EPA和DHA经Src 激酶调控中性粒细胞趋化反应介导的抗炎机制研究

The last few years, novel and unexpected roles of neutrophils have been discovered in diverse aspects of immunity and inflammation,and evidence is now accumulating that suggests considering them as therapeutic targets.A number of studies on fish oil supplementation demonstrated a decrease in chemotaxis of human neutrophils towards various chemoattractants. The omega-3 fatty acids (FA) found in these oils are mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). However, the mechanism of their targets and molecular mechanisms are not fully understood. In this study, we will focus on investigating the role of the Src family kinases (SFKs) in EPA and DHA-mediated neutrophil chemotaxis. First, we will observe the effect of EPA and DHA treatment on neutrophil chemotaxis using Zigmond chamber and Transwell. The impact of dietary (n-3) PUFA on both SFKs (Lyn, Fgr and Hck) will be examined in neutrophil chemotaxis using immunofluorescence stain, GST pull down and western blot.Secondly, we will explore the association between SFKs and lipid rafts in EPA and DHA regulating neutrophil polarization and chemotaxis by means of confocal microscopy and sucrose gradient ultracentrifugation At last,we will confirm the effect of EPA and DHA on neutrophil chemotaxis use transgenic zebrafish(Tg:mpx-GFP) ,in which neutrophil were labeled by green fluorescence protein(GFP).The present study will not only provide a new possible immunomodulatory mechanism by EPA and DHA during neutrophil chemotaxis, but also a novel scientific evidence for the treatment of neutrophil-mediated autoimmune disorders by fish oil.

近年来中性粒细胞(PMNs)成为抗炎的新靶标，健康人补充鱼油（主要成分EPA和DHA）抑制其趋化反应，但机制未明。本研究以EPA和DHA为受试物，结合体内外实验，首先观察PMNs趋化能力和胞内聚合F-actin的分布，检测Src家族激酶pSFKs,pLyn,pFgr,pHck,和GTP-Rac2蛋白表达水平，分析PMNs趋化反应中EPA和DHA对Src家族激酶磷酸化的影响。其次用confocal和超高速冷冻离心法观察和检测SFKs,Lyn,Fgr和Hck在脂筏中的表达水平，分析PMNs趋化反应中EPA和DHA对Src家族激酶膜定位的影响。最后建立GFP标记PMNs的转基因斑马鱼炎症模型，定量炎症部位PMNs数量并检测pSKFs蛋白表达水平，验证PMNs是鱼油抗炎的靶标。本研究系统阐明EPA和DHA经Src激酶调控PMNs趋化反应的分子机制，为鱼油防治PMNs介导的炎性疾病提供新的理论依据。

慢性非传染性疾病(noncommunicable diseases，NCDs)是全球的第一位死因，近年来以中性粒细胞为靶标成为防治NCDs的研究热点。流行病学研究表明健康人补充鱼油（主要成分EPA和DHA）抑制中性粒细胞趋化反应，但机制未明。本研究用EPA和DHA处理人原代中性粒细胞,发现Src家族激酶酪氨酸416磷酸化水平降低，活化Rac2蛋白表达水平受到抑制，细胞骨架分子单体F-actin聚合减少，细胞极性化和趋化能力减弱，且DHA的作用浓度低于EPA，该结果表明EPA和DHA通过抑制Src家族激酶蛋白磷酸化水平影响中性粒细胞的趋化反应。同时采用confocal发现EPA和DHA破坏脂筏后，Src家族激酶在中性粒细胞脂筏域中荧光减弱，提示长链多不饱和脂肪酸可通过影响Src 家族激酶在膜上不同区域的分布调节中性粒细胞趋化反应。已建立稳定的红色荧光标记Tg（lyz:dsred）中性粒细胞的斑马鱼炎症模型，可进一步从整体动物实验阐明n-3长链多不饱和脂肪酸的抗炎机理。本研究初步阐明EPA和DHA 经调控Src家族激酶磷酸化蛋白表达水平和不同胞膜区的分布影响中性粒细胞趋化反应，揭示了EPA和DHA 以中性粒细胞为靶标发挥免疫调节作用的分子机制,为膳食补充鱼油防治中性粒细胞介导的炎性疾病提供了重要理论依据。