【摘要】：背景腦血管疾病是指因腦部血管內(nèi)血液循環(huán)出現(xiàn)障礙而導致腦組織損傷損害的一類疾病,臨床上以急性腦血管病為主,分為缺血性和出血性兩類。作為世界上最常見的嚴重地威脅人類生命健康的疾病之一,腦血管疾病具有發(fā)病率高、死亡率高等特點,數(shù)據(jù)顯示,每年全世界約有1500萬人死于該病,居各種死因的首位。缺血性腦血管疾病(Ischemic cerebrovascular disease, ICVD)是指因主要為大腦供血的動脈出現(xiàn)閉塞或者狹窄而引起腦部組織缺血缺氧不足或中斷,最終導致局部腦區(qū)組織出現(xiàn)壞死等相關的臨床癥狀。有效的方法就是實施再灌注,通過溶栓或機械疏通的方式恢復缺血區(qū)的血液流通。但是很多時候,患者在缺血進行再灌注后,腦組織不但沒有因此而實現(xiàn)功能恢復,反而使其結構和功能進一步加重,這就是所謂的腦缺血再灌注損傷(Cerebral ischemia-reperfusion injury, CIRI)。腦缺血再灌注損傷的病理過程及機制十分復雜,此過程是一個涉及多個環(huán)節(jié)的快速的級聯(lián)反應,包括細胞凋亡基因的激活、興奮性氨基酸的釋放、細胞內(nèi)鈣離子穩(wěn)態(tài)失衡、能量代謝出現(xiàn)障礙、氧化自由基的生成等。這些環(huán)節(jié)之間聯(lián)系緊密、相互重疊、互為因果,形成了惡性循環(huán),最終導致了腦組織細胞的壞死或凋亡。針對CIRI復雜的病理生理機制,目前已有重大研究進展,但有效的治療方法仍然有限,許多研究人員希望通過藥物實現(xiàn)治療。一種思路就是使用溶栓藥物,如重組組織型纖維蛋白酶原激活劑(recombinant tissue plasminogen activator, RTPA),但其具有易造成出血并加重損傷的風險。另一種思路就是采用神經(jīng)保護劑,在缺血后級聯(lián)反應的關鍵環(huán)節(jié)找到靶點加以阻斷,以達期到有效減小腦梗體積并改善神經(jīng)功能的目的,但經(jīng)多次嘗試,此種保護劑并未找到。常壓氧療法(normobaric oxygen therapy, NOT)又稱為常壓高濃度氧療法,是指在常壓環(huán)境利用特定的吸氧裝置,在不加壓的情況下使患者佩戴吸氧罩呼吸高濃度氧氣的治療方法。近年來許多研究發(fā)現(xiàn),采用常壓氧療法處理大鼠急性缺血再灌注損傷模型,能明顯減少模型鼠腦組織梗死體積及減輕腦水腫與炎癥反應,可用于搶救急性腦缺血組織。但常壓氧療法是否能夠真的有效地保護急性缺血再灌注損傷的腦組織,其具體機制如何,是亟待解決的問題,需進一步更加深入地研究。目的采用常壓氧療法處理急性大鼠腦缺血再灌注損傷模型,從行為學水平、組織學水平及細胞分子生物學水平上探討常壓氧療法干預對大鼠局灶性腦缺血再灌注損傷的保護作用及其可能機制,為尋找有效治療CIRI的臨床方法提供依據(jù)。方法采用改良線栓塞法制備5只假手術組模型與10只大鼠腦缺血再灌注損傷模型,參照Zea-Longa的評分標準對模型大鼠進行神經(jīng)功能學缺陷評分,用以評價造模是否成功；將70只清潔級同月齡的健康雄性SD大鼠隨機分為假手術組,腦缺血再灌注模型組,NOT 3h組、6h組、9h組、12h組、24h組,假手術組只對頸總動脈與頸外動脈進行結扎處理,腦缺血再灌注模型組在腦缺血1.5h后拔出線栓實施血流再灌注24h,其余各組于腦缺血1.5h后拔線栓再灌注并放置于自制密閉常壓高氧供應裝置中分別持續(xù)處理3h、6h、9h、12h、24h,相應供氧時間結束即把大鼠放置于空氣中；再灌注24h后,對各組大鼠進行神經(jīng)功能學缺陷評分；取出腦組織,測定各組大鼠腦梗死體積與含水量；檢測各組大鼠腦組織勻漿中超氧化物歧化酶(Superoxide dismutase, SOD)活性與丙二醛(Malondialdehyde, MDA)含量變化；利用實時熒光定量PCR (quantitative Real-time PCR, qRT-PCR)方法檢測各組大鼠腦組織中炎癥因子TNF-α與IL-β1轉(zhuǎn)錄表達變化；利用酶聯(lián)免疫法(Euzyme linked immunosorbent assay, ELISA)與免疫印跡法(Western blot, WB)檢測腦組織中炎癥因子TNF-α與IL-β1蛋白水平表達變化。結果1、假手術組5只大鼠神經(jīng)功能學缺陷評分均0分,模型組10只大鼠均出現(xiàn)對側前爪不能完全伸展,向?qū)葍A倒或轉(zhuǎn)圈等癥狀,評分與假手術組相比差異顯著(P=0.000),表明模型組神經(jīng)系統(tǒng)已受損傷,造模成功。2、假手術組10大鼠神經(jīng)功能學缺陷評分均為O分,與假手術組相比,其他各組評分顯著增加(P0.05),與模型組相比,NOT 3h組與6h組評分明顯降低P0.05),差異具有統(tǒng)計學意義。3、除假手術組外,其余各組均有不同程度的腦梗死,程度不一,與模型組相比,NOT 3h組、6h組與9h組腦梗死體積明顯變小,含水量明顯減少(P0.05),差異顯著,NOT 12h組與24h組與模型組相比差異不明顯(P0.05)。4、與假手術組相比,其余各組腦組織SOD活性明顯下降、MDA含量明顯升高(P0.05),與模型組相比,NOT 3h組與6h組SOD活性顯著升高、MDA含量顯著降低(JF)0.05)。5、與假手術組相比,其余各組TNF-α與IL-B 1轉(zhuǎn)錄水平明顯升高(P0.05),與模型組相比,NOT 3h組、6h組與9h的兩個炎癥因子轉(zhuǎn)錄水平有下降趨勢,其中NOT 6h組下降最為顯著(P0.05)。6、ELISA與wB結果顯示,各組TNF-α與IL-β1蛋白表達水平均高于假手術組P0.05,相比于模型組,兩個炎癥因子在NOT 3h組下降最為顯著(P0.05)。結論常壓氧療法對大鼠急性腦缺血再灌注損傷具有保護作用,NOT處理時間點不同與持續(xù)時間的長短對腦缺血再灌注損傷的保護療效有非常重要的影響,在缺血1.5h內(nèi)實施再灌注并采用NOT干預處理3h至9h均有一定的療效,且干預3-6h效果最佳。常壓氧療法治療CIRI可能是通過降低腦損傷區(qū)氧化應激刺激,下調(diào)促炎因子的表達末實現(xiàn)的,這就為臨床上治療CIRI提供了一定的依據(jù)。
[Abstract]:Background cerebrovascular disease is a kind of disease which is caused by the impairment of blood circulation in the blood vessels of the brain. It is mainly divided into two types of ischemic and hemorrhagic diseases, which are mainly acute cerebral vascular diseases. As one of the most common and serious diseases threatening human life and health in the world, the incidence of cerebrovascular diseases is high and death is high. High death rate, data show that about 15 million people all over the world die of the disease every year, the leading cause of all kinds of death. Ischemic cerebrovascular disease (Ischemic cerebrovascular disease, ICVD) refers to the insufficiency or interruption of cerebral ischemia and hypoxia caused by the occlusion or stenosis of the arteries supplying the blood of the brain, which eventually leads to the local brain. The effective method is to carry out reperfusion