臂丛神经根性撕脱伤对脊髓前角Nrf-2和HO-1表达的影响

发布时间：2018-04-27 20:52

本文选题：臂丛神经 + 根性撕脱　；参考：《河北医科大学》2017年硕士论文

【摘要】：臂丛神经损伤是较常见的致残性高的周围神经损伤之一,臂丛神经损伤尤其是根性撕脱伤常引起患者上肢运动、感觉功能障碍,严重影响了患者的生活质量。如何减轻并改善臂丛神经损伤后患者上肢的运动及感觉功能障碍,始终是国内外临床研究的重点。目前临床治疗臂丛神经损伤的主要措施是外科手术+药物治疗,这些治疗可使患者的肩肘功能得到部分恢复,但患者前臂、腕关节及手部的症状均未见明显缓解。其原因是臂丛神经根性撕脱伤数分钟后,机体立即程序性启动一系列“瀑布”式的病理生理生化反应,如微脉管系统损伤、炎性反应、自由基的生产、兴奋性氨基酸过度释放、脂质过氧化(LP)、神经纤维脱髓鞘和胶质瘢痕形成等,其中氧化应激引起的二次损伤是导致患肢根性撕脱伤后运动和感觉功能恢复效果差的主要因素之一。氧化应激是内环境中的氧化和抗氧化系统之间失衡,导致机体自由基产生增多和(或)清除能力下降。过多的氧自由基(ROS)在组织或细胞内蓄积并启动氧化链式反应,如使生物大分子物质蛋白质、脂质、DNA失去生物活性或降解、抑制线粒体功能、离子平衡失调及兴奋性毒性等,最终导致神经元的裂解死亡。与此同时,机体启动了多种防御系统抵抗氧化应激对细胞和组织的损伤。Nrf-2是机体调控细胞对抗内外环境中氧化应激损伤的转录因子之一。生理状态下,大部分Nrf-2与Keap1结合而处于失活状态。在创伤、炎症等刺激下,Nrf-2与Keap1解离并从胞浆转移至胞核,与抗氧化反应元件(antioxidant response element,ARE)结合,启动其下游的血红素加氧酶-1(heme oxygenase 1,HO-1)、Ⅱ相解毒酶、抗氧化酶、抗炎因子和钙离子调节蛋白等的表达以增强机体细胞的抗氧化能力。HO-1是机体内广泛存在的一种诱导酶,能够分解血红素,减轻其对细胞的损伤作用,并消耗机体内的游离氧,从而减少氧自由基的生成和蓄积。研究表明,上调Nrf-2-ARE通路能够减少缺血缺氧部位自由基的生成,加快自由基的清除,恢复损伤部位细胞内氧化及抗氧化系统之间的稳态,进而降低缺血缺氧对机体组织及细胞的损伤。因此,激活nrf-2/are通路可能成为治疗臂丛神经损伤的一个新靶点。本研究中通过he染色检测大鼠臂丛神经根性撕脱伤后脊髓前角运动神经元的存活率,判断臂丛神经根性撕脱伤对脊髓前角运动神经元的影响;利用qpcr和免疫印迹观察大鼠臂丛神经根性撕脱伤后不同时间点脊髓前角nrf-2和ho-1的表达改变情况,探究nrf-2-are通路在臂丛神经根性撕脱伤后表达变化的时序性意义,期望为临床通过抗氧化治疗臂丛神经根性撕脱损伤提供了新的靶点和理论依据。目的:观察大鼠臂丛神经根性撕脱伤后不同时间点前角运动神经元的存活率和nrf-2及ho-1表达的时序性改变,期望为临床通过抗氧化治疗臂丛神经根性撕脱损伤提供了新的靶点和理论依据。方法:1大鼠臂丛神经根性撕脱模型的制备参照郑圣鼐等人的颈后入路方式制备大鼠臂丛神经根性撕脱模型。2测定指标及检查方法通过he染色观察大鼠损伤侧脊髓前角运动神经元形态结构和存活率的改变;westernblot及qpcr检测大鼠损伤侧脊髓前角运动神经元nrf-2、ho-1的表达变化。结果:1大体观察术后存活所有模型大鼠一般情况良好,均表现为右前肢瘫痪症状,左前肢活动正常,各组动物均未出现后肢瘫痪或排便障碍等表现。2大鼠脊髓前角运动神经元形态学和存活率的改变大鼠脊髓前角运动神经元形态学改变:正常对照组脊髓组织:光学显微镜下可见脊髓前角灰白质交界清楚,白质形态正常,结构完整清晰,轴突排列规则;无出血、水肿灶,运动神经元丰富,胞体形态正常,细胞核及核周尼氏体清晰可见,染色正常。手术撕脱侧脊髓:光学显微镜下可见造模后各时间点手术侧白质明显水肿,出现大量空泡,轴索扭曲部分断裂;可见散在出血灶,脊髓前角内运动神经元数量减少甚至缺如,胞核不清,核周尼氏体消失,胞浆浓缩,染色加深。此外可见固缩运动神经元,染色加深。大鼠脊髓前角运动神经元计数及存活率:统计学分析发现,各对照组大鼠脊髓前角运动神经元存活率无明显区别;对照组、臂丛神经损伤1 h、3 h、6 h、12 h、24 h、2 d、3 d、7 d组大鼠脊髓前角运动神经元存活率存在明显不同。两组间比较发现,臂丛神经损伤1 h、3 h、6 h、12 h、24 h、2 d组大鼠脊髓前角运动神经元存活率与对照组相比无明显区别;与对照组相比,臂丛神经损伤3 d、7 d组大鼠脊髓前角运动神经元存活率明显降低,具有统计学意义。3大鼠损伤侧脊髓前角运动神经元内Nrf-2、HO-1的表达改变臂丛神经根性撕脱损伤后各时间点均可见Nrf-2和HO-1的阳性表达,Nrf-2和HO-1的含量随损伤时间的延长呈一定的梯度变化。臂丛神经根性撕脱损伤后脊髓前角Nrf-2浆蛋白含量明显降低;Nrf-2核蛋白和HO-1含量在臂丛神经根性撕脱损伤后表达逐渐升高,Nrf-2核蛋白损伤后1 d含量最高,2 d开始下降,7 d明显降低,HO-1损伤后1-2 d含量最高,3d开始下降,7 d明显降低。臂丛神经根性撕脱损伤后脊髓前角Nrf-2 mRNA和HO-1 mRNA的表达改变与蛋白变化基本一致,也具有一定的时序性,Nrf-2 mRNA和HO-1 mRNA在臂丛神经根性撕脱损伤后表达逐渐升高,损伤后分别在24 h和1-2 d表达达到最高,随后开始下降,7 d明显降低。结论:1通过颈、胸椎后入路手术方式可成功建立大鼠臂丛神经根性撕脱模型。2大鼠臂丛神经根性撕脱损伤后,脊髓前角运动神经元的存活率随时间的延长而逐渐下降。3大鼠臂丛神经根性撕脱后Nrf-2和HO-1含量逐渐升高,分别在2 d和24h达到高峰,之后逐渐下降,具有一定的时序性。4大鼠臂丛神经根性撕脱损伤后脊髓前角伴有Nrf-2/ARE通路的激活,这为通过抗氧化治疗臂丛神经根性撕脱损伤提供了一定的理论基础。
[Abstract]:Brachial plexus injury is one of the most common and high disability peripheral nerve injuries. Brachial plexus injury, especially root avulsion, often causes upper limb movement and sensory dysfunction, which seriously affects the quality of life of the patients. How to reduce and improve the motor and sensory dysfunction of the upper limb after brachial plexus injury is always domestic The main measures for the clinical study of the brachial plexus injury are surgical and drug therapy. These treatments can partially restore the patient's shoulder and elbow function, but the symptoms of the forearm, wrist and hand are not significantly relieved. The reason is the immediate procedure of the body after a few minutes of brachial plexus root avulsion. A series of "waterfall" types of pathophysiological reactions, such as microvascular system damage, inflammatory reaction, free radical production, excessive release of excitatory amino acids, lipid peroxidation (LP), nerve fiber demyelination and glial scar formation, are introduced in a series of "waterfall" and physiological and biochemical reactions. The two damage caused by oxidative stress is the movement and sensation after the root avulsion of the affected limbs. One