神经肽Y在斑马鱼脊髓损伤修复中的作用
发布时间：2018-08-22 19:18
【摘要】：脊髓损伤(spinal cord injury,SCI)后,成年斑马鱼的脊髓组织能够再生且恢复运动能力,这与哺乳动物中枢神经系统损伤后有限的修复能力形成鲜明对比。因此,探索促进成年斑马鱼脊髓损伤修复的机制将为哺乳动物脊髓损伤的研究提供新思路,有利于脊髓损伤的治疗。已有研究发现神经肽Y(neuropeptide Y,NPY)在大鼠脊髓中有表达,且在脊髓损伤后表达升高,然而这一表达变化对脊髓损伤的作用却未有研究。同时NPY是一种神经保护因子,它能通过与Y1受体的结合发挥神经保护性作用。本课题研究了NPY对脊髓损伤后斑马鱼的游泳能力、轴突再生和运动神经元增殖表达的影响。在SCI后12小时、6天、11天和21天,通过定量实时聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)发现,与sham组(假手术组)相比,NPY mRNA的表达在SCI后12小时、6天、11天和21天明显下降。原位杂交(in situ hybridization,ISH)实验检测到NPY mRNA在sham组和脊髓损伤后的斑马鱼脊髓中均有表达,且表达变化与qRT-PCR检测结果一致。免疫荧光实验发现NPY蛋白质表达在沿脊髓中央管分布的细胞胞质中,且表达变化与RNA检测结果一致。吗啉代(morpholino,MO)抑制NPY表达后的结果显示,与对照MO组相比,NPY MO组斑马鱼脊髓中的轴突再生表达(与对照组相比降低了25.3%)和运动能力的恢复(第2周减少68%,第3周减少57.8%,第4周减少48.5%,第6周减少36.6%)水平都明显下降。NPY和运动神经元的免疫荧光共定位染色结果显示NPY主要分布于脊髓运动神经元的胞质中,同时,脊髓损伤后运动神经元表达了细胞核增殖抗原(proliferating cell nuclear antigen,PCNA),抑制NPY后运动神经元的增殖表达减少。此外,实时荧光定量PCR结果显示,与假手术组相比,Y1受体mRNA的表达在斑马鱼脊髓损伤后12小时、6天、11天和21天均显著降低。且Y1受体的原位杂交与NPY的免疫荧光共定位结果显示,Y1受体与NPY在对照组和脊髓损伤后的脊髓组织中均有共表达。Y1受体的原位杂交与运动神经元的免疫荧光共定位结果显示,Y1表达于运动神经元胞质中。因此NPY可能通过与Y1受体亚型的相互作用促进斑马鱼脊髓损伤后运动神经元的增殖。综上实验结果得出结论:NPY有助于成年斑马鱼脊髓损伤后运动功能的恢复和轴突的再生,且这种保护作用可能通过与Y1受体的相互结合促进了运动神经元的增殖来实现的。
[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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2
[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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2
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