本文選題：臂叢神經 + 根性撕脫　； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：臂叢神經損傷是較常見的致殘性高的周圍神經損傷之一,臂叢神經損傷尤其是根性撕脫傷常引起患者上肢運動、感覺功能障礙,嚴重影響了患者的生活質量。如何減輕并改善臂叢神經損傷后患者上肢的運動及感覺功能障礙,始終是國內外臨床研究的重點。目前臨床治療臂叢神經損傷的主要措施是外科手術+藥物治療,這些治療可使患者的肩肘功能得到部分恢復,但患者前臂、腕關節(jié)及手部的癥狀均未見明顯緩解。其原因是臂叢神經根性撕脫傷數分鐘后,機體立即程序性啟動一系列“瀑布”式的病理生理生化反應,如微脈管系統損傷、炎性反應、自由基的生產、興奮性氨基酸過度釋放、脂質過氧化(LP)、神經纖維脫髓鞘和膠質瘢痕形成等,其中氧化應激引起的二次損傷是導致患肢根性撕脫傷后運動和感覺功能恢復效果差的主要因素之一。氧化應激是內環(huán)境中的氧化和抗氧化系統之間失衡,導致機體自由基產生增多和(或)清除能力下降。過多的氧自由基(ROS)在組織或細胞內蓄積并啟動氧化鏈式反應,如使生物大分子物質蛋白質、脂質、DNA失去生物活性或降解、抑制線粒體功能、離子平衡失調及興奮性毒性等,最終導致神經元的裂解死亡。與此同時,機體啟動了多種防御系統抵抗氧化應激對細胞和組織的損傷。Nrf-2是機體調控細胞對抗內外環(huán)境中氧化應激損傷的轉錄因子之一。生理狀態(tài)下,大部分Nrf-2與Keap1結合而處于失活狀態(tài)。在創(chuàng)傷、炎癥等刺激下,Nrf-2與Keap1解離并從胞漿轉移至胞核,與抗氧化反應元件(antioxidant response element,ARE)結合,啟動其下游的血紅素加氧酶-1(heme oxygenase 1,HO-1)、Ⅱ相解毒酶、抗氧化酶、抗炎因子和鈣離子調節(jié)蛋白等的表達以增強機體細胞的抗氧化能力。HO-1是機體內廣泛存在的一種誘導酶,能夠分解血紅素,減輕其對細胞的損傷作用,并消耗機體內的游離氧,從而減少氧自由基的生成和蓄積。研究表明,上調Nrf-2-ARE通路能夠減少缺血缺氧部位自由基的生成,加快自由基的清除,恢復損傷部位細胞內氧化及抗氧化系統之間的穩(wěn)態(tài),進而降低缺血缺氧對機體組織及細胞的損傷。因此,激活nrf-2/are通路可能成為治療臂叢神經損傷的一個新靶點。本研究中通過he染色檢測大鼠臂叢神經根性撕脫傷后脊髓前角運動神經元的存活率,判斷臂叢神經根性撕脫傷對脊髓前角運動神經元的影響;利用qpcr和免疫印跡觀察大鼠臂叢神經根性撕脫傷后不同時間點脊髓前角nrf-2和ho-1的表達改變情況,探究nrf-2-are通路在臂叢神經根性撕脫傷后表達變化的時序性意義,期望為臨床通過抗氧化治療臂叢神經根性撕脫損傷提供了新的靶點和理論依據。目的:觀察大鼠臂叢神經根性撕脫傷后不同時間點前角運動神經元的存活率和nrf-2及ho-1表達的時序性改變,期望為臨床通過抗氧化治療臂叢神經根性撕脫損傷提供了新的靶點和理論依據。方法:1大鼠臂叢神經根性撕脫模型的制備參照鄭圣鼐等人的頸后入路方式制備大鼠臂叢神經根性撕脫模型。2測定指標及檢查方法通過he染色觀察大鼠損傷側脊髓前角運動神經元形態(tài)結構和存活率的改變;westernblot及qpcr檢測大鼠損傷側脊髓前角運動神經元nrf-2、ho-1的表達變化。結果:1大體觀察術后存活所有模型大鼠一般情況良好,均表現為右前肢癱瘓癥狀,左前肢活動正常,各組動物均未出現后肢癱瘓或排便障礙等表現。2大鼠脊髓前角運動神經元形態(tài)學和存活率的改變大鼠脊髓前角運動神經元形態(tài)學改變:正常對照組脊髓組織:光學顯微鏡下可見脊髓前角灰白質交界清楚,白質形態(tài)正常,結構完整清晰,軸突排列規(guī)則;無出血、水腫灶,運動神經元豐富,胞體形態(tài)正常,細胞核及核周尼氏體清晰可見,染色正常。手術撕脫側脊髓:光學顯微鏡下可見造模后各時間點手術側白質明顯水腫,出現大量空泡,軸索扭曲部分斷裂;可見散在出血灶,脊髓前角內運動神經元數量減少甚至缺如,胞核不清,核周尼氏體消失,胞漿濃縮,染色加深。此外可見固縮運動神經元,染色加深。大鼠脊髓前角運動神經元計數及存活率:統計學分析發(fā)現,各對照組大鼠脊髓前角運動神經元存活率無明顯區(qū)別;對照組、臂叢神經損傷1 h、3 h、6 h、12 h、24 h、2 d、3 d、7 d組大鼠脊髓前角運動神經元存活率存在明顯不同。兩組間比較發(fā)現,臂叢神經損傷1 h、3 h、6 h、12 h、24 h、2 d組大鼠脊髓前角運動神經元存活率與對照組相比無明顯區(qū)別;與對照組相比,臂叢神經損傷3 d、7 d組大鼠脊髓前角運動神經元存活率明顯降低,具有統計學意義。3大鼠損傷側脊髓前角運動神經元內Nrf-2、HO-1的表達改變臂叢神經根性撕脫損傷后各時間點均可見Nrf-2和HO-1的陽性表達,Nrf-2和HO-1的含量隨損傷時間的延長呈一定的梯度變化。臂叢神經根性撕脫損傷后脊髓前角Nrf-2漿蛋白含量明顯降低;Nrf-2核蛋白和HO-1含量在臂叢神經根性撕脫損傷后表達逐漸升高,Nrf-2核蛋白損傷后1 d含量最高,2 d開始下降,7 d明顯降低,HO-1損傷后1-2 d含量最高,3d開始下降,7 d明顯降低。臂叢神經根性撕脫損傷后脊髓前角Nrf-2 mRNA和HO-1 mRNA的表達改變與蛋白變化基本一致,也具有一定的時序性,Nrf-2 mRNA和HO-1 mRNA在臂叢神經根性撕脫損傷后表達逐漸升高,損傷后分別在24 h和1-2 d表達達到最高,隨后開始下降,7 d明顯降低。結論:1通過頸、胸椎后入路手術方式可成功建立大鼠臂叢神經根性撕脫模型。2大鼠臂叢神經根性撕脫損傷后,脊髓前角運動神經元的存活率隨時間的延長而逐漸下降。3大鼠臂叢神經根性撕脫后Nrf-2和HO-1含量逐漸升高,分別在2 d和24h達到高峰,之后逐漸下降,具有一定的時序性。4大鼠臂叢神經根性撕脫損傷后脊髓前角伴有Nrf-2/ARE通路的激活,這為通過抗氧化治療臂叢神經根性撕脫損傷提供了一定的理論基礎。
[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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R688

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