本文關(guān)鍵詞： 早期運(yùn)動(dòng)訓(xùn)練 腦缺血損傷 血腦屏障 神經(jīng)元凋亡 腦保護(hù)　出處：《復(fù)旦大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景：缺血性腦卒中是具有嚴(yán)重破壞性的急性腦血管疾病,它可以激活腦缺血瀑布,破壞血腦屏障,引發(fā)免疫炎癥反應(yīng)以及缺血區(qū)域神經(jīng)元凋亡,從而導(dǎo)致腦缺血繼發(fā)性神經(jīng)功能損傷。大量的基礎(chǔ)和臨床研究證實(shí)早期運(yùn)動(dòng)訓(xùn)練促進(jìn)卒中患者的神經(jīng)功能恢復(fù),但其所涉及的分子機(jī)制尚未完全闡明。本研究著眼于腦缺血繼發(fā)性損傷的兩個(gè)重要病理過程——血腦屏障破壞和神經(jīng)元凋亡,擬利用大鼠大腦中動(dòng)脈栓塞的腦缺血模型(MCAO),在缺血早期介入強(qiáng)度逐步增加的運(yùn)動(dòng)康復(fù)訓(xùn)練,綜合運(yùn)用行為學(xué)、組織學(xué)、形態(tài)學(xué)、分子生物學(xué)和蛋白質(zhì)組學(xué)方法研究早期運(yùn)動(dòng)訓(xùn)練對(duì)腦缺血損傷大鼠血腦屏障完整性和神經(jīng)元凋亡的影響,探討早期運(yùn)動(dòng)訓(xùn)練的腦保護(hù)作用及其潛在的分子機(jī)制。研究方法：采用SD成年雄性大鼠,隨機(jī)分為假手術(shù)組、缺血對(duì)照組(non-exercise)和早期運(yùn)動(dòng)組(early-exercise),建立90分鐘缺血時(shí)程的MCAO模型,在腦缺血再灌注24小時(shí)進(jìn)行強(qiáng)度漸增的跑臺(tái)運(yùn)動(dòng)訓(xùn)練干預(yù),每天30分鐘。在腦缺血損傷后相應(yīng)時(shí)間點(diǎn),采用神經(jīng)功能缺失評(píng)分法評(píng)估大鼠神經(jīng)功能缺失癥狀,分別使用foot-fault和beam balance方法評(píng)估大鼠的運(yùn)動(dòng)協(xié)調(diào)能力和平衡能力。此外,通過TTC染色和CV染色法檢測(cè)大鼠腦梗死體積；利用透射電鏡技術(shù)和伊文思藍(lán)染色研究血腦屏障結(jié)構(gòu)和功能的變化；同時(shí)采用Real-Time PCR、western blot和明膠酶譜法分析MMPs、TIMP-1、occludin、TLR4、NF-κB、IL-18的表達(dá)變化,研究早期運(yùn)動(dòng)對(duì)血腦屏障的保護(hù)機(jī)制；最后,我們采用TUNEL和F-J-B染色觀察大鼠損傷側(cè)皮層的神經(jīng)元凋亡情況；western blot檢測(cè)神經(jīng)元凋亡相關(guān)蛋白caspase-3和bcl-2的表達(dá)情況,進(jìn)一步探索早期運(yùn)動(dòng)的腦保護(hù)作用機(jī)制。研究結(jié)果：早期運(yùn)動(dòng)訓(xùn)練顯著減輕了腦缺血損傷引起的神經(jīng)功能缺失。與non-exercise相比,early-exercise組大鼠神經(jīng)功能缺失癥狀得到顯著緩解,運(yùn)動(dòng)協(xié)調(diào)能力也明顯恢復(fù),腦梗死體積減小。電鏡結(jié)果和伊文思藍(lán)染色顯示早期運(yùn)動(dòng)減輕腦缺血損傷對(duì)血腦屏障的破壞。早期運(yùn)動(dòng)抑制了腦缺血損傷引起的MMPs蛋白表達(dá)及其活性,促進(jìn)TIMP-1表達(dá),減少緊密連接蛋白o(hù)ccludin缺失,抑制TLR4、NF-κB、IL-18炎性相關(guān)介質(zhì)的表達(dá),從而發(fā)揮了對(duì)血腦屏障的保護(hù)作用。另外,早期運(yùn)動(dòng)通過促進(jìn)抗凋亡蛋白bcl-2的表達(dá)下調(diào)caspase-3的核裂解作用,并且運(yùn)用TUNEL和F-J-B染色也觀察到早期運(yùn)動(dòng)大鼠損傷側(cè)皮層的神經(jīng)元凋亡減少,進(jìn)一步發(fā)揮腦保護(hù)作用。結(jié)論：腦卒中早期運(yùn)動(dòng)訓(xùn)練具有腦保護(hù)作用,其作用機(jī)制可能與抑制TLR4/NF-κB信號(hào)傳導(dǎo)通路,下調(diào)促炎性因子,調(diào)控MMPs及其組織抑制因子TIMPs表達(dá),減少occludin缺失,減輕血腦屏障損傷有關(guān)；也與早期運(yùn)動(dòng)調(diào)控凋亡相關(guān)蛋白caspase-3和bcl-2,抑制神經(jīng)元凋亡密切相關(guān)。早期運(yùn)動(dòng)腦保護(hù)作用完整的分子機(jī)制仍需要進(jìn)一步的深入研究,為缺血性腦卒中的臨床治療提供新的思路和理論基礎(chǔ)。
[Abstract]:Background: ischemic stroke is a severe and destructive acute cerebrovascular disease. It can activate cerebral ischemic waterfall, destroy blood-brain barrier, induce immune inflammation and neuronal apoptosis in ischemic region. A large number of basic and clinical studies have confirmed that early exercise training promotes the recovery of neurological function in stroke patients. However, the molecular mechanism involved has not been fully elucidated. This study focuses on two important pathological processes of secondary injury of cerebral ischemia-blood-brain barrier damage and neuronal apoptosis. The model of cerebral ischemia in rats with middle cerebral artery embolism (MCAO) was used to train the exercise rehabilitation of increasing interventional intensity in the early stage of ischemia, and to use behavior, histology and morphology. Molecular biology and proteomics methods were used to study the effects of early exercise training on the integrity of blood-brain barrier