血小板体外活化后高表达P选择素(CD62P)抑制细菌增殖并诱导其凋亡样改变

High expression of P-selectin(CD62P)after platelet activation in vitro inhibits bacterial proliferation and induces apoptosis-like changes

目的对比两种不同细菌活化血小板P选择素(CD62P)分子表达水平及血小板对金黄色葡萄球菌(SA)、大肠杆菌(E.coli)增殖的抑制作用,并探索血小板抑制细菌增殖的分子机制。方法选取浓度为10^(6)菌落形成单位(CFU)/mL的SA、E.coli分别与2×10^(11)/L洗涤纯化的血小板共培养后,使用全自动酶标仪检测菌液吸光度(A_(600))值;倍比稀释后,涂板计数各组细菌数量;流式细胞术检测两种细菌与血小板共培养2、4 h后,血小板CD62P水平;ELISA检测血小板活化后释放胞内蛋白血小板因子4(PF4)含量;流式细胞术检测两种细菌与血小板共培养12 h后其分裂增殖的变化、流式细胞术检测两种细菌磷脂酰丝氨酸(PS)外翻和细菌细胞膜电位改变情况。结果血小板与SA共培养6 h后,菌液浊度明显降低;与E.coli共培养6 h后,菌液浊度略有降低;与对照组相比,两种细菌与血小板共培养后,细菌涂板计数均降低;SA、E.coli与血小板共培养2、4 h后,CD62P水平均升高,且血小板与SA共培养组的CD62P水平明显高于与E.coli共培养组;血小板活化后其胞内蛋白PF4释放量明显增加;血小板处理细菌后其分裂增殖速度均降低,且两种细菌均出现细胞膜PS外翻、细胞膜电位去极化。结论血小板体外活化后高表达CD62P,抑制SA、E.coli的增殖并诱导其发生细胞凋亡样改变,且血小板对SA的抑制作用优于对E.coli的抑制作用。

Objective To compare the expression level of P-selectin(CD62P)on platelets surface under the stimulation of Staphylococcus aureus(SA)and Escherichia coli(E.coli),explore the inhibitory effects of platelets on the their proliferation,and further investigate the molecular mechanism by which platelets inhibit the proliferation of bacteria.Methods 10^(6) CFU/mL SA and E.coli were co-cultured with 2×10^(11)/L purified platelets,and the A_(600) values of the two groups were detected;The CD62P of platelets was detected by flow cytometry after platelets co-cultured with SA and E.coli for 2 hours and 4 hours.The platelet factor 4(PF4)released by platelets was detected by ELISA;After co-cultured with SA and E.coli for 12 hours,the proliferation,phosphatidylserine(PS)eversion and cell membrane potential of SA and E.coli were analyzed by flow cytometry.Results After platelets co-cultured with SA and E.coli for 6 hours,the turbidity of SA decreased significantly and the turbidity of E.coli showed a slight decrease.Compared with the control group,the counts of bacterial plates decreased after two kinds of bacteria co-cultured with platelets.After co-cultured with SA and E.coli for 2 hours and 4 hours,the CD62P levels of platelets increased.In particular,the CD62P level of platelets co-cultured with SA was significantly higher than that of platelets co-cultured with E.coli.The release of intracellular protein PF4 of platelet increased significantly after bacteria stimulation.The proliferation rate of SA and E.coli decreased after co-cultured with platelets,and SA and E.coli exhibited PS eversion and depolarization of cell membrane potential.Conclusion High expression of CD62P inhibits the proliferation and induces apoptotic changes of SA and E.coli after platelets activation in vitro,and the inhibitory effect of platelets on SA was better than that of E.coli.