本文關(guān)鍵詞： 全腦缺血模型 血腦屏障 GPER-1 VEGF-A　出處：《第四軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：背景:缺血性腦卒中是全球死亡率的第二大原因。系統(tǒng)研究腦缺血的治療和機(jī)制需要一個(gè)穩(wěn)定的、可復(fù)制性高的全腦缺血模型。盡管在許多研究模型中使用小鼠和沙鼠,但是可靠的全腦缺血大鼠模型也是實(shí)驗(yàn)所需要的,并且有待進(jìn)一步改善。四血管阻斷法是一種廣泛使用的全腦缺血模型。手術(shù)需要灼燒大鼠寰椎的翼狀孔,永久性阻斷雙側(cè)椎動(dòng)脈。但是,術(shù)者往往無法直視下電凝椎動(dòng)脈使其完全閉塞。因此,建立成功的模型需要依賴觀察大鼠的翻正反射和腦電圖表現(xiàn)。本課題需要建立一種穩(wěn)定的全腦缺血模型,為后續(xù)實(shí)驗(yàn)打好基礎(chǔ)。血腦屏障是血漿與腦細(xì)胞之間的屏障,對中樞神經(jīng)系統(tǒng)的內(nèi)穩(wěn)態(tài)和正常功能的維持至關(guān)重要。血腦屏障內(nèi)皮細(xì)胞的功能和形態(tài)與非腦組織毛細(xì)血管內(nèi)皮細(xì)胞截然不同。在許多神經(jīng)系統(tǒng)疾病中,血腦屏障的完整性會(huì)遭到破壞,例如缺血性腦卒中。腦缺血對血腦屏障的損傷作用已被廣泛研究。腦缺血缺氧會(huì)引起腦內(nèi)一系列的分子變化,導(dǎo)致內(nèi)皮細(xì)胞緊密連接破壞,血腦屏障通透性增加。許多研究證明腦缺血后雌激素可通過阻止血腦屏障的破壞從而保護(hù)腦組織。雌激素的腦保護(hù)作用是由雌激素受體介導(dǎo)的(ERα、ERβ、GPER-1)。有研究表明ERα和ERβ可介導(dǎo)雌激素對腦缺血后血管內(nèi)皮細(xì)胞連接蛋白的保護(hù)作用。但是,GPER-1在腦缺血導(dǎo)致的血腦屏障損傷中的作用目前還不清楚。目的:1.探索大鼠椎動(dòng)脈與頸椎的位置關(guān)系,尋找阻斷大鼠椎動(dòng)脈的最佳部位。2.建立穩(wěn)定性高的新型全腦缺血模型,探索最佳缺血時(shí)間和最佳缺血部位。3.探索全腦缺血對去勢大鼠海馬CA1區(qū)血腦屏障以及GPER-1和VEGF-A表達(dá)的影響。4.探索激動(dòng)GPER-1受體是否可通過降低VEGF-A表達(dá)來保護(hù)全腦缺血大鼠CA1區(qū)血腦屏障。方法:1.采用CTA、DSA、血管乳膠灌注法明確大鼠椎動(dòng)脈走行以及與頸椎的解剖關(guān)系。尋找阻斷大鼠椎動(dòng)脈的最佳部位。2.建立新型全腦缺血模型。根據(jù)全腦缺血10、20、30分鐘后大鼠的存活率,得出大鼠全腦缺血的最佳持續(xù)時(shí)間。隨后進(jìn)行灌注、固定、取腦,采用尼氏染色和TUNEL標(biāo)記染色法觀察腦缺血后大鼠各個(gè)腦區(qū)神經(jīng)元的死亡情況,評估新型全腦缺血模型的穩(wěn)定性。最后根據(jù)大鼠神經(jīng)功能評分量表和膠帶實(shí)驗(yàn)結(jié)果,觀察全腦缺血大鼠的行為學(xué)變化,進(jìn)一步評估模型對大鼠行為學(xué)的損害情況。3.采用Western blot實(shí)驗(yàn)技術(shù)觀察全腦缺血20min后大鼠CA1區(qū)血腦屏障通透性的變化(Ig G漏出率)和連接蛋白(Occludin、Claudin-5)的表達(dá)變化。同時(shí)也對腦缺血后的CA1區(qū)GPER-1受體和VEGF-A蛋白的表達(dá)變化進(jìn)行觀察。4.采用Western blot和免疫熒光雙標(biāo)染色觀察激動(dòng)GPER-1受體對全腦缺血后大鼠CA1區(qū)血腦屏障通透性(Ig G漏出率)和連接蛋白(Occludin、Claudin-5)表達(dá)的影響。隨后采用Western blot和免疫組織化學(xué)染色法探索激動(dòng)GPER-1受體是否可通過降低VEGF-A表達(dá)來保護(hù)全腦缺血大鼠CA1區(qū)血腦屏障。結(jié)果:1.CTA、DSA、血管乳膠灌注法結(jié)果發(fā)現(xiàn),在頸1-頸2橫突間隙內(nèi)椎動(dòng)脈位于樞椎上關(guān)節(jié)突的外側(cè)深面,并且不受上關(guān)節(jié)突遮擋,頸后路手術(shù)可以在顯微鏡直視下輕松阻斷椎動(dòng)脈。2.生存分析結(jié)果顯示:10min全腦缺血大鼠術(shù)后7天的生存率達(dá)93%;20min全腦缺血大鼠術(shù)后7天的生存率達(dá)80%;30min全腦缺血大鼠術(shù)后7天的生存率僅為50%。制定20min為新型全腦缺血模型的最佳缺血時(shí)間。3.尼氏染色結(jié)果顯示:20min全腦缺血大鼠CA1區(qū)神經(jīng)元數(shù)量在術(shù)后第1天無統(tǒng)計(jì)學(xué)差異,但在術(shù)后第3天出現(xiàn)明顯下降。在隨后的3-7天中,CA1區(qū)神經(jīng)元數(shù)量沒有進(jìn)一步下降。制定CA1區(qū)為新型全腦缺血模型的最佳缺血部位。4.膠帶實(shí)驗(yàn)和TUNEL染色結(jié)果證明全腦缺血大鼠在術(shù)后7天內(nèi)皮層區(qū)和紋狀體區(qū)神經(jīng)元均存在凋亡。新型全腦缺血模型可以導(dǎo)致大鼠運(yùn)動(dòng)整合能力相關(guān)腦區(qū)神經(jīng)元損傷,并且這些神經(jīng)元的損傷可以通過膠帶實(shí)驗(yàn)進(jìn)行觀察評價(jià)。5.Western blot檢測結(jié)果顯示:全腦缺血大鼠CA1區(qū)Occludin和Claudin-5蛋白含量在缺血后6小時(shí)開始降低,24小時(shí)明顯降低。后續(xù)實(shí)驗(yàn)可以采用全腦缺血后24小時(shí)為觀察時(shí)間點(diǎn)。6.Western blot和免疫熒光雙標(biāo)染色結(jié)果顯示:激動(dòng)GPER-1受體可以阻止缺血后OVX大鼠CA1區(qū)連接蛋白表達(dá)(Occludin和Claudin-5)的下降。另外還發(fā)現(xiàn)給予G-1激動(dòng)劑后全腦缺血OVX大鼠CA1區(qū)VEGF-A表達(dá)明顯下降。結(jié)論:1.新型全腦缺血模型可以穩(wěn)定的造成大鼠海馬CA1區(qū)神經(jīng)元遲發(fā)性死亡,并引起大鼠神經(jīng)功能出現(xiàn)異常。20min的全腦缺血為最佳缺血時(shí)間,CA1區(qū)為最佳缺血部位。2.新型全腦缺血模型可以導(dǎo)致大鼠海馬CA1區(qū)血腦屏障的破壞,并且在缺血后24小時(shí),CA1區(qū)血腦屏障破壞最為明顯。3.激動(dòng)GPER-1受體可在腦缺血早期起到穩(wěn)定血腦屏障通透性的作用。激動(dòng)GPER-1受體可以通過減少VEGF-A的表達(dá)來穩(wěn)定缺血CA1區(qū)血腦屏障的通透性。
