【摘要】：脊髓損傷(spinal cord injury,SCI)后,成年斑馬魚的脊髓組織能夠再生且恢復運動能力,這與哺乳動物中樞神經系統損傷后有限的修復能力形成鮮明對比。因此,探索促進成年斑馬魚脊髓損傷修復的機制將為哺乳動物脊髓損傷的研究提供新思路,有利于脊髓損傷的治療。已有研究發(fā)現神經肽Y(neuropeptide Y,NPY)在大鼠脊髓中有表達,且在脊髓損傷后表達升高,然而這一表達變化對脊髓損傷的作用卻未有研究。同時NPY是一種神經保護因子,它能通過與Y1受體的結合發(fā)揮神經保護性作用。本課題研究了NPY對脊髓損傷后斑馬魚的游泳能力、軸突再生和運動神經元增殖表達的影響。在SCI后12小時、6天、11天和21天,通過定量實時聚合酶鏈反應(quantitative real-time polymerase chain reaction,qRT-PCR)發(fā)現,與sham組(假手術組)相比,NPY mRNA的表達在SCI后12小時、6天、11天和21天明顯下降。原位雜交(in situ hybridization,ISH)實驗檢測到NPY mRNA在sham組和脊髓損傷后的斑馬魚脊髓中均有表達,且表達變化與qRT-PCR檢測結果一致。免疫熒光實驗發(fā)現NPY蛋白質表達在沿脊髓中央管分布的細胞胞質中,且表達變化與RNA檢測結果一致。嗎啉代(morpholino,MO)抑制NPY表達后的結果顯示,與對照MO組相比,NPY MO組斑馬魚脊髓中的軸突再生表達(與對照組相比降低了25.3%)和運動能力的恢復(第2周減少68%,第3周減少57.8%,第4周減少48.5%,第6周減少36.6%)水平都明顯下降。NPY和運動神經元的免疫熒光共定位染色結果顯示NPY主要分布于脊髓運動神經元的胞質中,同時,脊髓損傷后運動神經元表達了細胞核增殖抗原(proliferating cell nuclear antigen,PCNA),抑制NPY后運動神經元的增殖表達減少。此外,實時熒光定量PCR結果顯示,與假手術組相比,Y1受體mRNA的表達在斑馬魚脊髓損傷后12小時、6天、11天和21天均顯著降低。且Y1受體的原位雜交與NPY的免疫熒光共定位結果顯示,Y1受體與NPY在對照組和脊髓損傷后的脊髓組織中均有共表達。Y1受體的原位雜交與運動神經元的免疫熒光共定位結果顯示,Y1表達于運動神經元胞質中。因此NPY可能通過與Y1受體亞型的相互作用促進斑馬魚脊髓損傷后運動神經元的增殖。綜上實驗結果得出結論:NPY有助于成年斑馬魚脊髓損傷后運動功能的恢復和軸突的再生,且這種保護作用可能通過與Y1受體的相互結合促進了運動神經元的增殖來實現的。
[Abstract]:After spinal cord injury (spinal cord injury-sci), adult zebrafish spinal cord can regenerate and restore motor ability, which is in contrast to the limited repair ability of mammalian central nervous system injury. Therefore, exploring the mechanism of promoting the repair of adult zebrafish spinal cord injury will provide a new idea for the study of mammalian spinal cord injury, which is beneficial to the treatment of spinal cord injury. It has been found that neuropeptide Y (neuropeptide YNPY) is expressed in the spinal cord of rats and increased after spinal cord injury. However, the effect of this change on spinal cord injury has not been studied. At the same time, NPY is a neuroprotective factor, it can play a neuroprotective role by binding to Y 1 receptor. The purpose of this study was to investigate the effects of NPY on the swimming ability, axon regeneration and motor neuron proliferation in zebrafish after spinal cord injury. After 12 hours and 6 days after SCI, the expression of mRNA in sham group was significantly lower than that in sham group (12 hours, 6 days, 11 days and 21 days after SCI) by quantitative real-time polymerase chain reaction (QRT-PCR). The expression of NPY mRNA was detected in sham group and spinal cord of zebrafish after spinal cord injury by in situ hybridization (in situ hybridization ish. The change of NPY mRNA expression was consistent with that of qRT-PCR. Immunofluorescence assay showed that NPY protein was expressed in the cytoplasm distributed along the central canal of spinal cord, and the change of expression was consistent with the result of RNA detection. The inhibitory effect of morpholinoimo on the expression of NPY showed that, The expression of axon regeneration in zebrafish spinal cord (decreased by 25.3% compared with control group) and the recovery of motor ability (decreased by 68% in the second week, 57.8% in the third week, 48.5% in the fourth week and 36.6% in the sixth week) compared with the control MO group. The results of immunofluorescence co-localization staining showed that NPY was mainly distributed in the cytoplasm of spinal motoneurons. At the same time, the motoneurons after spinal cord injury expressed proliferating antigen (proliferating cell nuclear antigen-PCNA, and inhibited the decrease of proliferation and expression of motoneurons after NPY. In addition, real-time fluorescence quantitative PCR showed that the expression of Y1 receptor mRNA was significantly decreased in 12 hours after spinal cord injury, 11 and 21 days after spinal cord injury in zebrafish compared with sham-operated group. The results of in situ hybridization of Y1 receptor and immunofluorescence of NPY showed that there was coexpression of Y1 receptor and NPY in control group and spinal cord tissue after spinal cord injury. In situ hybridization of Y1 receptor and immunofluorescence codetermination of motoneuron. The results showed that Y1 was expressed in the cytoplasm of motoneurons. Therefore, NPY may promote the proliferation of motoneurons after spinal cord injury in zebrafish by interacting with Y1 receptor subtype. From the above results, it is concluded that NPY is beneficial to the recovery of motor function and regeneration of axons after spinal cord injury in adult zebrafish, and this protective effect may be achieved by the interaction with Y1 receptor to promote the proliferation of motor neurons.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R651.2

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本文編號：2198058