of the major factors for the poor effect of functional recovery. Oxidative stress is an imbalance between oxidation and antioxidant systems in the internal environment, resulting in an increase in the production of free radicals and (or) reduced capacity of the body. Excessive oxygen free radicals (ROS) accumulate in tissues or cells and initiate oxidative chain reactions, such as biological macromolecules protein, lipid, and D NA loses biological activity or degradation, inhibits mitochondrial function, imbalance of ion balance and excitotoxicity, and eventually leads to neuronal cracking and death. At the same time, the body initiates a variety of defense systems to resist oxidative stress to cell and tissue damage,.Nrf-2 is the transcriptional cause of the body's regulation of cells against oxidative stress injury in the internal and external environment. Under the physiological state, most Nrf-2 and Keap1 are inactive. Under the stimulation of trauma and inflammation, Nrf-2 and Keap1 are dissociated and transferred from the cytoplasm to the nucleus, combining with the antioxidant response element (antioxidant response element, ARE), and starting its downstream hemoglobin oxygenase -1 (heme oxygenase 1, HO-1), phase II detoxification enzyme, and anti detoxification enzyme. The expression of oxidase, anti-inflammatory factor and calcium regulation protein to enhance the antioxidant capacity of the body cells.HO-1 is an inducible enzyme that exists widely in the body. It can decompose heme, reduce its damage to cells, and consume free oxygen in the body, thus reducing the formation and accumulation of oxygen free radicals. Research shows that up regulation of Nrf-2 -ARE pathway can reduce the formation of free radicals at the ischemic and anoxic sites, accelerate the free radical scavenging, restore the homeostasis of the oxidative and antioxidant systems within the injured site, and then reduce the damage to the tissues and cells of the body. Therefore, the activation of the nrf-2/are pathway may be a new target for the treatment of brachial plexus injury. The survival rate of the motor neurons in the anterior horn of the spinal cord after the brachial plexus root avulsion in rats was detected by HE staining, and the effect of the brachial plexus root avulsion on the motor neurons in the anterior horn of the spinal cord was determined. The expression of nrf-2 and HO-1 in the anterior horn of the spinal cord was observed by qPCR and Western blot. To explore the temporal significance of nrf-2-are pathway in the expression changes after brachial plexus root avulsion, hoping to provide a new target and theoretical basis for clinical antioxidation treatment of brachial plexus root avulsion injury. Objective: To observe the survival rate and nrf-2 of the anterior horn motoneurons at different time points after brachial plexus root avulsion in rats. And the temporal change of HO-1 expression is expected to provide a new target and theoretical basis for clinical anti oxidative treatment of brachial plexus root avulsion. Methods: the preparation of the brachial plexus root avulsion model in 1 rats was made with reference to Zheng Shengnai et al's posterior approach to the preparation of the.2 measurement index and the test side of the rat brachial plexus root avulsion model. The morphological structure and survival rate of motor neurons in the anterior horn of the injured side of the rat were observed by HE staining, and the expression of nrf-2 and HO-1 in the motor neurons of the anterior horn of the spinal cord was detected by Westernblot and qPCR. Results: 1 the general condition of all the rats survived after the operation was generally good, all of which showed the paralysis of the right forelimb, left