白细胞介素17A和干细胞因子对小鼠大脑中动脉缺血再灌注损伤的影响
发布时间：2018-08-01 15:29
【摘要】：背景缺血性脑卒中因其高发病率、高致残率和高死亡率而严重威胁人类健康。目前已超过肿瘤和心血管病,在全世界死亡原因中排名第二,在我国已成为第一位的疾病死亡原因和致残原因。目前公认的治疗急性缺血性脑卒中最有效的办法是发病4.5h内的重组人组织型纤溶酶原激活物(recombinant tissue plasminogen activator,rt-PA)静脉溶栓治疗。但迄今为止,静脉溶栓率一直很低,发达国家仍不到10%,我国则只有2%左右,因此,可以从静脉溶栓获益的患者仍很少。近年来通过血管内机械再通治疗急性缺血性脑卒中有了较大的发展,但也仅仅将脑梗死的治疗时间窗延长至6h之内,且从多模式血管内机械再通相关的临床研究发现,虽再通率高达90%,但良好转归却只有50%左右,这说明血管再通率与临床良好预后存在不匹配现象,提示很大一部分患者出现了无效再灌注或再灌注损伤。因此探索脑梗死急性期时间窗以外的治疗方法仍然尤为迫切。近年研究显示,炎症反应在脑缺血再灌注损伤的发生发展中起着关键性作用。γδT细胞分泌的IL-17A可诱导单核细胞、中性粒细胞等前往缺血脑组织增强炎症反应,该作用是导致脑缺血再灌注损伤炎症级联放大反应的关键因素之一。γδT细胞主要有Vγ1 T细胞和Vγ4 T细胞两个亚群,脑缺血再灌注损伤后IL-17A的高表达主要来源于Vγ1 T细胞亚群还是Vγ4 T细胞亚群至今仍不清楚。有研究者发现在小鼠大脑中动脉缺血再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)术后3 h给予抗白细胞介素-17A(anti-IL-17A)抗体可明显抑制炎症反应及减少脑梗死体积,还可同时促进脑缺血后神经功能的恢复。在脑缺血再灌注损伤治疗方面,近年关于干细胞因子(stem cell factor,SCF)的研究逐渐增多,SCF是一种重要的造血生长因子。研究发现小鼠MCAO/R术后3 h至7天,连续每天给予SCF可诱导脑组织缺血区周边神经发生、血管再生和改善神经功能,从而促进神经元的修复和侧支循环的建立。但脑缺血再灌注损伤的过程十分复杂,当前有效治疗方法仍有限。因此,进一步研究脑缺血再灌注损伤过程,寻找新的治疗方法来有效地减轻脑梗死、降低死亡率及致残率仍是临床研究的一大课题。因目前尚少见anti-IL-17A联合SCF治疗脑缺血再灌注损伤相关报道,且anti-IL-17A和SCF在治疗脑缺血再灌注损伤时发挥的修复作用各自不同,所以本实验旨在通过观察anti-IL-17A联合使用SCF相比单独使用anti-IL-17A或SCF能否为小鼠脑缺血再灌注损伤带来更好的治疗效果。此外,本实验还进一步探索脑缺血再灌注损伤后IL-17A的高表达主要来源于γδT细胞的Vγ1 T细胞亚群还是Vγ4 T细胞亚群,以及清除相关细胞亚群后脑缺血再灌注损伤能否得到相应改善。争取为临床脑梗死患者治疗提供新的研究思路。一、小鼠大脑中动脉缺血再灌注模型的建立与评价目的:建立小鼠大脑中动脉缺血再灌注模型并予以评价。方法:40只成年雄性C57BL/6小鼠随机分为假手术组(Sham)10只,手术组(MCAO/R)30只。通过线栓法建立小鼠大脑中动脉缺血再灌注(MCAO/R)模型,缺血60min后恢复右侧大脑中动脉血流。于术后24h观察两组小鼠神经功能评分、脑梗死体积、脑组织病理学改变等情况。结果:术后24h观察,Sham组神经功能评分为(0±0)分,脑梗死体积为(0±0)mm3,脑组织病理学检查未见明显异常。MCAO/R组小鼠均出现了不同程度的神经功能损伤,神经功能评分为(2.79±0.52)分,脑梗死体积为(72.46±23.61)mm3,且脑组织病理学检查出现了典型的脑梗死病理改变。结论:线栓法制备小鼠大脑中动脉缺血再灌注损伤模型成功率较高,脑梗死体积稳定、可靠,可控性及重现性好,与临床脑缺血再灌注损伤患者的症状表现、影像学和病理学改变相接近,可用于后续研究白细胞介素17A和干细胞因子对小鼠脑缺血再灌注损伤的影响。二、联合应用抗白细胞介素17A抗体和干细胞因子对小鼠大脑中动脉缺血再灌注损伤的影响目的探讨联合应用抗白细胞介素17A(anti-IL-17A)抗体和干细胞因子(stem cell factor,SCF)对小鼠大脑中动脉缺血再灌注损伤的影响。方法通过线栓法对成年雄性C57BL/6小鼠建立大脑中动脉缺血再灌注(MCAO/R)模型,缺血60min后恢复右侧大脑中动脉血流。根据小鼠MCAO/R术后处理因素的不同,分为生理盐水(normal saline,NS)组(n=5)、SCF组(n=30)、anti-IL-17A组(n=30)及anti-IL-17A+SCF组(n=30)。在不同时间点分别观察各组小鼠MCAO/R术后神经功能评分、脑梗死体积、脑水肿程度、脑组织病理损伤、神经细胞凋亡以及炎症反应等各项指标的变化。结果与NS组相比,其余各组神经功能评分得到明显改善(P0.05),脑梗死体积、脑水肿程度及脑组织病理损伤均显著减轻(P0.05),神经细胞凋亡明显减少(P0.05),且anti-IL-17A+SCF组显著优于anti-IL-17A组及SCF组(P0.05)。此外,anti-IL-17A还能显著降低脑组织IL-17AmRNA和IL-1βm RNA的表达(P0.05)。结论联合应用anti-IL-17A和SCF能更好地减轻小鼠大脑中动脉缺血再灌注损伤以及促进神经功能的恢复。三、探索脑缺血再灌注损伤后白细胞介素17A的主要来源细胞目的探索脑缺血再灌注损伤后白细胞介素17A的主要来源细胞。方法通过线栓法对成年雄性C57BL/6小鼠建立大脑中动脉缺血再灌注(MCAO/R)模型,缺血60min后恢复右侧大脑中动脉血流。小鼠分为野生型小鼠组(WT,n=30)、TCRδ-/-小鼠组(n=30)、IL-17A-/-小鼠组(n=30)、Vγ1 T细胞清除小鼠组(Vγ1 Depletion,n=30)、Vγ4 T细胞清除小鼠组(Vγ4 Depletion,n=30)。于MCAO/R术后第3天观察各组小鼠神经功能评分、脑梗死体积、IL-17A含量等变化。结果MCAO/R术后第3天,与WT组相比,TCRd-/-组、IL-17A-/-组、Vγ4 Depletion组神经功能评分、脑梗死体积均得到明显改善(P0.05),但WT组与Vγ1 Depletion组相比较无显著差异。Vγ4 Depletion组相较Control IgG组IL-17A阳性细胞数、IL-17A和IL-17AmRNA表达量均显著减少(P0.05)。结论在小鼠大脑中动脉缺血再灌注损伤后脑组织IL-17A的高表达主要来源于γδT细胞的Vγ4 T细胞亚群,且清除Vγ4 T细胞亚群后可显著减轻小鼠的神经功能损伤和脑梗死体积。
[Abstract]:Background ischemic stroke is a serious threat to human health because of its high incidence, high disability rate and high mortality. It is now more than cancer and cardiovascular disease. It ranks second in the world's cause of death and is the first cause of death and disability in China. The most effective treatment for acute ischemic stroke is the most effective treatment. The method is the recombinant human tissue type plasminogen activator (recombinant tissue plasminogen activator, rt-PA) intravenous