模拟缺血预适应对原代培养乳鼠窦房结细胞缺血再灌注损伤的保护作用

Protective effect of mimic ischemic preconditioning on viability of cultured sinoatrial node cells of neonatal rats

目的 通过观察不同时间、不同次数的模拟短暂缺血刺激对随后较长时间缺血 /再灌注乳鼠窦房结细胞活性的影响 ,以探讨模拟缺血预适应 (IP)对原代培养乳鼠窦房结细胞的保护作用。方法 取培养 2d的窦房结细胞 ,分为对照组、模拟缺血 /再灌注 (I/R)组及IP1(缺血 5min/再灌注 5min)组、IP2 (5min/ 5min× 2 )组、IP3 (5min/ 5min× 3 )组、IP4(10min/ 10min)组、IP5 (10min/ 10min× 2 )组、IP6(10min/ 10min× 3 )组、IP7(2 0min / 2 0min)组、IP8(2 0min/ 2 0min× 2 )组、IP9(2 0min/ 2 0min× 3 )组 ,在模拟缺血 3h/再灌注 4h结束后 ,通过PI染色及MTT比色法检测各组乳鼠窦房结细胞PI染色阳性率及D490 值的变化。结果 ①各实验组乳鼠窦房结细胞D490 值均较对照组明显降低 (P <0 0 1) ,但IP2组、IP3组、IP4组、IP5组及IP7组D490 值却较I/R组明显升高 (P <0 0 1)。②各实验组PI染色阳性窦房结细胞百分率均较对照组明显增高 (P <0 0 1) ,但IP2组、IP3组、IP4组、IP5组及IP7组PI染色阳性率却较I/R组显著降低 (P <0 0 1)。结论 模拟IP对随后较长时间I/R的乳鼠窦房结细胞具有保护效应 ,而这种保护作用的产生可能与累计的模拟缺血刺激“总时程”有关。

Objective To compare the effects of different ischemic preconditioning (IP) protocols on the viability of primary cultured sinoatrial node cells from neonatal rats for investigating the protective effect of IP. Methods The cells were randomized into eleven groups: control, ischemia/reperfusion (I/R), IP1 (preconditioned with ischemia/reperfusion for 5 min), IP2 (preconditioned with 2 cycles of ischemia/ reperfusion for 5 min), IP3 (preconditioned with 3 cycles of ischemia/reperfusion for 5 min), IP4 (preconditioned with ischemia/reperfusion for 10 min), IP5 (preconditioned with 2 cycles of ischemia/reperfusion for 10 min), IP6 (preconditioned with 3 cycles of ischemia/reperfusion for 10 min), IP7 (preconditioned with ischemia/reperfusion for 20 min), IP8 (preconditioned with 2 cycles of ischemia/reperfusion for 20 min), and IP9 (preconditioned with 3 cycles of ischemia/reperfusion for 20 min). PI positive staining rate and changes of D 490 after 3 h ischemia/4 h reperfusion were determined by PI staining and MTT chromatometry. Results ① D 490 value in sinoatrial node cells in each experimental group was significantly lower than that in the control group, but that in IP2, IP3, IP4, IP5, and IP7 groups was significantly higher than that in I/R group (P<0.01). ② The percentage of PI positively stained sinoatrial node cells in each experimental group was significantly higher than that in the control group, but that in IP2, IP3, IP4, IP5, and IP7 groups was significantly lower than that in I/R group (P<0.01). Conclusion IP can offer protection to sinoatrial node cells exposed to severe I/R and the protection may be determined by the total period of ischemic pretreatment.