ω-3多不饱和脂肪酸对人外周血树突状细胞的影响

发布时间：2018-05-27 07:42

本文选题：树突状细胞 + 细胞因子　；参考：《泸州医学院》2010年硕士论文

【摘要】： 目的：ω-3多不饱和脂肪酸(ω-3 polyunsaturated fatty acids,ω-3 PUFAs)是营养免疫中小分子物质的重要组成部分,其主要成分包括二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)。树突状细胞(Dendritic cells, DCs)具有激活免疫排斥和诱导免疫耐受的双重潜力,两者此消彼长。经DCs诱导耐受是延长移植物存活的有效方法。本研究通过人外周血单个核细胞来源的树突状细胞的体外培养,对DCs进行形态学观察、细胞因子及免疫表型的检测,探讨ω-3多不饱和脂肪酸对树突状细胞成熟状态及免疫学功能的影响。方法：取新鲜健康成人外周血,经淋巴细胞分离液梯度分离,取中间白膜层细胞,通过贴壁处理,获得单个核细胞,加入含10%胎牛血清的RPMI 1640完全培养基、人重组集落刺激因子(rhGM-CSF)、人重组白细胞介素-4(rhIL-4),使其终浓度为：rhGM-CSF 500U/ml, rhIL-4250U/ml,隔日半量换液,诱导5天,获得未成熟树突状细胞(immature DC, imDC),然后以ω-3多不饱和脂肪酸二十碳五烯酸(EPA),二十二碳六烯酸(DHA)或饱和脂肪酸硬脂酸(SA)处理细胞24h,并将其分为5组：未成熟DCs组、成熟DCs组、EPA处理组、DHA处理组和SA处理组。内毒素脂多糖(LPS)诱导细胞成熟后,细胞爬片Wright-Giemsa染色观察各组树突状细胞形态；采用半定量反转录聚合酶链式反应(RT-PCR)检测树突状细胞共刺激分子CD80 mRNA、CD86 mRNA及表面抗原提呈分子人类白细胞抗原-DR(HLA-DR) mRNA的表达；采用酶联免疫吸附试验(ELISA)法检测各组细胞上清中白细胞介素-12(IL-12)及肿瘤坏死因子-α(TNF-α)的含量。结果：未成熟DCs组CD80 mRNA.CD86 mRNA及HLA-DR mRNA的相对表达量为0.369±0.023、0.573±0.033及0.467±0.020,成熟DCs组这3种分子高表达,其相对表达量为0.767±0.017、1.160±0.047及0.801±0.016,成熟DCs组与未成熟DCs组之间相比,有显著性差异(P0.01)；EPA处理组CD80 mRNA.CD86mRNA及HLA-DRmRNA的相对表达量分别为0.547±0.034、0.849±0.043及0.657±0.023,DHA处理组CD80mRNA.CD86mRNA及HLA-DRmRNA的相对表达量分别为0.465±0.032.0.875±0.042及0.619±0.019,EPA和DHA处理组与成熟DCs组相比有统计学意义(P0.01),而SA处理组CD80mRNA.CD86mRNA及HLA-DRmRNA的相对表达量分别为0.757±0.020、1.132±0.064及0.792±0.018,与成熟组相比差异无统计学意义(P0.05)。细胞上清IL-12的含量,5个组分别为190.73±16.26、768.05±62.73.358.71±52.79.412.63±62.64.709.48±47.81 pg／ml,细胞上清TNF-α的含量,5个组分别为637.61±38.40、2405.73±125.48、1605.64±138.39.1807.38±140-34.2353.34±194.74 pg／ml,EPA及DHA处理组与成熟DCs组相比差异有统计学意义(P0.01),而SA组与成熟DCs组相比差异无统计学意义(P0.05)。结论：ω-3多不饱和脂肪酸可以在体外下调树突状细胞共刺激分子CD80.CD86及表面抗原提呈分子HLA-DR的表达,降低其细胞因子白细胞介素-12及肿瘤坏死因子-α的释放,减弱树突状细胞的抗原提呈能力,且对树突状细胞的体外成熟有抑制作用,从而增强免疫耐受性,提示ω-3多不饱和脂肪酸可能减轻器官移植中的排斥反应。
[Abstract]:Objective: Omega -3 polyunsaturated fatty acids (omega -3 polyunsaturated fatty acids, Omega -3 PUFAs) are important components of medium and small molecular substances in nutritional immunization. Their main components include twenty carbon five enoic acid (EPA) and twenty-two carbon six enoic acid (DHA). Dendritic cells (Dendritic cells, DCs) have double immunological rejection and induction of immune tolerance. DCs induced tolerance is an effective method for prolonging the survival of the grafts. This study was conducted through the culture of dendritic cells derived from human peripheral blood mononuclear cells, morphological observation, cytokine and immunophenotype detection of DCs, and the study of Omega -3 polyunsaturated fatty acids on the mature state and immunity of dendritic cells. Methods: the influence of the function of the epidemic. Methods: the fresh healthy adult peripheral blood was extracted by the gradient separation of lymphocyte separation liquid, and the interleukin cells were obtained. The single nucleus cells were obtained by adherent treatment, and the RPMI 1640 complete medium containing 10% fetal bovine serum was added. The recombinant human colony stimulating factor (rhGM-CSF) and human recombinant interleukin -4 (rhIL-4) were reorganized. The concentration