【作者】 朱滕滕；

【导师】 张树义；

【作者基本信息】 华东师范大学 ， 生态学， 2014， 博士

【摘要】 哺乳动物的冬眠具有低代谢率以及低水分消耗等特征,并且跟长寿、种群低灭绝率、延迟发育等有密切关系；具有冬眠能力的哺乳动物还可以为缺血再灌注、缓冲低温损害、体温和心率的迅速恢复、抗肌肉萎缩和骨质疏松的相关研究提供动物模型,因此冬眠这一特殊生理现象一直都是学者研究的热点。本论文围绕着冬眠现象并以蝙蝠这一具有众多特殊特征的哺乳动物为模型展开研究：主要对实验室条件下非冬眠季节诱导蝙蝠进入冬眠、Leptin(瘦素)与冬眠的直接关联以及冬眠前蝙蝠存储的脂肪在其不能进入冬眠时的消耗变化等问题进行了深入研究。自然条件下哺乳动物大都在冬季进入冬眠,因此对冬眠样品的采集以及生理现象的观察记录只能在冬季进行,这在时间上限制了对冬眠课题的研究。在本论文中,我们利用人工气候箱模拟野外冬眠环境,分别于春季、夏季与冬季成功的诱导了八只大蹄蝠(Hipposideros armiger)进入冬眠状态,并实时记录了蝙蝠的体温和心率变化数据。数据显示,当环境温度为9℃时,蝙蝠的体温能从正常状态的380C左右降低到10℃,心率从活跃时期的最高水平600多次每分钟降低到了最低6次每分钟。此外,我们将冬眠季节和非冬眠季节诱导的蝙蝠分为两组进行统计方差分析(one-way ANO VA),数据没有显著差异性(F1=2.850,P=0.102)。这说明了实验室条件下的冬眠诱导技术科学可靠,而且不受季节限制,从而打破了对冬眠现象研究的时间限制。此前研究Leptin通过调节能量代谢、饮食摄入以及脂肪分解等方式对哺乳动物的异温性具有重要意义,且Leptin在冬眠物种上的进化速率要明显快于在非冬眠物种上的进化速率,冬眠物种合成的Leptin对脂肪的分解效率也显著高于非冬眠物种合成的,但是Leptin对于冬眠的具体调节机制尚不清楚。基于以上研究以及下丘脑在温度以及能量代谢调控上发挥的中枢作用,我们猜测答案可能存在于Leptin在下丘脑发挥作用的调节通路上。Leptin作用于下丘脑主要是通过其在下丘脑区域表达的唯一长型功能受体OB-R1来实现的。随后我们利用RNA干扰技术将anti-OB-Rl慢病毒活体微注射到大蹄蝠的下丘脑区域,实验结束后的原位杂交结果显示慢病毒介导的anti-OB-Rl shRNA成功敲减了下丘脑区域的OB-Rl表达,对主要功能区域弓状核、腹内侧核区域和背内侧核区域的敲减效率分别高达80%、86.6%和80.4%。通过实时记录冬眠诱导条件下蝙蝠的体温和心率等数据,结果表明Leptin通路被阻断的大蹄蝠不能进入冬眠状态。这直观地证明了Leptin是蝙蝠进入冬眠的必要条件,研究认为Leptin的存在可能为哺乳动物提供了一个“脂肪已存储”的冬眠信号。哺乳动物冬眠前期会存储大量的脂肪以作为其冬眠期间的主要能量来源,从而保证冬眠动物在恶劣的寒冬季节存活下来。我们在大蹄蝠和大足鼠耳蝠(Myotis ricketti)诱导冬眠的过程中进行不定期的干扰,阻止它们进入冬眠状态。在10℃或者15℃环境条件下暴露一周之后,活跃的大蹄蝠体内的白色脂肪发生了明显的棕样化,即脂肪形态出现了棕色脂肪类似的多室多状态,UCP1和PGC-1α的表达量明显升高,最高可达对照组的750倍左右；而“慵懒”的大足鼠耳蝠体内的白色脂肪却没有明显变化。此外,与传统模型的皮下白色脂肪(sWAT)比腹腔白色脂肪(aWAT)更容易发生棕样化相反,大蹄蝠体内的aWAT却比sWAT更容易发生棕样化。这为白色脂肪棕样化的研究提供了新的模型,为治疗肥胖及肥胖相关疾病,尤其是腹腔脂肪相关疾病如脂肪肝炎、肠系膜疾病以及动脉粥样化等心血管疾病提供了更为宽广的视野。更多还原

