苏氨酸对鱼肠道紧密连接蛋白结构的影响及其调控机制研究

Intestine is not only important to nutrient digestion and absorption, but also is one of the nucleus immunization barrier for fish. Tight Junctions pay the key role in the integrity and barrier function of fish intestine. Threonine is the most abundant amino acid in the intestinal mucosa of mouse and newborn infant. Study in our lab showed threonine promoted the proliferation, differentiation and integrity of fish enterocyte membrane,however, the mechanisms remains unknown. whether threonine can regulate the formation of intestinal tight junctions to promote the integrity of fish intestine and the mechanisms are retained unclear. Thus, the project will firstly clone the genes of grass carp, including tight junction proteins like Claudin, Occludin, ZO and their subtypes, related signaling molecules such as ERK, MEK, Raf, PKC and their subtypes. Secondly, using immunohistochemisty and gene silencing technologies, we will carry out the study about the potential regulation role of signal molecules ERK, MEK, Raf and PKC on the assembly of fish intestinal tight junctions. Thirdly, we will study the relationship of threonine and the gene expression of fish intestinal tight junction proteins, related signal molecules, and their subtypes. After that, we will study the regulation effect of threonine on assembly of fish intestinal tight junctions, and whether it regulate the assembly of tight junction through the signaling pathway of PKC and Raf / MEK / ERK. At last, we will confirm if PKC could activate the Raf / MEK / ERK signaling to regulate the formation of tight junctions of fish treated by threonine. Summarily, the study will reveal the mechanisms of threonine-related regulation on formation of fish intestinal tight junctions, which will provide some theoretical supports to the mechanism of threonine-induced fish intestinal health and has important academic significance.

肠道不仅是鱼类消化吸收营养物质的重要场所，也是最重要的免疫屏障之一。紧密连接蛋白(TJs)在保证肠道结构完整和屏障功能上发挥至关重要的作用。苏氨酸（Thr）是大鼠和新生婴儿肠粘膜中含量最高的氨基酸，能促进鱼肠细胞增殖分化，保证鱼肠道和细胞膜结构完整，但其作用机制不清楚。Thr是否能调控鱼肠TJs结构保证肠道结构功能完善及其作用机制未见研究。因此，本项目拟在克隆草鱼肠TJs(Claudins、Occludin、ZOs)和信号分子PKC、Raf、MEK、ERK及其亚型的基础上，利用免疫组化和基因沉默技术率先研究PKC等信号分子在鱼肠道TJs结构中的调控作用；再探索Thr对鱼肠道TJs结构的影响；率先研究Thr是否通过PKC和Raf/MEK/ERK信号途径调控鱼肠道TJs结构，进一步确定PKC能否激活ERK参与Thr对鱼肠TJs的调控，将为揭示Thr保证肠道结构完整性的机制奠定理论基础。

肠道不仅是鱼类消化吸收营养物质的重要场所，也是最重要的免疫屏障之一。紧密连接蛋白(TJs)在保证肠道结构完整和屏障功能上发挥至关重要的作用。苏氨酸（Thr）是大鼠和新生婴儿肠粘膜中含量最高的氨基酸，能促进鱼肠细胞增殖分化，保证鱼肠道和细胞膜结构完整，但其作用机制不清楚。本项目克隆了草鱼肠道紧密连接蛋白相关蛋白及调控紧密连接大白相关的信号分子及其亚型基因18个；在此基础上，通过体内体外研究发现，苏氨酸能显著改善幼草鱼肠道紧密连接蛋白结构，保证细胞间结构的完整性；同时也能通过提高肠道上皮细胞抗氧化损伤能力、缓解肠道上皮细胞过度凋亡、抑制肠道炎症的发生等共同保证草鱼肠道健康。研究结果为揭示苏氨酸调控鱼类肠道健康的作用机制奠定了重要的理论基础。