Gln对肉鸡肠黏膜屏障的保护及调控

NLR family, CARD domain containing 5 (NLRC5) receptors is a kind of pattern recognition receptor, and the relationship between NLRC5 and nutrition is not clear. Glutamine is an conditionally essential amion acid, which can play an improtant role in the gut immune protection. However, the mechanisms of action are not well-documented. The objective of this study is to determine whether salmonella pollorum is associated with the mimmun diseases of broiler, and NLRC5-NF-κB signaling pathway plays a key role in the activity of intestinal epithelial cells to defend itself from immune stress. The luc report assay and western blot technology to evaluate that glutamine on gene and protein expression of NLRC5, NF-κB on the intestinal cell membrane surface and its signal pathway of broiler under the immune stress in vivo and to preliminarily reveal the mechanisms in vitro, then to study flurescein enzyme report gene to duplicate by the plasmid constructed, transfect, NLRC5-Knock-down and fluorescene research thchniques, to evaluate the effects of glutmine on the NLRC5 and NF-κB mRNA and protein expression levels in the intestinal epithelial cells by the Real-time PCR and western blot technology, respectively. It is expected to clarify the molecular mechanism of glutamine inhibits NLRC5-NF-κB expression and the inhibition in salmonella pollorum-induced immune stress of broiler, and to provide theoretical datas for the using of glutamine in the immune stress of broiler.

NLRC5是一类通过病原关联分子模式识别细菌和病毒等有害物质，来介导机体免疫反应的受体,其效应中心是半胱氨酸蛋白酶聚集结构域。谷氨酰胺作为一种由半胱氨酸合成的条件必需氨基酸，具有肠道免疫保护的效果，但其免疫保护的过程中是否有NLRC5受体的参与尚待研究证实。本项目以“肠黏膜上皮细胞-NLRC5-NF-κB信号通路”为轴线，以沙门菌引起免疫应激为出发点，利用体外肠上皮细胞和肉鸡为实验对象，通过基因检测、Western blot、载体构建、基因沉默等技术，分别对肠上皮细胞分泌的NLRC5, NF-κB mRNA和蛋白表达水平进行测定，研究谷氨酰胺在体内外对NLRC5-NF-κB的调节机制；可望阐明谷氨酰胺在发挥免疫保护时与NLRC5-NF-κB 的关系，为谷氨酰胺的合理利用及寻求改善肉鸡免疫能力的营养措施依据。

谷氨酰胺(glutamine，Gln)作为动物机体内条件性必需氨基酸，是一些快速分裂增殖细胞，如肠上皮细胞和淋巴细胞的主要能量来源。研究表明，Gln对应激或疾病状态下机体免疫功能的维持和恢复以及维持肠道正常功能方面有着重要作用。为了研究谷氨酰胺对肉鸡肠道功能的调控作用，利用细胞体外培养、ELISA、荧光定量PCR、用Westernblot等技术，本课题开展了谷氨酰胺对不同模型肉鸡肠黏膜屏障调控机理的研究。结果表明：应激可引起不同日龄肉鸡消化酶活性、肠道形态、杯状细胞数量、微生物数量、挥发性脂肪酸浓度、黏膜上皮形态学、D-木糖浓度、黏膜免疫球蛋白（IgA、IgM和IgG）和炎性因子（IL-2、TNF-α、IL-4和IL-6）浓度、黏膜通透性（DAO）、外周血T、B淋巴细胞转化率，以及营养成分转运载体（SGLT1, CaBP-D28k和L-FABP）、氧化调节蛋白（SOD，CAT和Nrf2)）、肠道紧密连接蛋白（ZO1, claudin-1和occluding ）、凋亡蛋白（Caspase-3）和炎症因子的mRNA表达量(IL-1β、IL-2 、IL-6、TNF-α、IκBα和NF-κB 和NLRC5)发生变化，进而引起肠黏膜炎症变化，最终导致肉仔鸡肠屏障损害。添加谷氨酰胺提高肉鸡消化酶活性，改善肠黏膜形态、促进了细胞增殖、抑制凋亡；改变了外周血T、B淋巴细胞转化率、肠黏膜免疫球蛋白和炎性因子浓度；通过调节紧密连接分子ZO1, claudin-1和occluding的表达，抑制NF-κB、IκBα 和NLRC5通路，有效改善肉鸡生产性能并激活机体及肠黏膜的免疫机能，缓解了肉鸡因应激引起的肠道炎症，促进了肉鸡肠道的损伤修复，为进一步阐明谷氨酰胺调节肉鸡肠黏膜屏障的保护提供理论参考。体外细胞培养试验表明，谷氨酰胺可以诱导肠上皮细胞NLRC5、 TNF-α的表达，活化NF-κB，这表明NLRC5-NF-κB信号通路对谷氨酰胺诱导的免疫调控是至关重要的。