高迁移率族蛋白B1对人T淋巴细胞功能性极化的调节作用

目的观察高迁移率族蛋白B1（HMGB1）对人外周血T淋巴细胞功能性极化的影响，并对其作用机制进行初步探讨。方法分离健康人外周血单个核细胞后接种于24孔培养板（2×10^6／ml），以植物血凝素激活T淋巴细胞体外增殖后，给予不同剂量重组人HMGB1（0、1、10、100和1000ng／m1）进行刺激12、24或48h。采用四色流式细胞术分析CD3^＋淋巴细胞CD4表达和胞内细胞因子干扰素（IFN）-γ、白细胞介素4-（IL-4）阳性（Th1、Th2）的比例。结果不同HMGB1刺激时间和作用剂量对CD4^＋T淋巴细胞和Th1亚群比例未造成明显改变，但1000ng／ml HMGB1刺激48h后CD4^＋／CD3^＋比值显著低于刺激12h组（P〈0．05）。较大剂量HMGB1（100、1000ng／m1）能显著增加Th2亚群比例（P〈0．05或P〈0．01），并因此降低Th1／Th2比值，出现Th1优势向Th2优势偏移。结论HMGB1剂量蓄积和持续刺激可诱导T细胞功能亚群从促炎反应向抗炎优势转化。HMGB1的这一免疫调控效应可能是机体免疫应答陷人抑制状态，并最终出现免疫麻痹的重要原因之一。

Effects of high mobility group box-1 protein on human peripheral T lymphocyte polarization

Objective To investigate the effect of high mobility group box-1 protein （HMGB1） on polarization of the peripheral blood T lymphocytes and its potential regulating function in healthy human. Methods Peripheral blood mononuclear cells （PBMCs） were isolated by Ficoll-Hypaque technique from 8 healthy adult volunteers and were suspended in RPMI 1 640 medium with 10% fetal calf serum at the concentration of 2×10^6 cells/ml. The PBMC suspension were inoculated in a 24 well cell culture plate, cultured with 20 μg/ml phytohemagglutinin in 5% CO2 at 37℃, and added with recombinant human HMGB1 （rhHMGBI） at the concentrations of 0, 1, 10, 100, and 1 000 ng/ml for 12 h, 24 h, and 48 h, respectively. Four-color flow cytometry （FCM） was used to determine the CD3^＋ / CD4^＋ ratio and levels of interleukin （IL）-4 and interferon （IFN）-5,. Results There were no significant changes in CD4 expression and the number of IFN-γ positive cells （Th1） of CD3^＋ T lymphocytes among the groups stimulated by rhHMGB1 of different concentrations with different stimulating durations. Treatment with higher doses of rhHMGB 1 （100 and 1 000 ng/ml） markedly increased the proportion of Th2 subset and decreased the ratio of Th 1 to Th2 （P〈0.05 and P〈0.01）, thereby resulting in the polarization of T-helper lymphocyte activity from Thl shifting to Th2. Conclusion With the increase of stimulating doses and duration, HMGB1 down-regulates T-cell-mediated immunity as a Th2-polarizing stimulus. HMGB1, as a ubiquitous novel eytokine in sepsis, may contribute to the anti-inflammatory immunosuppressive status.