發(fā)布時(shí)間：2018-05-05 04:44

  本文選題：秀麗隱桿線(xiàn)蟲(chóng) + GABA神經(jīng)突觸�。� 參考：《東北師范大學(xué)》2016年碩士論文

【摘要】：γ-氨基丁酸(GABA)神經(jīng)遞質(zhì)是一種神經(jīng)發(fā)生過(guò)程中對(duì)于神經(jīng)產(chǎn)物的形成與神經(jīng)環(huán)路的構(gòu)建都非常重要的調(diào)控物質(zhì),該類(lèi)型的神經(jīng)突觸對(duì)于哺乳動(dòng)物神經(jīng)系統(tǒng)的發(fā)育與功能起到非常重要的調(diào)控作用。有研究發(fā)現(xiàn),哺乳動(dòng)物生命早期中出現(xiàn)的焦慮癥以及抑郁癥是由于GABA神經(jīng)傳遞的損傷導(dǎo)致的。另也有研究發(fā)現(xiàn),皮質(zhì)及邊緣的GABA神經(jīng)環(huán)路的功能紊亂對(duì)于人類(lèi)多種神經(jīng)發(fā)育類(lèi)疾病有至關(guān)重要的影響,包括精神分裂癥、自閉癥、癲癇癥等一系列疾病的發(fā)生都與GABA神經(jīng)突觸的功能紊亂有著密不可分的關(guān)系。秀麗線(xiàn)蟲(chóng)一直是研究神經(jīng)發(fā)育的先鋒生物。在雌雄同體的秀麗線(xiàn)蟲(chóng)中只有302個(gè)神經(jīng)元,其中有2000個(gè)神經(jīng)突觸是以神經(jīng)肌肉接頭(NMJ)的形式存在。雖然秀麗線(xiàn)蟲(chóng)的神經(jīng)系統(tǒng)較為簡(jiǎn)單,但是其神經(jīng)系統(tǒng)的相關(guān)基因與哺乳動(dòng)物具有一定的同源性,其中包括GABA神經(jīng)突觸相關(guān)基因。micro RNAs(miRNAs)是近幾年來(lái)發(fā)現(xiàn)的一種廣泛存在的非編碼的小RNA,通過(guò)與靶基因的3’UTR結(jié)合抑制基因表達(dá)。有研究表明miRNAs在神經(jīng)突觸發(fā)育的各個(gè)階段都具有作用,比如在樹(shù)突基因組成、突觸發(fā)生以及突觸成熟方面。但對(duì)于miRNAs影響GABA神經(jīng)突觸功能方面的研究,現(xiàn)在還比較缺乏。為了解決這個(gè)問(wèn)題,本實(shí)驗(yàn)室前期利用miRNAs缺失的線(xiàn)蟲(chóng)突變品系,通過(guò)GABA遞質(zhì)的拮抗劑PTZ與乙酰膽堿酯酶的抑制劑aldicarb的雙重超敏模型,對(duì)參與調(diào)控GABA神經(jīng)突觸的miRNAs進(jìn)行了篩選。發(fā)現(xiàn)mir-83突變體線(xiàn)蟲(chóng)對(duì)于以上兩種篩選藥物都表現(xiàn)出比較敏感的反應(yīng),這說(shuō)明mir-83很可能對(duì)線(xiàn)蟲(chóng)的GABA神經(jīng)突觸功能具有調(diào)控作用。有文獻(xiàn)報(bào)道,mir-83在哺乳動(dòng)物中的同源物mir-29,也與神經(jīng)系統(tǒng)的發(fā)育以及神經(jīng)細(xì)胞的死亡、失調(diào)共濟(jì)以及細(xì)胞凋亡的發(fā)生相關(guān)。另也有報(bào)道,發(fā)現(xiàn)mir-29與阿茲海默癥相關(guān)的β-位點(diǎn)淀粉樣先驅(qū)蛋白裂解酶1(BACE1)表達(dá)增加有關(guān)。為了進(jìn)一步確定mir-83調(diào)控GABA神經(jīng)突觸功能的分子機(jī)制。我們首先利用生物信息學(xué)方法,通過(guò)RT-PCR以及熒光素酶雙報(bào)告檢測(cè)分析,確定了unc-52是被mir-83調(diào)控的靶基因。接下來(lái),我們通過(guò)雜交獲得mir-83;unc-52雙突變體線(xiàn)蟲(chóng),并發(fā)現(xiàn)unc-52基因突變能夠明顯抑制mir-83突變體線(xiàn)蟲(chóng)對(duì)PTZ和aldicarb的敏感反應(yīng),這說(shuō)明mir-83是通過(guò)抑制其靶基因unc-52的表達(dá)參與調(diào)控GABA神經(jīng)突觸功能的。然后我們利用能夠特異性標(biāo)記位于GABA神經(jīng)突觸后膜的GABAA受體的unc-49::GFP標(biāo)簽,發(fā)現(xiàn)mir-83突變體線(xiàn)蟲(chóng)中GABA神經(jīng)突觸后膜上的GABAA受體密度明顯減少,而unc-52基因突變對(duì)這種密度的減少具有部分恢復(fù)作用,說(shuō)明mir-83通過(guò)抑制unc-52表達(dá)調(diào)控了GABAA受體在GABA神經(jīng)突觸后膜上的分布。另外,利用unc-47p::GFP熒光標(biāo)簽,我們還發(fā)現(xiàn)mir-83突變體線(xiàn)蟲(chóng)中的GABA神經(jīng)元的軸突生長(zhǎng)出現(xiàn)了缺陷。但unc-52基因突變對(duì)這種缺陷并沒(méi)有恢復(fù)作用。因此,我們認(rèn)為mir-83雖然可以調(diào)控GABA神經(jīng)元的軸突生長(zhǎng),但該調(diào)控作用并不依賴(lài)于其靶基因unc-52。本論文通過(guò)揭示秀麗線(xiàn)蟲(chóng)mir-83調(diào)控GABA神經(jīng)突觸的分子機(jī)制,為研究哺乳動(dòng)物miRNAs如何調(diào)控GABA神經(jīng)突觸功能奠定了一定的基礎(chǔ),也為GABA神經(jīng)突觸功能相關(guān)的神經(jīng)性疾病的發(fā)病機(jī)制研究提供了新的方向。
[Abstract]:Gamma aminobutyric acid (GABA) neurotransmitter is an important regulatory substance for the formation of nerve products and the construction of neural circuits during neurogenesis. This type of synapse plays a very important role in regulating the development and function of mammalian nervous system. Current anxiety and depression are caused by damage to GABA neurotransmitters. Other studies have found that the dysfunction of the GABA nerve loop in the cortex and edge has a vital effect on a variety of neurodevelopmental diseases in humans, including schizophrenia, autism, and epilepsy, which are associated with the GABA synapse. The disorder has an inseparable relationship. Elegans has been a pioneer of neurodevelopment. There are only 302 neurons in the hermaphroditic Caenorhabditis elegans, of which 2000 synapses are