慢性挤压伤后鼠神经生长因子对鼠腰脊神经节内AQP-2表达影响的研究

发布时间：2018-01-19 19:15

  本文关键词： 鼠神经生长因子(mNGF) 水通道蛋白-2(AQP-2) 脊神经节 免疫组化　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的:鼠神经生长因子(mNGF)分子量约为26.5×103,它是一种大分子蛋白质,具有生物活性,mNGF最初是在小鼠颌下腺中提取而获得的,它具有营养人类正常细胞的作用,在神经损伤后,尤其是对外周神经具有促进恢复和再生的作用,目前在临床上已被广泛用于治疗中枢及外周神经损伤,并且获得了比较理想的的临床疗效。腰脊神经节与神经根相邻,该部位是感觉神经的胞体集中区域,当炎症或者外来暴露对其造成损伤后,患侧肢体会出现感觉功能的异常。随着人类年龄增长,腰椎会逐渐发生退行性的改变,如椎间盘突出,椎管狭窄等,这些退变常可导致腰脊神经节(Dorsal root ganglion,DRG)的损伤,因此腰脊神经节损伤后的修复对临床具有重要的意义。本研究中利用结扎实验大鼠的坐骨神经来模拟建立腰脊神经节损伤的动物模型,通过鼠神经生长因子(mNGF)的治疗来研究其在腰脊神经节慢性损伤后促进神经功能恢复的作用,并研究其对脊神经节内水通道蛋白-2(AQP-2)含量变化的影响,了解临床治疗过程中脊神经节内分子水平的变化情况,为治疗提供理论依据。方法:选取健康雄性成年SD大鼠90只,体重200-250g,将这90只实验鼠随机分为三组,假手术组,手术组,mNGF治疗组,每组平均30只,在假手术组中将大鼠左侧坐骨神经给予暴露,不给予结扎;手术组暴露左侧坐骨神经,在距神经起始处附近用4.0含铬羊肠线结扎坐骨神经4道,每道间隔约1-2 mm,被结扎的神经长约4.5 mm,然后逐层缝合,mNGF组处理同手术组,三组分别于手术前,术后第3天,7天,14天行热缩足潜伏期、及机械缩足反应阈测试。并与术后第3天,第7天,和第14天对三个实验组中的部分大鼠进行处死并留取脊神经节标本,然后对标本进行免疫组织化学染色观察脊神经节内神经元染色情况,Western blot分析来定量分析AQP-2量。结果:1行为学测试术后3天三组间热缩足潜伏期分别为13.5±0.6s,5.4±0.3s,6.4±0.4s,第7天分别为13.5±0.6s,5.8±0.3s,8.3±0.7s,第14天分别为13.3±0.6s,5.8±0.3s,10.7±0.8s;术后第3天、7天、14天三组间差异均有统计学意义(P0.05)。术后3天三组间机械缩足反应阈分别为34.3±1.3s,15.9±0.8s,22.5±0.8s,第7天分别为34.3±0.9s,15.9±1.0s,24.8±0.8s,第14天分别为34.3±0.5s,15.6±0.6s,28.6±1.3s;术后第3天、7天、14天三组间差异均有统计学意义(P0.05)。2免疫组化染色假手术组的脊神经节免疫组化染色术后第3天,第7天,第14天染色较浅,随时间增长染色变化不大;手术组于术后第3天进行免疫组织化学染色可见大量点状的DRG细胞被染成棕黄色,随着时间增长,在第7天,第14天,阳性细胞染色变化不大;mNGF组于术后3天和第7天染色程度与手术组相差不大,第14天可见染色细胞数明显减少,染色变浅。3 Western blot分析分别与术后3天,7天,14天对假手术组,手术组,以及mNGF组脊神经节内AQP-2表达量行定量分析,结果以积分光密度(IOD)值的平均值±标准差表示,术后3天假手术组与手术组和mNGF组分别为328±20,2744±77,2173±110,三组间有统计学差异(P0.05),术后第7天三组IOD值分别为323±16,2299±93,1664±54、三组间有统计学差异(P0.05),术后第14天三组IOD值分别为323±16,1948±40,841±46,三组间有统计学差异(P0.05)。结论:腰脊神经节损伤后给予应用mNGF进行治疗,可明显增长大鼠热缩足潜伏期,提高机械缩足反应阈,减轻感觉过敏症状,同时腰脊神经节慢性挤压伤可造成腰脊神经节内AQP-2表达增多,mNGF可降低AQP-2的表达量。
[Abstract]:Background and objective: mouse nerve growth factor (mNGF) molecular weight is about 26.5 * 103, it is a kind of macromolecular protein with biological activity, mNGF was extracted and obtained in the mouse submandibular gland, it has the nutrition of human normal cells, after nerve injury, especially with peripheral nerve promote the recovery and regeneration, is currently in clinical practice has been widely used in the treatment of central and peripheral nerve injury, and has obtained the ideal clinical curative effect. Spinal ganglion and nerve root adjacent to the site of somatic sensory nerve area when inflammation or damage to the external exposure after suffering limb abnormalities occur in sensory function. With the growth of age, will gradually lumbar degenerative changes, such as disc herniation, spinal stenosis, which often leads to degeneration of lumbar spinal nerve section (Dorsal root ganglion, DRG) damage, so the spinal ganglion injury repair has important significance for clinical. To simulate the animal model of spinal ganglion injury in rats by ligation of the sciatic nerve in this study, the rat nerve growth factor (mNGF) treatment to study the recovery of neurological function in patients with chronic injury spinal ganglion, and to study the spinal ganglion of aquaporin -2 (AQP-2) content changes, understand the changes in the molecular level in the spinal ganglia in the course of clinical treatment, and provide theoretical basis for treatment. Methods: healthy adult male SD rats 90, weight 200-250g, the 90 experimental rats were randomly divided into three groups, sham operation group, operation group, mNGF treatment group, each group had an average of 30, exposed in the sham group, rats with left sciatic nerve ligation surgery group, not given; the left sciatic nerve was exposed. 璺濈�炵粡璧峰�嬪�勯檮杩戠敚�

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