孤独症相关基因影响抑制性神经递质的研究

论文总字数：63076字

摘 要

孤独症是一种以社交障碍，兴趣狭隘，活动刻板为主要特征的广泛性发育障碍疾病，到目前仍未能完全阐释其致病机理。果蝇是神经科学研究的重要模型。对GABA进行免疫染色研究发现：与野生型果蝇CS相比，除dnl4以外，dnl3，dnrx，dnl3/dnrx突变型果蝇内GABA的密度均有不同程度改变：dnl3中GABA密度升高，而dnrx，dnl3/dnrx中则相反而且dnrx中GABA密度下降程度较dnl3/dnrx更高。而对5-HT的免疫染色发现与野生型果蝇CS相比，dnl3中第一对，第六对，第十一对5-HT能神经元分支数目均下降，dnrx中第六对5-HT能神经元分支数目少量增加，而第十一对5-HT能神经元分支数目却显著下降，dnl4中第十一对5-HT能神经元分支数显著减少，dnl3/dnrx双突变果蝇中仅仅在第一对5-HT能神经元分支数目上有一定程度增加。而通过电镜观察统计腹神经索混合型型突触扣结（MV）中致密型突触囊泡发现与野生型相比，dnrx与dnl3中致密型突触囊泡密度有轻微的下降，其余两种突变型果蝇中致密型突触囊泡密度不变；而对于致密性突触囊泡占总囊泡比分析中发现dnl3中致密型突触囊泡占比略微提高，但在dnl4中这种比例显著提高，相反，在dnrx中致密型突触囊泡占比显著下降，而在dnl3/dnrx中致密型突触囊泡占比与野生型相比没有统计学差异。综上所述，通过这些研究表明孤独症相关基因的突变会导致抑制性神经递质表达失调，这可能是孤独症发病主要原因之一，并为孤独症的治疗提供一个新的思路。

关键词：孤独症，果蝇，抑制性神经递质，MV型突触扣结

ABSTRACT

Autism is a widespread developmental disorder with social disorders, narrow interests, and stereotyped activities. The pathogenesis of autism is still not fully understood. Drosophila is a important model of neuroscience. The immunohistochemical study revealed that compared with wild-type(CS) except for dnl4 mutant Drosophila, the dnl3, dnrx, dnl3/dnrx mutant Drosophila showed varying degrees of GABA density compared to the wild-type(CS). In the dnl3-mutant Drosophila, the GABA density increased, whereas dnrx, dnl3/dnrx are reversed and dnrx medium density is more degraded. However, for the 5-HT staining, the number of branches in the first pair, the sixth pair, and the eleventh pair of 5-HT neurons in dnl3 were all decreased. The sixth pair of 5-HT neurons in dnrx increased slightly, while the number of branches in the eleventh pair of 5-HT energy neurons decreased significantly. The number of branches of the eleventh pair of 5-HT neurons in dnl4 was significantly reduced, and the number of the first pair of 5-HT neurons in the dnl3/dnrx double mutant Drosophila increased to a certain extent. Through the observation of densified synaptic vesicles in Mixed vesicle (MV) synaptic boutons of abdominal neuromuscular strain by electron microscopy, something interesting are found: the density of dense synaptic vesicles in dnrx and dnl3 was slightly decreased compared with that in the wild type, and density of dense synaptic vesicles in the two mutant Drosophila are remaining. However, for the analysis of dense synaptic vesicles in total vesicles, the proportion of dense synaptic vesicles in dnl3 was slightly increased but increased significantly in dnl4. In contrast, the proportion of dense synaptic vesicles in dnrx was significantly decreased. However, there was no statistically significant difference in the proportion of dense synaptic vesicles in dnl3/dnrx compared with wild-type. In conclusion, these studies demonstrate that mutations in autism-related genes can lead to dysregulated expression of neurotransmitters, which may be one of the leading causes of autism and provide a new way of thinking for the treatment of autism.

KEY WORDS: autism, Drosophila, inhibitory neurotransmitter, MV synaptic bouton

目 录

摘要············································································································I

Abstract·······································································································II

第一章 绪论·································································································1

1.1引言······························································································1

1.2 NRXNS家族研究现状·······································································2

1.2.1 NRXN1与孤独症相关性研究························································2

1.2.2 NRXN2与孤独症相关性研究························································2

1.2.3 NRXN3与孤独症相关性研究························································3

1.3 NLGNS家族研究现状·····································································3

1.3.1 NLGN1与孤独症相关性研究························································4

1.3.2 NLGN2与孤独症相关性研究························································4

1.3.3 NLGN3与孤独症相关性研究························································5

1.3.4 NLGN4与孤独症相关性研究························································6

1.4本文的研究目的及主要研究内容···························································7

1.4.1抑制性神经递质在腹神经索的整体分布研究·····································7

1.4.2抑制性神经递质超微结构观察研究·················································7

第二章 实验材料与方法···················································································8

2.1实验材料·························································································8

2.1.1果蝇品系···················································································8

2.1.2实验试剂及溶液··········································································8

2.1.3实验仪器及设备··········································································8

2.2实验方法·························································································8

2.2.1果蝇的饲养················································································9

2.2.2果蝇三龄幼虫腹神经索免疫标记·····················································9

2.2.2.1解剖与固定·······································································9

2.2.2.2漂洗与封闭·······································································9

2.2.2.3免疫荧光标记····································································9

2.2.2.4封片与观察·······································································9

2.2.3果蝇三龄幼虫腹神经索常规电镜样本的制备······································9

2.2.3.1解剖与固定·······································································9

2.2.3.2漂洗与固定·······································································9

2.2.3.3铀染过夜··········································································9

2.2.3.4脱水与渗透·······································································9

2.2.3.5包埋与聚合·······································································9

2.2.3.6修块，切片，染色和包埋·····················································9

2.2.4果蝇三龄幼虫腹神经索半薄切片染色················································9

2.2.4.1解剖与固定······································································10

2.2.4.2漂洗与固定······································································10

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