生命早期维生素D缺乏对食物过敏发生的表观遗传学和VDR信号通路机制及其干预研究

Latest studies show that vitamin D deficiency might be one of the important risk factors contributing to the high prevalence of food allergy in modern society, and the underlying mechanisms are still unknown. It is suggested that the physiological functions of vitamin D might be related with the vitamin D receptor (VDR) expression, and the modulation of vitamin D on immune balance and intestinal barrier function. In this project, we aim to study the effect of vitamin D deficiency during early life (fetus and infant) on the development of food allergy and the related mechanisms. Firstly, we will establish food allergy animal model by using young mice with vitamin D deficiency at early life, and study the relationship of vitamin D concentration, VDR expression level and the occurrence of food allergy and its severity. Secondly, we will analyze the methylation level of the DNA promoters of key cytokines and transcription factors in immune modulation pathway to illustrate the epigenetic modification mechanism of vitamin D deficiency on the development of food allergy. Thirdly, we will use the wide-type and VDR knock-out mice and VDR-siRNA transfection in Caco-2 cells in vitro culture to study the effect of vitamin D deficiency on intestinal barrier function (including the colonic microflora composition, tight junction proteins level and the secretion of mucosal antigen-specific antibodies) by VDR signal pathway. Fourthly, we will study the stimulation of vitamin D to dendritic cells (DC) maturation and CD4+T cells differentiation through VDR signal pathway by using VDR knock-out mouse and DC-CD4+ T cells co-culture system. Furthermore, we also observe the effect of vitamin D supplementation at early life on the development of food allergy in animal models. By this project, we will clarify whether vitamin D nutritional condition at early life will influence the development of food allergy and the complicated mechanism, and the study will present strong evidence for early food allergy prevention by dietary measures.

近期研究发现，维生素D缺乏可能是婴幼儿食物过敏发病率增加的重要危险因素之一，但相关机制不明，可能与维生素D受体（VDR）表达、维生素D对免疫平衡和肠屏障功能的调节有关。本课题将研究生命早期不同阶段（胎儿期、哺乳期、断乳后）维生素D 缺乏对小鼠食物过敏发生的影响及其作用机制。一方面拟检测模型动物免疫调节通路中重要基因的甲基化水平，探讨维生素D缺乏对食物过敏发生的表观遗传学机制；另一方面将利用VDR基因敲除小鼠、VDR-siRNA体外细胞转染、抗原递呈细胞-效应T细胞共培养等技术手段，研究维生素D浓度、VDR表达水平与食物过敏发生之间的相关性，以及维生素D通过VDR信号通路影响肠黏膜屏障功能、调节免疫细胞分化与平衡在防治食物过敏中的作用机制；同时研究早期给予维生素D干预的作用效果。本课题的实施为阐明早期维生素D营养状况影响食物过敏发生的作用机制，和通过早期营养措施预防食物过敏提供重要理论依据。

维生素D缺乏和不足已成为一个日益严重的全球性问题，近期研究提示生命早期维生素D缺乏可能是婴幼儿食物过敏发病率增加的重要危险因素之一，但相关机制不明。本课题研究生命早期不同阶段（胎儿期、哺乳期、断乳后）维生素D 缺乏以及给予维生素D补充干预对小鼠食物过敏发生的影响及其初步作用机制。以BALB/c小鼠为实验动物，分别给予正常饲料和维生素D缺乏饲料，建立维生素D缺乏动物模型。检测血清25(OH)维生素D3水平确定母代小鼠维生素D缺乏模型建立成功后，将母鼠与正常饲料喂养雄性小鼠合笼，子代小鼠断乳后分别给予正常饲料和维生素D缺乏饲料，喂养至6周龄时以乳清蛋白为过敏原建立食物过敏模型。结果显示，通过维生素D缺乏饲料喂养BALB/c小鼠，可以有效地建立维生素D缺乏小鼠模型，二代动物模型可以更加有效的降低体内血清维生素D水平，生命早期不同阶段（孕哺期和断乳后）缺乏维生素D均可导致血清维生素D水平下降，孕哺期和断乳后全程缺乏组小鼠体内维生素D水平最低。对OVA-IgE抗体水平的检测发现，孕哺期维生素D缺乏子代小鼠血清OVA-IgE浓度显著升高，而断乳后缺乏维生素D没有引起子代小鼠血清OVA-IgE浓度显著升高，说明孕哺期维生素D缺乏是造成子代小鼠食物过敏发生的关键期，孕哺期维生素D缺乏对可能长期甚至终生影响子代免疫平衡。对免疫细胞分化和免疫活性因子水平的检测发现，维生素D缺乏食物过敏组小鼠IFN-γ/IL-4比例与阴性对照组和正常对照组相比均无变化，提示在生命早期维生素D缺乏致食物过敏进程中，Th1/Th2免疫应答失衡可能不是导致食物过敏的主要机制。食物过敏组脾和肠系膜淋巴结CD4+CD25+Foxp3+Treg细胞百分比较阴性对照组均显著降低，提示Treg细胞在维生素D缺乏致食物过敏小鼠模型中发挥重要作用，维生素D在生命早期尤其是孕哺期对CD4+CD25+Foxp3+Treg细胞分化、发育、成熟和迁移发挥重要作用，维生素D缺乏可能抑制Treg细胞的产生和发育，改变其调节活性，从而影响食物过敏的发生。