本文選題：雷帕霉素 + 腦缺血再灌注損傷��； 參考：《南華大學(xué)》2014年碩士論文

【摘要】：目的： 研究大鼠腦缺血再灌注損傷后溶酶體組織蛋白酶D及p-Akt表達(dá)的變化情況，并初步探討雷帕霉素干預(yù)處理后的腦保護(hù)作用相關(guān)機(jī)制。 方法： 選擇清潔級(jí)10~12周齡、體重為280±20g的健康雄性Sprague-Dawley(SD)大鼠100只，隨機(jī)分為正常組（n=10）、假手術(shù)對(duì)照組（Sham組，n=30）、腦缺血再灌注模型組（I/R組，n=30）和雷帕霉素干預(yù)組（Rap組，n=30）。采用改良Long線栓法制備右側(cè)大腦中動(dòng)脈梗塞（middle cerebral artery occlusion，MCAO）模型，缺血2小時(shí)后恢復(fù)腦組織灌注，干預(yù)組在側(cè)腦室注射Rap5μL（10mmol/L）30min后行MCAO手術(shù)，Sham組及I/R組于相應(yīng)時(shí)間點(diǎn)側(cè)腦室注射等量的DMSO溶液，Longa’s5級(jí)標(biāo)準(zhǔn)評(píng)分法進(jìn)行大鼠神經(jīng)功能缺損評(píng)分，分別于缺血再灌注后3、6、12、24及48小時(shí)處死動(dòng)物。TTC染色法觀察腦組織的病理學(xué)變化，末端標(biāo)記法（TUNEL法）原位檢測(cè)神經(jīng)細(xì)胞凋亡，免疫組織化學(xué)法分別檢測(cè)缺血側(cè)頂葉大腦皮層的Cathepsin D、p-Akt蛋白表達(dá)變化。 結(jié)果： 一、本實(shí)驗(yàn)造模成功率為72.29%，與I/R組相比，Rap組各時(shí)間點(diǎn)動(dòng)物模型神經(jīng)功能缺損評(píng)分均有明顯改善（P0.05）。 二、TTC染色示再灌注損傷24小時(shí)后白色缺血梗死灶主要位于腦組織皮層和基底節(jié)；正常組及Sham組大鼠均未見(jiàn)梗死灶，I/R組梗死灶明顯，Rap干預(yù)后腦梗死體積較前明顯縮小（P0.05）。 三、正常組及Sham組可檢測(cè)到少量的凋亡細(xì)胞；I/R組凋亡細(xì)胞表達(dá)呈時(shí)間依賴性，再灌注6小時(shí)凋亡細(xì)胞明顯增多，，并隨時(shí)間點(diǎn)延長(zhǎng)逐漸增高，48小時(shí)達(dá)高峰；Rap干預(yù)后，再灌注6h、12h、24h、48h凋亡細(xì)胞數(shù)較I/R組均有較明顯減少（P0.05），缺血再灌注后3h凋亡細(xì)胞數(shù)雖也有所減少，但P0.05。 四、正常組及Sham組可以檢測(cè)到Cathepsin D蛋白的少量表達(dá)，I/R組Cathepsin D蛋白表達(dá)6小時(shí)開(kāi)始上升，24h達(dá)到高峰，48h仍保存高水平，與模型組比較，雷帕霉素干預(yù)組Cathepsin D蛋白表達(dá)在各時(shí)間點(diǎn)均明顯減弱（p0.05）。 五、正常組及Sham組頂葉皮層可見(jiàn)少量p-Akt蛋白的表達(dá)；I/R組3h缺血側(cè)p-Akt蛋白的表達(dá)達(dá)高峰，隨時(shí)間延長(zhǎng)逐漸降低，與正常組及假手術(shù)組相比差異有統(tǒng)計(jì)學(xué)意義（P0.05）；Rap干預(yù)后，缺血再灌注后3小時(shí)p-Akt蛋白表達(dá)與I/R組3h組相比，蛋白表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05），6小時(shí)明顯上升，12小時(shí)達(dá)峰，48小時(shí)有較高表達(dá)，差異有顯著性(P0.05)。 結(jié)論： Rap可能通過(guò)下調(diào)Cathepsin D蛋白、上調(diào)p-Akt蛋白，減輕大鼠缺血再灌注損傷后大腦中動(dòng)脈梗塞的面積及神經(jīng)細(xì)胞凋亡，發(fā)揮其腦保護(hù)作用。
[Abstract]:Objective: To study the changes of lysosomal cathepsin D and p-Akt expression after cerebral ischemia-reperfusion injury in rats and to explore the mechanism of brain protection after rapamycin intervention. Methods: A total of 100 healthy male Sprague-Dawley SD rats of 10 ~ 12 weeks old, weighing 280 鹵20 g, were randomly divided into three groups: normal group (n = 10), sham group (n = 30), cerebral ischemia-reperfusion model group (n / R) (n = 30) and rapamycin intervention group (Rap) (n = 30). The middle cerebral artery occlusion (MCAO) model of right middle cerebral artery infarction was established by modified Long thread occlusion method. The cerebral perfusion was restored 2 hours after ischemia. The intervention group received MCAO operation after intracerebroventricular injection of Rap5 渭 L(10mmol/L)30min and the I / R group received the same amount of DMSO solution at the same time point. The histopathological changes of brain tissue were observed by TTC staining at 24 and 48 hours after ischemia and reperfusion, and neuronal apoptosis