【Abstract】 Hibernation patterns have been extensively studied because of their relevance to longevity, low extinction rate, delayed development, low metabolic rate, and low water loss, resistance to muscle atrophy and osteoporosis. We studied mammal hibernation in this article, especially focused on inducing Hipposideros armiger into hibernation under laboratory condition during non-hibernation season, the regulatory mechanism of Leptin conducted on hibernation and cold-induced white adipose tissue browning using bat as a model.Natural conditions restrict hibernation studies because sampling could only be conducted during winter, which hinders development in hibernation research. In the present study, we induced eight Great Roundleaf bats(Hipposideros armiger) into hibernation in winter, spring, and summer under laboratory conditions. Body temperature and heart rate were recorded as index parameters of hibernation. All bats entered a state of hibernation. Body temperature decreased from the normal38℃to10℃, and heart rate decreased from the highest at600beats per min to the lowest at6beats per min when the bats were placed inside the artificial climate incubator with ambient temperature of9℃. In addition, no significant difference in heart rate of torpid bats during the hibernation season and non-hibernation season (spring and summer) was observed (F1=2.850, P=0.102). In summary, hibernation in bats could be induced outside the hibernation season, which provides significant convenience for further research on hibernation in this species.Previous studies have shown that the hormone Leptin has a key function in mammalian heterothermy by regulating metabolism and food intake via lipolysis, as well as selected pressure, which results in the increase in Leptin lipolysis activity in mammals. However, the mechanism by which Leptin functions in heterothermic regulation in mammals is unknown. By combining previous results, we speculated that the answer may lie in the Leptin signaling pathway in the hypothalamus. OB-R1(Leptin receptor long form) is mainly distributed in the hypothalamus, and has a key function in mediating Leptin function, In this study, we used OB-R1as a molecular marker to explore the mechanisms of Leptin in heterothermic regulation. We used RNA interference technology and physiological/molecular analyses with Hipposideros armiger as an animal model. Results of in situ hybridization proved that anti-OB-R1shRNA lentivirus could significantly decreased OB-R1expression in the hypothalamus, especially in the arcuate nucleus, ventromedial hypothalamic nucleus and dorsomedial nucleus. Physiological analysis demonstrated that the thermological ability of bats (e.g., low core body temperature and heart rate) was significantly depressed after OB-RL gene silencing in the hypothalamus. Thus, animals could not enter a state of hibernation. Our study first proved that Leptin pathway blocking via OB-R1silencing inhibited heterothermic regulation. Leptin may be provided a signal of "fat were enough" for inducing mammals into hibernation.We described two species-Hipposideros armiger and Myotis ricketti, belonged to the second largest group model to study cold-induced response in morphology and molecular expression in WAT and BAT. After Exposed to10℃or15℃for7days, Harmiger showed an obvious change in morphology, with increasing multilocular adipocytes and vascular density, and the remarkably high expression of UCP1and PGC-1α in WAT, but that did not happen in M.ricketti. What is more, it was contrary to what happened in mice, the abdominal adipose tissue showed a rapid and significant browning compared to the subcutaneous adipose tissue in H.armiger. These data offer a novel strategy to research the abdominal adipose tissue for disease, such as steatohepatitis, mesenteric diseases and atheroma, as well as an unusual insight into exploration applications on human beings based on the study of this H.armiger model.更多还原