抑制磷酸二酯酶5的短发夹RNA重组腺病毒载体对缺氧缺糖乳鼠心肌细胞的保护作用

Protective effects of phosphodiesterase 5 shRNA on cardiomyocytes cultured under oxygen glucose deprivation

目的观察磷酸二酯酶5(PDE5)shRNA对缺氧缺糖条件下乳鼠心肌细胞的保护作用。方法培养新生C57BL/6J小鼠心肌细胞,通过缺氧缺糖建立小鼠心肌细胞缺氧缺糖损伤模型。随机分2组:实验组(shPDE5)通过构建PDE5 shRNA,将PDE5 shRNA转染心肌细胞;对照组(Null):将普通腺病毒载体转染心肌细胞。用原位末端标记分析(TUNEL)检测2组细胞凋亡情况;免疫蛋白印迹检测PDE5蛋白的表达;用酶联免疫吸附法测定各组心肌细胞cGMP和PKG的水平。结果与对照组比较,实验组PDE5表达明显下降,cGMP和PKG的活性明显上调(P<0.05);实验组细胞凋亡数明显减少(P<0.05)。结论 PDE5 shRNA可明显拮抗缺氧缺糖诱导的心肌细胞凋亡,可能与PDE5 shRNA持续抑制心肌细胞PDE5基因表达、显著上调cGMP和PKG活性有关。

Objective To study the protective effects of PDE5 shRNA on cardiomyocytes cultured under oxygen glucose deprivation. Methods Cardiomyocytes were isolated from neonatal C57BL/6J mice. Cardio- myocytes were randomly assigned to test group （shPDE5） ： transfection of cultured cardiomyocytes with adenoviral phosphodiesterase 5 （PDE5） short hairpin RNA （PDE5 shRNA）, and control group （Null） ： transfec- tion of cultured cardiomyocytes with adenoviral vector without therapeutic PDE5 shRNA. After oxygen and glucose deprivation （OGD） treatment, the apoptosis was evaluated by TdT- mediated dUTP nick -end labeling （TUNEL）, the level of PDE5 expression was detected by Western blot. The level of cGMP or PKG activity in the ceils was evaluated by enzyme linked immunosorbent assay （ ELISA ）. Results Compared to control group, the level of PDE5 was significantly decreased and the levels of eGMP and PKG were significantly increased in the test group. Conclu- sion The present study suggests the protective effects of PDE5 shRNA on cardiomyoeytes cultured under oxygen glucose deprivation, significant- ly attenuating cardiomyocyte apoptosis through possible activating cGMP/ PKG signaling pathway.