合成红藻氨酸对星形胶质细胞微丝表达及缝隙连接的影响

Effects of synthetic kainic acid on microfilament expression and gap junction in astrocytes

目的:研究合成红藻氨酸(synthetic kainic acid,SKA)对星形胶质细胞(astrocytes,AST)细胞骨架微丝表达及缝隙连接的影响。方法:取1日龄Wistar乳鼠大脑,差速贴壁法去除成纤维细胞,并利用振荡分离法去除少突胶质细胞,获得高纯度的AST细胞。将SKA和其它干涉剂与AST细胞共培养24 h后,用激光扫描共聚焦显微镜观察SKA对骨架蛋白中纤丝状肌动蛋白(F-actin)形态学及表达量的影响,并观察缝隙连接通道阻滞剂1-庚醇(1-heptanol,1-Hep)对SKA作用的影响。结果:和对照组相比,KCl组和KCl+SKA组的荧光强度均明显增强(P<0.01),后者更明显(P<0.01);镜下这2组的F-actin细丝状物较对照组明显增多、增粗、密集,KCl+SKA组更甚。1-Hep可明显降低KCl和SKA所致的F-actin表达,镜下所有1-Hep组可见细胞内部分丝状断裂,有些呈横切状。结论:SKA诱发癫痫的机制可能与其诱发AST细胞中F-actin的增多有关,细胞间缝隙连接可能参与了SKA诱发癫痫的过程。

AIM ： To study the effects of synthetic kainic acid （SKA） and 1-heptanol （ 1-Hep）, a gap junction blocker, on the cytoskeletal filament expression in the astrocytes. METHODS： The neonatal rat brain was obtained from the Wistar rats （ 1 day old） and primary purified astrocytes were obtained by differential attachment for removing filamentoblasts and orbital shaker for removing the oligodendrocytes. The effects of SKA and other interventions on the morpbologic changes and expression levels of skeleton protein filamentous actin （F-actin） were observed in the astrocytes after 24 h of the exposures by laser scanning confocal microscopy. The effect of 1-Hep on the expression of F-actin was also explored. RESULTS ： Compared with control group, the fluorescence intensities in KCl group and KCl ＋ SKA group were increased, and highly increased in KCl ＋ SKA group. The F-actin filaments in the above 2 groups were more intensive, thickened and concentrated than those in control group, and more obvious in KCl ＋ SKA group. 1-Hep significantly decreased the expres-sion of F-actin in KC1 group and KCl ＋ SKA group as compared with control group, and parts of the filamentous fracture were seen in the astrocytes in all 1-Hep-treated groups, in which some of the filamentous lines were crosscut. CONCLU-SION ： Increase in the expression of F-actin in the astrocytes affects the structure and function of the intercellular gap junctions, which may be involved in the mechanism of SKA-induced epilepsy.