摘要目的 观察应用RNA干扰阻断Ryanodine受体2(RyR2)合成是否对心肌缺血再灌注(I/R)损伤有保护作用.方法 分离培养大鼠原代心肌细胞并建立I/R模型.应用RNA干扰技术阻断RyR2表达,分别检测对照组、RNA干扰组、I/R模型组、RNA干扰+IYR模型组细胞凋亡率、RyR2 mRNA、细胞内钙离子浓度、培养上清中乳酸脱氢酶(LDH)水平和线粒体膜电位变化.结果 与对照组相比.I/R组细胞发生明显凋亡和损伤(60.1%比5.5%.P<0.05),细胞内钙离子浓度显著上升(平均荧光强度21.2比7.6,P<0.05),RyR2表达变化不明显(20.1比22.7,P>0.05),线粒体膜电位显著下降(平均荧光强度37.2比85.1,P<0.05).相对于I/R组,siRNA干预组细胞RyR2表达显著下降(6.8比20.1,P<0.05),LDH水平、凋亡率、细胞内钙离子浓度均有明显下降(上清LDH为48 IU/L比125 IU/L,Annexin V阳性细胞31.2%比60.1%,钙离子浓度为平均荧光强度8.6比21.2,均P<0.05),线粒体膜电位显著升高(55.8比37.2,P<0.05).结论 在大鼠原代心肌细胞中RyR2基因沉默对心肌I/R损伤有一定保护作用.

abstractsObjectives To block the synthesis of ryanodine receptor 2 (RyR2) in myocardial cells by RNA interference and to investigate its biological impact on ischemia-reperfusion (I/R)in rat myocardial cells. Methods Rat myocardial cells were isolated and cultured for an I/R model in vitro. RNA interference technique was used to block the synthesis of RyR2 in myocardial cells. Changes of LDH level, apoptasis, RyR2 mRNA expression and cytosolic Ca2+ concentration were analyzed accordingly. Results Myocardial cells after I/R manipolation were severely injuried (LDH leakage, 125 IU/L vs 12 IU/L, P<0.05), apoptosis (60.1% vs 5.5%, P<0.05), significant cytosolic Ca2+ overload (21.2 vs 7.6, P< 0.05) and remarkable mitochondrial membrane potential loss (37.2 vs 85.1, P < 0.05). However, no visible change of RyR2 was observed (20.1 vs 22. 7,P>0.05). Pre-treatment with RyR2 specified siRNA demonstrated suppressed expression of RyR2 ( 6.8 vs 20.1, P< 0.05), increased mitochondrial membrane potential(55.8 vs 37.2, P<0.05), attenuated cytosolic Ca2+ overload (8.6 vs 21.2) and cellular apoptosis (31.2% vs 60. 1%, P < 0.05). Conclusion RyR2 gene silencing enables to protect myocardial ceils from I/R injury in vitro.