5-脂氧合酶在体外培养的大鼠腹腔巨噬细胞中的表达

Expression of 5-lipoxygenase in the peritoneal macrophages of rat cultured in vitro

目的 探讨 5 脂氧合酶 (5 LO)在体外培养的大鼠腹腔巨噬细胞中的表达。方法 分离大鼠腹腔巨噬细胞培养一定时间 ,用高效液相色谱、Westernblot和RT PCR分别观察 5 LO催化的代谢产物的生物合成、5 LO和 5 脂氧合酶激活蛋白 (FLAP)表达的变化 ,并用毛细管电泳定量分析RT PCR产物。结果 体外培养 2 4h对 5 LO以及FLAP的表达没有明显影响 ,5 LO酶活性没有明显的变化 ;培养到第 72小时 ,5 LOmRNA和蛋白表达没有明显的影响 ,但FLAP几乎检测不到 ,mRNA的量明显降低 ,检测不到白三烯B4(LTB4)和 5 羟二十碳四烯酸 (5 HETE)。进一步培养到第 12 0小时 ,5 LO的mRNA和蛋白表达明显下降 ,FLAP的mRNA和蛋白检测不到。毛细管电泳定量分析结果显示 ,培养 1、2 4和 72h ,5 LO的相对表达量没有明显变化 ,培养到 12 0h则下降了约 80 %。结论 5 LO可以在体外培养的大鼠腹腔巨噬细胞中稳定表达 72h。

Objective To elucidate the expression of 5 lipoxygenase (5 LO) in the peritoneal macrophages of rat cultured in vitro. Methods The peritoneal macrophages of rat were isolated and cultured for indicated time. The pro ducts of 5 LO were detected by HPLC. The protein and mRNA of 5 LO and FLAP were detected by Western blot and RT PCR respectively, and the products of the RT PCR were quantified with capillary electrophoresis (CE). Results The results of Western blot and RT PCR proved that there were no significant changes on the expressions of 5 LO and FLAP when the peritoneal macrophages of rat were cultured for 24 h. The activity of 5 LO of the cells almost remained normal. When the cells were cultured for 72 h, the protein and mRNA of 5 LO almost remained normal while the protein of FLAP decreased to be almost undetectable and the mRNA of FLAP significantly decreased. And leukotriene B 4 and 5 hydroxy 6,8,11,14 eicosatetraenoic acid (5 HETE) could not be detected by HPLC. The protein and mRNA of 5 LO significantly decreased when the cells were cultured for 120 h in vitro, however the protein and mRNA of the FLAP were not detected. The relative gene expression of 5 LO decreased for about 80% by CE when the cells were cultured for 120 h even though it was almost unchanged at 1, 24 and 72 h. Conclusions 5 LO is able to express normally in the peritoneal macrophages of rat cultured for 72 h in vitro.