常压氧疗法干预对大鼠脑缺血再灌注损伤的保护作用及其机制研究
发布时间：2018-07-31 07:30
【摘要】：背景脑血管疾病是指因脑部血管内血液循环出现障碍而导致脑组织损伤损害的一类疾病,临床上以急性脑血管病为主,分为缺血性和出血性两类。作为世界上最常见的严重地威胁人类生命健康的疾病之一,脑血管疾病具有发病率高、死亡率高等特点,数据显示,每年全世界约有1500万人死于该病,居各种死因的首位。缺血性脑血管疾病(Ischemic cerebrovascular disease, ICVD)是指因主要为大脑供血的动脉出现闭塞或者狭窄而引起脑部组织缺血缺氧不足或中断,最终导致局部脑区组织出现坏死等相关的临床症状。有效的方法就是实施再灌注,通过溶栓或机械疏通的方式恢复缺血区的血液流通。但是很多时候,患者在缺血进行再灌注后,脑组织不但没有因此而实现功能恢复,反而使其结构和功能进一步加重,这就是所谓的脑缺血再灌注损伤(Cerebral ischemia-reperfusion injury, CIRI)。脑缺血再灌注损伤的病理过程及机制十分复杂,此过程是一个涉及多个环节的快速的级联反应,包括细胞凋亡基因的激活、兴奋性氨基酸的释放、细胞内钙离子稳态失衡、能量代谢出现障碍、氧化自由基的生成等。这些环节之间联系紧密、相互重叠、互为因果,形成了恶性循环,最终导致了脑组织细胞的坏死或凋亡。针对CIRI复杂的病理生理机制,目前已有重大研究进展,但有效的治疗方法仍然有限,许多研究人员希望通过药物实现治疗。一种思路就是使用溶栓药物,如重组组织型纤维蛋白酶原激活剂(recombinant tissue plasminogen activator, RTPA),但其具有易造成出血并加重损伤的风险。另一种思路就是采用神经保护剂,在缺血后级联反应的关键环节找到靶点加以阻断,以达期到有效减小脑梗体积并改善神经功能的目的,但经多次尝试,此种保护剂并未找到。常压氧疗法(normobaric oxygen therapy, NOT)又称为常压高浓度氧疗法,是指在常压环境利用特定的吸氧装置,在不加压的情况下使患者佩戴吸氧罩呼吸高浓度氧气的治疗方法。近年来许多研究发现,采用常压氧疗法处理大鼠急性缺血再灌注损伤模型,能明显减少模型鼠脑组织梗死体积及减轻脑水肿与炎症反应,可用于抢救急性脑缺血组织。但常压氧疗法是否能够真的有效地保护急性缺血再灌注损伤的脑组织,其具体机制如何,是亟待解决的问题,需进一步更加深入地研究。目的采用常压氧疗法处理急性大鼠脑缺血再灌注损伤模型,从行为学水平、组织学水平及细胞分子生物学水平上探讨常压氧疗法干预对大鼠局灶性脑缺血再灌注损伤的保护作用及其可能机制,为寻找有效治疗CIRI的临床方法提供依据。方法采用改良线栓塞法制备5只假手术组模型与10只大鼠脑缺血再灌注损伤模型,参照Zea-Longa的评分标准对模型大鼠进行神经功能学缺陷评分,用以评价造模是否成功；将70只清洁级同月龄的健康雄性SD大鼠随机分为假手术组,脑缺血再灌注模型组,NOT 3h组、6h组、9h组、12h组、24h组,假手术组只对颈总动脉与颈外动脉进行结扎处理,脑缺血再灌注模型组在脑缺血1.5h后拔出线栓实施血流再灌注24h,其余各组于脑缺血1.5h后拔线栓再灌注并放置于自制密闭常压高氧供应装置中分别持续处理3h、6h、9h、12h、24h,相应供氧时间结束即把大鼠放置于空气中；再灌注24h后,对各组大鼠进行神经功能学缺陷评分；取出脑组织,测定各组大鼠脑梗死体积与含水量；检测各组大鼠脑组织匀浆中超氧化物歧化酶(Superoxide dismutase, SOD)活性与丙二醛(Malondialdehyde, MDA)含量变化；利用实时荧光定量PCR (quantitative Real-time PCR, qRT-PCR)方法检测各组大鼠脑组织中炎症因子TNF-α与IL-β1转录表达变化；利用酶联免疫法(Euzyme linked immunosorbent assay, ELISA)与免疫印迹法(Western blot, WB)检测脑组织中炎症因子TNF-α与IL-β1蛋白水平表达变化。