摘 要mTOR/S6K1是调控乳蛋白合成的主要信号通路。有研究证实mTOR主要通过氨基酸调控翻译水平，但是mTOR接收氨基酸的机制还不是很清楚。我们推测T1R1/T1R3二聚体通过对mTOR信号通路的调控作用从而指导乳蛋白翻译。本实验用T1R3抑制剂（Lactisole）对小鼠乳腺外植体进行处理,从中培养并提取出蛋白质,然后通过Western blot方法以检测抑制剂对小鼠乳蛋白合成的影响。本实验首先采集小鼠乳腺外植体，并从处理过的小鼠乳腺中提取蛋白质，通过Western blot方法将蛋白质条带转移到PVDF膜上。采用化学发光法进行显影,对显影结果中不同处理的蛋白质条带进行对比分析。对比分析后可观察到Lactisole对mTOR信号通路和S6产生了抑制作用，初步揭示Lactisole对蛋白的合成有影响，能初步验证T1R3通过mTOR信号通路调控乳蛋白合成机制。本次通过对小鼠外植体泌乳机制的实验研究，可以为接下来进一步研究牛泌乳机制提供理论和实践指导。69166

毕业论文关键词：小鼠乳腺  T1R3  泌乳机制  mTOR  Lactisole

The Influence of mTOR Signaling Pathways In Mice Mammary Gland Explant After Using Lactisole

Abstract

The mTOR/S6K1 signaling pathway is the primary regulator of milk protein synthesis. While mTOR is known to be regulated at the translational level by amino acids, the mechanism by which mTOR accepts the amino acid signal is not yet clear. We hypothesized that T1R1 / T1R3 dimers guiding the milk protein translation through the regulation of mTOR signaling pathways. The purpose of this experiment is to treat breast gland explant from mice with T1R3 inhibitors (Lactisole), to develop and extract protein from them and then using Western blot method to test the effects of inhibitor on the mice mammary mechanism. Breast gland explant were first collected from mice, and then extract protein from the treated gland explant, to transfer protein bands on PVDF membrane with Western blot method. We analyze the different protein bands which were treated by the multicolor fluorescence enhancement. After analysis, we can make a preliminary judgment that inhibitors have an impact on the mTOR/S6K1 signaling pathway. It can explain that Lactisole affect the protein synthesis. It can also verify that T1R3 can regulate the milk protein synthesis through mTOR signaling pathways. This experiment based on the research of the mouse mammary mechanism. It can be the next further research guidance for further study of bovine mammary mechanism. It can make guidance for the further research and make bedding for further study of bovine mammary mechanism.

Key words: Breast in mice  T1R3  Mechanism of lactation  mTOR  Lactisole 

目 录

摘 要 I

Abstract II

目 录 III

1引言 1

2材料与方法 2

2.1材料 2

2.2方法 3

3 结果与分析 5

3.1 BCA定量结果 5

3.2 显影图分析 5

4 讨论 7

5 结论 8

参考文献 10

致 谢 12

1引言

随着科学技术的发展，人们为了满足自身的需要，每天都要摄入大量的乳制品，而乳制品中含有大量乳蛋白。具有高营养蛋白质的乳蛋白，可以为人类和新生动物提供高品质的蛋白质和氨基酸。此外，具有特定生理功能的一些活性肽类物质是由某些乳蛋白组分及乳蛋白消化产生的。因此人们对乳制品的需求量越来越大，对乳制品的质量要求也越来越高。现在的乳制品大都是牛奶制品，但是现在的奶牛由于易患病或者产奶质量低、产量少等原因已不能满足人们对于乳制品的需求，因此，提高奶用动物乳蛋白的产量、改善乳蛋白的质量是动物营养学家和动物育种学家迫切需要解决的问题。而利用细胞培养技术、分子生物学技术、基因工程等其它技术对奶牛泌乳机制进行改造就是关键所在。但是,关于奶牛泌乳调控机制的研究还不是很完善，而科学家们对小鼠信号通路生物研究较多，因此通过研究小鼠乳腺外植体泌乳调控机制可为研究牛乳腺外植体泌乳调控机制提供理论和技术指导，对于进一步研究奶牛乳腺mTOR信号通路有着重要的指导意义。通过对mTOR信号通路功能进行深入研究，进一步了解细胞的生命活动规律，为获得更优质的乳制品提供技术指导与理论依据。