thrombolytic therapy in 4.5H. But so far, the rate of venous thrombolysis has been very low, the developed countries are still less than 10%, and our country is only about 2%. Therefore, the patients who can benefit from venous thrombolysis are still very few. Mechanical recanalization has a great development in the treatment of acute ischemic stroke, but it only extends the time window of the cerebral infarction to within 6h, and the clinical study of the multimode intravascular mechanical recanalization has found that the recanalization rate is up to 90%, but the good prognosis is only about 50%. This shows that the rate of vascular recanalization is not in good condition with the clinical prognosis. The matching phenomenon suggests a large number of patients with ineffective reperfusion or reperfusion injury. Therefore, it is still particularly urgent to explore the treatment methods outside the acute phase window of cerebral infarction. In recent years, the inflammatory reaction plays a key role in the occurrence and development of cerebral ischemia reperfusion injury. The IL-17A secreted by gamma Delta T cells can be induced to be single. Nuclear cells and neutrophils go to the ischemic brain tissue to enhance the inflammatory response, which is one of the key factors leading to the cascade of inflammation in the cerebral ischemia reperfusion injury. There are two subgroups of V gamma 1 T cells and V gamma 4 T cells. The high expression of IL-17A is mainly derived from the V gamma 1 T cell subgroup after cerebral ischemia-reperfusion injury. The V gamma 4 T cell subgroup is still unclear. Some researchers found that the anti leukocyte -17A (anti-IL-17A) antibody can obviously inhibit the inflammatory response and reduce the volume of cerebral infarction in the 3 h of middle cerebral artery occlusion/reperfusion, MCAO/R, and can also promote the nerve function after cerebral ischemia. The study of stem cell factor (SCF) is increasing in recent years. SCF is an important hematopoietic growth factor. The study found that SCF can induce peripheral neurogenesis, vascular regeneration and improvement of nerve in the ischemic area of brain group from 3 to 7 days after MCAO/R operation. Function, thus promoting the repair of neurons and the establishment of collateral circulation, but the process of cerebral ischemia reperfusion injury is very complex, and the current effective treatment is still limited. Therefore, further study of the process of cerebral ischemia reperfusion injury, search for new treatment methods to reduce the death of cerebral infarction and reduce the mortality and disability rate is still a clinical study. A major topic. There are few reports of anti-IL-17A combined with SCF in the treatment of cerebral ischemia reperfusion injury, and the effect of anti-IL-17A and SCF in the treatment of cerebral ischemia reperfusion injury is different. Therefore, the purpose of this experiment is to observe whether anti-IL-17A or SCF can be used separately for brain deficiency in mice by using anti-IL-17A combined with SCF. Blood reperfusion injury brings better therapeutic effect. In addition, this experiment further explores the high expression of IL-17A in V gamma 1 T cells or V gamma 4 T cell subsets, which are mainly derived from gamma delta T cells after cerebral ischemia-reperfusion injury, and can improve the cerebral ischemia reperfusion injury after