SYK基因对奶牛乳腺上皮细胞增殖的影响研究

发布时间：2018-06-27 06:53

本文选题：奶牛 + 乳腺　；参考：《东北农业大学》2015年硕士论文

【摘要】：牛是畜牧业中最重要的经济动物,牛奶的产量和质量是衡量畜牧业水平和经济效益的一个重要标准。我国虽是世界奶牛养殖大国,但奶牛的平均单产世界的平均水平低。牛奶中的乳脂和乳蛋白的含量相对较低,对消费者的消费需求产生严重影响。乳汁是由乳腺腺泡的分泌细胞产生的。每个乳腺腺泡都由一层分泌上皮细胞构成,这些高度特化的腺泡上皮细胞是奶牛乳腺泌乳的基本单位。腺泡上皮细胞一直持续增殖直达泌乳早期,乳腺分泌乳汁的能力由腺泡上皮细胞的数量决定。奶牛乳腺组织的发育及产生乳汁的过程当中,一些重要基因的表达变化是调节腺泡上皮细胞分化、增殖以及乳汁合成、分泌、转运等生理过程的基础。因此,筛选对奶牛乳腺组织的发育和产生乳汁有调节作用的基因以及鉴定这些基因的功能对提高奶牛乳腺的泌乳能力、促进我国乳品业的发展有重要作用。脾源性酪氨酸激酶(spleen tyrosine kinase,SYK)是一种非受体型的蛋白酪氨酸激酶,在造血细胞、成纤维细胞、上皮细胞、肝细胞、血管内皮细胞以及乳腺组织中都有所表达。SYK是一些细胞信号传导途径中关键的激酶,主要有调节细胞分化和增殖等作用。对于SYK基因的研究在乳腺癌案例中较多,但在正常的乳腺中研究较少。为探讨SYK基因的表达与奶牛乳腺发育和泌乳功能之间的关系,本实验选取泌乳期高乳品质、泌乳期低乳品质和干乳期的中国荷斯坦奶牛,采用蛋白免疫印迹法和激光共聚焦显微技术研究SYK基因在不同生理状态和泌乳性状的奶牛乳腺中的表达含量与表达部位变化;采用基因干扰和过表达技术研究SYK基因对奶牛乳腺上皮细胞增殖以及增殖相关信号分子表达的影响;采用双荧光素酶报告基因系统研究SYK基因调节奶牛乳腺上皮细胞增殖的分子机制。Western blotting和激光共聚焦结果显示干乳期奶牛乳腺组织中SYK的表达显著高于泌乳期的奶牛乳腺组织。在干乳期的荷斯坦奶牛乳腺中,SYK主要在乳腺导管上皮细胞的胞质中表达。而在泌乳期,SYK主要在奶牛乳腺腺泡上皮细胞的胞质中表达。基因干扰和过表达结果显示SYK表达降低时,奶牛乳腺上皮细胞的增殖能力下降,β-酪蛋白的表达量降低,同时与细胞增殖相关信号分子AKT1和MAPK等蛋白表达量也相对降低;而SYK过表达时,细胞增殖增加、β-酪蛋白、AKT1和MAPK的蛋白表达量上调。双荧光素酶报告基因系统研究结果显示奶牛乳腺上皮细胞中SYK可以激活AKT1启动子的活性,其作用位点位于AKT1启动子区-439~+83bp的序列范围。这些结果表明SYK是奶牛乳腺上皮细胞增殖与分化的调节因子,SYK基因通过调节AKT1的转录活性促进奶牛乳腺上皮细胞增殖,为干乳期乳腺组织重建做准备。这项研究结果可以丰富泌乳生物学理论,为提高牛奶产量和质量提供一定的理论依据,对促进我国乳产业的进一步发展具有重要的理论意义和生产实际意义。
[Abstract]:Cattle are the most important economic animals in animal husbandry. The yield and quality of milk are an important standard to measure the level of animal husbandry and economic benefits. Although China is a big country in the world, the average level of the average yield of dairy cows is low. The content of milk fat and milk protein is relatively low, and the consumption demand of consumers is produced. "Milk is produced by the secretory cells of the mammary glands. Each mammary gland is made up of a layer of epithelial cells. These highly specialized acinar epithelial cells are the basic units of milk secretion in the cow's mammary glands. The acinar epithelial cells continue to proliferate directly to the early lactation, and the ability of the mammary glands to secrete milk from the acinar epithelial cells." Quantitative determination. During the development of mammary gland tissue and the process of milk production, the expression of some important genes is the basis for regulating the differentiation and proliferation of acinar epithelial cells, and the basis of physiological processes such as milk synthesis, secretion, transport and other physiological processes. Therefore, the screening of genes that regulate the development of mammary gland tissue and the regulation of milk production and identification of milk cow's mammary gland tissue and identification of this The function of some genes plays an important role in improving the lactating ability of dairy cows and promoting the development of dairy industry in China. Spleen tyrosine kinase (SYK) is a kind of non receptor protein tyrosine kinase, which is found in hematopoietic cells, fibroblasts, epithelial cells, hepatocytes, vascular endothelial cells, and mammary tissues. The expression of.SYK is the key kinase in some cell signaling pathways, which mainly regulate cell differentiation and proliferation. The study of SYK gene is more in breast cancer cases, but less in normal mammary gland. In order to explore the relationship between the expression of SYK gene and the mammary gland development and lactation function of dairy cows, this experiment selected secretion. Chinese Holstein cows with high milk quality, low lactation quality and dry milk stage were studied by protein immunoblotting and laser confocal microscopy to study the expression and expression of SYK gene in dairy cows with different physiological status and lactation traits, and SYK gene was used to study the milk of milk in the milk of dairy cows with different physiological States and lactation traits. The effect of the proliferation of bovine mammary epithelial cells and the expression of proliferation related signal molecules; the molecular mechanism of SYK gene regulating the proliferation of mammary epithelial cells by double luciferase reporter gene system.Western blotting and laser confocal results showed that the expression of SYK in the mammary tissues of dairy cows with dry milk period was significantly higher than that of lactating cows. SYK is mainly expressed in the cytoplasm of the mammary ductal epithelial cells during the dry milk period of Holstein cows. While in lactation, SYK is mainly expressed in the cytoplasm of the mammary gland epithelial cells of the dairy cows. The results of gene interference and overexpression show that the proliferation of milk gland epithelial cells in dairy cows decreased when the expression of SYK decreased, and the beta casein was reduced. At the same time, the expression of AKT1 and MAPK related to cell proliferation related signal molecules also decreased; while SYK overexpression, the proliferation of cells increased, the protein expression of beta casein, AKT1 and MAPK was up-regulated. The results of the double luciferase reporter gene system study showed that SYK could activate the AKT1 promoter in milk bovine mammary epithelial cells. The activity, its site of action, is located in the range of -439~+83bp in the promoter region of AKT1. These results suggest that SYK is a regulator of the proliferation and differentiation of mammary epithelial cells in dairy cows. The SYK gene promotes the proliferation of mammary epithelial cells in dairy cows by regulating the transcriptional activity of AKT1. The results of this study can enrich the secretion of mammary glands in the dry milk. The theory of milk biology provides a certain theoretical basis for improving the production and quality of milk. It is of great theoretical significance and practical significance to promote the further development of dairy industry in China.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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