缺氧早期大鼠心肌细胞微管损害的观察

Study on injury to microtubule of cardiomyosites at early post-hypoxia stage

目的了解缺氧早期心肌细胞微管损害程度。方法将分离培养的Wistar大鼠心肌细胞分为正常组、缺氧组(建立缺氧细胞模型并设缺氧10、20、30、60 min为观察时相点)。用激光共聚焦显微镜及扫描电镜观察2组细胞微管分布、形态变化,对微管蛋白荧光强度进行半定量分析,用蛋白质印迹法检测2组细胞游离α微管蛋白的表达。结果与正常组比较,缺氧10 min后,缺氧组细胞微管念珠状结构消失,但排列尚有规律、数量无明显减少;缺氧20 min不仅念珠状结构消失,而且微管排列散乱,远离胞核区出现微管缺失;缺氧30、60 min时微管发生扭曲、断裂,纹理紊乱,完全丧失规律性。缺氧组心肌细胞微管蛋白荧光强度较正常组下降,且随缺氧时间延长愈加明显;缺氧组心肌细胞内游离的α微管蛋白表达(缺氧10 min为46 644±145)高于正常组(13 357±98),两组比较,差异有统计学意义(P<0.01),随缺氧时间的延长此升高趋势愈加明显。结论在缺氧状态下,心肌细胞微管发生解聚时间较早,其结构和分布规律被破坏。微管解聚在缺氧所致心肌细胞早期病理损害中的作用值得深入研究。

To investigate the degree of injury to microtubule of myocardium at early posthypoxia stage. Methods Cadiomyocytes from Wistar rats were isolated and cultured, and they were then divided into normal control and hypoxia groups. The distribution and morphological changes in microtubules were observed with laser confocal microscopy and scanning electron microscope at 10, 20, 30 post-hypoxia minutes（PHM） and 1 post-hypoxia hour（PHH）. Then the fluorescence intensity of α-microtubulin was detected with RT-PCR, the morphology of microtubule was observed, and the expression of dissociative α-microtubulin was determined by Western blot. Results Compared with normal control group, the bead-like structure of the microtubule in hypoxia group disappeared at 10 PHM, but no obvious change was observed in the distribution and number of microtubules. Despite the disappearance of bead-like structure of the microtubule, the microtubule derangement and loss of microtubule at the edge of cell were observed at 20 PHM. The fragmentation, derangement of texture, and loss of regularity in cardiomyocytes were observed at 30 PHM and 1 PHH. The fluorescence intensity of α-microtubulin in hypoxia group was evidently decreased than that in normal group in a time-dependent manner. The expression of dissociative α-microtubulin in hypoxia group at 10 PHM （46 644 ± 145 ） was obviously higher than that in normal group（ 13 357 ± 98, P 〈 0.01 ） , and its increase was maintained with elapse of time. Conclusion Microtubule injury to cadiomyocytes occurs at early stage of post-hypoxia, with destruction of its structure and distribution.