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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 a method to detect the chemotactic function of macrophages using Boyden chamber method and to explore the effect of corticosterone on the chemotactic function of peritoneal macrophages in rats. Methods Adult Sprague-Dawley (SD) rat peritoneal macrophages were isolated. 48-well Boyden chamber chemotaxis assay chemotactic function. In the lower chamber, 27 μL of 10 nM FMLP was added as a chemotactic agent or RPMI-1640 containing 1% bovine serum albumin as a negative control, and 50 μL of rat peritoneal macrophage suspension of different cell densities (3, 6, 12 × 10 6 / mL) was placed in the upper chamber to determine the optimal cell density. The chemotactic device was incubated in a 5% CO2, 37 ° C cell incubator for 2, 3 h to determine the optimal incubation time. At the same time, rat peritoneal macrophages were treated with 0, 10, 501, 000 ng / mL corticosterone, and the chemotactic function of macrophages was detected by Boyden chamber method. Results Macrophages increased density dependently on 10 nM FMLP chemotactic agent and the optimal cell density was 6 × 106 / mL in the upper chamber. The optimum incubation time was 3 h. The chemotactic index of peritoneal macrophages treated with low concentration of corticosterone (10,50ng / mL) was significantly higher than that of the control group (P <0.01), while that of macrophages treated with high concentration of corticosterone (1 000ng / mL) Differentiation index compared with the control group, the difference was not obvious. Conclusion The modified Boyden chamber method was successfully established to detect the chemotaxis of macrophages. Corticosterone can affect the chemotactic function of macrophages in a dose-dependent manner. Under the condition of no immunostimulation, low concentrations of corticosterone can enhance the tendency of macrophages Function.