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目的 :探讨K+ ATP通道开放剂diazoxide对模拟缺血 再灌注 (I/R)时培养乳鼠窦房结细胞的保护作用及其可能机制。方法 :分离乳鼠窦房结细胞 ,纯化培养 2d后进行实验。随机分为对照组、模拟I/R组、dia zoxide干预 (D +I/R)组及K+ ATP通道阻断剂 5 HD干预——— 5 HD +D +I/R组及 5 HD +I/R组。以流式细胞术检测各组窦房结细胞存活率 ;用激光共聚焦显微镜测定各组窦房结细胞内Ca2 + 。并采用全细胞膜片钳技术测定各组细胞L型钙电流 (L ICa)密度。结果 :①D +I/R组窦房结细胞存活率 [(6 1.4 3± 5 .14 ) % ]较I/R组 [(5 1.79±6 .2 8) % ]增加 (P <0 .0 1) ;5 HD +D +I/R组 [(5 2 .35± 4 .94 ) % ]及 5 HD +I/R组 [(5 3.16± 5 .35 ) % ]明显增高 ,均P <0 .0 1;②D +I/R组窦房结细胞相对荧光值较I/R组、5 HD +D +I/R组及 5 HD +I/R显著降低 ,均P <0 .0 1;③D +I/R组窦房结细胞L ICa密度较I/R组及 5 HD +D +I/R组明显增加 ,均P <0 .0 1。结论 :diazoxide降低窦房结细胞内钙负荷 ,对模拟I/R时的培养乳鼠窦房结细胞有保护作用 ,并可对抗模拟I/R对培养乳鼠窦房结细胞L ICa的影响 ,该作用可能与细胞线粒体K+ ATP通道的开放有关。
Objective: To investigate the protective effect of diazoxide, a K + ATP channel opener, on sinoatrial node cells cultured neonatal rats with simulated ischemia / reperfusion (I / R) and its possible mechanism. Methods: The sinoatrial node cells of neonatal rats were isolated and cultured for 2 days. Randomly divided into control group, simulated I / R group, diazoxide intervention (D + I / R) group and K + ATP channel blocker 5 HD intervention --- 5 HD + D + I / R group and 5 HD + I / R group. The survival rate of sinoatrial node cells in each group was detected by flow cytometry. The intracellular Ca2 + of sinoatrial node cells in each group was detected by laser scanning confocal microscopy. Whole cell patch clamp technique was used to determine the L-type calcium current (L ICa) density in each group. RESULTS: ① The survival rate of sinoatrial node cells in D + I / R group was significantly higher than that in I / R group [(6.143 ± 6.28)%] (P <0. 0 1 in 5 HD + D + I / R group and 5 HD + I / R group were significantly higher than those in 5 HD + I / R group [(5 3.16 ± 5 .35)%] 0 .0 1; ② The relative fluorescence of sinoatrial node cells in D + I / R group was significantly lower than those in I / R group, 5 HD + D + I / R group and 5 HD + I / R group ; ③ The density of L ICa of sinoatrial node cells in D + I / R group was significantly higher than those in I / R group and 5 HD + D + I / R group (P <0.01). Conclusion: diazoxide can reduce the intracellular calcium load of sinoatrial node cells and protect sinoatrial node cells cultured in simulated I / R rats. It can also antagonize the effect of simulated I / R on L ICa of cultured sinoatrial node cells, This effect may be related to the opening of cellular mitochondrial K + ATP channels.