scavenging related subsets. The purpose of establishing and evaluating the model of middle cerebral artery ischemia reperfusion in mice was to establish and evaluate the model of middle cerebral artery ischemia reperfusion in mice. Methods: 40 adult male C57BL/6 mice were randomly divided into 10 rats in the sham operation group (Sham), and 30 in the operation group (MCAO/R). Cerebral ischemia reperfusion (MCAO/R) model of rat middle cerebral artery and recovery of right middle cerebral artery blood flow after ischemic 60min. After 24h, the neurological function score of two groups of mice, cerebral infarction volume, and pathological changes of brain tissue were observed. Results: after 24h observation, the score of nerve function in group Sham was (0 + 0), cerebral infarction volume was (0 + 0) mm3, brain histopathology There was no obvious abnormal abnormal nerve function injury in the.MCAO/R group. The score of nerve function was (2.79 + 0.52), the volume of cerebral infarction was (72.46 + 23.61) mm3, and the pathological changes of cerebral infarction appeared. Conclusion: the ischemic reperfusion injury model of middle cerebral artery in mice was prepared by the thread thrombus method. The success rate is high, the volume of cerebral infarction is stable, reliable, controllable and reproducible, it is close to the symptoms of the patients with cerebral ischemia reperfusion injury, and the changes of imaging and pathology are close. The effect of interleukin 17A and stem cell factor on the injury of cerebral ischemia reperfusion in mice can be studied. Two, the combination of anti leukin 17A The effect of antibodies and stem cell factors on ischemia reperfusion injury in the middle cerebral artery in mice objective to explore the effect of combined application of anti leukocyte 17A (anti-IL-17A) antibody and stem cell factor (SCF) on the ischemia reperfusion injury in the middle cerebral artery of mice. Methods the brain of adult male C57BL/6 mice was established by the thread thrombus method. Ischemia and reperfusion (MCAO/R) model of middle artery and recovery of the right middle cerebral artery blood flow after 60min ischemia. According to the different treatment factors after MCAO/R operation in mice, it was divided into normal saline (NS) group (n=5), SCF group (n=30), anti-IL-17A group (n=30) and anti-IL-17A group. Function score, cerebral infarction volume, cerebral edema degree, brain tissue pathological injury, nerve cell apoptosis and inflammatory reaction. Compared with group NS, the scores of other groups were significantly improved (P0.05), cerebral infarction volume, brain edema degree and brain tissue pathological damage were significantly reduced (P0.05), nerve cell withering The mortality was significantly decreased (P0.05), and group anti-IL-17A+SCF was significantly better than group anti-IL-17A and SCF (P0.05). In addition, anti-IL-17A could significantly reduce the expression of IL-17AmRNA and IL-1 beta m RNA in brain tissue (P0.05). Conclusion combined use of anti-IL-17A and minerals can better reduce ischemia reperfusion injury in the middle cerebral artery of mice and promote the recovery of nerve function. Complex. Three, explore the main source of interleukin 17A after cerebral ischemia reperfusion injury to explore the main source of interleukin 17A after cerebral ischemia reperfusion injury. Methods the middle cerebral artery