本文選題：攝食行為 + 神經(jīng)環(huán)路��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：探究動(dòng)物的攝食行為,不論是對(duì)畜牧業(yè)生產(chǎn)效益的提升,還是對(duì)人類代謝相關(guān)疾病的研究都有重大的科研價(jià)值。以往的研究表明,中樞神經(jīng)系統(tǒng)在動(dòng)物攝食行為產(chǎn)生的過(guò)程中發(fā)揮了重要的調(diào)控作用,而下丘腦是中樞神經(jīng)系統(tǒng)中調(diào)節(jié)動(dòng)物代謝的關(guān)鍵部位。本課題的目的旨在通過(guò)改造狂犬病毒,將其作為神經(jīng)環(huán)路示蹤工具,對(duì)下丘腦中攝食相關(guān)核團(tuán)的神經(jīng)環(huán)路進(jìn)行探究。本實(shí)驗(yàn)主要分為三個(gè)部分:第一,利用缺失病毒毒力基因G蛋白、帶有熒光蛋白的狂犬病毒,針對(duì)下丘腦區(qū)域的五個(gè)攝食相關(guān)核團(tuán)——下丘腦室旁核、下丘腦背內(nèi)側(cè)核、下丘腦腹內(nèi)側(cè)核、弓狀核、下丘腦外側(cè)區(qū),在全腦范圍內(nèi)示蹤對(duì)其有軸突投射的神經(jīng)元。實(shí)驗(yàn)結(jié)果顯示,主要有額眶部皮層、外側(cè)隔核、視前內(nèi)側(cè)區(qū)、視前內(nèi)側(cè)核、終紋床核、杏仁核、腹側(cè)被蓋區(qū)、藍(lán)斑、臂旁核、水管周灰質(zhì)等核團(tuán)將其軸突投射到了下丘腦這五個(gè)核團(tuán);第二,構(gòu)建Env A/TVA病毒示蹤體系,實(shí)現(xiàn)狂犬病毒“定位、跨突觸可受調(diào)控”的感染模式,最終該示蹤體系構(gòu)建成功并在鼠腦上得到驗(yàn)證;第三,為了構(gòu)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細(xì)胞系,從而實(shí)現(xiàn)狂犬病毒對(duì)注射區(qū)域神經(jīng)元的胞體集中且高效的感染模式。我們利用CRISPR/Cas9技術(shù)手段,改造B7GG細(xì)胞——將該細(xì)胞系基因組中的G蛋白變?yōu)榍逗螱蛋白,并將藍(lán)色熒光蛋白替換掉綠色熒光蛋白,最終利用潮霉素壓力篩選和流式細(xì)胞儀對(duì)細(xì)胞系進(jìn)行了純化,后期可通過(guò)進(jìn)一步擴(kuò)大培養(yǎng)完成細(xì)胞系的建立。對(duì)動(dòng)物攝食行為有調(diào)控作用的神經(jīng)系統(tǒng)錯(cuò)綜復(fù)雜,利用靈活高效的嗜神經(jīng)性病毒作為示蹤工具來(lái)揭示腦功能性神經(jīng)環(huán)路的結(jié)構(gòu)和聯(lián)系一直以來(lái)是神經(jīng)科學(xué)領(lǐng)域研究的熱點(diǎn),也是解決機(jī)體代謝異常相關(guān)疾病的有效途徑之一。對(duì)于本實(shí)驗(yàn)中揭示出的與下丘腦有投射關(guān)系的神經(jīng)位點(diǎn)有望對(duì)之后攝食環(huán)路的探究起到一定的啟示作用;同時(shí),本實(shí)驗(yàn)中構(gòu)建的狂犬病毒示蹤工具的應(yīng)用為之后神經(jīng)環(huán)路的探究和新型示蹤工具的開發(fā)奠定了一定的實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

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本文編號(hào)：1899169