Diazoxide对乳鼠窦房结细胞模拟缺血-再灌注时的保护作用研究  

The protective effects of diazoxide on sinoatrial node cells in cultured neonatal rat during simulated ischemia/reperfusion periods

作　　者：仝识非[1] 宋治远[1] 钟理[1] 何国祥[1] 

机构地区：[1]第三军医大学附属西南医院心内科,重庆400038

出　　处：《临床心血管病杂志》2004年第8期473-475,共3页Journal of Clinical Cardiology

基　　金：国家自然科学基金资助课题 (No :30 0 70 314 )

摘　　要：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 effects of diazoxide, a ATP-sensitive potassium channel opener, on primary cultured sinoatrial node(SAN) cells during simulated ischemia and evaluate the mechanism of protection. Method: The SAN cells were isolated from newborn rats and purified. The 48 h cultured cells were cultivated in following mediums: simulated reperfusion solution as normal control, simulated ischemia/reperfusion solution(I/R), diazoxide+I/R(D+I/R), 5-HD+D+ I/R and 5-HD+I/R. The survival rate was measured by flow cytometer; Cytosolic Ca 2+ was measured by confocal laser scanning microsopy after cells were loaded with the esterified form of Ca 2+-sensitive fluorescent probe fluo-4; L-I Ca was recorded by the ruptured-patch whole-cell technique. Result: ①D+I/R had a higher survival rate ( 61.43± 5.14)% than I/R (P< 0.01); 5-HD completely reversed the protective effects of diazoxide (P< 0.01). ②The relative fluorescent values in each group were as follows: I/R (374±52)%, D+I/R (147±18)% (vs I/R, P< 0.01), (403±63)% (5-HD+D+I/R, vs D+I/R, P< 0.01) and (379±44)% (5-HD+I/R, vs D+I/R, P< 0.01).③Compared with that in I/R, the if density of the sinoatrial cells in D+I/R increased significantly under the same commanding voltage (P< 0.01), and it was decreased in 5-HD+D+I/R (vs D+I/R, P< 0.01). Conclusions: K+-ATP channel opener diazoxide reduce cytosolic Ca 2+ loading, protecte cultured SAN cells during I/R and antagonize the effects of simulated ischemia/reperfusion on L-type calcuim current, it may be correlative with the opening of K+-ATP channels in mitochondrion in SAN cells.

关 键 词：窦房结 缺血-再灌注 L型钙电流 K^--ATP通道开放剂 

分 类 号：R322.1[医药卫生—人体解剖和组织胚胎学]