ischemia reperfusion (MCAO/R) model of adult male C57BL/6 mice was established by the thread thrombus method, and the right cerebral middle movement was restored after ischemic 60min. Blood flow. The mice were divided into wild type mice (WT, n=30), TCR Delta / - mouse group (n=30), IL-17A-/- mice group (n=30), V gamma 1 T cells scavenging mice group (V 1 Depletion, n=30), V gamma 4 cells cleared the mice group (n=30). After third days, the neurological function scores, cerebral infarction volume, and other changes were observed. Third days after MCAO/R, compared with group WT, the neurological function score of group TCRd-/-, IL-17A-/- group and V gamma 4 Depletion group was significantly improved (P0.05), but there was no significant difference between WT group and V gamma 1 Depletion group. 0.05). Conclusion the high expression of IL-17A in the brain tissue after ischemia reperfusion injury of the middle cerebral artery in mice is mainly derived from the V gamma 4 T cell subgroup of the gamma delta T cells, and the scavenging of V gamma 4 T cell subsets can significantly reduce the impairment of neural function and the volume of cerebral infarction in mice.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
本文编号：2158017
[Abstract]:Background ischemic stroke is a serious threat to human health because of its high incidence, high disability rate and high mortality. It is now more than cancer and cardiovascular disease. It ranks second in the world's cause of death and is the first cause of death and disability in China. The most effective treatment for acute ischemic stroke is the most effective treatment. The method is the recombinant human tissue type plasminogen activator (recombinant tissue plasminogen activator, rt-PA) intravenous thrombolytic therapy in 4.5H. But so far, the rate of venous thrombolysis has been very low, the developed countries are still less than 10%, and our country is only about 2%. Therefore, the patients who can benefit from venous thrombolysis are still very few. Mechanical recanalization has a great development in the treatment of acute ischemic stroke, but it only extends the time window of the cerebral infarction to within 6h, and the clinical study of the multimode intravascular mechanical recanalization has found that the recanalization rate is up to 90%, but the good prognosis is only about 50%. This shows that the rate of vascular recanalization is not in good condition with the clinical prognosis. The matching phenomenon suggests a large number of patients with ineffective reperfusion or reperfusion injury. Therefore, it is still particularly urgent to explore the treatment methods outside the acute phase window of cerebral infarction. In recent years, the inflammatory reaction plays a key role in the occurrence and development of cerebral ischemia reperfusion injury. The IL-17A secreted by gamma Delta T cells can be induced to be single. Nuclear cells and neutrophils go to the ischemic brain tissue to enhance the inflammatory response, which is one of the key factors leading to the cascade of inflammation in the cerebral ischemia reperfusion injury. There are two subgroups of V gamma 1 T cells and V gamma 4 T cells. The high expression of IL-17A is mainly derived from the V gamma 1 T cell subgroup after cerebral ischemia-reperfusion injury. The V gamma 4 T cell subgroup is still unclear. Some researchers found that the