【作者】 朱莉萍；

【导师】 王政昆；

【作者基本信息】 云南师范大学 ， 环境科学， 2003， 硕士

【摘要】 本论文从分子水平对中缅树鼩的冷适应性产热进行研究。中缅树鼩是分布于我国南方的典型热带、亚热带小型哺乳动物。本实验室的工作主要涉及到UCP1蛋白质及基因序列的研究。 本论文主要内容有三个部分： 1、中缅树鼩褐色脂肪组织解偶联蛋白质-1的纯化。 2、中缅树鼩UCP1免疫学分析 3、中缅树鼩UCP1高效液相色谱分析 主要研究结果和结论如下： 1、在lin等人纯化UCP1的方法基础，利用SDS银染法成功的分离出了UCP1。中缅树鼩UCP1分子量为33.142kDa。大约由306个氨基酸残基组成。与其它动物体内的UCP1相比较分子量相近。冷驯化后，中缅树鼩UCP1含量、线粒体总蛋白质含量及褐色脂肪组织重量显著增加。所以，冷驯化是影响哺乳动物UCP1含量变化的有效因子。 2、我们酶联免疫实验结果表明UCP1浓度与冷驯化时间呈正相关。我们选择的时间梯度为0、0.1、0.8、1、7、14、21、28天。UCP1在前三天内增加不明显。在七天以后显著增加。21到28天增加开始平缓。28天以后，UCP1趋于稳定状态。同时实验室结果还证明了UCP1与兔抗中缅树鼩抗血清为单位位点结合，而抗血清浓度在1000～10000倍检测效果最佳。所以，UCP1具有组织特异性。仅存在于BAT中，是中缅树鼩适应性产热的分子机制所在。 3、高效液相色谱图谱表明，我们所提纯的蛋白质溶液中含有两种成分。这两种成分分子量接近、吸收峰都在218和271左右。而UCP1和UCP3的氨基酸序列有71％的同源性，分子量接近，且都是线粒体内膜上的载体蛋白。虽然两者都可以产生质子泄露，但是机制不同。UCP1是通过氧化磷酸化与呼吸链解偶联。UCP3仅是打开质子通道，影响基础代谢。所以其含量较低。在冷驯化过程中，UCPI含量增加为线粒体总蛋白质增加的主要原因。 以上的结果及结论表明:在持续冷胁迫条件下，中缅树的主要依赖于其褐色脂肪组织中的UCPI解偶联活动来产生热量维持体温。UCP3虽然分布于B八T和骨骼肌中，但是，对于非颇抖性产热并不产生影响。与基础代谢率有关的UCPZ分布与身体的各个部分，具有调节局部温度的功能。更多还原

【Abstract】 The article is about the cold adaptative characters of the UCP1 on the molecular level during the cold exposure . The Tree Shrew, which was selected as the experimental animal in our research, is he representative tropic and sub-tropic small mammal distributed in the south of China . Our main work is to purify the UCP1 and analysis its sequences.This thesis is consists of three parts:1. The purification of UCP-l(Uncoupling protein -1) from BAT(brown adipose tissue) of the tree shrew.2. The preparation of antisersum of rabbit anti -UCP1 from brown adipose tissue in the tree shrew and enzyme linked immunosorbent assays.3. Analysis by the mean of high performance liquit chromatography .(HPLC). The main results and conclusions are show as follow:1. On the basis of the method of purification for UCP-1 in rodent by Lin et al. The UCP1 from adipose tissue in tree shrew was isolated and purified successfully. The MW of UCP-1 was about 33.142kDa, which is consisted of 306 amino acids. We measured the physical effect of UCP-1 of the BAT under the coldexposure. We found it increased abviously about 7 days cold acclimation. And after 28 day in the cold acclimation (4 ) , the content of the UCP-1 in the mitochondrion, the weight of the BAT and the total content of the protein in the mitochondrion began to smooth. So we conclude that cold exposure is an efficient factor which affect the content of UCP-1 in small mammal animal.2. The antiserum of rabbit anti-UCP1 from brown adipose tissue of tree shrew was prepared successfully . And the enzyme linked immunsorbent assays was established too. In our experiment, we select 0, 0.1, 0.8, 1- 3, 7, 14, 21, 28 days as our gradient by this means . We found the content of the UCP1 and the time of the cold exposure have the positive relation . The content of UCP1 keep stability after 7 days . And between 21 days and 28 days, it was liable to smooth. At the same time , we can conclude from our result: UCP1 and the rabbit anti-UCPl of tree shrew have a single tide-site . The content of rabbit anti-UCPl was about 1000-1000, which was the better content. We know now , UCP1 only can be found in the BAT .UCP2 exist in the tissue all of the body. But UCP3 only exist in BAT and muscle.3. From the map of the HPLC(high performance liquit chromatography), we can know that the thing we get from experiment maybe has 2 kinds of component o The characteristic of them were similar. The have the absorb peak are similar , which was about 218 and 271. Otherwise , the sequences of amino acids of UCP1 and UCP3 have the same sequences about 71 % , and also have the similar MW. UCP1 and UCP3 both the inner protein-carriers in the mitochondrion . Though they both can do the carry work of H+ , but the mechanism are different, UCP1 can uncouple the correlation between theoxidation phophorylation and the respiratory chain or the flux of H+. But, UCP3 only form a protein transporter,which only affect the base metabolism . So in the cold acclimated time, the content of UCP1 increased, but not UCP3. Which is the molecular mechanism of the adaptive thermogenesis.Conclusion:In the course of cold acclimation, the main way of the tree shrew to keep the temperature is based on the UCP1(Uncoupling Protein-1) of the BAT. Although, UCP3 exist in BAT, but is has no the same function , UCP3 also exist in muscle . It has the effect on the base metablism. UCP2 has the extensive display, which maybe regulate the temperature of the special parts.更多还原