丁硫氨酸硫酸亚胺早期处理抑制单核细胞向树突状细胞的分化

 目的 研究细胞氧化-还原态对树突状细胞（DC）分化和功能的影响。 方法 细胞氧化-还原态的调控应用特异性谷胱甘肽（GSH）合成抑制剂丁硫氨酸硫酸亚胺（BSO）。自健康人外周血单个核细胞（PBMC）分离单核细胞，在含重组人粒细胞-巨噬细胞集落刺激因子和 IL-4的RPMI 1640 培养液中诱导分化 DC。将单核细胞分为 BSO 早期（培养第 1 天）用药组、BSO 晚期（培养第 5 天）用药组和不予 BSO 处理的对照组。BSO 用药剂量为终浓度 1 mmol/L，换液时补加相应剂量的 BSO。培养第 5 天，分别给予加入脂多糖（LPS）100 ng/ml（LPS刺激组）或重组人干扰素 （rhIFN- ）20 U/ml（IFN- 刺激组）或不予刺激（无刺激组）的处理；培养第 7 天收集各组悬浮细胞进行表型分析和功能检测，并在倒置显微镜下观察 BSO 早期用药组和对照组贴壁细胞。表型分析采用特异性荧光标记单抗及流式细胞仪。功能检测采用混合淋巴细胞反应试验，将经丝裂霉素C 处理的 DC（刺激细胞）与异基因非贴壁 PBMC（反应细胞）混合（刺激细胞:反应细胞分别为 1:25、1:50、1:100）培养 5 d 后，用3H-胸腺嘧啶核苷法检测淋巴细胞增殖情况。 结果 培养第 7 天，镜下观察显示 BSO早期用药组贴壁细胞明显多于对照组；流式细胞仪检测显示 BSO 早期用药组 CD86 和CD1a表达明显低于对照组，CD86 平均荧光强度（MFI）分别为311.3 ± 97.3、552.0 ± 97.9（P = 0.01），CD1a 分别为52.2 ± 22.2、121.2 ± 4.2（P = 0.03）；BSO早期用药组及对照组3H-胸腺嘧啶核苷掺入量（cpm）在刺激细胞:反应细胞＝1:25时分别为 1587 ± 1458 和9336 ± 1333（P=0.015）；加入rhIFN- 20 U/ml 刺激后二组 CD86 MFI 分别为 495.9 ± 143.9 和 800.4 ± 27.7（P = 0.06），而加入 LPS 100 ng/ml 刺激后二组CD86 MFI 分别为 1736.7 ± 375.7 和 1960.8 ± 494.5（P > 0.05）。表明单核细胞分化早期加入 BSO 对 DC 的分化和功能有抑制作用，并对 IFN- 诱导DC成熟有抑制作用。BSO 晚期用药组 DC 抗原提呈功能分子的表达及与对照组比较均无明显差异，对 LPS 或 rhIFN- 刺激的 DC成熟也无明显影响。 结论 BSO 早期持续处理能抑制单核细胞向 DC 细胞的分化，细胞内氧化-还原态水平可能是影响 DC 分化成熟的关键因子。

Early treatment with buthionine sulfoximine inhibits differentiation of monocytes into dendritic cells

Objective To investigate the effect of buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, on differentiation and maturation of human dendritic cells (DCs). Methods Monocytes isolated from peripheral blood monuclear cells (PBMCs) of health donors were cultured with GM-CSF and IL-4 in RPMI 1640 medium to produce DCs. The monocytes were divided into early treatment (added with BSO 1 mmol/L from day 1) late treatment (added with BSO 1 mmol/L from day 5), and control (cultured without BSO) groups. The medium was changedevery other day with the BSO complemented to a same concentration (1 mmol/L). On day 7, the unattached cells were collected to analyze their phenotypes and function; the attached cells in the early treatment and control groups were collected and observed under an inverted microscope. In another experiment, the unattached cells were transferred to a new plate on day 5, cultured with LPS (100 ng/ml), IFN- (20 U/ml), or without any stimulation for 48 hours, and then collected for phenotyping with specific monoclonal antibodies and flow cytometry. In the functional assay, mitomycin C-treated DCs were co-cultured with allogeneic unattached PBMCs (1 105) in ratios of 1:25, 1:50, or 1:100 for 5 days. The proliferation of the lymphocytes was analyzed by measuring the value of 3H-thymidine incorporation with -counter. Results On day 7, the attached cells in the early treatment group was significantly more than that in the control group. Flow cytometry showed that the CD86+ and CD1a+ cells in the early treatment group were less than those in the control group, mean fluorescence intensity (MFI) value of CD86 was 311.3 ± 97.3 and 552.0 ± 97.9 (P = 0.01), while MFI value of CD1a was 52.2 ± 22.2 and 121.2 ± 4.2 (P = 0.03), respectively. The value of 3H-thymidine incorporation in the early treatment group was significantly lower than that in the control group when the ratio of DCs to nonadherent PBMCs was 1:25 (1587 ± 1458 vs. 9336 ± 1333, P = 0.015). After being cultured with IFN- (20 U/ml), the MFI value of CD86 in the early treatment and control groups was 495.9 ± 143.9 and 800.4 ± 27.7, respectively, while it was 1736.7 ± 375.7 and 1960.8 ± 494.5 (P > 0.05) in those treated with LPS (100 ng/ml), indicating that both the differentiation of monocytes into DCs and IFN- -induced maturation of DCs was inhibited by the early treatment with BSO. No significant difference was detected in the expression of the antigen presenting-related molecules among the cells treated with LPS, IFN- , or none of them in the late treatment group, and the control group. Conclusions Early treatment with BSO could inhibit the differentiation of monocytes into DCs and the maturation of DCs. Cellular redox state at early stage might be a pivotal decision factor for the differentiation and maturation of DCs.