The changes in the morphology and survival rate of the motor neurons in the anterior horn of the spinal cord of.2 rats were not found in all the animals. The morphological changes of the motor neurons in the anterior horn of the spinal cord were changed in rats. The spinal cord tissue of the normal control group: the gray matter junction of the anterior horn of the spinal cord was clear under the optical microscope, and the white matter was normal. The structure was complete and clear, and the axon was arranged regularly. There was no bleeding, edema, motoneurons, normal cell morphology. The nuclei and periuclear Nissl bodies were clearly visible, and the staining was normal. The operation avulsed lateral spinal cord: visible edema, large number of vacuoles, and axonal twisted part fracture in the operation side after the optical microscope. The number of motor neurons in the anterior horn of the spinal cord was reduced or even absent, the nucleus was not clear, the Nissl body disappeared, the cytoplasm concentration, and the dyeing deepened. In addition, the motor neurons of the spinal cord and the survival rate of the motor neurons in the anterior horn of the spinal cord were also seen. In the control group, the brachial plexus nerve injury was 1 h, 3 h, 6 h, 12 h, 24 h, 2 D, 3 D, and 7 d group, and the survival rate of the motor neuron in the anterior horn of spinal cord was significantly different. The comparison of the two groups found that the brachial plexus nerve injury was 1 h, 3 h, 6 h, 12 h, 24 24, and there was no significant difference between the spinal cord anterior horn motoneuron survival rate compared with the control group. Compared with the control group, the injury of the brachial plexus was 3 D, the survival rate of the motor neurons in the anterior horn of the spinal cord in the 7 d rats was significantly reduced. The positive expression of Nrf-2 and HO-1 in the injured lateral spinal cord motor neurons in the injured side of the.3 rats was observed and the content of Nrf-2 and HO-1 were observed with the expression of HO-1. The content of Nrf-2 plasma protein in the anterior horn of the spinal cord decreased significantly after the brachial plexus root avulsion injury, and the expression of Nrf-2 nuclear protein and HO-1 increased gradually after the brachial plexus root avulsion injury. The content of 1 d after the Nrf-2 nuclear protein injury was the highest, the 2 D began to decrease, 7 d decreased obviously, and 1-2 d after HO-1 injury. The amount of 3D began to decrease, and the 7 d decreased obviously. The expression of Nrf-2 mRNA and HO-1 mRNA in the anterior horn of the spinal cord after brachial plexus root avulsion was basically consistent with the changes of protein, and the expression of Nrf-2 mRNA and HO-1 mRNA increased gradually after the brachial plexus root avulsion injury. After injury, the expression was reached in 24 h and 1-2 D. The highest, then began to decline, 7 d decreased significantly. Conclusion: 1 through the neck, thoracic spinal posterior approach can successfully establish rat brachial plexus root avulsion model.2 rats brachial plexus root avulsion injury, the survival rate of motor neurons in the anterior horn of the spinal cord decreased with time and gradually decreased Nrf-2 and HO-1 after the root avulsion of the brachial plexus in.3 rats The content gradually increased and reached the peak at 2 D and 24h, then decreased gradually. The anterior horn of the brachial plexus root avulsion in.4 rats was activated by the Nrf-2/ARE pathway, which provided a theoretical basis for the treatment of the root avulsion injury of the brachial plexus.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R688

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