摘要目的 探讨核因子(NF)kB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-kB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、36 h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24 h后,在上清液中加入不同浓度(0.5、1、2、4、8 mg/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、36 h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、36 h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、36 h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24 h后加入抗RANTES抗体浓度为0.5、1、2、4、8 mg/L时,单核细胞的趋化抑制率分别为5%、23%、40%、62%和61%.结论 NF-kB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.

abstractsObjective To study the inhibitory effect on the expression of regulated upon activation,normal T cell expressed and secreted (RANTES) and monocyte ehemotactic activity of ectopic endometrial stromal cells by nuclear factor(NF)-kB decoy oligonucleotides (ODN). Methods The stromal cells of ectopic endometrium were divided into 3 groups. Two groups were cultured with or without 10 μg/L of interleukin (IL)-1β. Another group was transfected with NF-kB decoy ODN with the aid of a lipofectamine reagent. After 4 h of transfection, 10 μg/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, 4and 8 mg/L) were added into IL-1β induced medium of 24 h to detect the monocyte chemotactic activity of RANTES in supernatant. 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-kB decoy ODN, evidently lower than that of stromal cells stimulated with IL-1β alone. The monocyte ehemotactic 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-kB 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.