β3-肾上腺素能受体基因过表达促进心肌细胞肥大

Overexpression of β3-adrenoreceptor aggravates cardiac hypertrophy

目的:通过慢病毒转染使心肌细胞过表达β3-肾上腺素能受体(β3-adrenoreceptor,β3-AR)基因,研究其对体外培养鼠原代心肌细胞肥大的影响。方法:体外培养新生SD大鼠心室肌细胞,细胞培养24 h后,分别以转染复数(multiplicity of infection,MOI)20、50、80、100转染只携带绿色荧光蛋白(GFP)慢病毒(空病毒),病毒转染72 h后,在倒置荧光显微镜下观察绿色荧光蛋白GFP表达情况,以确定最佳转染复数。心肌细胞以最佳MOI值转染携带β3-AR基因慢病毒24 h后,去甲肾上腺素(norepinephrine,NE)诱导心肌细胞48 h,建立转染后体外心肌肥厚模型。蛋白免疫印迹法(Western Blot)检测β3-AR、原癌基因c-myc、c-fos的蛋白表达水平。结果:用不同MOI的慢病毒转染心肌细胞,转染效率不同,以MOI=50转染效率最高;NE诱导心肌细胞48 h后,随β3-AR表达增加,c-myc、c-fos蛋白水平表达增加(Control组:0.098±0.012、0.105±0.008 vs NE组:0.174±0.012、0.164±0.015 vsβ3-AR转染组:0.268±0.010、0.325±0.014,P<0.05),心肌肥厚加重。结论:以携带β3-AR的慢病毒转染心肌细胞可有效增加β3-AR表达,随β3-AR表达增加,心肌肥厚加重。

Objective To investigate the effect of β3-adrenoreceptor（β3-AR）overexpression on cardiac hypertrophy.Method Sprague-Dawley rat neonatal ventricular cardiomyocytes（NRVMs） were isolated and cultured in vitro.The infection of lentiviruswas examined after cardiomyocytes were infected with lentivirus at different multiplicity of infection（MOI） of 20、50、80 and 100.Fluorescence microscopy was used to observe the expression of GFP and to confirm the best MOI for lentivirus infection.Following the enforced expression of β3-AR by lentivirus,2u M norepinephrine（NE） was used to treatment the infected cardiomyocytes for 48 h.Expressions of β3-AR、c-myc and c-fos protein in cardiomyocytes were detected by Western Blot.Results The results of fluorescence microscopy indicated that the best MOI was 50.The protein level of β3-AR was significantly increased in β3-AR overexpression group compared with the control group and the NE treatment group（P〈0.05）.Following the treatment of NE,the expressions of c-myc and c-fos were also significantly increased in β3-AR overexpression group compared with the control group（P〈0.05）.Conclusion Over-expression of β3-AR can aggravate cardiomyocyte hypertrophy.