【作者】 姚政；

【导师】 王政昆；

【作者基本信息】 云南师范大学 ， 环境科学， 2002， 硕士

【摘要】 哺乳动物在低温环境下的适应性产热及其调节机理研究是当前生理生态学研究领域中的热点问题之一，其机理已深入到分子水平上。与动物冷适应密切相关的解偶联蛋白（Uncoupling Protein，UCP）与动物对的能量的分配与利用，以及与人类肥胖症和非胰岛素依赖性糖尿病具有密切的关系。本论文以分布于我国南方的典型热带、亚热带小型哺乳动物，中缅树鼩（Tupaia belangeri）为研究对象，对其冷适应机制的进行了基因学初步研究，以深入探讨其冷适应产热机制，从而帮助解释动物的生存适应机制和进化对策，并为人类肥胖症和非胰岛素依赖性糖尿病的治疗提供一定的实验依据。 本论文的主要内容有四个部分：1．冷驯化条件下不同肾上腺能受体激动剂对中缅树鼩产热能力的影响；2．冷驯化对中缅树鼩线粒体呼吸的影响；3．冷驯化对中缅树鼩肝脏和BAT RNA含量的影响；4．中缅树鼩UCP1，UCP2的RT-PCR及UCP1基因测序。 具体的研究结果和结论如下： 1．与对照组（未冷驯化）相比较，冷驯化后中缅树鼩的RMR有所提高，β₃-NST（注射β₃-肾上腺素激动剂BEL37344）和NST（注射NE）均极显著增加。无论是未驯化还是冷驯化动物，在注射NE和BRL37344后其耗氧量均有极显著的增加。未冷驯化中缅树鼩的NST高于β₃-NST，但并不显著，冷驯化后，中缅树鼩NST与β3-NST相比较具有差异极显著。表明冷驯化可使中缅树鼩的产热能力显著增强，中缅树鼩采用的是通过增加RMR和NST来适应寒冷的对策，其BAT中可能存在有β₃-AR，并且β₃-AR并不是BAT中唯一的肾上腺能受体；NE可能通过多种肾上腺能途径来增加产热。NST主要是由BAT，肝脏和骨骼肌线粒体内膜上的UCPs所产生的，其中BAT所占的成分较大。肾上腺能受体通路在中缅树鼩适应性产热中起到了重要作用，肝脏线粒体质子泄露对决定静止代谢率的大小具有重要影响。 2．急性冷暴露导致中缅树鼩肝脏线粒体状态Ⅲ和状态Ⅳ呼吸显著增强。但与持续冷暴露的情况不同，短期急性冷暴露条件下中缅树鼩RCR和P/O与对照组相比较却显著增加。短期急性冷暴露条件下中缅树鼩肝脏线粒体状态Ⅳ呼吸的显著增加。这些结果表明中缅树鼩肝脏对冷适应产热的增加起着重要的作用，冷暴露情况下中缅树鼩肝脏线粒体中有明显的质子漏情况出现，UCP2确实与质子漏有密切关系。短期急性冷暴露条件下中缅树阈RCR和P／O的显著增加，这是否是由于短期急性冷暴露条件下动物体内的各种生理变化较为激烈，反映在线粒体水平上各项指标变动较大，呈现出不稳定，甚至是与持续冷暴露情况下相反的实验结果，还是因为在短期急性冷暴露导致动物体内出现的一系列生理变化需要大量ATP，从而出现氧化磷酸化作用的迅速加强，导致了RCR的迅速提高，尚不得而知，需要更多的实验证据证明。 3．中缅树嗣在冷驯化过程中体重显著增加，肝脏和BAT重量均呈先下降后上升的趋势，肝脏和BAT总RNA含量的变化趋势均呈逐渐上升的趋势，其中肝脏总NA含量开始增加的时间要比BAT总RNA提前，但BAT总KNA的总体增加幅度较肝脏的大。持续冷驯化条件下，中缅树酮肝脏和 BAT总 RNA含量均能维持在较高的水平，但出现缓慢下降的趋势。这些结果表明冷驯化能够诱导中缅树跑肝脏、BAT细胞增加和UCP基因表达活性显著增强，肝脏对急性冷暴露的反应要比BAT快，但BAT的产热能力强于肝脏。动物体内具有使 UCP mRNA在受冷刺激时爆发性上调，细胞中 UCP浓度迅速提高，一段时间后 UCP InRNA 或UCP 处于适当的生理需求水平的双重调节机制（Rehnlnark，1992）。 4．以大鼠 UC＊和 UCPZ基因为参照设计引物，采用中缅树酮 BAT总 RNA进行 UCP的 RT－PCR，肝脏总 RNA进行 UCPZ的 RT－PCR，均得到单一清晰的特异扩增带。分子量大小与理论预期①Cpl：483hp；UCpZ：1015hp)相符。我们克隆获得的中缅树酮UCP基因CDNA通过测序得到了450hp的序列，包含有五个完整的开放阅读框，主要涵盖了 UCP基因的外显子 2和 3的全部，以及外显子4的一部分。RT－PCR的成功首次证明了中缅树阈BAT中有UCPI存在，肝脏中有UCPZ存在，表明UCPS家族的保守序列在不同的物种中的序列相似度较高，同时也暗示着中缅树跑与大鼠在进化关系上可能比较接近。我们得到的中缅树酮 UCP基因 cDNA序列与其它一些动物的 UCPs基因序列相比较，具有较高的同源性，具有 UCPS基因所共有的 DNA序列和 UCP所特有的一些氨基酸残基，据此可判断该基因确实属于UCPS基因家族。 以上实验结果表明，低温是诱导中缅树酮的产热能力显著增强的环境因子，肝脏和 BAT是小型哺乳动物体内重要的产热器官。在冷暴露条件下，中缅树嗣从个体水平、细胞水平到分子水平都产生了一系列与冷适应产热有关的变化。这些变化都与位于线粒体内膜上的一种特殊产热蛋白——解偶联蛋白有密切的关系。更多还原

