改造狂犬病毒探索小鼠下丘脑摄食相关核团的神经环路

发布时间：2018-05-17 00:46

本文选题：摄食行为 + 神经环路　；参考：《华中农业大学》2017年硕士论文

【摘要】：探究动物的摄食行为,不论是对畜牧业生产效益的提升,还是对人类代谢相关疾病的研究都有重大的科研价值。以往的研究表明,中枢神经系统在动物摄食行为产生的过程中发挥了重要的调控作用,而下丘脑是中枢神经系统中调节动物代谢的关键部位。本课题的目的旨在通过改造狂犬病毒,将其作为神经环路示踪工具,对下丘脑中摄食相关核团的神经环路进行探究。本实验主要分为三个部分:第一,利用缺失病毒毒力基因G蛋白、带有荧光蛋白的狂犬病毒,针对下丘脑区域的五个摄食相关核团——下丘脑室旁核、下丘脑背内侧核、下丘脑腹内侧核、弓状核、下丘脑外侧区,在全脑范围内示踪对其有轴突投射的神经元。实验结果显示,主要有额眶部皮层、外侧隔核、视前内侧区、视前内侧核、终纹床核、杏仁核、腹侧被盖区、蓝斑、臂旁核、水管周灰质等核团将其轴突投射到了下丘脑这五个核团;第二,构建Env A/TVA病毒示踪体系,实现狂犬病毒“定位、跨突触可受调控”的感染模式,最终该示踪体系构建成功并在鼠脑上得到验证;第三,为了构建含有水泡性口炎病毒和狂犬病毒嵌合G蛋白的B7B-VSV SAD G细胞系,从而实现狂犬病毒对注射区域神经元的胞体集中且高效的感染模式。我们利用CRISPR/Cas9技术手段,改造B7GG细胞——将该细胞系基因组中的G蛋白变为嵌合G蛋白,并将蓝色荧光蛋白替换掉绿色荧光蛋白,最终利用潮霉素压力筛选和流式细胞仪对细胞系进行了纯化,后期可通过进一步扩大培养完成细胞系的建立。对动物摄食行为有调控作用的神经系统错综复杂,利用灵活高效的嗜神经性病毒作为示踪工具来揭示脑功能性神经环路的结构和联系一直以来是神经科学领域研究的热点,也是解决机体代谢异常相关疾病的有效途径之一。对于本实验中揭示出的与下丘脑有投射关系的神经位点有望对之后摄食环路的探究起到一定的启示作用;同时,本实验中构建的狂犬病毒示踪工具的应用为之后神经环路的探究和新型示踪工具的开发奠定了一定的实验基础。
[Abstract]:It is of great value to explore the feeding behavior of animals, not only to improve the efficiency of animal husbandry, but also to study the metabolic diseases of human beings. Previous studies have shown that the central nervous system (CNS) plays an important role in the production of animal feeding behavior, and the hypothalamus is the key part of the central nervous system (CNS) to regulate animal metabolism. The purpose of this study is to investigate the neural loop of feeding related nuclei in the hypothalamus by modifying rabies virus as a tracer of neural loop. This experiment is mainly divided into three parts: first, using the virus virulence gene G protein and rabies virus with fluorescent protein, the hypothalamic paraventricular nucleus and dorsal medial hypothalamus nucleus were targeted at the five feeding related nuclei in the hypothalamus region, the hypothalamic paraventricular nucleus, and the hypothalamic dorsal medial nucleus. The ventromedial hypothalamic nucleus arcuate nucleus and lateral hypothalamic area trace axonal projecting neurons throughout the brain. The results showed that there were mainly frontal orbital cortex, lateral septal nucleus, preoptic medial area, preoptic medial nucleus, bed nucleus of final stria, amygdala, ventral tegmental area, locus coeruleus, parabrachial nucleus. The periaqueductal gray matter and other nuclei project their axons to the five hypothalamic nuclei. Secondly, the Env A/TVA virus tracer system is constructed to realize the rabies virus "localization, transsynaptic and regulated" infection pattern. Finally, the tracer system was successfully constructed and verified in the brain. Thirdly, in order to construct B7B-VSV SAD G cell line containing chimeric G protein of vesicular stomatitis virus and rabies virus, In order to achieve rabies virus to the injection area of neurons concentrated and efficient mode of infection. We used CRISPR/Cas9 technology to transform B7GG cells into chimeric G proteins in the genome of the cell line and replace green fluorescent proteins with blue fluorescent proteins. Finally, hygromycin pressure screening and flow cytometry were used to purify the cell line. The nervous system which can regulate the feeding behavior of animals is complicated. It has been a hot spot in the field of neuroscience to use the flexible and efficient neuroviruses as a tracer tool to reveal the structure and connection of the functional neural loop in the brain. It is also one of the effective ways to solve metabolic disorders. It is expected that the neural sites which are related to the hypothalamus in this study may be helpful to the exploration of the feeding loop. At the same time, The application of rabies virus tracer tool in this experiment has laid a certain experimental foundation for the later exploration of neural loop and the development of new tracer tool.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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