高迁移率族蛋白B1对小鼠巨噬细胞细胞因子和黏附分子表达的影响

Differential expression of tmnor necrosis factor-α, interleukin-lO, and intercellular adhesion molecule-1 in nmrine peritoneal macrophage in response to HMGB1

目的观察高迁移率族蛋白B1（HMGB1）对小鼠腹腔巨噬细胞肿瘤坏死因子（TNF）-α、白细胞介素（IL）-10及细胞间黏附分子-1（ICAM-1）表达的影响。方法分离小鼠腹腔巨噬细胞，经不同质量浓度（0、1、10、100、1000ng／m1）HMGB1的刺激，于不同时间点（0、6、12、24、48、72h）采用实时荧光定量PCR方法测定细胞TNF-α、IL-10及ICAM-1mRNA表达的变化，同时应用酶联免疫吸附试验检测细胞培养上清液中TNF-α、IL-10以及sICAM-1蛋白水平的变化。数据采用单因素方差分析。结果1～1000ng／ml的HMGB1作用24h，可使巨噬细胞的TNF-α、ICAM-1基因表达增加，与对照组比较差异具有统计学意义（P〈0．01），其中1000ng／ml HMGB1的作用最强，呈剂量一效应关系。而IL-10仅在1000ng／ml HMGB1作用时表达有所增加。以100ng／ml的 HMGB1作用不同时问后，培养上清液TNF-α与sICAM-1的浓度48h达高峰（P〈0．01），呈时间一效应关系，HMGB1对巨噬细胞TNF-α的分泌呈“双峰”特征，而IL-10水平无明显变化。结论一定浓度的HMGB1可诱导巨噬细胞合成、释放促炎细胞因子与黏附分子，具有显著的促炎特性。

Objective To investigate the differential expression of tumor necrosis faetor-α （TNF-α）, interleukin-10 （IL-10）, and intereellular adhesion molecule-1 （ICAM-1） in response to high mobility group box-1 protein （HMGB1） in murine peritoneal macrphage. Meethod The peritoneal maerophages were obtained from female BALBe mice. After exposure to various concentration of HMGB1 （0, 1, 10, 100, 1000 ng/ml）, four various lengths of time （0, 6, 12, 24, 48, 72 hours）, the maerophages were denatured in cell culture plates to determine mRNA expressions of TNF-α, 1L-10 as well as ICAM-1 by using real-time quantitative PCR, and cell supernatants were harvested to determine the protein levels of TNF-α, IL-10 as well as sICAM-1 by ELISA. Statistical evaluation of data was performed by one-way ＇analysis of variance （ ANOVA）. Results The protein and gene expressions of TNF-α and ICAM-1/sICAM-1 in peritonael maccrphage were markedly up-regulated by treatment with HMGB1, except for IL-10. Effects of HMGB1 on TNF-α and ICAM-1/sICAM-1 expressions were in a dose-dependent manner, ranged from 1 ng/ml to 1000 ng/ml （ P 〈 0.01 ）. The effects of HMGB1 on TNF-α and ICAM-1/sICAM-1 protein and gene expressions were also time-dependent with a significant increase at 48 hours （P 〈 0.01）. HMGB1 induced a biphasie response of TNF-α, the first peak approximately 6 hours and the second peak at 24 hours after exposure. However, there was no marked changes in IL-10 gene and protein expressions noted in peritoneal macrophages stimulated with HMGB1. Conclusions HMGB1 appears to be a proinflammatory cytokine, and promotes the synthesis as well as release of TNF-α and ICAM-1, except for IL-10.