【Abstract】 The studies of the adaptive thermogenic capacity and mechanisms of small mammals in cold were one of the hottest issues in ecophysiology, and had gone deeply the molecular level. The uncoupling protein (UCP) played an important role in the adaptive thermogenesis of animals. And it also tightly associated with energy allocating and utilizing and human’s adiposity and NIDDM.Tree shrew (Tupaia belangeri), which was the representative tropic and sub-tropic small mammal distributed in the south of China, had studied on cold adaptive characters of genie level in cold stress as the experimental animal in our research. This study would help to probe into adaptive thermogenic mechanism, survival mechanism and evolution strategy of small mammals at low temperature. The study also offered the substantial basis to the cure of adiposity and diabetes which not tying on insulin.This thesis consisted of four parts: 1. The effects of different p-AR agonist selectivity on the thermogenic capacity of tree shrews; 2. The effects of cold exposure on mitochondrial respiration of liver in tree shrews; 3. The effects of cold exposure on total RNA in liver and BAT of tree shrews; 4. The RT-PCR of UCP 1 and UCP2 and the sequencing of UCP 1 of tree shrews.The detailed results and conclusions were as follow as:1. Compared with control (out of cold exposure), the RMR of tree shrews after cold exposure increased, the Pa-NST and NST increased mighty significantly. Whether out of or under cold exposure, the Ps-NST and NST both increased mighty significantly. The NST was higher than P3-NST in control, but not significant., The NST was significantly higher than P3-NST in cold exposure. These indicated that the thermogenic capacity of tree shrews could increase significantly after cold exposure. Tree shrews could adopt the strategy that increased thermogenic capacity to adapt the cold stress. There were Ps-AR in the BAT of tree shrews and the p3-AR was not the only AR in BAT. Perhaps, NE enhanced thermogenesis by P-AR approach. UCPs which lay in the mitochondrial inner membrane of EAT^ liver and skeletal6muscle brought on the most NST, and the BAT gave the most contributions among the all. The proton leak was very important to BMR. The AR approach played an important role in the adaptive thermogenesis of tree shrews.2. The state III and IV of nutochondrial respiration of liver in tree shrews were enhanced significantly in cold. Different with persistent cold exposure compared with control, the RCR and P/O increased significantly in acute cold exposure. The respiration of state IV also increased significantly in acute cold exposure. These facts suggested that the liver indeed played an important role in the adaptive thermogenesis of tree shrews. There were obvious proton leak in live mitochondrial in cold exposure and the proton leak nearly connected with UCP2. As for the reason of the RCR and P/O increased significantly in acute cold exposure, whether drastic physiologic changes induced big changes in mitochondrial level, so the experiment results became instability even opposition to that in persistent cold exposure, or oxidative phosphorylation came into being a plenty of ATP witch needed by the physiologic change and induced the increasing of RCR. Now all of these were not clear and needed more experiments to testify.3. During cold exposure, the body weight of tree shrews increased significantly and the mass of liver and BAT decreased firstly and increased subsequently. The total RNA contents of liver and BAT increased gradually. The time that the total RNA of liver began increasing was earlier than that of BAT, but the increasing range of total RNA content of BAT was bigger than that of liver. In persistent cold exposure. The total RNA contents of liver and BAT were at high levels, but fell slowly. All these results indicated that the cells of liver and BAT could add and the expression activity of UCPs would enhance significantly in cold exposure. It suggested that the liver of tree shrews responded more rapidly to cold e更多还原