小肽介导的肉仔鸡小肠上皮细胞信号传导通路研究

The intestine is not only a place for digestion and absorption of nutrients, but also an important immune barrier. Therefore, it is significant for livestock to keep intestine in health to achieve normal growth and development. The small peptides, the bioactive molecule as well as nutrients, have dual functions of nutritional and physiological regulation. Based on our previous works, the current project is conducted to clarify the effects of small peptides including L-Leu-L-Leu, Gly-Gly and Gly-L-Leu on growth, proliferation and apoptosis of intestinal epithelial cells（IEC）of broilers and molecular mechanism within above process using in vitro cell cultures, RNA-Seq, qRT-PCR and western blotting technologies. The project will investigate the effects of small peptides on growth, proliferation and apoptosis of broilers IEC, and the transcriptome and proteome of IEC, as well as abundance of proteins within TOR (Target of rampamycin) signaling pathway and in the upstream and downstream of TOR. These results will be helpful to elucidate signaling pathway mediated by small peptides in IEC of broilers, complete small peptide nutrition theory, and thus provide a basic guideline for small peptide nutritional regulation technology.

肠道是动物消化吸收养分的重要场所，也是机体重要的免疫屏障，维护肠道健康对于保障动物的正常生长发育具有极其重要的意义。小肽既是营养物质，又是生物活性分子，对机体具有营养和生理调节的双重功能。为了阐明小肽对肉仔鸡小肠上皮细胞（IEC）生长、增殖和凋亡的影响及其分子机理，本项目在前期研究基础上，以亮氨酰亮氨酸肽、甘氨酰甘氨酸肽和甘氨酰亮氨酸肽为实验材料，采用体外细胞培养、高通量测序、qRT-PCR和western blotting等技术，拟研究：小肽对IEC细胞生长、增殖和凋亡的影响；小肽对IEC基因表达的影响；小肽对IEC中差异信号蛋白表达的影响；小肽对体外IEC中雷帕霉素靶蛋白（TOR）及其上下游信号蛋白表达的影响。拟初步阐释小肽介导IEC信号传导通路，为完善“小肽营养理论”体系和建立小肽营养调控技术提供基础理论。

小肽理论体系是蛋白质理论体系重要的组成部分。本项目通过成功分离到肠道上皮细胞，并进行鉴定及传代培养，经免疫荧光鉴定细胞纯度达到90%以上。在此基础上开展L-Leu、Gly、L-Leu-L-Leu、Gly-Gly、L-Leu-Gly等5种氨基酸及小肽培养液分别干预细胞生长，培养48h后观测细胞生长曲线及细胞分裂指数、细胞周期、克隆形成率和细胞凋亡等指标，并采用转录组测序和蛋白质测序技术筛选获得差异表达基因和差异表达蛋白，通过Go和KGGE方法进行通路富集分析。研究结果显示：（1）L-Leu-L-Leu、Gly-Gly、L-Leu-Gly三种小肽均可以促进细胞的增殖和延缓细胞衰老，其中以L-Leu-L-Leu效果最优；（2）L-Leu与对照组比较筛选出40个差异表达基因，L-Leu-L-Leu与对照组比较筛选出30个差异表达基因，L-Leu-L-Leu与L-Leu组比较筛选出30个差异表达基因，主要参与免疫调节和能量代谢信号转导通路；（3）L-Leu组与对照组比较共筛选出90个差异表达蛋白，L-Leu-L-Leu与对照组比较共筛选出221个差异表达蛋白，L-Leu与L-Leu-L-Leu组比较共筛选出47个差异表达蛋白，大部分上调和下调差异表达蛋白主要参与免疫调节和能量代谢途径。