星形胶质细胞微丝表达与细胞缝隙连接的关系及其在癫痫发病中的意义

Relation between microfilament expression and gap junction of astrocyte and its meaning in epileptogenesis

目的研究模拟神经元兴奋环境下即高浓度KCl环境中马桑内酯（coriaria lactone,CL）对培养的星形胶质细胞（astrocyte,AST）肌动蛋白（actin）表达的影响及其与缝隙连接通道的关系,探讨它们在癫痫发病中的意义。方法利用改良McCarthy的方法培养星形胶质细胞,制备细胞爬片,并对培养的细胞进行不同干预分组：A（正常对照组）;B（高浓度的KCl作用组）;C（KCl＋CL组）;D（KCl＋1-hep）组;E（KCl＋CL＋1-hep）组。每组再分为24~48h作用时间亚组。采用直接免疫荧光法,用BODIPY FL phallacdin对星形胶质细胞F-actin进行荧光染色。用激光扫描共聚焦显微镜（laser scan-ning confocal microscope,LSCM）观察各组细胞骨架F-actin的结构及分布,同时测定F-actin含量。结果发现高浓度的KCl作用于星形胶质细胞24,48h后,F-actin的荧光强度较对照组升高（P〈0.01）,同时给予KCl＋CL作用于星形胶质细胞24,48h后F-actin的荧光强度较KCl组明显升高（P〈0.01）,同时给予KCl＋1-hep作用于星形胶质细胞24,48h后F-actin的荧光强度较KCl组明显降低（P〈0.01）,而同时给予KCl＋CL＋1-hep组荧光强度较KCl＋CL组明显降低（P〈0.01）。同一因素作用下24h作用组与48h作用组荧光强度未见统计学差异（P〉0.05）。结论CL可引起星形胶质细胞内F-actin含量的升高,而1-庚醇可引起F-actin含量的降低,这种变化影响细胞骨架微丝装配和星形胶质细胞胞间信号传导,可能在癫痫的产生中发挥重要作用。

Objective To study the influence of CL （Coriaria Lactone） to F actin expression of cultured astrocyte under the simulated neuron excitement environment, i. e. , high concentration of KCl, and the relationship with the gap junction passage, and to explore the meaning in epileptogenesis. Methods We cultured astrocytes with McCarthy＇s methed, and prepared creep slice. These slices were divided into 5 groups, including the control group （A）, the group dealed with KCl （B）, the group dealed with KCl＋CL （C）, The group dealed with KCl＋1-hep （D）, and the group dealed with KCl＋CL＋1-hep （E）. Each group was divided into two subgroups dealed for 24 hours and 48 hours. Using the direct immunofluorescence, the F actin of cultural astrocytes was stained using BODIPY FL phallacdin. Then, the structure and distribution of the F--actin of cultural astrocytes were observed with LSCM through DIF, and the content of F actin was determinated simultaneously. Results The cultural astrocytes were 90% positive verificated by immunocytochemistry. The content of F--actin was observed with LSCM through DIF. In the groups dealed for 24 hours, the fluorescence intensity of normal control was （0. 886 45±0. 013 56） ; the group dealed with KCI（0. 932 33±0. 015 62） ; the group dealed with KCI＋ CL（0. 989 63±0. 012 42） ; the group dealed with KCl＋1-hep （0. 813 70±0. 009 56） ; the group dealed with KCI＋CL＋1-hep（0. 865 69±0. 026 54）. In the groups dealed for 48 hours, the fluorescence intensity of normol control was（0. 887 02±0. 010 22） ; the group dealed with KCI（0. 936 23±0. 013 24） ; the group dealed with KCI＋CL（0. 992 32±0. 001 46） ; the group dealed with KCl＋1-hep（0. 825 36±0. 015 86）; the group dealed with KCI＋CL＋1-hep（0. 874 24±0. 014 42）. We （ound the fluorescence intensity after astrocytes were dealed in high concentration KCI for 24h, 48h increased compared with the control group （P 〈 0.01）. F actin fluorescence intensity after astrocytes were dealed with KCI＋ CL highly increased compared with the group dealed only with KCI （P 〈 0.01）. F--actin fluorescence intensity after astrocytes were dealed in KCl＋1 hep highly decreased compared with the group dealed only with KCl （P 〈 0.01）, while KCl＋CL＋1-hep group decreased compared with the KCl＋CL group （P 〈 0.01）. There was no significant diffence bwteen the groups dealed with the same factor for 24 hours and 48 hours （P 〉 0.05）. Conclusions CI. can increase the level of F--actin in astroeyte, while 1 --hepatal can do the opposite. This change may influence the signal transmission between cytoskeletal filament and astrocyte, and this may do something important in epilepgenic.