秀丽线虫miR-83通过抑制靶基因UNC-52表达调控GABA神经突触功能的研究

发布时间：2018-05-05 04:44

本文选题：秀丽隐杆线虫 + GABA神经突触　；参考：《东北师范大学》2016年硕士论文

【摘要】：γ-氨基丁酸(GABA)神经递质是一种神经发生过程中对于神经产物的形成与神经环路的构建都非常重要的调控物质,该类型的神经突触对于哺乳动物神经系统的发育与功能起到非常重要的调控作用。有研究发现,哺乳动物生命早期中出现的焦虑症以及抑郁症是由于GABA神经传递的损伤导致的。另也有研究发现,皮质及边缘的GABA神经环路的功能紊乱对于人类多种神经发育类疾病有至关重要的影响,包括精神分裂症、自闭症、癫痫症等一系列疾病的发生都与GABA神经突触的功能紊乱有着密不可分的关系。秀丽线虫一直是研究神经发育的先锋生物。在雌雄同体的秀丽线虫中只有302个神经元,其中有2000个神经突触是以神经肌肉接头(NMJ)的形式存在。虽然秀丽线虫的神经系统较为简单,但是其神经系统的相关基因与哺乳动物具有一定的同源性,其中包括GABA神经突触相关基因。micro RNAs(miRNAs)是近几年来发现的一种广泛存在的非编码的小RNA,通过与靶基因的3’UTR结合抑制基因表达。有研究表明miRNAs在神经突触发育的各个阶段都具有作用,比如在树突基因组成、突触发生以及突触成熟方面。但对于miRNAs影响GABA神经突触功能方面的研究,现在还比较缺乏。为了解决这个问题,本实验室前期利用miRNAs缺失的线虫突变品系,通过GABA递质的拮抗剂PTZ与乙酰胆碱酯酶的抑制剂aldicarb的双重超敏模型,对参与调控GABA神经突触的miRNAs进行了筛选。发现mir-83突变体线虫对于以上两种筛选药物都表现出比较敏感的反应,这说明mir-83很可能对线虫的GABA神经突触功能具有调控作用。有文献报道,mir-83在哺乳动物中的同源物mir-29,也与神经系统的发育以及神经细胞的死亡、失调共济以及细胞凋亡的发生相关。另也有报道,发现mir-29与阿兹海默症相关的β-位点淀粉样先驱蛋白裂解酶1(BACE1)表达增加有关。为了进一步确定mir-83调控GABA神经突触功能的分子机制。我们首先利用生物信息学方法,通过RT-PCR以及荧光素酶双报告检测分析,确定了unc-52是被mir-83调控的靶基因。接下来,我们通过杂交获得mir-83;unc-52双突变体线虫,并发现unc-52基因突变能够明显抑制mir-83突变体线虫对PTZ和aldicarb的敏感反应,这说明mir-83是通过抑制其靶基因unc-52的表达参与调控GABA神经突触功能的。然后我们利用能够特异性标记位于GABA神经突触后膜的GABAA受体的unc-49::GFP标签,发现mir-83突变体线虫中GABA神经突触后膜上的GABAA受体密度明显减少,而unc-52基因突变对这种密度的减少具有部分恢复作用,说明mir-83通过抑制unc-52表达调控了GABAA受体在GABA神经突触后膜上的分布。另外,利用unc-47p::GFP荧光标签,我们还发现mir-83突变体线虫中的GABA神经元的轴突生长出现了缺陷。但unc-52基因突变对这种缺陷并没有恢复作用。因此,我们认为mir-83虽然可以调控GABA神经元的轴突生长,但该调控作用并不依赖于其靶基因unc-52。本论文通过揭示秀丽线虫mir-83调控GABA神经突触的分子机制,为研究哺乳动物miRNAs如何调控GABA神经突触功能奠定了一定的基础,也为GABA神经突触功能相关的神经性疾病的发病机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.

【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R741;Q78

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