红肉中免疫原性唾液酸N-羟乙酰神经氨酸（Neu5Gc）的作用机制及调控方法研究

This project is aimed to study the level and the functional mechanism of the non-human sialic acid N-glycolylnuraminic acid (Neu5Gc) in red meats. It will also attempt to discover the possible approaches that can reduce or control the adverse effects that Neu5Gc poses on human. Neu5Gc widely presents in most mammals except for human, as the CMAH gene responsible for Neu5Gc synthesis in human has been mutated 2-3 million years ago. However, Neu5Gc is often found in human and this is due to the consumption of Neu5Gc-containing foods. Unlike Neu5Ac which plays very important benificial functions in human, Neu5Gc is shown to be related to the development of many diseases such as cancers, cardiovascular diseases and diabetes. It has been found that red meats are the foods that contain the highest level of Neu5Gc compared to other foods and this therefore brought the concern upon to the safety of consumption of red meats. Until now, the studies related to this issue have been focused on the free Neu5Gc or individual pure Neu5Gc containing protein (e.g. porcine submaxillary mucin, PSM). No report has been available about the effect of Neu5Gc ingested through red meats in which Neu5Gc exists in more complex forms. This study will first establish and optimize the UHPLC-MS based and immunal method for Neu5Gc detection and use the methods to detect Neu5Gc in different meats，meat products and other biological samples. The processing technologies will be also investigated for the first time for their effects on Neu5Gc in meats. In order to understand the function mode of exogenous Neu5Gc from red meats, the CMAH-gene-knockout mouse fed with crude red meat proteins will be employed and the level of Neu5Gc in different body parts including blood, urine, heart, liver and kidney will be investigated. Additionally, the health state of the mouse will be also checked. Based on the function mode, attempt will be also made on the control or reduction of the effect of Neu5Gc to the body. This will be carried out through two ways which include the sialidase treatment of meat to release the sialic aicd from the glycoconjugates, and the addition of excessive Neu5Ac into the meat for the competition with Neu5Gc over the synthesis of sialic acid containing glycoproteins. It can be expected that with the results of this study, the more comprehensive picture will be drawn to elucidate the functioning mechanism of Neu5Gc from whole red meat and the effect of processing of meat on Neu5Gc for the first time. Together with the apporaches which can effectively reduce the adverse effect of Neu5Gc, The entire outcome will provide the meat industry very useful and trustful information for their future development.

本项目拟对红肉中的有害唾液酸N-羟乙酰神经氨酸（Neu5Gc）的含量和存在形式、作用机理及调控方法进行研究。Neu5Gc是一种致病性非人源唾液酸，人类不能合成，主要通过膳食途径进入人体，能够引发免疫反应，促进多种疾病的发展。而膳食中又以红肉含Neu5Gc最多，引起人们对红肉摄入安全性的重视，是肉品研究的一个新动向。本项目拟对不同种类红肉中Neu5Gc的含量和存在形式以及加工工艺对肉中Neu5Gc的影响进行分析；以红肉中含Neu5Gc的糖蛋白为对象，以Neu5Gc合成酶基因CMAH敲除小鼠为模型，研究日常膳食下红肉所含Neu5Gc在机体内的代谢和影响；同时探索降低Neu5Gc对人体有害影响的可能途径。本项目将是首次对日常膳食条件下摄入红肉中Neu5Gc的作用机理进行系统深入的研究，也是首次对降低膳食中Neu5Gc影响的方法进行新探索，研究结果将对肉品研究和行业发展起到积极的推动作用。

Neu5Gc 是一种致病性非人源唾液酸，人类不能合成，主要通过膳食途径进入人体，能够引发免疫反应，促进多种疾病的发展。而膳食中又以红肉含 Neu5Gc 最多，近年来引起人们对红肉摄入安全性的重视。但关于Neu5Gc 致病性的研究都局限于细胞或是抗体诱导实验，至于日常膳食模式下摄入红肉的影响尚未见报道。本项目即在正常膳食模式下研究红肉摄入对机体的影响。. 本项目首先建立了更为准确灵敏的唾液酸检测方法，利用该方法对不同种类红肉、白肉、海产品、蛋中的Neu5Gc 进行了系统测定。在此基础上，考察了不同加工方式对红肉中Neu5Gc的影响。以 Neu5Gc 合成酶基因 CMAH 敲除小鼠为模型模拟人体，研究日常膳食下红肉所含 Neu5Gc 对机体代谢生化指标、免疫因子及肠道微生物等的影响；同时探索添加Neu5Ac对降低 Neu5Gc影响的效果。. 研究结果表明：在食物中红肉所含有的Neu5Gc最高（其中小肠和胃中Neu5Gc的含量比肉中要高出很多），且主要以结合态存在。加工方式对红肉中Neu5Gc的含量基本没有影响。当以红肉蛋白替代小鼠日粮中的蛋白开展为期10个月的饲养实验后，实验组小鼠的血液生化代谢指标（血糖、甘油三酯、肌酐、尿素氮）、血细胞指标（白细胞、中性粒细胞、淋巴细胞、嗜酸性粒细胞、嗜碱性粒细胞、单核细胞）、细胞免疫因子（IL-6、IL-8、TNF-α）等指标都在正常值范围内，与鸡肉（白肉）蛋白组和兔肉（红肉但不含Neu5Gc）蛋白组以及大豆蛋白组相比，除了甘油三脂比大豆蛋白组和鸡肉组低，嗜酸性粒细胞和嗜碱性粒细胞比其它组高以及增重比其它组低以外，其它指标都没有显著差异。此外，牛肉蛋白添加Neu5Ac组的各指标与牛肉蛋白组相比也没有显著差异。肝脏病理切片结果显示一切正常。肠道微生物的测定结果表明，每个组之间肠道菌群都有差异。总而言之，本项目结果表明，正常膳食模式下CMAH-/-小鼠饲喂牛肉蛋白10个月后没有对小鼠的健康产生任何不良影响。. 本项目是首次对日常膳食模式下摄入红肉中 Neu5Gc 的影响进行系统深入的研究，结果证明在每日摄入红肉至中年的情况下，小鼠没有任何不良反应，推翻了一直以来认为食用红肉容易导致体内炎症及相关疾病的观点。