ETEC诱导的小鼠腹泻模型中小肠水通道蛋白表达水平的检测

发布时间：2018-06-10 19:17

本文选题：水通道蛋白 + 小肠　；参考：《吉林农业大学》2017年硕士论文

【摘要】：水通道蛋白(Aquaporin,AQP)是广泛存在于原核和真核细胞膜上快速转运水分子的特异性孔道蛋白,目前已经从哺乳动物中克隆出13种水通道蛋白(AQP0-AQP12)。研究表明水通道蛋白在机体体液转运生理及病理过程(包括尿浓缩、外分泌腺功能、眼球房水代谢、脑水肿形成以及脑脊液分泌和吸收)中发挥重要作用。肠道作为动物体内仅次于肾脏的第二大水盐代谢器官,水通道的表达和功能的研究一直是该领域国际研究的前沿和热点。然而,截至目前对于肠道中水通道的研究主要集中在人和大鼠结肠,而关于小鼠肠道,特别是小肠各段水通道蛋白的表达和功能的研究却鲜有报道。本研究首先应用PCR和免疫印迹技术检测正常小鼠十二指肠、空肠和回肠中水通道蛋白的表达情况。结果显示AQP3在小鼠空肠中存在基因和蛋白表达,AQP4在小鼠回肠中存在基因和蛋白表达。为进一步明确这两种水通道蛋白在小肠中的表达位置,我们通过免疫组织化学的方法证实,AQP3主要表达在小鼠空肠黏膜上皮细胞,而AQP4主要表达在小鼠回肠隐窝细胞基底膜。产肠毒素型大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起人和动物,特别是低龄动物腹泻的主要病原菌之一。目前对于ETEC的致病机理的研究主要集中在其产生的两种肠毒素(LT和ST型)通过影响小肠上皮细胞CFTR的功能,加强小肠对离子的分泌而导致腹泻。但是,ETEC对于小肠上皮细胞水转运功能的影响,目前还是研究的盲点。本研究通过建立ETEC诱导的小鼠腹泻模型,应用RT-PCR、qPCR、ELISA等实验方法检测了感染ETEC产生腹泻后小鼠AQPs基因和蛋白表达量的变化。结果显示,小鼠感染ETEC导致腹泻后,空肠中AQP3和回肠中AQP4的基因及蛋白表达量与正常对照组相比都明显下降。因此我们推测,ETEC附着小肠后,可能通过释放肠毒素使AQP3和AQP4表达量降低,影响小肠上皮细胞的水转运功能,从而引起腹泻。
[Abstract]:Aquaporin aqup is a special pore protein which widely transports water molecules on prokaryotic and eukaryotic cell membranes. At present, 13 aquaporin (AQP0-AQP12) proteins have been cloned from mammals. Studies have shown that aquaporins play an important role in the physiological and pathological processes of body fluid transport, including urine concentration, exocrine gland function, aqueous humor metabolism, cerebral edema formation, and cerebrospinal fluid secretion and absorption. Intestinal tract is the second largest water-salt metabolic organ in animals after kidney. The research on the expression and function of water channel has been the frontier and hotspot in this field. However, up to now, the study of the water channel in the intestinal tract is mainly focused on human and rat colon, but there are few reports on the expression and function of aquaporin in the intestine of mice, especially in the small intestine. In this study, the expression of aquaporin in duodenum, jejunum and ileum of normal mice was detected by PCR and Western blot. The results showed that there was gene and protein expression of AQP3 in the jejunum of mice. AQP4 expressed genes and proteins in mouse ileum. In order to further clarify the expression of these two aquaporins in the small intestine, we confirmed by immunohistochemical method that AQP3 was mainly expressed in mouse jejunal mucosal epithelial cells, and AQP4 was mainly expressed in the basement membrane of mouse ileum crypt cells. Enterotoxigenic Escherichia coli (ETEC) is one of the main pathogens causing diarrhea in humans and animals, especially in young animals. At present, the pathogenesis of ETEC is mainly focused on the two enterotoxins (LT and St type), which affect the function of CFTR in intestinal epithelial cells and enhance the secretion of ions in the small intestine and lead to diarrhea. However, the effect of ETEC on the water transport function of intestinal epithelial cells is still a blind spot. In this study, the mouse diarrhea model induced by ETEC was established, and the changes of AQPs gene and protein expression in mice with diarrhea induced by ETEC were detected by RT-PCR- qPCR- Elisa. The results showed that the gene and protein expression of AQP3 in jejunum and AQP4 in ileum were significantly decreased in mice infected with ETEC. Therefore, we speculate that the expression of AQP3 and AQP4 may be decreased by releasing enterotoxin, which may affect the water transport function of intestinal epithelial cells and cause diarrhea.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S856.4

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