ATP活化的小鼠巨噬细胞通讯功能的研究

INVESTIGATION OF COMMUNICATED FUNCTION ON ATP ACTIVATED MACROPHAGES

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 Mφ process. Methods Mouse peritoneal macrophages (Mφ)were used in this work. Experimental group (Mφ) was added with ATP into peritoneal Mφ solution, control group was used with peritoneal Mφ solution. Two group's Mφ cells were seeded in 96 well tissue culture plate or glass slide respectively and cultured in CO 2 incubator 37℃ for 15', activity of Mφ 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 Mφ cells obviously.(2) Increase of GJIC was observed in ATP stimulated Mφ cells.(3) CX43 expression of ATP stimulated Mφ cells was increased. (4) Improved of organization of vinculin positive adhesion plaques was observed in ATP stimulated Mφ cells.(5) The ATP stimulated Mφ 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 Mφ cells by ATP stimulation was correlated closely with GJIC, CX43 expression and cytoskeleton reorganization.