奶牛小肽转运蛋白的免疫组织定位及其转运调控研究

The main end products of protein digestion are not free amino acids but small peptide. Dipeptide and tripetide can be directly absorbed into blood circulation, the absorption and protein synthesis rates of di/tripetide are 70% and 30% higher than that of free amino acids, respectively, which plays an important role in protein nutrition. Regrading to the work about transporter family of di/tripetide (SLC15), much effort has been made for PepT1 and PepT2 in monogastric animals. There are seldom reports about PHT1 and PHT2. Moreover, there has been little research about SLC15 in ruminant. Using body tissue and cells of dairy cattle as research materials, we plan to study the expression sites in tissue or cells, transport efficiency and characteristics of the members of SLC15 as well as the factors (such as animal developing period, feed composition, feed intake time, et al ) affecting the expression by the techniques of immunohistochemistry, confocal microscopy, 15N isotope tracer and proteomics so as to find the expression sites in tissue and cells of the members (PepT1,PepT2,PHT1 and PHT2) of SLC15 and the transporting types and quantity of small peptide and discover the relationship between the members of SLC15 and factors affecting small peptide transportation. The expected results of the study could lay a foundation for the mechanism research of small peptide absorption and utilization in tissue or cells and also provide theoretical basis for regulation of the small peptide absorption and utilization in dairy cattle.

蛋白质的消化终产物大部分是小肽而非游离氨基酸，小肽以二肽和三肽被吸收进入血液循环，吸收速率较氨基酸快70%，合成蛋白质的效率较氨基酸高26%，在蛋白质营养中有重要意义。关于二肽和三肽转运蛋白家族（SLC15）成员研究多限于单胃动物的PepT1，少许PepT2，对PHT1、PHT2少之又少。对反刍动物SLC15成员的研究更为罕见。本项目以奶牛的体组织、细胞为材料，利用免疫组化技术、共聚焦显微镜技术、15N同位素示踪和蛋白质组学技术，对SLC15成员在组织或细胞的表达部位、转运效率与特性、影响其表达量的因素（如动物发育时期、饲料、采食时间）进行研究，得出SLC15成员PepT1、PepT2、PHT1和PHT2在组织或细胞的表达部位、转运小肽的种类和数量、SLC15各成员间的相互关系和调控转运因素。为奶牛的组织或细胞的小肽吸收利用机理研究奠定基础，也为调控小肽在奶牛体内的吸收利用提供理论依据。

蛋白质的消化终产物大部分是小肽而非游离氨基酸，小肽以二肽和三肽吸收进入血液循环，吸收速率较氨基酸快70%，合成蛋白质效率较氨基酸高26%，在蛋白质营养中有重要意义。小肽转运蛋白家族（SLC15）PepT1、PepT2、PHT1和PHT2在吸收利用小肽的过程中发挥着至关重要的作用。目前SLC15研究多限于单胃动物的PepT1，少许PepT2，对PHT1、PHT2少之又少。对反刍动物SLC15的研究更为罕见。有必要探究SLC15在奶牛组织存在及数量的多少，表达数量受哪些因素影响。本项目对奶牛PepT1、PepT2、PHT1和PHT2的免疫组织学定位，不同生长阶段奶牛的组织和小肠上皮细胞的表达量；小肠上皮细胞对可能调节PepT1、PepT2、PHT1和PHT2的调控因子的种类和数量等进行研究。PepT1分布于奶牛MG小叶间结缔组织；PepT2分布于MG叶间导管及腺泡腔内侧上皮细胞。PepT1和PepT2分布于BMEC的细胞质。PepT1 mRNA在奶牛空肠和十二指肠表达量极显著高于回肠、瘤胃、肺脏、结肠、瓣胃、肝脏、盲肠、网胃、胸腺、脾脏、皱胃、肾脏、乳腺、心脏和肌肉；PepT2 mRNA在乳腺中表达量极显著高于肾脏、肝脏、肺脏、脾脏、回肠、皱胃、胸腺、十二指肠、网胃、盲肠、心脏、空肠、结肠、瓣胃、肌肉和瘤胃；PHT1 mRNA在脾脏、盲肠和肺脏中表达量极显著高于心脏和肌肉；PHT2 mRNA在肺脏、盲肠和胸腺表达量极显著高于瘤胃、皱胃、网胃、心脏和肌肉。PepT1、PepT2、PHT1和PHT2在成年奶牛体组织广泛表达，但表达水平差异较大。PepT1 mRNA在BIECs中表达量最高，主要是通过Cdx2和PPAR-α基因调控。Ala-Gln能上调小肽转运载体表达，且能上调PI3K/AKT传导信号中关键基因的表达。二肽与氨基酸混合培养可促进乳κ-酪蛋白和蛋白合成相关基因转录。Met、Val和Leu可促进BMEC对Met-Met、Val-Met和Leu-Met二肽的转运。BIECs细胞中PepT1 mRNA的表达量极显著高于PepT2、PHT1和PHT2。PepT1蛋白的表达量最高，极显著高于PepT2和PHT2。犊牛饲喂精粗比75:25的饲粮更能促进其肠道PepT1、PepT2、PHT1和PHT2 mRNA的表达。研究结果为深入探讨奶牛小肽转运吸收与调控利用提供理论依据。