摘要
目的 探索间隙连接通讯功能、间隙连接蛋白CX43表达、微丝、纽蛋白在巨噬细胞活化过程中所起的重要作用。 方法 ATP作用于由小鼠腹腔获取的巨噬细胞(M),用MTT法检测ATP对巨噬细胞的活化作用,用罗氏黄(Ly)荧光染料示踪术检测ATP对小鼠腹腔巨噬细胞间隙连接通讯功能的影响,用免疫荧光技术检测ATP作用下的M连接蛋白Cx43 的表达和粘着斑-纽蛋白表达的影响,用罗丹明-鬼笔环肽-F-actin 专一标记技术,观察ATP作用下的M微丝蛋白(F-actin)的变化。 结果 (1)ATP对M有明显的活化作用;(2)ATP上调M的间隙连接通讯功能;(3)ATP增强连接蛋白Cx43的表达;(4)ATP增强粘着斑主要粘着蛋白-纽蛋白的表达;(5)ATP改善M微丝束的组装。 结论 在ATP促进M的活化过程中,间隙连接通讯功能、连接蛋白、纽蛋白。
Objective\ In order to investigate the major effects of gap junctional intercellular communication(GJIC),connexin(CX43) and cytoskeleton protein(vinculin and F actin) expression in ATP activated Mφ process. Methods Mouse peritoneal macrophages (Mφ)were used in this work. Experimental group (Mφ) was added with ATP into peritoneal Mφ solution, control group was used with peritoneal Mφ solution. Two group's Mφ cells were seeded in 96 well tissue culture plate or glass slide respectively and cultured in CO 2 incubator 37℃ for 15', activity of Mφ cells were examined by using MTT method. Expression of CX43 was demonstrated by using immunofluorescent cytochemistry method, GJIC was assessed by examining transfer of lucifer yellow dye after loading in the medium, microfilament cytoskeleton was studied by using double staining with rhodamine phalloidin for F actin and immunofluorescence for vinculin. Results It was found that (1) ATP stimulated Mφ cells obviously.(2) Increase of GJIC was observed in ATP stimulated Mφ cells.(3) CX43 expression of ATP stimulated Mφ cells was increased. (4) Improved of organization of vinculin positive adhesion plaques was observed in ATP stimulated Mφ cells.(5) The ATP stimulated Mφ cells displayed reorganization of F actin from dot like structure of controlled group to well assembled filamentous stress fibre structure. Conclusion These results indicated that activative Mφ cells by ATP stimulation was correlated closely with GJIC, CX43 expression and cytoskeleton reorganization.
出处
《解剖学报》
CAS
CSCD
北大核心
1999年第4期334-336,I012,共4页
Acta Anatomica Sinica
基金
国家自然科学基金
北京市自然科学基金
关键词
ATP
巨噬细胞
活化
间隙连接通讯
ATP
Macrophages cells
Activated
GJIC
CX43
Vinculin
F actin