大黄鱼白细胞介素-4功能的初步研究

Interleukin-4 (IL-4), an important member of the class I cytokine family, is secreted predominantly by activated Th (T-helper) 2 cells that promotes a variety of Th2-mediated humoral immune responses, including B cell proliferation and activation, and specific antibody production. Moreover, IL-4 is also involved in Th cell development and macrophage activation. Recent studies suggest that IL-4 homologues exist in fish. However, the functions of most fish IL-4 genes remain unknown. In the previous study, IL-4 was identified in the large yellow croaker (Larimichthys crocea). Based on this result, we will study the tissue distribution and gene expression pattern of IL-4 after bacterial infection, and focus on the functional identification of IL-4 in Th2 response and Th1/Th2 development by detecting B cell proliferation, specific antibody production, and expression levels of Th1/Th2-related transcription factors and cytokines. We will also demonstrate the roles of IL-4 in macrophage activation through respiratory burst activity analysis, and expression level analysis of inflammatory factors and growth factors in macrophages. These results may reveal the functions of IL-4 in large yellow croaker more comprehensively, providing valuable resources for further understanding the process of Th2 immune response in fish, and exploitation of IL-4 as immune adjuvant of large yellow croaker in the future.

白细胞介素-4（IL-4）属于I型细胞因子，在哺乳动物中主要由活化的Th（T-helper）2细胞产生，通过促进B细胞增殖与分化、特异性抗体产生等方式介导Th2免疫应答。此外，IL-4还参与Th细胞发育和巨噬细胞活化等过程。近期的研究显示鱼类存在IL-4同源基因，但是其功能还不明确。本项目拟在已获得大黄鱼IL-4基因的基础上，进一步研究IL-4的组织分布和细菌感染后的表达模式；通过检测大黄鱼IL-4在B细胞增殖和特异性抗体产生中的作用，及对Th1和Th2细胞相关转录因子和细胞因子表达的影响，明确其在Th2免疫反应和Th1/Th2细胞发育中的功能；通过检测IL-4诱导后巨噬细胞的呼吸爆发活性、炎症因子和生长因子的表达变化，明确其在巨噬细胞活化中的作用。这些结果将全面地揭示大黄鱼IL-4的功能，对深入认识鱼类Th2免疫反应具有重要意义，也为大黄鱼IL-4作为佐剂开发提供理论基础。

本项目完成了大黄鱼（Larimichthys crocea）白细胞介素4（LcIL-4）分子特征与功能研究，发现LcIL-4基因在健康大黄鱼各组织或器官中呈组成型表达，在心脏和鳃中表达量最高；灭活的三联弧菌疫苗诱导后，大黄鱼头肾组织中LcIL-4基因的转录水平显著上调，说明LcIL-4可能参与大黄鱼抗细菌免疫应答。LcIL-4基因在大黄鱼外周血淋巴细胞，头肾单核/巨噬细胞和粒细胞，以及头肾细胞系中也均有表达，在外周血淋巴细胞中表达量最高。利用大肠杆菌系统重组表达了IL-4蛋白（rLcIL-4），rLcIL-4体内注射显著促进大黄鱼T淋巴细胞的增值，并诱导辅助T细胞1 (Th1)、Th2和Th17细胞相关的细胞因子和转录因子表达，表明大黄鱼IL-4在Th1、Th2和Th17细胞应答中发挥重要作用。大黄鱼IL-4重组蛋白还显著促进B淋巴细胞增殖，与弧菌亚单位疫苗DLD共同免疫大黄鱼，大黄鱼血清中抗DLD的特异性抗体效价显著提高，表明LcIL-4显著促进大黄鱼体液免疫应答。此外，体外实验发现，rLcIL-4蛋白显著抑制头肾单核/巨噬细胞中超氧阴离子、活性氧族（ROS）和一氧化氮（NO）的产生，下调单核/巨噬细胞中炎症因子（IL-1β和TNF-α）的表达水平，并上调细胞生长相关基因（TGF-β和VEGF）的表达水平，从而诱导单核/巨噬细胞向M2型极化。这些结果不仅对于深入认识IL-4在鱼类Th2免疫反应中的功能具有重要意义，也为大黄鱼IL-4作为免疫佐剂的开发奠定了基础。