储存红细胞代谢产物持续影响巨噬细胞功能的初步研究

Preliminary study of stored red blood cell metabolites persistently affecting macrophage function

目的探究储存红细胞代谢产物持续影响巨噬细胞功能表达的情况。方法从人外周血分离诱导培养巨噬细胞,采用流式细胞术检测巨噬细胞CD14和CD68分子的表达情况,选择储存21 d悬浮少白细胞红细胞与其共培养,分别在0 h、12 h、24 h和36 h检测培养液上清中的白介素4(IL-4)和白介素6(IL-6)的含量,并采用免疫荧光的方法检测巨噬细胞CD68蛋白的表达情况。结果贴壁法和磁珠纯化法均可培养出符合条件的巨噬细胞,但磁珠纯化获得的巨噬细胞纯度更高。巨噬细胞与红细胞共培养后,不同时间的巨噬细胞CD68蛋白表达情况不同,其中24 h组CD68的表达最强。上清中IL-4和IL-6的分泌也不相同,其中IL-4含量在0 h、12 h、24 h和36 h分别为31.21 pg/mL、31.55 pg/mL、31.77 pg/mL和31.73 pg/mL,差异无统计学意义(P>0.05);IL-6含量在0 h、12 h、24 h和36 h的检测分别为2.34 pg/mL、25.56 pg/mL、65.15 pg/mL和35.47 pg/mL,差异有统计学意义(P<0.05)。结论人外周血分离培养巨噬细胞方法可靠性高。对储存红细胞刺激巨噬细胞功能变化及炎症因子研究发现储存红细胞代谢产物对机体内环境有促炎作用,可为进一步研究储存红细胞对巨噬细胞功能的影响提供理论参考和数据支持。

Objective To investigate the effect of metabolites of stored red blood cells(RBCs)on the function of macrophages.Methods The macrophages were isolated and cultured from human peripheral blood,then CD14 and CD68 macrophages were detected by flow cytometry.The contents of IL-4 and IL-6 in the supernatant which the RBCs stored for 21 days and co-cultured with the macrophages were detected,and the expression of CD68 protein in macrophages was detected by immunofluorescence method at 0 h,12 h,24 h and 36 h,respectively.Results Suitable macrophages could be cultured by both adherence method and magnetic beads method,but the purity of macrophages obtained by magnetic beads was higher.The expression of CD68 protein in macrophages was different after different time of co-culture with RBCs,and the expression of CD68 was the highest in the 24-hour group.The secretion of IL-4 and IL-6 in supernant was also different,and the content of IL-4 was 31.21 pg/mL,31.55 pg/mL,31.77 pg/mL and 31.73 pg/mL at 0 h,12 h,24 h and 36 h,respectively,with no statistical difference(P>0.05).The content of IL-6 was 2.34 pg/mL,25.56 pg/mL,65.15 pg/mL and 35.47 pg/mL at 0 h,12 h,24 h and 36 h,respectively,with significant differences(P<0.05).Conclusion The reliability of the method for isolating and culturing macrophages in peripheral blood of humans was high.The study on the functional changes of macrophages stimulated by stored red blood cells and inflammatory factors found that metabolic products of stored red blood cells had pro-inflammatory effects on the internal environment of the body,which could provide theoretical references and data support for further research on the impact of stored red blood cells on macrophage function.