[Abstract]:Background: ischemic stroke is the second leading cause of mortality worldwide. The treatment and mechanism of cerebral ischemia research requires a stable, reproducible model of global cerebral ischemia is high. Although the use of mice and gerbils in many models, but the cerebral ischemia rats model is reliable experiment. And it needs to be further improved. The four vessel occlusion method is a widely used model of global cerebral ischemia. The rats of hole surgery requires cautery of atlas, the permanent occlusion of bilateral vertebral artery. However, patients are often unable to open electrocoagulating the complete occlusion of the vertebral artery. Therefore, a successful model relies on turning reflection and electroencephalogram of rats were observed. The need to establish a stable model of global cerebral ischemia, to lay a good foundation for the subsequent experiments. The blood-brain barrier is between plasma and brain cells of the central nervous system barrier. Critical to maintain homeostasis and normal function. The endothelial cells of blood brain barrier function and morphology and non brain capillary endothelial cells are quite different. In many nervous system diseases, the integrity of the blood-brain barrier will be destroyed, such as ischemic stroke. The damage effect of cerebral ischemia on blood brain barrier in the brain has been widely studied. Hypoxia ischemia will cause a series of molecular changes in the brain, leading to endothelial cell tight junction damage, increase the permeability of blood brain barrier after cerebral ischemia. Many studies have shown that estrogen can prevent the destruction of the blood-brain barrier to protect the brain. Cerebral protective effect of estrogen is mediated by estrogen receptors (ER alpha, ER beta. GPER-1). Studies have shown that ER alpha and ER beta can mediate the protective effects of estrogen on vascular endothelial cell junction protein after cerebral ischemia. However, blood brain barrier damage in cerebral ischemia caused by GPER-1 The role is unclear. Objective: To explore the relationship between the 1. position and cervical vertebral artery of rats, to find.2. best position to block rat vertebral artery to establish high stability model of cerebral ischemia, to explore the best time and the best part of ischemia.3. ischemia cerebral ischemia exploration of ovariectomized rat hippocampal CA1 region blood brain barrier and the expression of GPER-1 and VEGF-A.4. to explore whether activation of GPER-1 receptor can reduce the expression of VEGF-A to protect cerebral ischemia area CA1 blood brain barrier. Methods: 1. using CTA, DSA, vascular latex perfusion clear rat vertebral artery and cervical spine anatomy and.2.. Looking for the best position to block rat the vertebral artery to establish a new model of global cerebral ischemia. According to the survival rate of cerebral ischemia in rats after 10,20,30 minutes, the optimal duration of cerebral ischemia rats. Then perfusion fixed, brain was removed by Nissl staining Observation of cerebral ischemia and TUNEL color marker staining in various brain regions of neurons in rats after death, assessing the stability of whole brain ischemia model model. Finally, according to the neural function of rats and tape scale experimental results, to observe the pathological change of cerebral ischemia in rats, further evaluation model by changes in rats CA1 in the permeability of blood brain barrier after cerebral ischemia 20min observation of Western blot experimental technique on the behavior of rats damage.3. (Ig G leakage rate) and connexin (Occludin, Claudin-5). The changes of the expression and expression of CA1 after cerebral