ATF6促进成年斑马鱼脊髓损伤后神经修复的实验研究

发布时间：2018-03-10 14:53

本文选题：激活转录因子6　切入点：脊髓损伤　出处：《延安大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本论文利用成年斑马鱼脊髓横断损伤模型,研究激活转录因子6(activating transcription factor 6,ATF6)对斑马鱼脊髓损伤后神经修复的影响作用和相关机制。方法:我们在4-6月龄的成年斑马鱼上建立脊髓横断损伤模型,在损伤后4小时、12小时、3天、7天取损伤点下段4mm的脊髓组织进行实时定量聚合酶链反应和原位杂交,检测ATF6 m RNA水平变化和表达定位,同时在脊髓损伤后4小时、12小时、3天、7天对ATF6与运动神经元或与放射状胶质细胞进行共标。斑马鱼脊髓损伤后,用ATF6 morpholino(MO)抑制ATF6蛋白质表达,检测斑马鱼运动能力恢复情况以及穿过损伤位点神经轴突修复的水平变化。同时再用实时定量聚合酶链反应的方法检测内质网应激标志性因子免疫重链结合蛋白(binding immunoglobulin protein,BIP)BIP和C/EBP同源蛋白(C/EBP homologus protein,CHOP)m RNA的表达水平变化以探究内质网应激对修复的影响。结果:1.ATF6 m RNA水平在斑马鱼脊髓损伤后的12小时和3天维持在较高的表达水平,而在损伤后7天,其恢复到假手术组的表达水平。2.ATF6蛋白表达在运动神经元中,并且在脊髓损伤后12小时和3天表达水平增高。3.ATF6蛋白环绕在放射状胶质细胞中,并且在脊髓损伤后不同时间和对照组都不共表达。4.斑马鱼脊髓损伤后第2和第3周,MO抑制ATF6蛋白表达处理组斑马鱼运动能力比对照组分别降低了52%和63%。5.通过神经示踪技术发现,抑制脊髓ATF6表达六周后,神经轴突修复被抑制。6.BIP m RNA表达水平在脊髓损伤后7天时呈显著性升高至假手术组的2倍。7.CHOP m RNA在脊髓损伤后4小时明显高表达于假手术组,且此时CHOP m RNA表达水平是假手术组的2倍,而在脊髓损伤后的7天CHOP m RNA表达水平呈显著性下降到假手术组的38%。结论:ATF6有助于斑马鱼脊髓损伤后运动能力的恢复和轴突的再生。而这种保护性的作用可能是通过活化后的ATF6促进内质网应激促存活分子BIP和抑制CHOP的表达保护运动神经元内环境稳态后来实现的。
[Abstract]:Objective: to use adult zebrafish spinal cord injury model. To study the effect of activating transcription factor 6 ATF6 on nerve repair after spinal cord injury in zebrafish. Methods: we established a spinal cord transection injury model on adult zebrafish aged 4-6 months. The spinal cord tissue of 4 mm lower segment of the injured point was taken at 4 hours and 12 hours after injury for real-time quantitative polymerase chain reaction (PCR) and in situ hybridization to detect the changes of ATF6 m RNA level and its expression localization. At the same time, ATF6 was co-labeled with motor neurons or radial glial cells at 4 hours and 12 hours and 3 days and 7 days after spinal cord injury. After spinal cord injury in zebrafish, the expression of ATF6 protein was inhibited by ATF6 morpholinoprotein. To detect the recovery of motor ability of zebrafish and the changes of nerve axon repair through the damaged site. Meanwhile, real-time quantitative polymerase chain reaction was used to detect the immunoreactive heavy chain binding protein of the iconic factor of endoplasmic reticulum stress. In order to explore the effect of endoplasmic reticulum stress on repair, the expression level of C / EBP homologus protein and C / EBP homologous protein C / EBP homologus protein Chopm RNA were observed to explore the effect of endoplasmic reticulum stress on repair. Results: 1. ATF6 m RNA level was maintained at a high level at 12 hours and 3 days after spinal cord injury in zebrafish. On the 7th day after injury, the expression level of ATF6 protein returned to the level of sham operation group. 2. ATF6 protein was expressed in motor neurons, and the expression level increased 12 hours and 3 days after spinal cord injury. 3. ATF6 protein was surrounded by radial glial cells. Moreover, there was no co-expression of .4.The motor ability of zebrafish treated with the inhibition of ATF6 protein expression at 2 and 3 weeks after spinal cord injury decreased by 52% and 63.5.The results of nerve tracing technique showed that the motility of zebrafish was significantly lower than that of control group. After 6 weeks of inhibition of ATF6 expression in spinal cord, the expression of BIPm RNA in axon repair was significantly increased at 7 days after spinal cord injury. The expression of chop m RNA was significantly higher than that in sham operation group at 4 hours after spinal cord injury, and the expression of BIP m RNA in sham operation group was significantly higher than that in sham operation group at 4 hours after spinal cord injury. The expression level of CHOP m RNA was twice as high as that in sham operation group. On the 7th day after spinal cord injury, the expression of CHOP m RNA decreased significantly to 38 in sham-operated group. Conclusion: ATF6 is helpful to the recovery of motor ability and regeneration of axons after spinal cord injury in zebrafish. Activation of ATF6 promotes endoplasmic reticulum stress to promote survival molecule BIP and inhibit the expression of CHOP to protect homeostasis in motor neurons.
【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

【参考文献】