OB-RL基因介导瘦素参与大蹄蝠冬眠期脂肪代谢的研究

The ability of mammalian hibernation, with dramatic changes in physiology, such as body temperature, neural activity and metabolic activity, is considered key to their evolutionary success and allows them to survive in adverse climates and during periods when food is scarce. This physiological ability is precisely controlled by internal molecular driven mechanisms and represents a natural model for studying human-related disease, including diabetes, obese, ischemia-reperfusion injuries and muscle disuse, etc. Combining with the privious studies, which showed that the periodic changes of the level of serum Leptin and other related molecules involved in the regulate of metabolism and food intake in hibernation and the positive selection drived the the evolution of Leptin in the heterothermic bats, all these results indicated the key role of Leptin in hibernating regulation of mammals. Thus, in-deeply investigating the molecular mechanism of Leptin in the regulation of hibernation will pave the way of theory of mammalian hibernation and provide clues for human medicine. As the most important Leptin receptor, OB-RL mainly distributes in hypothalamus, which is one of key brain slices in hibernating regulation, and plays the key role on mediating the function of Leptin in metabolism and neuroendocrine, etc. Moreover, in privious study, we have cloned the OB-RL complete CDS of Hipposideros armiger (a hibernating bat species). Thus, we design a in-directly way to explore the mechanism of Leptin on hibernation by interfering the bio-function of OB-RL in hypothalamus with H. armiger as animal model. The main approaches employed in this study includ: (a) design and construct the OB-RL-siRNA lentivirus plasmid; (b) microinjection the OB-RL-siRNA lentivirus into hypothalamus of H. armiger, with PBS microinjection as control; (c) induce H. armiger into the heterothermic state, collect the physiological and endocrinological data changes and other functional variables, and compare the difference between two groups, OB-RL-siRNA lentivirus injection and PBS injection; (d) prepare brain slices and and carry out the in situ hybridization to assay the expressive level of OB-RL in hypothalamus after RNA interfering; (e) combine the physiological data and in situ hybridization results to evaluate the role of OB-RL blocking on hibernation and clarify the mechanism of Leptin in the regulation of hibernation.

冬眠是哺乳动物在进化过程中获得的独特的生存能力，其调控机制为研究许多威胁人类健康的疾病提供了线索，一直是国内外科学家研究的热点。大量研究结果表明，瘦素（Leptin）是冬眠调控的关键因子。然而，由于Leptin靶基因众多、代谢网络复杂，其参与冬眠调控的分子机制一直没有被阐明。OB-RL是介导Leptin生物功能的主要受体，并且在冬眠调控的主要脑区（下丘脑）特异的表达，是深入研究Leptin调节冬眠状态下脂肪利用机制的首选靶标。 本研究旨在前期工作基础上，以大蹄蝠为研究对象，利用RNAi技术特异性阻断下丘脑中的Leptin受体OB-RL基因；人工诱导大蹄蝠冬眠并测定其体温、心率等生理指标的变化；比较沉默OB-RL对大蹄蝠冬眠能力的影响，探讨OB-RL在蝙蝠冬眠调控通路中的作用，阐明Leptin参与蝙蝠冬眠调控的分子机制，为研究哺乳动物冬眠机制提供理论依据。

本项目以大蹄蝠为研究对象，利用RNAi技术特异性阻断下丘脑中的Leptin受体OB-RL基因表达，通过比较分析OB-RL沉默对大蹄蝠冬眠能力的影响，研究Leptin及OB-RL在蝙蝠冬眠调控通路中的作用，阐明Leptin参与蝙蝠冬眠调控的分子机理。研究结果表明：大蹄蝠下丘脑OB-RL表达被特异阻断后，其变温调控能力受到显著的影响。由于OB-RL是瘦素（Leptin）在下丘脑中的特异受体，因此可以推断：大蹄蝠下丘脑区域OB-RL表达被特异阻断后其变温调控能力降低是由于Leptin的功能被阻断而引起的，从而间接阐明Leptin是调控蝙蝠冬眠状态的关键因子。本研究结果为进一步探讨哺乳动物冬眠机制奠定基础。此外，本研究团队深入开展了小分子非编码RNA筛选及功能研究、蝙蝠携带病原体筛查等相关工作，并取得一定的原创性科研成果，超额完成本项目任务指标。