【作者】 于义娟；

【导师】 郭定宗；

【作者基本信息】 华中农业大学 ， 临床兽医学， 2011， 硕士

【摘要】 斯钙素-1(STC-1)是一种最早在硬骨鱼特有的斯坦尼氏小体发现的多肽类激素,后来在人和多种哺乳动物体内也发现STC-1的基因表达并有一些蛋白定位的报道。STC-1参与钙磷代谢,抑制钙的吸收和促进磷酸盐的吸收、重吸收,抑制磷酸盐的排泄。在分子和组织水平探索STC-1在奶牛体内的表达,对研究奶牛钙磷代谢的机理具有重要的意义,同时为探索防治奶牛钙磷代谢病提供新的思路。为从基因水平检测在中华荷斯坦奶牛肾脏和十二指肠中是否有STC-1基因的表达,本试验选择1日龄中华荷斯坦犊牛,通过提取试验奶牛肾脏和十二指肠组织的总RNA,建立PCR反应体系,克隆STC-1 cDNA的部分序列。结果显示,STC-1基因在中华荷斯坦奶牛肾脏和十二指肠组织中有表达。将所克隆的STC-1 cDNA序列连接T载体、转化、测序。同源分析显示与Bos taurus的STC-1基因相应序列的核苷酸序列同源性达到100%。在钙磷代谢过程中,十二指肠是钙和磷的最重要的吸收部位,而肾脏是重要的重吸收和排泄的器官。为探索STC-1蛋白在奶牛钙磷代谢中的生理作用,本试验运用免疫组化技术对STC-1蛋白在1日龄中华荷斯坦犊牛肾脏和十二指肠的分布进行检测,结果显示在肾脏的近曲小管和远曲小管上皮细胞有明显的棕黄色阳性染色,主要集中在上皮细胞的胞浆内,十二指肠的肠腺和杯状细胞也呈现出明显的棕黄色；运用原位杂交技术对STC-1 mRNA在肾脏和十二指肠进行检测,结果显示在奶牛肾脏的近端小管和皮质升支粗段有明显的杂交信号,在远端小管有微弱的杂交信号。在肾小球区域则见不到棕黄色的染色。在十二指肠的杯状细胞边缘和肠腺有明显的杂交信号。本实验首次证实了中华荷斯坦奶牛肾脏和十二指肠中有STC-1基因的表达,首次在中华荷斯坦奶牛肾脏和十二指肠组织中进行了STC-1蛋白和mRNA的定位研究。本试验为进一步研究STC-1的基因结构、在不同组织中的表达及表达模式提供了新的材料,并为研究STC-1的生理作用、探索预防和治疗奶牛钙磷代谢病提供新的资料。更多还原

【Abstract】 Stanniocalcin-1 (STC-1) is a peptide hormones that was firstly idendified in the bony fish specific corpuscle of stannius(CS). Later, it was reported that STC gene express in human and a variety of mammals and had some protein and mRNA distribution reports. STC-1 participates in calcium and phosphorus metabolism. STC-1 inhibits the absorption of calcium, promotes the absorption of phosphate, inhibits the excretion of phosphate. The structure and function of muscle and blood vessel formation and reproductive process were also affected by STC-1. In dairy cows, exploring STC-1 expression in the horizontal of molecular and tissue level is significant to study the mechanism of calcium and phosphorus metabolism. On the other hand, it may provide a new way to explore prevention and control of calcium and phosphorus metabolism disease.To test STC-1 expression or not, and distribution in kidney and duodenum of Chinese Holstein cows from the gene level, the total RNA of kidney and duodenum of experimenting one day old dairy cows was extracted, the purpose cDNA was synthesized through the established PCR reaction system. The result showed that, STC-1 gene expressed in kidney and duodenal of the Chinese Holstein cows. The cloned purpose cDNA was connected to T vector, and was transformed and sequenced. Sequence analysis showed that, the partial sequence of STC-1 gene nucleotide sequence homology is 100% corresponding with Bos taurus.In calcium and phosphorus metabolism, the duodenum was the most important calcium and phosphorus absorption site, and kidney was important organ of re-absorption and excretion. To explore the physiological role of STC-1 protein in diary cow’s calcium and phosphorus metabolism, STC-1 protein was tested in the kidney and duodenum of one day old Chinese Holstein cows. The results of immunohistochemistry showed that the epithelial cells of kidney proximal tubule and distal tubule have significant brown staining, mainly in the cytoplasm of epithelial cells. And glands and duodenal goblet cells of the intestinal showed clear brown staining. STC-1 mRNA was tested in the kidney and duodenum by in situ hybridization. The results showed that, the epithelial cells in kidney proximal tubule, cortical thick ascending limb have conspicuous hybridization signals, and with weak hybridization signals in the distal tubule. And no positive brown staining was showed in the glomerular regions. In the duodenum, it is significant hybridization signal in the edge of goblet cells and intestinal glands. In this experiment, it was the first time prove that STC-1 gene expressed in kidney and duodenum of the Chinese Holstein cows, and also was the first localization of STC-1 protein and mRNA in the kidney and duodenal tissues of Chinese Holstein cows. The results provided new materials for further studying of STC-1 gene structure, expression in different tissues and expression patterns, created the conditions for researching the physiological role of STC-1, and provided new informations for exploring prevention and treatments of calcium and phosphorus metabolic diseases of dairy cows.更多还原