结果1、假手术组5只大鼠神经功能学缺陷评分均0分,模型组10只大鼠均出现对侧前爪不能完全伸展,向对侧倾倒或转圈等症状,评分与假手术组相比差异显著(P=0.000),表明模型组神经系统已受损伤,造模成功。2、假手术组10大鼠神经功能学缺陷评分均为O分,与假手术组相比,其他各组评分显著增加(P0.05),与模型组相比,NOT 3h组与6h组评分明显降低P0.05),差异具有统计学意义。3、除假手术组外,其余各组均有不同程度的脑梗死,程度不一,与模型组相比,NOT 3h组、6h组与9h组脑梗死体积明显变小,含水量明显减少(P0.05),差异显著,NOT 12h组与24h组与模型组相比差异不明显(P0.05)。4、与假手术组相比,其余各组脑组织SOD活性明显下降、MDA含量明显升高(P0.05),与模型组相比,NOT 3h组与6h组SOD活性显著升高、MDA含量显著降低(JF)0.05)。5、与假手术组相比,其余各组TNF-α与IL-B 1转录水平明显升高(P0.05),与模型组相比,NOT 3h组、6h组与9h的两个炎症因子转录水平有下降趋势,其中NOT 6h组下降最为显著(P0.05)。6、ELISA与wB结果显示,各组TNF-α与IL-β1蛋白表达水平均高于假手术组P0.05,相比于模型组,两个炎症因子在NOT 3h组下降最为显著(P0.05)。结论常压氧疗法对大鼠急性脑缺血再灌注损伤具有保护作用,NOT处理时间点不同与持续时间的长短对脑缺血再灌注损伤的保护疗效有非常重要的影响,在缺血1.5h内实施再灌注并采用NOT干预处理3h至9h均有一定的疗效,且干预3-6h效果最佳。常压氧疗法治疗CIRI可能是通过降低脑损伤区氧化应激刺激,下调促炎因子的表达末实现的,这就为临床上治疗CIRI提供了一定的依据。
[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.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.3
本文编号：2154865
[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.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.3
【参考文献】
相关期刊论文 前10条
1 曹秋云,江开达,潘旭东;大鼠脑缺血再灌注损伤IL-8变化的研究[J];放射免疫学杂志;2001年02期
2 范瑞明;余昌胤;杨丽;詹剑;;糖尿病大鼠缺血再灌注脑组织核转录因子-κB的表达及意义[J];贵州医药;2006年11期
3 高艳;;小胶质细胞生理特点及在脑缺血损伤中的作用[J];中国组织化学与细胞化学杂志;2012年06期
4 田士强,王任直,李桂林,王欣,张波,姚勇,窦万臣,孔燕国;大鼠局灶性脑缺血模型的改进[J];基础医学与临床;2004年04期
5 韩英,刘楠,陈荣华,郑安,黄华品;白细胞介素8在大鼠脑缺血再灌注损伤中的变化[J];中国动脉硬化杂志;2005年02期
6 冷耀明;毛卫未;殷小平;詹忠明;方欢欢;蒋钦;;糖尿病脑梗死患者血清IL-18、MMP-9水平与脑缺血损伤的关系[J];山东医药;2010年17期
7 狄政莉,万琪,来华安,王洪典;IL-1β和IL-6在脑缺血再灌注微血管内皮细胞炎症反应中的作用[J];西安医科大学学报;2001年05期
8 张红星;刘灵光;周利;黄浩;李璇;杨敏;;头针对急性脑缺血再灌注大鼠炎症反应的影响[J];中西医结合学报;2007年06期
9 吴建华;方云祥;;法舒地尔对大鼠脑缺血再灌注损伤炎症反应的影响[J];中国临床药理学与治疗学;2008年04期
10 方舒东;朱也森;;脑缺血再灌注损伤的病理生理研究进展[J];医学综述;2006年18期
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