Ryanodine受体2基因沉默对大鼠心肌细胞缺血再灌注损伤的影响

Effect of ryanodine receptor 2 gene silencing on ischemia-reperfusion injury of rat myocardial cells

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＋I／R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48IU／L比125IU／L，AnnexinV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。

Objectives 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, apoptosis, RyR2 mRNA expression and cytosolie Ca^2＋ 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 Ca^2＋ 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 Ca^2＋ 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 cells from I/R injury in vitro.