and neuronal apoptosis in rats with cerebral ischemia injury. Methods: Sprague-Dawley adult male rats were randomly divided into sham-operated group, non-exercise control group and early exercise group to establish a 90-minute ischemic MCAO model. After 24 hours of cerebral ischemia-reperfusion, the rats were treated with increased treadmill exercise training for 30 minutes a day. At the corresponding time points after cerebral ischemia injury, the neurological deficit score was used to evaluate the neurological deficit symptoms in rats. Foot-fault and beam balance were used to evaluate the ability of motor coordination and balance in rats. In addition, the volume of cerebral infarction was detected by TTC staining and CV staining. The changes of blood-brain barrier structure and function were studied by means of transmission electron microscopy and Evans blue staining, and the expression of IL-18 in TLR4NF- 魏 B was analyzed by Real-Time PCR western blot and gelatin zymography to study the protective mechanism of early exercise on blood-brain barrier. TUNEL and F-J-B staining were used to observe the neuronal apoptosis in the injured cortex of rats. The expression of apoptosis-related protein caspase-3 and bcl-2 was detected by western blot. The results showed that early exercise training significantly alleviated the neurological deficit caused by cerebral ischemia injury. Compared with non-exercise, the neurological deficit symptoms of rats in the early exercise group were significantly alleviated. The ability to coordinate sports has also been significantly restored. The results of electron microscope and Evans blue staining showed that early exercise alleviated the damage of blood-brain barrier caused by cerebral ischemia. Early exercise inhibited the expression and activity of MMPs protein and promoted the expression of TIMP-1. By reducing the deletion of tight junction protein occludin and inhibiting the expression of IL-18 inflammatory mediators in TLR4, NF- 魏 B, IL-18 may play a protective role on the blood-brain barrier. In addition, early exercise down-regulates the nuclear cleavage of caspase-3 by promoting the expression of anti-apoptotic protein bcl-2. TUNEL and F-J-B staining were also used to observe the decrease of neuronal apoptosis in the injured lateral cortex of early exercise rats, and to further exert the protective effect of brain. Conclusion: early exercise training has protective effect on brain after stroke. The mechanism may be related to the inhibition of TLR 4 / NF- 魏 B signal transduction pathway, the down-regulation of pro-inflammatory factors, the regulation of MMPs and its tissue inhibitor TIMPs expression, the reduction of occludin deletion and the reduction of blood-brain barrier damage. It is also closely related to the regulation of apoptosis-related proteins caspase-3 and bcl-2 and the inhibition of neuronal apoptosis. The complete molecular mechanism of early motor brain protection still needs to be further studied. To provide a new thinking and theoretical basis for the clinical treatment of ischemic stroke.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3

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本文編號(hào)：1551482