anti leukocyte -17A (anti-IL-17A) antibody can obviously inhibit the inflammatory response and reduce the volume of cerebral infarction in the 3 h of middle cerebral artery occlusion/reperfusion, MCAO/R, and can also promote the nerve function after cerebral ischemia. The study of stem cell factor (SCF) is increasing in recent years. SCF is an important hematopoietic growth factor. The study found that SCF can induce peripheral neurogenesis, vascular regeneration and improvement of nerve in the ischemic area of brain group from 3 to 7 days after MCAO/R operation. Function, thus promoting the repair of neurons and the establishment of collateral circulation, but the process of cerebral ischemia reperfusion injury is very complex, and the current effective treatment is still limited. Therefore, further study of the process of cerebral ischemia reperfusion injury, search for new treatment methods to reduce the death of cerebral infarction and reduce the mortality and disability rate is still a clinical study. A major topic. There are few reports of anti-IL-17A combined with SCF in the treatment of cerebral ischemia reperfusion injury, and the effect of anti-IL-17A and SCF in the treatment of cerebral ischemia reperfusion injury is different. Therefore, the purpose of this experiment is to observe whether anti-IL-17A or SCF can be used separately for brain deficiency in mice by using anti-IL-17A combined with SCF. Blood reperfusion injury brings better therapeutic effect. In addition, this experiment further explores the high expression of IL-17A in V gamma 1 T cells or V gamma 4 T cell subsets, which are mainly derived from gamma delta T cells after cerebral ischemia-reperfusion injury, and can improve the cerebral ischemia reperfusion injury after scavenging related subsets. The purpose of establishing and evaluating the model of middle cerebral artery ischemia reperfusion in mice was to establish and evaluate the model of middle cerebral artery ischemia reperfusion in mice. Methods: 40 adult male C57BL/6 mice were randomly divided into 10 rats in the sham operation group (Sham), and 30 in the operation group (MCAO/R). Cerebral ischemia reperfusion (MCAO/R) model of rat middle cerebral artery and recovery of right middle cerebral artery blood flow after ischemic 60min. After 24h, the neurological function score of two groups of mice, cerebral infarction volume, and pathological changes of brain tissue were observed. Results: after 24h observation, the score of nerve function in group Sham was (0 + 0), cerebral infarction volume was (0 + 0) mm3, brain histopathology There was no obvious abnormal abnormal nerve function injury in the.MCAO/R group. The score of nerve function was (2.79 + 0.52), the volume of cerebral infarction was (72.46 + 23.61) mm3, and the pathological changes of cerebral infarction appeared. Conclusion: the ischemic reperfusion injury model of middle cerebral artery in mice was prepared by the thread thrombus method. The success rate is high, the volume of cerebral infarction is stable, reliable, controllable and reproducible, it is close to the symptoms of the patients with cerebral ischemia reperfusion injury, and the changes of imaging and pathology are close. The effect of interleukin 17A and stem cell factor on the injury of cerebral ischemia reperfusion in mice can be studied. Two, the combination of anti leukin 17A The effect of antibodies