巨噬细胞趋化检测方法的建立及皮质酮对其趋化功能的影响

Establishment of chemotaxis assay of macrophage and effect of corticosterone on its chemotactic function

目的建立改良Boyden趋化小室(Boyden chamber)法检测巨噬细胞趋化功能的方法,探讨皮质酮对大鼠腹腔巨噬细胞趋化功能的影响。方法分离成年Sprague-Dawley(SD)大鼠腹腔巨噬细胞。应用48孔Boyden chamber趋化板检测趋化功能。下室加入27μL的10nM FMLP作为趋化剂或含1%牛血清清蛋白的RPMI-1640作为阴性对照,将50μL不同细胞密度的大鼠腹腔巨噬细胞悬液(3、6、12×106/mL)置于上室以确定最适细胞密度,将趋化装置置于5%CO2、37℃细胞培养箱内孵育2、3h以确定最适孵育时间。同时,以0、10、501、000ng/mL皮质酮处理大鼠腹腔巨噬细胞,应用Boyden chamber法检测巨噬细胞趋化功能。结果巨噬细胞对10nM FMLP趋化剂呈细胞密度依赖性增加,上室巨噬细胞密度为6×106/mL时为最适细胞密度,并且最适孵育时间为3h。低浓度皮质酮(10、50ng/mL)处理大鼠腹腔巨噬细胞其趋化指数显著高于对照组(P<0.01),而高浓度皮质酮(1 000ng/mL)处理后巨噬细胞趋化指数与对照组相比差异不明显。结论成功建立了改良Boyden chamber法检测巨噬细胞趋化功能方法,皮质酮可呈剂量依赖性地影响巨噬细胞趋化功能,在无免疫刺激状态下,低浓度皮质酮能增强巨噬细胞趋化功能。

Objective To establish the ehemotaxis assay of macrophage in modified Boyden chamber, to explore the eilect ol cor- ticosterone on the ehemotaxis of rat peritoneal macrophages. Methods Isolated peritoneal macrophages from adult male Sprague- Dawley （SD） rats. The chemotaxis assay was used the 48-well chemotaxis chamber. The lower wells contained 27μL of the che- moattractant 10nM FMI.P or RPMI-1640 with 1% bovine serum albumin （BSA） as negative control,and the upper wells contained 50μL of various cell density of rat peritoneal macrophages suspension （3,6,12 × 10^6/mL） to determine the optimum cell density. The entire apparatus was incubated at 37℃ in humidified air with 5 % CO2 for 2,3h for determination of optimum incubation time. Meanwhile, rat peritoneal macrophages were treated with corticosterone （0,10,50,1 000ng/mlD, respectively. Chemotaxis of mac- rophage using the Boyden chamber assay was assessed. Results Macrophages exhibited cell density dependent increase in the re- sponsiveness to chemoattractant 10aM FMLP and macrophage cell density of 6 × 10^6/mL in the upper well was found to be opti- mum,and the optimum incubation time was found to be 3h. Peritoneal macrophages incubated with low concentrations of corticos- terone （10,50ng/mL） showed a greater chemotaxis index than control group（P〈0.01）. After treated with high concentration of corticosterone （1 000ng/mL）, difference was not found between the data of high concentration of corticosterone （1 000ng/mL） group and control group. Conclusion The chemotaxis assay of macrophage in modified Boyden chamber was established successfully. Corticoterone could influence the chemotaxis of macrophage in a dose-dependent manner. Low concentrations of cortieosterone enhanced the chamotaxis of macrophage in the absence of immunologic stimulation.