论文部分内容阅读
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 investigate the effect and mechanism of curcumin (Cur)-induced tolerogenic dendritic cells (DCs). Methods Immature DCs from Wistar rats were treated with different concentrations of Cur (0, 10, 20, and 30 μmol/L), and their phenotypes were analyzed by flow cytometry. Then 30 μmol/L Cur was applied to immature DCs, stimulated with lipopolysaccharide (LPS) with or without lipopolysaccharide. The ability of phagocytosed dextran was detected by flow cytometry. DCs secreted by leucocytes were determined by enzyme-linked immunosorbent assay (ELISA). The ability of IL-12 (IL-12) to detect nuclear factor-κB (NF-κB) p65 and RelB nuclear translocation in DC, NF-κB DNA binding ELISA and luciferase reporter gene detection of nuclear NF-κB activity in Western blotting. Mixed lymphocyte reaction was used to examine its ability to stimulate T lymphocyte proliferation in Lewis rats. Results Cur could significantly inhibit the expression of DC co-stimulatory molecules CD80, CD86 and CD40 in a dose-dependent manner. When the concentration of Cur reached 30 μmol/L, the expression of costimulatory molecules was not statistically different from that of immature DCs. DC stimulated by LPS (LPS group), DCs phagocytosed (36.6 ± 7.2)%, IL-12 secretion (415.9 ± 42.7) pg / ml, its nuclear RelB and NF-κB p56 high Expression, RelB DNA binding activity and NF-κB p65 DNA binding activity were 0.65±0.08 and 0.74±0.07, respectively. Reporter gene luciferase activity was 435% of control cells. This DC has strong stimulation of allogeneic T lymphocyte proliferation. Ability. The first DC was treated with 30μmol/L Cur and then added to LPS (Cur+LPS group). The phagocytic DCs accounted for (78.6±14.2)%, which was significantly higher than that of LPS group (P<0.01); IL- The secreted amount of 12 was (97.5±19.6) pg/ml, which was significantly lower than that of LPS group (P<0.01); its nuclear RelB and NF-κB p56 were low; RelB DNA binding activity and NF-κB p65 DNA binding activity were respectively The viscosities of 0.15±0.06 and 0.29±0.06 were all significantly lower than those in the LPS group (P<0.01); the luciferase activity of the reporter gene was 197% of the control cells and significantly lower than that of the LPS group (P<0.01); the DC stimulated the same species. The ability of T lymphocytes to proliferate was significantly weaker than that of the LPS group. Conclusion Cur-induced production of tolerogenic DC may be achieved by inhibiting the activation of NF-κB in DC.