in the form of a neuromuscular junction (NMJ). Although the nervous system of the Caenorhabditis elegans is relatively simple, its nervous system phase The genes are homologous to mammals, including the GABA neural synapse related gene.Micro RNAs (miRNAs), a widely existing, non coded small RNA that has been found in recent years. It inhibits gene expression by binding to the 3 'UTR of the target gene. Studies have shown that miRNAs plays a role in all stages of synapse development. For example, the composition of dendrites, synapses, and synaptic maturation. But there is still a lack of research on the effect of miRNAs on GABA synaptic function. In order to solve this problem, we used the miRNAs deletion mutant lines in our laboratory to pass the GABA transmitter antagonist PTZ and the acetylcholinesterase inhibitor aldica The double hypersensitivity model of Rb screened the miRNAs that participates in the regulation of GABA synapses. It was found that the mir-83 mutant nematode showed a more sensitive response to the two screening drugs, suggesting that mir-83 is likely to regulate the GABA synaptic function of the nematode. It is reported that mir-83 is homologous in mammals. Substance mir-29 is also associated with the development of the nervous system and the death of nerve cells, ataxia and the occurrence of apoptosis. It is also reported that the expression of amyloid precursor protein lyase 1 (BACE1) in the beta loci of Alzheimer's disease is associated with the increase of the expression of mir-29. In order to further determine the molecular mechanism of mir-83 regulation of GABA synaptic function System. We first use bioinformatics method, RT-PCR and luciferase double report detection analysis to determine that unc-52 is the target gene regulated by mir-83. Next, we get mir-83, unc-52 double mutant nematode by hybridization, and find that the mutation of unc-52 gene can clearly inhibit the sensitivity of mir-83 mutant nematode to PTZ and aldicarb. This suggests that mir-83 is involved in the regulation of the GABA synaptic function by inhibiting the expression of its target gene unc-52, and then we use the unc-49:: GFP tag that specifically labeled the GABAA receptor in the GABA postsynaptic membrane, and we found that the GABAA receptor density on the GABA neuron postsynaptic membrane in the mir-83 mutant nematode is significantly reduced, and u. Nc-52 gene mutation has a partial recovery effect on the reduction of this density, indicating that mir-83 regulates the distribution of GABAA receptors on the postsynaptic membrane of GABA by inhibiting the expression of unc-52. In addition, we also found a defect in the axon growth of GABA Shen Jing in the mir-83 mutant nematode by using unc-47p:: GFP fluorescence label. But the unc-52 gene is found. Mutation has no effect on this defect. Therefore, we think that although mir-83 can regulate the axon growth of GABA neurons, this regulation does not depend on its target gene unc-52. in this paper by revealing the molecular mechanism of GABA synapses regulated by the Caenorhabditis elegans mir-83 in order to study how mammalian miRNAs regulates the GABA synapses. It has laid a foundation for the function and provided a new direction for the study of the pathogenesis of GABA related neurologic diseases.

【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R741;Q78

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