本文選題：激活轉(zhuǎn)錄因子6　切入點：脊髓損傷　出處：《延安大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:本論文利用成年斑馬魚脊髓橫斷損傷模型,研究激活轉(zhuǎn)錄因子6(activating transcription factor 6,ATF6)對斑馬魚脊髓損傷后神經(jīng)修復的影響作用和相關(guān)機制。方法:我們在4-6月齡的成年斑馬魚上建立脊髓橫斷損傷模型,在損傷后4小時、12小時、3天、7天取損傷點下段4mm的脊髓組織進行實時定量聚合酶鏈反應(yīng)和原位雜交,檢測ATF6 m RNA水平變化和表達定位,同時在脊髓損傷后4小時、12小時、3天、7天對ATF6與運動神經(jīng)元或與放射狀膠質(zhì)細胞進行共標。斑馬魚脊髓損傷后,用ATF6 morpholino(MO)抑制ATF6蛋白質(zhì)表達,檢測斑馬魚運動能力恢復情況以及穿過損傷位點神經(jīng)軸突修復的水平變化。同時再用實時定量聚合酶鏈反應(yīng)的方法檢測內(nèi)質(zhì)網(wǎng)應(yīng)激標志性因子免疫重鏈結(jié)合蛋白(binding immunoglobulin protein,BIP)BIP和C/EBP同源蛋白(C/EBP homologus protein,CHOP)m RNA的表達水平變化以探究內(nèi)質(zhì)網(wǎng)應(yīng)激對修復的影響。結(jié)果:1.ATF6 m RNA水平在斑馬魚脊髓損傷后的12小時和3天維持在較高的表達水平,而在損傷后7天,其恢復到假手術(shù)組的表達水平。2.ATF6蛋白表達在運動神經(jīng)元中,并且在脊髓損傷后12小時和3天表達水平增高。3.ATF6蛋白環(huán)繞在放射狀膠質(zhì)細胞中,并且在脊髓損傷后不同時間和對照組都不共表達。4.斑馬魚脊髓損傷后第2和第3周,MO抑制ATF6蛋白表達處理組斑馬魚運動能力比對照組分別降低了52%和63%。5.通過神經(jīng)示蹤技術(shù)發(fā)現(xiàn),抑制脊髓ATF6表達六周后,神經(jīng)軸突修復被抑制。6.BIP m RNA表達水平在脊髓損傷后7天時呈顯著性升高至假手術(shù)組的2倍。7.CHOP m RNA在脊髓損傷后4小時明顯高表達于假手術(shù)組,且此時CHOP m RNA表達水平是假手術(shù)組的2倍,而在脊髓損傷后的7天CHOP m RNA表達水平呈顯著性下降到假手術(shù)組的38%。結(jié)論:ATF6有助于斑馬魚脊髓損傷后運動能力的恢復和軸突的再生。而這種保護性的作用可能是通過活化后的ATF6促進內(nèi)質(zhì)網(wǎng)應(yīng)激促存活分子BIP和抑制CHOP的表達保護運動神經(jīng)元內(nèi)環(huán)境穩(wěn)態(tài)后來實現(xiàn)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.
【學位授予單位】：延安大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R651.2

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