脂多糖对人外周血树突状细胞成熟的影响

Effects of lipopolysaccharide on the maturation and secretion of human peripheral dendritic cells

目的 通过研究内毒素的不同作用方式,模拟慢性乙型肝炎肠源性内毒素血症,探讨脂多糖（LPS）对人外周血树突状细胞（DC）成熟的影响.方法 用重组人粒细胞集落刺激因子、重组人白细胞介素-4、酪氨酸激酶受体3配体和肿瘤坏死因子α体外诱导、培养人外周血单个核细胞.分为持续刺激组：于1、4、7、9 d加入LPS 1 μg/ml;短期刺激组：于7、8 d加入LPS 1 μg/ml;对照组：不加LPS.观察细胞形态,用流式细胞仪检测细胞表型,混合淋巴细胞反应检测DC刺激T淋巴细胞的能力,用酶联免疫吸附法检测DC分泌细胞因子的水平.组间比较采用SPSS10.0统计学软件进行单因素方差分析,进一步两两比较用SNK法.结果 DC表达人白细胞-DR抗原、CD86、CD80、CD83分子水平,持续刺激组分别为65.81%±10.96%、48.81%±18.13%、13.56%±5.48%、11.52%±5.09%,对照组分别为78.43%±20.34%、51.29%±15.75%、15.22%±5.53%、15.64%±5.26%,短期刺激组分别为89.83%±16.99%、69.90%±24.05%、25.97%±10.81%、25.96%±10.59%,持续刺激组DC表面分子表达水平低于短期刺激组和对照组,各组比较,F值分别为3.376、3.823、4.535、5.320,P值均＜0.05,差异均有统计学意义.3组DC诱导同种异体混合T淋巴细胞的增殖指数分别为1.593±0.303、1.949±0.240、1.548±0.365,各组比较,F=3.572,P=0.049,差异有统计学意义.3组DC表达干扰素γ水平短期刺激组为（40.52±11.38）pg/ml,高于对照组和持续刺激组[分别为（21.57±7.68）pg/ml、（15.6±5.83）pg/ml],各组比较,F=3.403,P=0.019,差异有统计学意义.3组DC表达白细胞介素12水平短期刺激组为（84.45±31.28）pg/ml,高于对照组和持续刺激组[分别为（54.42±20.34）pg/ml、（51.77±11.02）pg/ml],各组比较,F=2.212,P=0.088,差异有统计学意义.结论 LPS持续刺激可抑制DC的发育成熟,可能是肠源性内毒素血症患者细胞免疫功能低下的原因所在.

Objective To study the effects of Lipopolysaccharide （LPS） on the maturation and secretion of human peripheral dendritic cells （DCs）. Methods DCs from healthy human perpheral monocytes （PBMCs） were induced in vitro with rhGM-CSF, rhIL-4, Flt3-L and TNF α. The subjects were divided into 3 groups： the long-term group stimulated with LPS 1 μg/ml at day 1, 4, 7, 9 postculture; the short-term group stimulated with LPS 1 μg/ml at day 7 and 8 postculture, and the DCs without LPS stimulation was control group. After 10 days of culture, the morphologic features of DCs were observed by light and electron microscopes, the phenotypic patterns were charactericed by flow cytometry, the proliferation of T cell were evaluated with mixed leukocytes reaction （MLR） and the levels of IL-12 and IFN γ produced by DCs were analyzed with ELISA. Results Compared with the short-term group, the expressions of HLA-DR （65.81% ±10.96%）, CD86 （48.81% ± 18.13%）, CD80 （13.56% ± 5.48%）, CD83 （11.52% ± 5.09%）, the secretionsofIFN γ （15.60 ± 5.83 pg/ml） and IL-12 （51.77 ± 11.02 pg/ml） by the DCs in long-term group were decreased obviously （P 〈 0.05） and the proliferation of homogenic lymphcyte cells （1.548 ± 0.365） stimulated by DCs was also impaired （P 〈 0.05）. Conclusion Long-term LPS stimulation can suppress the maturation and secretion of DCs, which might be the reason of poor immunity in the patients with intestinal endotoxemia.