and stem cell factors on ischemia reperfusion injury in the middle cerebral artery in mice objective to explore the effect of combined application of anti leukocyte 17A (anti-IL-17A) antibody and stem cell factor (SCF) on the ischemia reperfusion injury in the middle cerebral artery of mice. Methods the brain of adult male C57BL/6 mice was established by the thread thrombus method. Ischemia and reperfusion (MCAO/R) model of middle artery and recovery of the right middle cerebral artery blood flow after 60min ischemia. According to the different treatment factors after MCAO/R operation in mice, it was divided into normal saline (NS) group (n=5), SCF group (n=30), anti-IL-17A group (n=30) and anti-IL-17A group. Function score, cerebral infarction volume, cerebral edema degree, brain tissue pathological injury, nerve cell apoptosis and inflammatory reaction. Compared with group NS, the scores of other groups were significantly improved (P0.05), cerebral infarction volume, brain edema degree and brain tissue pathological damage were significantly reduced (P0.05), nerve cell withering The mortality was significantly decreased (P0.05), and group anti-IL-17A+SCF was significantly better than group anti-IL-17A and SCF (P0.05). In addition, anti-IL-17A could significantly reduce the expression of IL-17AmRNA and IL-1 beta m RNA in brain tissue (P0.05). Conclusion combined use of anti-IL-17A and minerals can better reduce ischemia reperfusion injury in the middle cerebral artery of mice and promote the recovery of nerve function. Complex. Three, explore the main source of interleukin 17A after cerebral ischemia reperfusion injury to explore the main source of interleukin 17A after cerebral ischemia reperfusion injury. Methods the middle cerebral artery ischemia reperfusion (MCAO/R) model of adult male C57BL/6 mice was established by the thread thrombus method, and the right cerebral middle movement was restored after ischemic 60min. Blood flow. The mice were divided into wild type mice (WT, n=30), TCR Delta / - mouse group (n=30), IL-17A-/- mice group (n=30), V gamma 1 T cells scavenging mice group (V 1 Depletion, n=30), V gamma 4 cells cleared the mice group (n=30). After third days, the neurological function scores, cerebral infarction volume, and other changes were observed. Third days after MCAO/R, compared with group WT, the neurological function score of group TCRd-/-, IL-17A-/- group and V gamma 4 Depletion group was significantly improved (P0.05), but there was no significant difference between WT group and V gamma 1 Depletion group. 0.05). Conclusion the high expression of IL-17A in the brain tissue after ischemia reperfusion injury of the middle cerebral artery in mice is mainly derived from the V gamma 4 T cell subgroup of the gamma delta T cells, and the scavenging of V gamma 4 T cell subsets can significantly reduce the impairment of neural function and the volume of cerebral infarction in mice.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
【参考文献】
相关期刊论文 前6条
1 卜跃华;解芳;;脑脊液IL-17和IL-23检测在诊断细菌性脑膜炎中的意义[J];中国医科大学学报;2012年05期
2 王树;张力;;脑缺血及脑缺血再灌注损伤动物模型制备方法及评价[J];神经药理学报;2011年03期
3 何学令,尹海林;脑缺血动物模型的研究进展[J];中国比较医学杂志;2004年04期
4 赖先阳;γδT细胞的发育与其信号传导[J];国外医学(免疫学分册);2003年05期
5 王军;大鼠脑缺血模型研究进展[J];中医研究;2002年05期
6 刘红梅,高天明,佟振清;急性脑缺血动物模型实验研究进展[J];第一军医大学学报;1999年04期
相关博士学位论文 前1条
1 贺教江;IL-17在大脑皮质发育障碍癫痫发生中的作用研究[D];第三军医大学;2013年
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