ischemia GPER-1 receptor and VEGF-A protein were observed by Western.4. blot and immunofluorescence staining were used to observe the activation of GPER-1 receptor on CA1 blood brain barrier permeability in rats after cerebral ischemia (Ig G leakage rate) and connexin (Occludin, Claudin-5) on the expression of the Wes. Tern blot and immunohistochemical staining to explore whether activation of GPER-1 receptor can reduce the expression of VEGF-A to protect cerebral ischemia area CA1 blood brain barrier. Results: 1.CTA, DSA, vascular latex perfusion showed that the lateral aspect in carotid 1- cervical transverse process of the 2 gap within the vertebral artery is located in the upper joint axis process, and is not affected by the superior articular process of cervical posterior occlusion in the surgery under the microscope can easily block the vertebral artery.2. survival analysis showed: 10min cerebral ischemia rats 7 days after operation, the survival rate was 93%; 20min cerebral ischemia rats 7 days after operation, the survival rate was 80%; the best time of ischemia.3. 7 rats in the postoperative survival rate is only 50%. 20min for making model of whole brain ischemia cerebral ischemia 30min Nissl staining results show that: the number of neurons in CA1 area of rats with cerebral ischemia 20min on the first day after operation there was no significant difference, but on the third day after operation significantly Decreased. In the subsequent 3-7 days, the number of CA1 neurons decreased. No further develop CA1 area for the whole brain ischemia model best ischemia.4. tape test and TUNEL staining results showed that cerebral ischemia rats after 7 days in the cortex and striatum neuron apoptosis. There are new models of ischemia the brain can lead to neuronal damage in rat brain areas related to motion integration capabilities, and these neurons can observe the evaluation of.5.Western blot showed that by tape experiments: whole brain Occludin and Claudin-5 protein in ischemic rats CA1 content began to decrease at 6 hours after ischemia, 24 hours can be significantly reduced. The subsequent experiments using whole brain 24 hours after ischemia for observation time point.6.Western blot and immunofluorescence staining showed that activation of GPER-1 receptor can prevent OVX after ischemia in rat CA1 region binding protein table Da (Occludin and Claudin-5). It is also found that the decrease of cerebral ischemia in OVX rats of CA1 VEGF-A expression was significantly decreased after the administration of G-1 agonists. Conclusion: 1. new models of global cerebral ischemia can cause stability in rat hippocampal CA1 neurons delayed neuronal death, and cause the neurological function of rats with abnormal.20min full cerebral ischemia is the best time of ischemia, the CA1 district was the best site of ischemia.2. new model of global cerebral ischemia can lead to hippocampal CA1 region of rat blood brain barrier damage, and in 24 hours after ischemia, the most obvious.3. activation of GPER-1 receptor in the early stage of cerebral ischemia to stabilize the BBB permeability to blood brain area CA1 barrier damage. Activation of GPER-1 receptor can stabilize CA1 ischemia area of blood brain barrier permeability by decreasing the expression of VEGF-A.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R743.3
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本文編號：1501220