高迁移率族蛋白B1对小鼠腹腔巨噬细胞免疫活性的影响

Effect of high mobility group box-1 protein on immune function of peritoneal macrophages of BALB/c mice

目的观察高迁移率族蛋白B1(HMGB1)对离体小鼠腹腔巨噬细胞免疫活性的影响及其意义。方法分离小鼠腹腔巨噬细胞,经不同浓度(1、10、100、1000ng/ml)的HMGB1刺激,以未加HMGB1的培养细胞为对照组,于不同时间点(6、12、24、48、72h)观察巨噬细胞摄取中性红能力,噻唑蓝法测定其对L1210细胞的杀伤作用,应用Transwell小室趋化装置观察趋化活性变化,并采用流式细胞术检测细胞表面I-Ak抗原表达的变化。结果1、10、100ng/ml的HMGB1可使巨噬细胞的吞噬功能、杀伤活性、趋化活性及抗原呈递能力明显提高,其中100ng/ml时的作用最强,与对照组比较差异显著(P<0.01);100ng/ml的HMGB1刺激48h后,对细胞吞噬功能、杀伤活性及趋化活性的影响达高峰(P<0.01)。结论一定浓度的HMGB1可增强巨噬细胞的免疫功能。

Objective To investigate the effect of high mobility group box-1 protein （HMGB1） on immune function of peritoneal macrophages in mice. Methods The peritoneal macrophages were isolated from female BALBc mice in vitro. The macrophages were stimulated with different doses of HMGB1 （1, 10, 100, 1 000ng/ml） for 6 hours, or with 100ng/ml HMGB1 for different duration （0, 6, 12, 24, 48, 72 hours）. The phagocytosis ability of macrophages was determined by the neutral red dye uptake assay and optical densities of samples were read at 540nm The cytotoxicity to L1210 cells was measured with MTT assay and was expressed by absorbance at 570nm. The chemotaxis was assayed by using the Costar Transwell cell culture chamber inserts and the cells on the lower surface of the transwell membrane were fixed and counted. The cells were harvested at the indicated time points, fixed, and stained for indirect immunofluorescence, then the expressions of I-A^k MHC class Ⅱ alloantigen on macrophages were determined with flow cytometry. Results The effects of HMGB1 on phagocytic activities, cytotoxicity, chemotaxis and I-A^k antigen expression of macrophages were enhanced in a dose-dependent manner, starting at doses as low as 1ng/ml and with a maximal response at 100ng/ml （P〈0. 01）. The effects of HMGB1 on macrophages were also time-dependent, with a significant increase in phagocytosis ability, chemotaxis and cytoxicity at 48 hours following stimulation with 100ng/ml HMGB1 （ P〈0. 01）. The peak expressions of I-A^k antigen occurred at 12 hours （ P〈0. 01）. Conclusion The present study suggested that extracellular HMGB1 in certain dosage could be capable of enhancing immune function of macrophages of mice.