【摘要】：背景缺血性腦卒中因其高發(fā)病率、高致殘率和高死亡率而嚴重威脅人類健康。目前已超過腫瘤和心血管病,在全世界死亡原因中排名第二,在我國已成為第一位的疾病死亡原因和致殘原因。目前公認的治療急性缺血性腦卒中最有效的辦法是發(fā)病4.5h內(nèi)的重組人組織型纖溶酶原激活物(recombinant tissue plasminogen activator,rt-PA)靜脈溶栓治療。但迄今為止,靜脈溶栓率一直很低,發(fā)達國家仍不到10%,我國則只有2%左右,因此,可以從靜脈溶栓獲益的患者仍很少。近年來通過血管內(nèi)機械再通治療急性缺血性腦卒中有了較大的發(fā)展,但也僅僅將腦梗死的治療時間窗延長至6h之內(nèi),且從多模式血管內(nèi)機械再通相關(guān)的臨床研究發(fā)現(xiàn),雖再通率高達90%,但良好轉(zhuǎn)歸卻只有50%左右,這說明血管再通率與臨床良好預(yù)后存在不匹配現(xiàn)象,提示很大一部分患者出現(xiàn)了無效再灌注或再灌注損傷。因此探索腦梗死急性期時間窗以外的治療方法仍然尤為迫切。近年研究顯示,炎癥反應(yīng)在腦缺血再灌注損傷的發(fā)生發(fā)展中起著關(guān)鍵性作用。γδT細胞分泌的IL-17A可誘導(dǎo)單核細胞、中性粒細胞等前往缺血腦組織增強炎癥反應(yīng),該作用是導(dǎo)致腦缺血再灌注損傷炎癥級聯(lián)放大反應(yīng)的關(guān)鍵因素之一。γδT細胞主要有Vγ1 T細胞和Vγ4 T細胞兩個亞群,腦缺血再灌注損傷后IL-17A的高表達主要來源于Vγ1 T細胞亞群還是Vγ4 T細胞亞群至今仍不清楚。有研究者發(fā)現(xiàn)在小鼠大腦中動脈缺血再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)術(shù)后3 h給予抗白細胞介素-17A(anti-IL-17A)抗體可明顯抑制炎癥反應(yīng)及減少腦梗死體積,還可同時促進腦缺血后神經(jīng)功能的恢復(fù)。在腦缺血再灌注損傷治療方面,近年關(guān)于干細胞因子(stem cell factor,SCF)的研究逐漸增多,SCF是一種重要的造血生長因子。研究發(fā)現(xiàn)小鼠MCAO/R術(shù)后3 h至7天,連續(xù)每天給予SCF可誘導(dǎo)腦組織缺血區(qū)周邊神經(jīng)發(fā)生、血管再生和改善神經(jīng)功能,從而促進神經(jīng)元的修復(fù)和側(cè)支循環(huán)的建立。但腦缺血再灌注損傷的過程十分復(fù)雜,當前有效治療方法仍有限。因此,進一步研究腦缺血再灌注損傷過程,尋找新的治療方法來有效地減輕腦梗死、降低死亡率及致殘率仍是臨床研究的一大課題。因目前尚少見anti-IL-17A聯(lián)合SCF治療腦缺血再灌注損傷相關(guān)報道,且anti-IL-17A和SCF在治療腦缺血再灌注損傷時發(fā)揮的修復(fù)作用各自不同,所以本實驗旨在通過觀察anti-IL-17A聯(lián)合使用SCF相比單獨使用anti-IL-17A或SCF能否為小鼠腦缺血再灌注損傷帶來更好的治療效果。此外,本實驗還進一步探索腦缺血再灌注損傷后IL-17A的高表達主要來源于γδT細胞的Vγ1 T細胞亞群還是Vγ4 T細胞亞群,以及清除相關(guān)細胞亞群后腦缺血再灌注損傷能否得到相應(yīng)改善。爭取為臨床腦梗死患者治療提供新的研究思路。一、小鼠大腦中動脈缺血再灌注模型的建立與評價目的:建立小鼠大腦中動脈缺血再灌注模型并予以評價。方法:40只成年雄性C57BL/6小鼠隨機分為假手術(shù)組(Sham)10只,手術(shù)組(MCAO/R)30只。通過線栓法建立小鼠大腦中動脈缺血再灌注(MCAO/R)模型,缺血60min后恢復(fù)右側(cè)大腦中動脈血流。于術(shù)后24h觀察兩組小鼠神經(jīng)功能評分、腦梗死體積、腦組織病理學(xué)改變等情況。