雷帕霉素对大鼠脑缺血再灌注损伤后Cathepsin D、p-Akt表达的研究

发布时间：2018-05-25 08:35

本文选题：雷帕霉素 + 脑缺血再灌注损伤　；参考：《南华大学》2014年硕士论文

【摘要】：目的：研究大鼠脑缺血再灌注损伤后溶酶体组织蛋白酶D及p-Akt表达的变化情况，并初步探讨雷帕霉素干预处理后的脑保护作用相关机制。方法：选择清洁级10~12周龄、体重为280±20g的健康雄性Sprague-Dawley(SD)大鼠100只，随机分为正常组（n=10）、假手术对照组（Sham组，n=30）、脑缺血再灌注模型组（I/R组，n=30）和雷帕霉素干预组（Rap组，n=30）。采用改良Long线栓法制备右侧大脑中动脉梗塞（middle cerebral artery occlusion，MCAO）模型，缺血2小时后恢复脑组织灌注，干预组在侧脑室注射Rap5μL（10mmol/L）30min后行MCAO手术，Sham组及I/R组于相应时间点侧脑室注射等量的DMSO溶液，Longa’s5级标准评分法进行大鼠神经功能缺损评分，分别于缺血再灌注后3、6、12、24及48小时处死动物。TTC染色法观察脑组织的病理学变化，末端标记法（TUNEL法）原位检测神经细胞凋亡，免疫组织化学法分别检测缺血侧顶叶大脑皮层的Cathepsin D、p-Akt蛋白表达变化。结果：一、本实验造模成功率为72.29%，与I/R组相比，Rap组各时间点动物模型神经功能缺损评分均有明显改善（P0.05）。二、TTC染色示再灌注损伤24小时后白色缺血梗死灶主要位于脑组织皮层和基底节；正常组及Sham组大鼠均未见梗死灶，I/R组梗死灶明显，Rap干预后脑梗死体积较前明显缩小（P0.05）。三、正常组及Sham组可检测到少量的凋亡细胞；I/R组凋亡细胞表达呈时间依赖性，再灌注6小时凋亡细胞明显增多，，并随时间点延长逐渐增高，48小时达高峰；Rap干预后，再灌注6h、12h、24h、48h凋亡细胞数较I/R组均有较明显减少（P0.05），缺血再灌注后3h凋亡细胞数虽也有所减少，但P0.05。四、正常组及Sham组可以检测到Cathepsin D蛋白的少量表达，I/R组Cathepsin D蛋白表达6小时开始上升，24h达到高峰，48h仍保存高水平，与模型组比较，雷帕霉素干预组Cathepsin D蛋白表达在各时间点均明显减弱（p0.05）。五、正常组及Sham组顶叶皮层可见少量p-Akt蛋白的表达；I/R组3h缺血侧p-Akt蛋白的表达达高峰，随时间延长逐渐降低，与正常组及假手术组相比差异有统计学意义（P0.05）；Rap干预后，缺血再灌注后3小时p-Akt蛋白表达与I/R组3h组相比，蛋白表达差异无统计学意义（P0.05），6小时明显上升，12小时达峰，48小时有较高表达，差异有显著性(P0.05)。结论： Rap可能通过下调Cathepsin D蛋白、上调p-Akt蛋白，减轻大鼠缺血再灌注损伤后大脑中动脉梗塞的面积及神经细胞凋亡，发挥其脑保护作用。
[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 卤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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

【参考文献】