and restore the blood circulation in the ischemic area by thrombolytic or mechanical dredging. But in many cases, after reperfusion, the brain not only does not restore the function, but further aggravates the structure and function of the brain tissue. It is called Cerebral ischemia-reperfusion injury (CIRI). The pathological process and mechanism of cerebral ischemia reperfusion injury is very complicated. This process is a rapid cascade reaction involving multiple links, including the activation of apoptosis genes, the release of excitatory amino acids, and the homeostasis of intracellular calcium ions. There is an obstacle in energy metabolism and the formation of free radicals. These links are closely linked, overlapping and cause and effect, forming a vicious cycle, which eventually leads to the necrosis or apoptosis of the brain cells. In view of the complex pathophysiological mechanism of CIRI, there have been significant advances in research, but the effective treatment methods are still limited and many studies have been studied. People want to be treated with drugs. One idea is to use thrombolytic drugs such as the recombinant recombinant tissue plasminogen activator (RTPA), but it has a risk of causing bleeding and aggravating damage. Another idea is to use neuroprotectant, the key ring of cascade reaction after ischemia. Normobaric oxygen therapy (NOT), also known as the normal pressure and high concentration oxygen therapy, refers to the use of specific oxygen inhalation devices in the ambient pressure ring, which is made under non pressure conditions. In recent years, many studies have shown that the treatment of acute ischemia reperfusion injury model in rats by atmospheric pressure oxygen therapy can reduce the infarct volume in the brain tissue of the model rats and reduce the brain edema and inflammation, and can be used to rescue the acute cerebral ischemia tissue. How to effectively protect the brain tissue of acute ischemia-reperfusion injury is an urgent problem and need to be further studied. Objective to deal with the model of cerebral ischemia reperfusion injury in acute rats by atmospheric pressure oxygen therapy, and discuss the normal pressure from the level of behavior, histology and cell molecular biology. The protective effect of oxygen therapy on focal cerebral ischemia reperfusion injury in rats and its possible mechanism provide the basis for finding a clinical method for effective treatment of CIRI. Methods 5 models of sham operation group and 10 rat models of cerebral ischemia-reperfusion injury were prepared by modified linear embolization method, and the model rats were evaluated according to the standard of Zea-Longa. 70 healthy male SD rats of the same month age were randomly divided into sham operation group, cerebral ischemia reperfusion model group, group NOT 3h, group 6h, group 9h, group 9h, 12h group, 24h group, and the sham operation group only ligation of the common carotid artery and the external carotid artery, and the cerebral ischemia reperfusion model group was in the brain deficiency. After the blood 1.5h was pulled out to carry out the blood flow reperfusion 24h, the rest of the other groups were placed in the self-made closed atmospheric high oxygen supply device after cerebral ischemia 1.5h and placed in the self-made high oxygen supply device of closed atmospheric pressure. The rats were treated with 3h, 6h, 9h, 12h, 24h, and the rats were placed in the air at the end of the corresponding oxygen supply time. The brain tissue was taken out and the volume and water content of cerebral infarction were measured in each group. The changes of Superoxide