is: rhGM-CSF 500U/ml, rhIL-4250U/ml, half a day for fluid replacement, induction for 5 days, to obtain immature dendritic cells (immature DC, imDC), and then use Omega -3 polyunsaturated fatty acids twenty carbon five enoic acid (EPA), twenty-two carbon six enoic acid (DHA) or saturated fatty acid hard fatty acid (SA) to treat cell 24h, and divide it into 5 groups: immature DCs group, mature Group DCs, EPA treatment group, DHA treatment group and SA treatment group. After endotoxin lipopolysaccharide (LPS) induced cell maturation, cell crawling Wright-Giemsa staining was used to observe the morphology of dendritic cells. Semi quantitative reverse transcriptional polymerase chain reaction (RT-PCR) was used to detect CD80 mRNA in dendritic cells, CD86 mRNA and surface antigen presented molecules. The expression of leucocyte antigen -DR (HLA-DR) mRNA and the content of interleukin -12 (IL-12) and tumor necrosis factor - alpha (TNF- alpha) in the supernatant of each group were detected by enzyme linked immunosorbent assay (ELISA). Results: the relative expression of CD80 mRNA.CD86 mRNA and HLA-DR mRNA.CD86 was 0.369 + 0.033 and 0.467 + 0.020 in the immature DCs group. The high expression of these 3 molecules in mature DCs group was 0.767 + 0.017,1.160 + 0.047 and 0.801 + 0.016. Compared with immature DCs group, the mature DCs group had significant difference (P0.01), and the relative expression of CD80 mRNA.CD86mRNA and HLA-DRmRNA in EPA treatment group was 0.547 + 0.034,0.849 + 0.043 and 0.657 + 0.023, DHA treatment group CD80mRNA The relative expressions of.CD86mRNA and HLA-DRmRNA were 0.465 + 0.032.0.875 + 0.042 and 0.619 + 0.019 respectively. Compared with the mature DCs group, the EPA and DHA treatment groups were statistically significant (P0.01), while the relative expressions of CD80mRNA.CD86mRNA and HLA-DRmRNA were 0.757 + 0.064 and 0.792 + 0.018 respectively in SA treatment group, and there were no differences compared with those of the mature group. Study significance (P0.05). The content of IL-12 in cell supernatant was 190.73 + 16.26768.05 + 16.26768.05 + 62.73.358.71 + 52.79.412.63 + 62.64.709.48 + 47.81 pg / ml, and the content of TNF- alpha in cell supernatant was 637.61 + 38.402405.73 + 125.481605.64 + 138.39.1807.38 + 194.74 194.74 The difference between group Cs and group SA was statistically significant (P0.01), but there was no significant difference between group SA and mature DCs group (P0.05). Conclusion: Omega -3 polyunsaturated fatty acids can reduce the expression of CD80.CD86 and surface antigen presentation of molecular HLA-DR in dendritic cells in vitro, and reduce the cytokines of interleukin -12 and the cause of cancer death. The release of sub - alpha reduces the antigen presenting ability of dendritic cells and inhibits the maturation of dendritic cells in vitro, thus enhancing immune tolerance, suggesting that Omega -3 polyunsaturated fatty acids may mitigate the rejection in organ transplantation.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.4

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