细胞骨架在原代培养乳鼠窦房结细胞模拟缺血预适应中的作用研究

观察细胞骨架在乳鼠窦房结细胞模拟缺血预适应(IP)中的作用。取培养 2d的乳鼠窦房结细胞,随机分为①对照组;②模拟缺血 /再灌注(I/R)组;③模拟IP组;④phalloidin(微丝聚合剂 ) +I/R组;⑤cytochalasinD(微丝解聚剂) +IP组:⑥Taxol(微管聚合剂 ) +I/R组;⑦colchicine(微管解聚剂 ) +IP组。以FITC phalloidin及SABC cy3试剂盒分别标记F actin及β tubulin,用激光共聚焦显微镜检测各组窦房结细胞荧光强度改变。结果:①phalloi din预处理能显著增强再灌注后窦房结细胞F actin及β tubulin的荧光强度,并维持其形态、结构的相对完整性,模拟IP效应。②cytochalasinD及colchicine均能阻断模拟IP的细胞骨架保护作用,显著降低窦房结细胞F actin及β tubulin的荧光强度,导致细胞骨架的解体。③Taxol未能对I/R窦房结细胞提供IP样保护作用。结论:维持微丝结构的相对完整性可以减轻窦房结细胞I/R损伤,模拟IP效应;维持细胞骨架的相对完整性是IP产生的重要前提。

Role of Cytoskeleton in Ischemic Preconditioning of Primarily Cultured Sinoatrial Node Cells of Neonatal Rat

To investigate the role of cytoskeleton in ischemic preconditioning(IP) of sinoatrial node cells, cells were randomized to seven groups: ①Control,②Ischemia/reperfusion(I/R),③IP,④Phalloidin(microfilament stabilizer)+I/R, ⑤ Cytochalasin D(microfilament destabilizer)+IP,⑥Taxol(microtubule stabilizer)+I/R and ⑦Colchicine(microtubule destabilizer)+IP. After labeled with FITC-phalloidin and SABC-cy3 Fluorescent kit at the end of the experiment, F-actin and β-tubulin were studied with laser confocal microscopy. Results:①Phalloidin pretreatment significantly enhanced the fluorescence intensity of F-actin and β-tubulin of sino-atrial node cells exposed to severe I/R as well as better maintained their structural integrity. ②Both cytochalasin D and colchicine pretreatment blocked the protection of IP by reducing the fluorescence intensity of F-actin and β-tubulin and led to the disintegration of microfilament and microtubule. ③No improvement of F-actin and β-tubulin was observed in the group pretreated with Taxol compared with the group of I/R. Conclusion: These studies demonstrate that maintaining the structural integrity of microfilament can mimic the protective effect of IP and the integrity of cytoskeleton is a key point of IP.