鲤鱼肠道Sglt1表达、结构与转运活性研究

Na+/glucose cotransporter (Sglt1) is a glycosylated integral brush-border membrane protein which is abundantly expressed in small intestine while at a lower level in kidney. The major function of Sglt1 is to accumulate glucose in intestinal or kidney epithelial cells with adverse concentration gradient and is served as the principal uptake pathway for glucose derived from diet. The expression level and transport activity of Sglt1 are considered as the decisive factor on the glucose metabolism of the body. In this study, based on the similar foreign and domestic researches and with the nutrition, immunology, histochemistry, cell biology and molecular biology technology, protein transport activity and mRNA and carrier protein expression of Sglt1 in Cyprinus carpio intestine will be determined by using BBMV, Real-time PCR, Western blot and ELISA methods, and then the circadian rhythm of them will be probed into with different time within 3 days. The relationship of mRNA and carrier protein expression between Sglt1 and Glut2 (glucose transporter 2) will be observed supplemented with siRNA and Sglt1 antibody，respectively. The regulation on the expression and transport activities of Sglt1 will be carried out with adding the different sodium concentration and glucose contents to the control diet. The carrier protein structure of Sglt1 will be obtained by using the atomic force micro-probe (AFM-Tip). In conclusion, the results of the study could partly reveal the glycometabolism of the fish on molecular level, enrich the nutrition theory of aquatic animal and promote the scientific use of glucose in the aquatic animal feeds.

钠/葡萄糖共转运载体(Na+/glucose cotransporter，Sglt1)是葡萄糖跨膜转运的重要载体蛋白，其表达水平及转运活性是机体葡萄糖代谢的决定因素；Glut2是位于上皮细胞基底膜上的易化扩散载体，Sglt1与Glut2协同，构成了葡萄糖代谢的主要途径。本项目在前期研究工作的基础上，参考国内外同类研究成果，以鲤鱼为实验对象，综合采用营养学、免疫学、组织化学、细胞生物学、分子生物学等实验技术，从基因转录、翻译和蛋白水平研究鲤鱼Sglt1 表达、结构、转运活性及Sglt1-Glut2的表达关联，阐明鲤鱼肠道Sglt1表达及其转运活性的昼夜节律，探讨日粮中Na+浓度、葡萄糖浓度对Sglt1表达及转运活性的调控规律。项目的研究结果能够部分揭示鱼类糖代谢的分子机理，丰富水产动物营养学理论，促进糖类在水产动物饲料中的科学使用。

鱼类对糖的需求和利用能力普遍较低。鱼类营养学研究领域的一个重要课题便是如何提高其对糖的利用能力。日粮中的葡萄糖通过肠上皮细胞进行吸收和转运，该过程可分为两个步骤：首先，葡萄糖由顶端膜Sglt1主动运输转运进入细胞内；然后，通过基底外侧膜上的易化扩散载体Glut2转运进入血液。Sglt1与 Glut2 的协同作用，构成了肠道葡萄糖吸收转运的主要途径。本研究取得的主要成果：（1）鲤肠道Sglt1和Glut2的mRNA和蛋白水平、BBMV葡萄糖转运活性呈现出夜间低白天高的特点，且在开始摄食时到达峰值；（2）首次发现了Sglt1和Glut2表达、BBMV转运活性在鲤肠道的分布规律，即前肠和中肠高于后肠，表明前肠和中肠是鲤肠葡萄糖吸收的主要部位；（3）通过饲养实验，首次证明日粮中糖和盐水平的升高，能够通过上调鲤肠道Sglt1的和Glut2的表达，增强鲤BBMV转运活性，促进了肠道葡萄糖的吸收转运；（4）日粮中含有不同浓度氯化钠和葡萄糖时，sglt1和glut2 mRNA在鲤前、中、后肠中的表达，均具有极强的正相关性；（5）饲料中糖水平的升高，可显著增加鲤肠绒毛的高度，有利于进一步增强肠道葡萄糖的吸收；（6）随着饲料中盐水平的升高，鲤肠绒毛的高度逐渐缩小，这可能是鲤在高盐饲料条件下产生的适应性变化，以减少肠上皮中Na+通道的数量。此外，我们还建立了鲤BBMV制备技术、BBMV荧光法检测鱼类肠道Sglt1转运活性、以及鲤肠上皮细胞分离等相关实验技术或模型，能够较好地用于鱼类肠道葡萄糖吸收、糖代谢等相关领域的研究。本课题的研究结果部分揭示了鱼类糖代谢的分子机理，丰富了水产动物营养学理论，为促进糖类在水产动物饲料中的科学使用奠定了理论基础。