佛波酯对猪中性粒细胞巨噬细胞表面分子抗原-1的作用及有关信号机制研究

Study on the effect of PMA on porcine neutrophil Mac-1 and the signaling mechanism

【目的】研究佛波酯诱导猪中性粒细胞黏附分子巨噬细胞表面分子抗原-1表达的作用及有关信号机制。【方法】采用密度梯度离心法分离纯化猪外周血中性粒细胞,以流式细胞术检测中性粒细胞巨噬细胞表面分子抗原-1表达,分别以实时荧光定量PCR、Western Blot法检测信号蛋白p38丝裂原活化蛋白激酶、胞外信号调控激酶的基因表达和磷酸化活性。【结果】20、30 nmol/L的佛波酯能极显著促进猪中性粒细胞巨噬细胞表面分子抗原-1的表达(P<0.01)。与对照相比,佛波酯在0.5、6 h可明显增加猪中性粒细胞p38丝裂原活化蛋白激酶的基因表达量(P<0.05,P<0.01);在0.5、3和6 h可极显著增加猪中性粒细胞胞外信号调控激酶的基因表达量(P<0.01)。与对照相比,佛波酯在1、5和30 min可极显著促进p38丝裂原活化蛋白激酶磷酸化活性(P<0.01);在0.5、1、5和30 min可明显促进胞外信号调控激酶磷酸化活性(P<0.05,P<0.01)。【结论】佛波酯能诱导猪中性粒细胞黏附分子巨噬细胞表面分子抗原-1的表达,其机制至少与上调p38丝裂原活化蛋白激酶、胞外信号调控激酶的基因表达与磷酸化活性有关。

【Objective】To investigate the role of PMA in the expression of macrophage surface molecular antigen-1(Mac-1),a neutrophil adhesion molecule,and the related signaling mechanism.【Methods】Porcine peripheral blood neutrophils were isolated and purified by density gradient centrifugation,and the expression of Mac-1 in neutrophils was detected by Flow Cytometry.The gene expression and phosphorylation activity of signaling proteins p38 MAPK and ERK were detected by Real-Time fluorescence quantitative PCR and Western Blot,respectively.【Results】PMA at 20 and 30 nmol/L promoted the expression of porcine neutrophil Mac-1 very significantly(P<0.01).Compared with the control,PMA significantly increased the gene expression of p38 MAPK in porcine neutrophils at 0.5 and 6 h(P<0.05,P<0.01);and highly significantly increased the gene expression of ERK in porcine neutrophils at 0.5,3 and 6 h(P<0.01).Compared with the control,PMA significantly promoted p38 MAPK phosphorylation at 1,5 and 30 min(P<0.01);and significantly promoted ERK phosphorylation at 0.5,1,5 and 30 min(P<0.05,P<0.01).【Conclusion】PMA induces the expression of porcine neutrophil adhesion molecule Mac-1,and the mechanism is at least related to the up-regulation of gene expression and phosphorylation of p38 MAPK and ERK.