磷在肉仔鸡小肠中的吸收规律及其分子机制

In the present project, series of trials half in vivo with in situ ligated small intestinal loops of broilers, in vivo with naturally feeding intact broilers,and in vitro with primarily cultured small intestinal cells of broilers in combination with the gene silence technique, will be conducted to investigate the kinetics of phosphorus (P) absorption in ligated duodenal,jejunal and ileal segments and primarily cultured small intestinal cells, and the effect of P treatment on plasma P contents from the hepatic portal vein of intact broilers, P absorptions in normal and typeⅡb sodium-phosphate cotransporter (NaP-Ⅱb),inorganic phosphate transporter 1 (PiT-1),inorganic phosphate transporter 2 (PiT-2) expression-silenced small intestinal cells in the primary culturre,and mRNA and protein expresstion levels of NaP-Ⅱb,PiT-1 and PiT-2 in ligated duodenal,jejunal and ileal segments as well as in these small intestinal segments of intact broilers, and normal and these transporters expression-silenced small intestinal cells in the primary culture, so as to systemicly and deeply elucidate absorption traits of P in the small intestine of broilers and its molecular mechanisms. The outcomes from this project will be of great theoretical and practical significances for how to improve dietary P utilization efficiency,save P resource and minimize the environmental pollution of P excreted by broilers in the broiler production.

本项目拟通过肉仔鸡半体内原位结扎灌注小肠段试验、全体内自然饲喂试验和体外原代培养小肠细胞并结合基因沉默技术试验，研究磷在肉仔鸡结扎十二指肠、空肠、回肠段和原代培养小肠细胞中吸收的动力学模式及磷对自然饲喂肉仔鸡肝门静脉血浆中磷含量、正常和钠磷协同转运载体Ⅱb型(NaP-Ⅱb)、无机磷转运载体1(PiT-1)、无机磷转运载体2(PiT-2)表达分别沉默的原代培养小肠细胞中的磷转运吸收与结扎十二指肠、空肠、回肠段、自然饲喂肉仔鸡十二指肠、空肠、回肠、正常及上述转运载体表达分别沉默的原代培养小肠细胞中NaP-Ⅱb、PiT-1、PiT-1 mRNA及其翻译蛋白表达水平的影响，从而系统、深入揭示磷在肉仔鸡小肠中的吸收规律及其分子机制，丰富和发展肉鸡磷营养的现有理论和知识，对于我国肉鸡生产中如何提高其饲粮磷利用效率，节约磷资源以及减少磷排放对环境的污染，都具有十分重要的理论与现实指导意义。

本项目通过肉仔鸡半体内原位结扎灌注小肠段试验、全体内自然饲喂试验和体外原代培养肉鸡鸡胚十二指肠上皮细胞并结合基因沉默技术试验，研究了磷在肉仔鸡小肠中的吸收规律及其分子机制，取得了国内、外文献中均未见报道的创新性研究新成果：(1)发现十二指肠是肉仔鸡小肠中磷吸收的主要部位，空肠和回肠次之，且磷在十二指肠中的吸收以饱和载体转运为主，而在空肠和回肠中的吸收则主要是非饱和扩散方式；(2)成功构建了原代培养肉鸡鸡胚十二指肠上皮细胞的物质吸收模型，即在细胞接种密度为3×10^6个/mL和培养时间为48 h时，细胞活力及其紧密连接度均达最佳，可用于物质吸收研究；(3)揭示了磷在肉仔鸡小肠中吸收的分子机制，即适宜水平非植酸磷可通过上调肉仔鸡十二指肠钠磷协同转运载体IIb型(NaP-IIb)和无机磷转运载体2(PiT-2)的蛋白表达水平，下调十二指肠无机磷转运载体1(PiT-1)的蛋白表达水平，进而促进磷的吸收；siRNA特异性沉默原代培养肉鸡鸡胚十二指肠上皮细胞的NaP-IIb基因表达后，降低了磷在十二指肠上皮细胞的吸收，表明NaP-IIb可能直接参与了磷在肉仔鸡小肠中的转运吸收。以上研究新成果揭示了磷在肉仔鸡小肠中的主要吸收部位、方式及其分子机制，丰富和发展了肉鸡磷营养的现有理论和知识，对于我国肉鸡生产中如何提高其饲粮磷利用效率，节约磷资源以及减少磷排放对环境的污染，都具有十分重要的理论与现实指导意义。