【作者】 林琳；

【导师】 林叶；

【作者基本信息】 东北农业大学 ， 生物化学与分子生物学， 2015， 硕士

【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。更多还原

【Abstract】 Dairy cows are one of the most important economic animal, the milk production and milk quality is the important to economic benefits. China is the superpower dairy farming of the world, but the average annual yield of cows is lower than the world average. The rate of butterfat and milk protein content is relatively low, and it will bring serious influence for the consumption demand of consumers. Milk is synthetic and secretion by the mammary gland itself. Every mammary bubble is formed by a layer of secretory epithelial cells. These highly specialized bubble epithelial cells are the basic unit of lactation. The mammary epithelial cells will keep proliferation until early lactation, and the number of mammary epithelial cell is very important for milk yield. The expression of genes involved lactation is important to mammary gland development and lactation. Therefore, research on genes involved mammary gland development and lactation has a positive role to improve dairy cattle genetic performance and promote the development of dairy industry in China.Spleen tyrosine kinase(SYK) is a non-receptor type protein tyrosine kinase, which expresses in hematopoietic cells, fibroblasts cells, epithelial cells, liver cells, endothelial cells and mammary glands. SYK is the most important kinase in B cell signal transduction and T cell signal transduction. It play a key role in cell differentiation and proliferation. There is some research about SYK on breast cancer, but not on normal mammary gland. In order to make clear the relationship between the expression of SYK and mammary gland development and lactation, lactation dairy cow mammary gland with high or low quality milk and dry period dairy cow mammary gland were used in this research. To explore the expression and localization of SYK in dairy cow mammary gland, western blotting and laser confocal microscope were used. To research the influence of SYK on mammary epithelial cell proliferation, RNA interference and gene overexpression technology was used. In the end, the regulation mechanism of SYK on mammary epithelial cells proliferation was detected by dual luciferase reporter gene system. The results showed that SYK expression in dry period mammary gland was significant higher than lactating mammary gland. SYK was mainly located in the cytoplasm of ductal epithelial cells in dry period mammary gland. In lactating mammary gland, SYK was existed in acinar epithelial cells. When SYK was silence in mammary epithelial cells, cell proliferation decreased, as well as the expression of β-casein and signaling molecules involved in proliferation, such as AKT1 and MAPK. When SYK was overexpression, mammary epithelial cells proliferation increased. In the meanwhile, the expression of β-casein, AKT1 and MAPK increased significantly.Dual luciferase reporter gene system results showed that SYK activated AKT1 promoter activity in dairy cow mammary epithelial cells, and the activitysite is located in the AKT1 promoter region of-439~+83bp. Taken together, all these results revealed that SYK promoted mammary gland epithelial cells proliferation and differentiation. SYK gene promoted dairy cow mammary epithelial cell proliferation by regulating the transcription activity of AKT1, which was important to mammary gland reconstruction in dry period. This research provide the theoretical basis to improve the yield and quality of milk. It is important to milk industry development in China.更多还原