結(jié)果:術(shù)后24h觀察,Sham組神經(jīng)功能評分為(0±0)分,腦梗死體積為(0±0)mm3,腦組織病理學(xué)檢查未見明顯異常。MCAO/R組小鼠均出現(xiàn)了不同程度的神經(jīng)功能損傷,神經(jīng)功能評分為(2.79±0.52)分,腦梗死體積為(72.46±23.61)mm3,且腦組織病理學(xué)檢查出現(xiàn)了典型的腦梗死病理改變。結(jié)論:線栓法制備小鼠大腦中動脈缺血再灌注損傷模型成功率較高,腦梗死體積穩(wěn)定、可靠,可控性及重現(xiàn)性好,與臨床腦缺血再灌注損傷患者的癥狀表現(xiàn)、影像學(xué)和病理學(xué)改變相接近,可用于后續(xù)研究白細胞介素17A和干細胞因子對小鼠腦缺血再灌注損傷的影響。二、聯(lián)合應(yīng)用抗白細胞介素17A抗體和干細胞因子對小鼠大腦中動脈缺血再灌注損傷的影響目的探討聯(lián)合應(yīng)用抗白細胞介素17A(anti-IL-17A)抗體和干細胞因子(stem cell factor,SCF)對小鼠大腦中動脈缺血再灌注損傷的影響。方法通過線栓法對成年雄性C57BL/6小鼠建立大腦中動脈缺血再灌注(MCAO/R)模型,缺血60min后恢復(fù)右側(cè)大腦中動脈血流。根據(jù)小鼠MCAO/R術(shù)后處理因素的不同,分為生理鹽水(normal saline,NS)組(n=5)、SCF組(n=30)、anti-IL-17A組(n=30)及anti-IL-17A+SCF組(n=30)。在不同時間點分別觀察各組小鼠MCAO/R術(shù)后神經(jīng)功能評分、腦梗死體積、腦水腫程度、腦組織病理損傷、神經(jīng)細胞凋亡以及炎癥反應(yīng)等各項指標的變化。結(jié)果與NS組相比,其余各組神經(jīng)功能評分得到明顯改善(P0.05),腦梗死體積、腦水腫程度及腦組織病理損傷均顯著減輕(P0.05),神經(jīng)細胞凋亡明顯減少(P0.05),且anti-IL-17A+SCF組顯著優(yōu)于anti-IL-17A組及SCF組(P0.05)。此外,anti-IL-17A還能顯著降低腦組織IL-17AmRNA和IL-1βm RNA的表達(P0.05)。結(jié)論聯(lián)合應(yīng)用anti-IL-17A和SCF能更好地減輕小鼠大腦中動脈缺血再灌注損傷以及促進神經(jīng)功能的恢復(fù)。三、探索腦缺血再灌注損傷后白細胞介素17A的主要來源細胞目的探索腦缺血再灌注損傷后白細胞介素17A的主要來源細胞。方法通過線栓法對成年雄性C57BL/6小鼠建立大腦中動脈缺血再灌注(MCAO/R)模型,缺血60min后恢復(fù)右側(cè)大腦中動脈血流。小鼠分為野生型小鼠組(WT,n=30)、TCRδ-/-小鼠組(n=30)、IL-17A-/-小鼠組(n=30)、Vγ1 T細胞清除小鼠組(Vγ1 Depletion,n=30)、Vγ4 T細胞清除小鼠組(Vγ4 Depletion,n=30)。于MCAO/R術(shù)后第3天觀察各組小鼠神經(jīng)功能評分、腦梗死體積、IL-17A含量等變化。結(jié)果MCAO/R術(shù)后第3天,與WT組相比,TCRd-/-組、IL-17A-/-組、Vγ4 Depletion組神經(jīng)功能評分、腦梗死體積均得到明顯改善(P0.05),但WT組與Vγ1 Depletion組相比較無顯著差異。Vγ4 Depletion組相較Control IgG組IL-17A陽性細胞數(shù)、IL-17A和IL-17AmRNA表達量均顯著減少(P0.05)。結(jié)論在小鼠大腦中動脈缺血再灌注損傷后腦組織IL-17A的高表達主要來源于γδT細胞的Vγ4 T細胞亞群,且清除Vγ4 T細胞亞群后可顯著減輕小鼠的神經(jīng)功能損傷和腦梗死體積。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R743.3

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