was detected by Tunel in situ. Immunohistochemical method was used to detect the expression of Cathepsin Dfp-Akt protein in the parietal cortex of ischemic side. Results: The results were as follows: 1. The successful rate of modeling was 72.29. Compared with the I / R group, the neurological deficit score of the rat model in Rap group was significantly improved at every time point (P 0.05). After 24 hours of reperfusion injury, white ischemic infarction was mainly located in cortex and basal ganglion of cerebral tissue, and no infarct focus was found in group I / R after reperfusion injury, and the volume of cerebral infarction in group I / R was significantly smaller than that in group I / R after Rap intervention (P 0.05). 3. In the normal group and Sham group, a small number of apoptotic cells were detected in a time-dependent manner in the I / R group, and the apoptotic cells increased significantly at 6 h after reperfusion, and gradually increased with the prolongation of the time point and reached the peak at 48 h after the intervention of Rap. Compared with the I / R group, the number of apoptotic cells decreased significantly at 6 h, 12 h, 24 h and 48 h after reperfusion, and the number of apoptotic cells at 3 h after ischemia and reperfusion was also decreased, but the number of apoptotic cells was also decreased (P 0.05). 4. A little expression of Cathepsin D protein was detected in normal group and Sham group. The expression of Cathepsin D protein in I / R group began to rise at 6 h and reached the peak at 24 h and remained at a high level at 48 h, compared with the model group. The expression of Cathepsin D protein in rapamycin intervention group was significantly decreased at each time point. 5. A small amount of p-Akt protein was found in parietal cortex of normal group and Sham group. The expression of p-Akt protein in ischemic side of I / R group reached the peak at 3 h, and gradually decreased with time. The difference was statistically significant compared with normal group and sham operation group. There was no significant difference in the expression of p-Akt protein between I / R group and I / R group at 3 hours after ischemia and reperfusion. There was no significant difference in the expression of p-Akt protein. The expression of p-Akt protein was significantly higher than that in I / R group at 6 hours and 12 hours after reperfusion, and there was a significant difference in the expression of p-Akt protein between I / R group and I / R group (P < 0.05). Conclusion: Rap may play a protective role by down-regulating Cathepsin D protein, up-regulating p-Akt protein, reducing the area of middle cerebral artery infarction and neuronal apoptosis after ischemia-reperfusion injury in rats.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3

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