褪黑素对大鼠急性脊髓损伤后自噬的影响

发布时间：2018-02-10 08:16

本文关键词： 脊髓损伤褪黑素自噬 LC3 Beclin-1　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的作为脊柱损伤最严重的并发症,急性脊髓损伤(acute spinal cord injury,ASCI)常引起躯体感觉与功能障碍的严重损害。对患者本人而言,其带来了严重的身体和心理伤害,对整个社会而言,其造成了巨大的经济负担。鉴于此,当今医学界对急性脊髓损伤的预防、治疗和康复给予了越来越多的重视和关注。随着病理生理学的发展,对于急性脊髓损伤的认识也逐渐加深,将其分为原发性和继发性脊髓损伤。由于目前临床无法干预原发性脊髓损伤,继发性脊髓损伤在ASCI中起着重要的作用,而神经细胞的凋亡与继发性脊髓损伤的发生密切相关。因此抑制神经元细胞的凋亡成为ASCI早期治疗的关键。细胞自噬是一种高度保守的生物自我保护机制,可以使变性的蛋白及丧失功能的细胞器降解并重新利用。近年来,研究证实细胞自噬与凋亡有着相互影响。褪黑素(MT)是由松果腺产生和分泌的两性分子,广泛存在于机体内,目前学者普遍认为,褪黑素通过减轻水肿、抑制凋亡、减少氧化应激等机制发挥其保护神经的作用。然而,褪黑素治疗脊髓损伤在抗凋亡方面的机制尚无统一结论。因此,在本研究中,我们通过在ASCI动物模型中予以褪黑素干预,观察急性脊髓损伤后自噬的发生并探讨褪黑素对自噬的影响,研究褪黑素的作用机制,为临床应用提供参考。材料与方法108只成年SD大鼠,按随机数字表法随机分为三组:假手术组(A组)、脊髓损伤组(B组)、褪黑素治疗组(C组)。A组仅行椎板切除,暴露T11-T12节段,不损伤脊髓神经。B组和C组用改良的Allen's法建立T11-T12脊髓损伤模型,并分别在脊髓损伤后10 min腹腔注射等体积的无水乙醇和100mg/kg的MT制剂。各组分别于术后6 h、12 h、24 h随机选取12只大鼠,取T11-T12节段脊髓标本,6个标本检测自噬相关蛋白的表达水平,6个标本HE染色和免疫荧光切片,观察脊髓损伤及修复情况。术后72h对3组大鼠进行Basso Beattie Bresnahan(BBB)运动功能评分。结果脊髓损伤后,B、C两组均出现了严重的下肢神经功能障碍,在术后72h,C组的BBB运动功能评分较B组明显上升(p㩳0.05);术后各时间点大鼠脊髓的微管相关蛋白1轻链3(LC3)表达水平比较:C组最高、B组次之、A组最低。在6h、12 h和24 h时间点上各组之间差异均有统计学意义(p㩳0.05);术后各时间点大鼠脊髓的Beclin-1表达水平比较:C组最高、B组次之、A组最低。在6h、12 h和24 h时间点上各组之间差异均有统计学意义(p㩳0.05);HE染色切片观察脊髓损伤程度:A组最轻、C组次之、B组最重,并且各组损伤时间越长,损伤程度越重;免疫荧光显示:C组LC3阳性神经元细胞最多、B组次之、A组最少。结论大鼠急性脊髓损伤后早期神经组织发生了自噬,应用褪黑素能增强自噬并在一定程度上起到保护神经功能的作用。
[Abstract]:Background and objective as the most serious complication of spinal injury, acute spinal cord injuryASCI often causes severe damage to somatosensory and functional disorders. In view of this, the medical profession has paid more and more attention to the prevention, treatment and rehabilitation of acute spinal cord injury. With the development of pathophysiology, The recognition of acute spinal cord injury is gradually deepened, which is divided into primary spinal cord injury and secondary spinal cord injury. Because it is difficult to intervene in primary spinal cord injury, secondary spinal cord injury plays an important role in ASCI. The apoptosis of neurons is closely related to the occurrence of secondary spinal cord injury. Therefore, inhibition of neuronal apoptosis is the key to early treatment of ASCI. Autophagy is a highly conservative biological self-protection mechanism. In recent years, studies have shown that autophagy and apoptosis interact with each other. Melatonin (MTT) is a amphoteric molecule produced and secreted by the pineal gland and is widely present in the body. At present, it is widely believed that melatonin protects nerves by reducing edema, inhibiting apoptosis and reducing oxidative stress. However, there is no uniform conclusion on the mechanism of anti-apoptosis of melatonin in spinal cord injury. In this study, we observed the occurrence of autophagy after acute spinal cord injury and studied the effect of melatonin on autophagy through the intervention of melatonin in ASCI animal model. Materials and methods 108 adult Sprague-Dawley rats were randomly divided into three groups: sham operation group (group A), spinal cord injury group (group B) and melatonin treatment group (group C). T11-T12 spinal cord injury model was established by modified Allen's method in group B and C without spinal cord injury. Anhydrous ethanol and 100 mg / kg MT were injected intraperitoneally 10 min after spinal cord injury. The expression level of autophagy related protein was detected in 6 spinal cord specimens from T11-T12 segment and HE staining and immunofluorescence sections were obtained from 6 specimens. The motor function score of Basso Beattie Bresnahanna BBB was evaluated 72 hours after operation. Results severe lower extremity neurological dysfunction appeared in group C after spinal cord injury. The BBB motor function score of group C was significantly higher than that of group B at 72 hours after operation. The expression level of microtubule associated protein 1 light chain 3 (LC3) in the spinal cord of rats at different time points after operation was lower than that in group B, the highest in group C, and the lowest in group A. there were significant differences in the expression of microtubule associated protein 1 between each group at 12 h and 24 h after operation. The expression level of Beclin-1 in spinal cord of rats at different time points after operation was lower than that in group B, the highest in group C, and the lowest in group A. the differences were statistically significant at the time points of 12 h and 24 h after operation. The degree of spinal cord injury in group A was the lighter than that in group B, and the longer the injury time was, the more serious the injury was. Immunofluorescence showed that the number of LC3 positive neurons was the most in group B and the lowest in group A. conclusion autophagy occurred in the early stage of spinal cord injury in rats. Melatonin can enhance autophagy and protect neural function to some extent.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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