摘要
目的探讨姜黄素(Cur)诱导耐受性树突状细胞(DC)的效果及其机制。方法分别用不同浓度Cur(0、10、20和30μmol/L)作用于Wistar大鼠来源的不成熟DC,流式细胞仪分析其表型。再以30μmol/L的Cur作用于不成熟DC,加或不加脂多糖(LPS)刺激,流式细胞仪检测其吞噬葡聚糖的能力,酶联免疫吸附试验(ELISA)测定DC分泌白细胞介素12(IL-12)的能力,Western印迹法检测DC中核因子κB(NF-κB)p65和RelB核转位,NF-κB DNA结合ELISA和荧光素酶报告基因检测核内NF-κB活性,混合淋巴细胞反应检测其刺激Lewis大鼠T淋巴细胞增殖的能力。结果Cur能显著抑制DC共刺激分子CD80、CD86、CD40的表达,呈剂量依赖性,当Cur的浓度达30μmol/L时,其共刺激分子的表达与不成熟DC比较,差异无统计学意义。DC在LPS刺激下(LPS组),吞噬葡聚糖的DC占(36.6±7.2)%,IL-12的分泌量达(415.9±42.7)pg/ml,其核内RelB及NF-κB p56高表达,RelB DNA结合活性和NF-κB p65 DNA结合活性分别为0.65±0.08和0.74±0.07,报告基因荧光素酶活性是对照细胞的435%,该DC有较强的刺激同种T淋巴细胞增殖的能力。而先以30μmol/L Cur作用于DC,然后再加入LPS者(Cur+LPS组),吞噬葡聚糖的DC占(78.6±14.2)%,明显高于LPS组(P<0.01);IL-12的分泌量为(97.5±19.6)pg/ml,明显低于LPS组(P<0.01);其核内RelB及NF-κB p56低表达;RelB DNA结合活性和NF-κB p65 DNA结合活性分别为0.15±0.06和0.29±0.06.均明显低于LPS组(P<0.01);报告基因荧光素酶活性是对照细胞的197%,明显低于LPS组(P<0.01);该DC刺激同种T淋巴细胞增殖的能力明显弱于LPS组。结论Cur诱导产生耐受性DC可能是通过抑制DC中NF-κB的活化实现的。
Objective To observe the induction of tolerogenic dendritic cells （DCs） by curcumin（Cur） and its mechanism. Methods After immature DCs from bone marrow cells of Wistar rats were treated with different concentrations of Cur （0, 10, 20 and 30μmol/L） respectively, and then the DCs were tested by flow cytometry for the surface molecules expression. After the immature DCs were treated by 30μmol/L Cur with or without stimulation of LPS, endocytosis of DCs to dextran was tested by flow cytometry. The production of IL-12 in DC culture supernatant was determined by ELISA. The levels of NF-κB p65 and RelB translocation to the nucleus were investigated by Westernblot. The activity of NF-κB was detected by NF-κB-binding ELISA and luciferase reporter gene analysis. The ability of DCs to stimulate the proliferation of T cells from Lewis rats were analyzed by mixed leukocyte reactions （MLR）. Results Cur suppressed LPS-induced cell-surface expression of costimu-latory molecules （CD80, CD86 and CD40） in a dose-dependent manner. When Cur was used at a concentration of 30μmol/L, there was no marked difference in the surface molecules expression of LPS- inducing DCs as compared with immature DCs. After DCs were induced by LPS （LPS group）, the positive rate of FITC-Dextran uptake was （36.6±7.2） %, and the secretory amounts of IL-12 were （415.9±42.7） pg/ml. In DCs of LPS group, the intranuclear RelB and p65 were highly expressed and their DNA binding activity was 0.65±0.08 and 0.74±0.07 respectively. The luciferase activity of reporter gene in LPS group DCs was remarkably increased to 435% as compared with that in the controls. DCs in LPS group showed strong capacity to stimulate T cells proliferation. When DCs were treated with 30μmol/L Cur followed by induction with LPS （Cur ＋ LPS group）, the positive rate of Dextran uptake was （78.6±14.2）% and remarkably higher than in LPS group （P〈0.01）. But the secretory amounts of IL-12 of DCs in Cur＋ LPS group were （97. 5±19. 6） pg/ml, remarkably lower than in LPS group （P〈0.01）. In DCs of Cur ＋ LPS group, the intranuclear RelB and p65 were lowly expressed and their DNA binding activity respectively was 0.15±0.06 and 0.29±0.06, which was remarkably higher than in LPS group （P〈0.01）. The luciferase activity of reporter gene in Cur ＋ LPS group was only 197% of the control group, and obviously less than in LPS group （P〈0.01）. DCs in Cur ＋ LPS group showed the decreased capacity to stimulate T cells proliferation as compared with that in LPS group （P〈0.01 ）. Conclusion Cur can induce tolerogenic DCs, and the mechanism may be associated with blockade of NF- κB signaling.
出处
《中华器官移植杂志》
CAS
CSCD
北大核心
2007年第11期651-654,共4页
Chinese Journal of Organ Transplantation
基金
国家自然科学基金(30671993)
武汉大学人才专项基金(502273106)
