ω-3多不饱和脂肪酸对树突状细胞生物物理学特性及迁移能力的影响
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1.贵州医科大学基础医学院;2.贵州医科大学生物与工程学院,生物与医学工程重点实验室

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R318.01

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Effects of ω-3 Polyunsaturated Fatty Acids on the Biophysical Properties and Migration of Dendritic Cells
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    摘要:

    目的 从生物物理学与免疫学交叉角度,分析二十碳五烯酸(eicosapentaenoic acid, EPA)和二十二碳六烯酸(docosahexaenoic acid, DHA)对鼠源树突状细胞(dendritic cells, DCs)生物物理学特性、细胞骨架及迁移能力的影响,以期探讨ω-3多不饱和脂肪酸(ω-3 polyunsaturated fatty acids, ω-3 PUFAs)对DCs免疫功能的影响及其潜在作用机制。方法 分离C57BL/6J小鼠骨髓来源单核细胞,经20 ng/mL重组鼠集落刺激因子(recombinant mouse granulocyte macrophage colony stimulating factor, rmGM-CSF)和10 ng/mL重组鼠白介素-4(recombinant mouse interleukin-4, rmIL-4)诱导分化为未成熟树突状细胞(immature dendritic cells, imDCs),第6 d加入100 ng/mL脂多糖诱导为成熟树突状细胞(mature dendritic cells, mDCs),进一步对imDCs及mDCs进行形态学观察及CD11c阳性率分析;然后通过细胞增殖检测(cell conting kit-8, CCK-8)试剂盒及流式细胞仪检测不同浓度EPA和DHA(浓度均为0~60 μM)作用下DCs细胞活力及凋亡情况;在确定最佳作用浓度后分别通过荧光偏振法、细胞电泳法及浓度梯度法检测分析DCs的膜流动性、电泳迁移率(electrophoretic mobility, EPM)及渗透脆性变化,并用免疫荧光法检测其细胞骨架纤维状肌动蛋白(filamentous actin, F-actin)表达,最后利用Transwell系统检测DCs迁移能力。结果 imDCs及mDCs的CD11c阳性率均在80%左右;不同浓度EPA和DHA均呈剂量依赖性降低DCs细胞活力(P<0.05或P<0.01),但并没有诱导其凋亡。在EPA和DHA(浓度均为50 μM)作用下,DCs生物物理学特性均发生改变,其中渗透脆性和EPM明显下降(P<0.05或P<0.01),膜流动性明显增大(P<0.05或P<0.01)。与此同时,DCs细胞骨架F-actin含量表达均明显上升(P<0.05或P<0.01),迁移率显著下降(P<0.05或P<0.01)。结论 ω-3 PUFAs可能会通过改变DCs细胞骨架结构及生物物理学特性,抑制细胞迁移能力,进而影响其免疫功能。这为深入了解ω-3 PUFAs对DCs免疫功能的影响机制提供理论支持,也为临床上自身免疫性疾病和器官移植中免疫抑制剂的使用提供可能。

    Abstract:

    Objective From the perspective of Biophysics and immunology, the effects of Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA)on the biophysical characteristics, cytoskeleton and migration ability of mouse derived dendritic cells (DCs) were analyzed, so as to explore the effect of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) on the immune function of DCs and its potential mechanism. Methods The bone marrow-derived monocytes from C57BL/6J mice were isolated and induced to differentiate into immature dendritic cells (imDCs) by 20 ng/mL recombinant mouse granulocyte colony stimulating factor (rmGM-CSF) and 10 ng/mL recombinant mouse interleukin-4 (rmIL-4). After 6 days, 100 ng/mL lipopolysaccharide was added to induce mature dendritic cells (mDCs). Further the morphological observation and positive rate of CD11c in imDCs and mDCs were analyzed, Then in different concentrations of EPA and DHA (0~60 μM), the cell viability and apoptosis of DCs were detected by cell conting kit-8 (CCK-8) kit and flow cytometry. After the optimal concentration of EPA and DHA were determined, the changes of the membrane fluidity, electrophoretic mobility (EPM) and osmotic fragility of DCs were separately detected by the fluorescence polarization, cell electrophoresis and concentration gradient. The expression of filamentous actin (F-actin) was detected by the immunofluorescence. Finally, the migration ability of DCs was detected by the Transwell system. Results The positive rate of CD11c in DCs was about 80%, the viability of DCs were decreased in a dose-dependent manner under the action of EPA and DHA of different concentrations (P<0.05 or P<0.01), which didn’t induce the apoptosis of DCs. Under the action of EPA and DHA(50 μM), the osmotic fragility and EPM of DCs were significantly decreased (P<0.05 or P<0.01), and the membrane fluidity were significantly increased (P<0.05 or P<0.01). Meanwhile, the expression of F-actin in DCs were obviously increased (P<0.05 or P<0.01), and the migration rate of cells were obviously decreased (P<0.05 or P<0.01). Conclusion ω-3 PUFAs may affect the cytoskeleton structure and biophysical characteristics of DCs, inhibit the migration, and then affect its immune function. It not only provides the theoretical backup to deeply understand the mechanism of ω-3 PUFAs on immune function of DCs, but also possibilities for the application of immunosuppressants in the autoimmune diseases and organs transplantation.

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  • 收稿日期:2021-05-06
  • 最后修改日期:2021-07-29
  • 录用日期:2021-08-03
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