dismutase (SOD) activity and the content of malondialdehyde (Malondialdehyde, MDA) were detected in the homogenate of each group, and the methods of real-time fluorescence quantitative PCR (quantitative Real-time PCR, qRT-PCR) were used to detect each group. The changes in the transcription and expression of inflammatory factors TNF- alpha and IL- beta 1 in the rat brain tissue, and the changes in the expression of TNF- alpha and IL- beta 1 in brain tissues by enzyme linked immunosorbent assay (Euzyme linked immunosorbent assay, ELISA) and Western blot (Western blot, WB). Results 1, the scores of neurological deficits of 5 rats in the sham operation group were 0 scores. In the 10 rats of the model group, the symptoms of the lateral front claw could not be fully extended, to the opposite side of the dump or turn circle. The score was significantly different from the sham group (P=0.000). It showed that the model group had been injured, the model was successful.2, and the scores of the neural functional defects in the sham operation group were all O points, compared with the sham operation group, the scores of the other groups were significantly higher than those in the sham operation group. In addition to the model group, compared with the model group, the NOT 3H group and the 6h group scored significantly lower P0.05), the difference was statistically significant.3. Except for the sham operation group, the other groups had different degrees of cerebral infarction, the degree was different. Compared with the model group, the NOT 3H group, the 6h group and the 9h group were significantly smaller, the water content decreased significantly (P0.05), the difference was significant, NOT was significant, the difference was significant, the difference was significant. Compared with the sham group, the SOD activity of the other groups was significantly decreased and the MDA content was significantly increased (P0.05). Compared with the model group, the SOD activity of the NOT 3H group and the 6h group was significantly higher than that in the sham group. Compared with the model group, the SOD activity of the NOT 3H group and the 6h group was significantly increased, and the MDA content was significantly reduced (0.05). Compared with the sham group, the SOD activity was significantly reduced (0.05), and the other groups were transcribed with 1 transcripts compared with the sham operation group. The level of two inflammatory factors in NOT 3H group, 6h group and 9h decreased, and NOT 6h group decreased most significantly (P0.05).6, and ELISA and wB showed that the expression level of TNF- alpha and beta 1 protein in each group was higher than that of the artificial hand group. Compared with the model group, the two inflammatory factors were compared with the model group. The decrease is most significant (P0.05). Conclusion atmospheric oxygen therapy has a protective effect on acute cerebral ischemia-reperfusion injury in rats. The different time points and duration of NOT treatment have a very important effect on the protective effect of cerebral ischemia-reperfusion injury. The application of reperfusion in ischemic 1.5h and the intervention of NOT to 3H to 9h have a certain therapeutic effect. Effect, and intervention in the best effect of 3-6h. Atmospheric oxygen therapy for the treatment of CIRI may be by reducing the oxidative stress stimulation in the brain damage area and down-regulation of the expression of proinflammatory factors, which provides a certain basis for clinical treatment of CIRI.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R743.3

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