyocytes transferred by adeno associated virus vector

作　　者：高文谦[1] 李小鹰[1] 吴小兵[2] 李梁[3] 裴雪涛[3] 

机构地区：[1]解放军总医院老年心内科,北京市100853 [2]中国预防医学科学院病毒学研究所 [3]军事医学科学院国家生物医学分析中心

出　　处：《中华老年医学杂志》2001年第4期275-278,共4页Chinese Journal of Geriatrics

基　　金：国家 973基金资助项目 (G2 0 0 0 0 5 6 90 6

摘　　要：目的　探讨充血性心力衰竭 (心衰 )及其他心脏疾病的基因治疗途径。　方法　构建携带人 β2 肾上腺素能受体 (β2 AR)和增强型绿色荧光蛋白 (EGFP)双基因的腺病毒相关病毒 (adeno associatedvirus ,AAV)载体 ,感染大鼠的心肌细胞 ,检测心肌细胞 β2 AR、EGFP的表达及环腺苷酸(cAMP)的含量。　结果　Northern印迹杂交显示感染的心肌细胞表达人 β2 ARmRNA ;Western免疫印迹杂交分析表明感染的心肌细胞有人 β2 AR ;在荧光显微镜下可观察到心肌细胞表达EGFP ;放射性配基检测显示感染的心肌细胞 β AR密度〔(2 0 4 0± 6 4)fmol/mg蛋白〕明显高于未感染的心肌细胞〔(76 0± 2 8)fmol/mg蛋白 ,P <0 0 1〕 ;经 10 μmol/L异丙肾上腺素作用 ,感染的心肌细胞的cAMP含量〔(116 2± 5 8)pmol/ 10 6cells〕明显高于对照组〔(5 8 4± 4 6 )pmol/ 10 6cells,P <0 0 1〕。　结论　人β2 AR、EGFP基因经AAV载体导入心肌细胞后能有效地表达 ,且表达的人 β2Objective To investigate the way of gene therapy on congestive heart failure and other heart diseases. Methods Recombinant adeno associated viruses (AAV) encoding the human β 2 adrenergic receptor(AR) and enhanced green fluorescent protein (EGFP) were constructed and the β 2 AR and EGFP cDNA were transfected in vitro to the neonate rat ventricular myocytes. The expression of β 2 AR and EGFP on cultured neonate rat ventricular myocytes and the ability of β 2 AR to potentiate the beta adrenergic signaling were assessed. Results The presence of human β 2 AR mRNA was demonstrated by Northern blot, the expression of the β 2 AR gene was demonstrated by Westernimmuno blot. The density of β AR in the myocytes was greater than that in the control 〔(204.0 ± 6.4) fmol/mg protein vs (76.0 ± 2.8) fmol/mg protein〕 according to a ligand binding assay. Human β 2 AR gene significantly increased isoproterenol stimulated cAMP as compared with the control〔(116.2±5.8) pmol/10 6 cell vs (58.4 ± 4.6) pmol/10 6 cell〕. EGFP was also expressed in the transfected cardiomyocytes. Conclusions Human β 2 AR gene could be transfected into cardiomyocytes by AAV vector and could potentiate the beta adrenergic signaling.

关 键 词：腺病毒相关病毒 肾上腺素能受体 荧光抗体技术 基因治疗 心肌细胞 心脏病 基因表达 

分 类 号：R541.6[医药卫生—心血管疾病]