核因子κB诱捕脱氧寡核苷酸对异位内膜基质细胞RANTES含量及趋化能力的影响

Influence of nuclear factor-κB decoy oligonucleotides on RANTES expression and monocyte chemotactic activity in stromal cells of ectopic endometrium

目的探讨核因子（NF）κB诱捕脱氧寡核苷酸（ODN）对子宫内膜异位症（内异症）患者异位内膜基质细胞中正常T淋巴细胞表达和分泌调节活化因子（RANTES）的含量，及其对单核细胞趋化能力的影响。方法分离、培养11例内异症患者的卵巢异位内膜基质细胞。实验分为3组，即刺激组[用10μg／L白细胞介素（IL）1β刺激培养]、转染后刺激组（NF-κB诱捕ODN转染异位内膜基质细胞后，用10μg／L IL-1β刺激培养）和空白对照组。各组刺激培养4、8、12、24、36h后分别取上清液，采用酶联免疫吸附试验检测RANTES蛋白的含量；采用Boyden小室趋化实验检测上清液中RANTES对单核细胞的趋化能力。另对刺激组培养24h后，在上清液中加入不同浓度（0.5、1、2、4、8mg／L）的抗RANTES抗体，采用Boyden小室趋化实验检测上清液中RANTES对单核细胞的趋化活性。结果（1）培养8、12、24、36h后RANTES蛋白的含量，刺激组分别为（58±10）、（150±35）、（360±46）、（586±42）ng／L，均高于空白对照组[分别为（32±6）、（32±7）、（31±6）、（33±8）ng／L]，两组分别比较，差异均有统计学意义（P〈0．05）；培养12、24、36h后RANTES蛋白的含量，转染后刺激组分别为（86±16）、（128±28）、（183±32）ng／L，均低于刺激组，两组分别比较，差异均有统计学意义（P〈0．05）。（2）刺激培养12、24、36h后RANTES对单核细胞的趋化指数，转染后刺激组分别为10．3±0．9、13．7±1．1、18．6±1．2，均低于刺激组（分别为15．3±1．2、24．3±1．4、32．4±1．6），两组分别比较，差异均有统计学意义（P〈0．05）。（3）刺激组培养24h后加入抗RANTES抗体浓度为0．5、1、2、4、8mg／L时，单核细胞的趋化抑制率分别为5％、23％、40％、62％和61％。结论NF-κB诱捕ODN可抑制IL-1β所诱导的异位内膜基质细胞中RANTES的含量，并降低其对单核细胞的趋化能力。在内异症病灶中，RANTES是单核-巨噬细胞的主要趋化因子。

Objective To study the inhibitory effect on the expression of regulated upon activation, normal T cell expressed and secreted （RANTES） and monocyte chemotactic activity of ectopic endometrial stromal cells by nuclear factor（NF）-κB decoy oligonucleotides （ODN）. Methods The stromal cells of ectopic endometrium were divided into 3 groups. Two groups were cultured with or without 10μg/L of interleukin （IL）-1β. Another group was transfected with NF-κB decoy ODN with the aid of a lipofectamine reagent. After 4 h of transfection, 10μg/L of IL-1β was added to induce the stromal cells to secrete RANTES. Concentration of RANTES in the supernatant at 4, 8, 12, 24 and 36 h was measured with the sandwich enzyme linked immunosorbent assay （ELISA）. U937 monocyte chemotactic activity was assayed in Boyden chambers. The specific RANTES-neutralizing monoclonal antibodies at serial doses （0. 5, 1, 2, 4 and 8 mg/L） were added into IL-1β induced medium of 24 h to detect the monocyte chemotactic activity of RANTES in supematant. Results The concentration of RANTES secreted by stromal cells was respectively （58 ± 10） , （ 150 ± 35 ）, （ 360 ± 46 ） and （ 586 ± 42 ） ng/L after IL-1β stimulation for 8,12,24 and 36 h, significantly higher than that of stromal cells cultured without IL-1β. The concentrations of RANTES were respectively （86 ± 16） , （128 ± 28 ） and （183 ± 32）ng/L after IL-1β stimulation for 12, 24 and 36 h in stromal cells transfected with NF-κB decoy ODN, evidently lower than that of stromal cells stimulated with IL-1β alone. The monocyte chemotactic index of 12, 24, 36 h in conditioned medium of stromal cells transfected with NF-KB decoy ODN was respectively 10. 3 ± 0. 9, 13.7 ± 1.1, 18. 6 ± 1.2, which was evidently lower than that of stromal cells stimulated with IL-1β alone. The anti-RANTES antibody at 0. 5, 1, 2, 4 and 8 mg/L inhibited respectively 5%, 23% , 40%, 62% and 61% of the chemotactic activity in 12 h medium treated with IL-1β. Conclusions RANTES accounts for the majority of the monocyte chemotactic activity in IL-1β induced medium of 24 h. NF-κB decoy ODN may influence the feed-forward inflammatory loop whereby IL-1β from activated macrophages can lead to RANTES production by ectopic implants and further monocyte chemotaxis.