自噬在小鼠脑出血后脑损伤中的作用及机制研究

发布时间：2018-07-11 09:38

本文选题：脑出血 + 自噬　；参考：《苏州大学》2014年硕士论文

【摘要】：目的：自噬是一个涉及到细胞自身结构通过溶酶体机制而被降解的过程，其中包括细胞器、衰老及错误折叠或破损的蛋白质等。在不同的状况下，自噬可以引起细胞的死亡，也可以促进细胞生存。研究表明，，自噬在脑出血（intracerebralhemorrhage, ICH）后被激活。造成脑出血后继发的神经功能缺损的主要原因包括脑出血后脑血肿周边脑水肿的形成和继发的神经细胞死亡。本研究中，我们分别应用了自噬的抑制剂3-methyladenine（3-MA）和诱导剂雷帕霉素（rapamycin，RAP）来调控自噬的水平，探索不同自噬水平对小鼠脑出血后脑损伤的作用，并通过对自噬和凋亡相关蛋白的监测来探讨自噬与凋亡的关系，以期为脑出血的治疗提供新的方向。方法：以ICR小鼠为实验动物，采用在纹状体部位注射胶原酶IV的方法建立ICH模型。在ICH前15min，小鼠侧脑室给予自噬的抑制剂3-MA或激活剂雷帕霉素（RAP），并以等量的配制溶剂（DMSO与生理盐水1:1混溶）注入侧脑室作为对照组；于ICH后1d-7d，应用行为学试验（Motor Test）评估ICH后各组试验动物的运动功能；取ICH后24h和72h作为检测时间点处死小鼠取全脑，用干-湿重法检测脑出血侧及对侧纹状体和脑皮质以及小脑的含水量；取ICH后24h和72h作为检测时间点，于处死前1h腹腔注射碘化丙啶（propidium iodide, PI）后取全脑，采用体视学显微镜观察小鼠大脑出血中心区及周边区PI阳性细胞数，研究不同的自噬激活水平对ICH引起的神经细胞死亡的影响。为了进一步探索不同自噬水平对ICH后脑损伤的作用机制，本研究中同样取ICH后24h和72h作为检测时间点处死小鼠取全脑，运用免疫印迹法分别检测ICH后24h和72h的自噬活性相关蛋白（Beclin-1，LC3-II，p62）和凋亡相关蛋白（Bcl-2，Bax，cleavedcaspase-3）的表达水平。结果：（1）3-MA预处理后，Beclin-1及LC3-II的蛋白表达水平均明显降低，p62蛋白水平增加；3-MA预处理组脑出血后的细胞死亡（PI阳性细胞数）显著减少，脑水肿减轻，运动功能的损害也得到明显恢复。相反，雷帕霉素预处理上调了Beclin-1和LC3-II的表达，降低了p62的蛋白水平，但增加了脑出血后的细胞死亡数量、加重了脑水肿及运动功能损害程度。（2）3-MA预处理显著降低了ICH引起的Bax和cleaved caspase-3的蛋白表达的上调，但逆转了ICH后Bcl-2的下调；相反，雷帕霉素预处理后，Bax和cleaved caspase-3的蛋白表达增加，同时进一步降低了Bcl-2的表达。结论：（1）ICH可引起自噬激活增强，3-MA预处理降低了ICH后的自噬表达水平，RAP预处理进一步升高了ICH后的自噬表达水平；（2）抑制自噬减轻了ICH引起的脑损伤，而上调自噬加重了ICH后的脑损伤程度；（3）在小鼠ICH模型中，自噬可以通过激活细胞凋亡通路调节ICH引起的神经细胞损伤。
[Abstract]:Objective: autophagy is a process involving the degradation of cell structures through lysosomal mechanisms, including organelles, senescence, and misfolded or damaged proteins. Autophagy can cause cell death and promote cell survival in different conditions. Studies have shown that autophagy is activated after intracerebral hemorrhage (ICH). The main causes of the secondary neurological deficit after ICH include the formation of cerebral edema around the intracerebral hematoma and the secondary nerve cell death. In this study, we used 3-methyladenine (3-MA), an inhibitor of autophagy, and rapamycin rap, an inducer, to regulate the level of autophagy and to explore the effects of different levels of autophagy on brain injury after intracerebral hemorrhage in mice. The relationship between autophagy and apoptosis was studied by monitoring autophagy and apoptosis-related proteins in order to provide a new direction for the treatment of intracerebral hemorrhage. Methods: ICH model was established by injecting collagenase IV into striatum of ICR mice. At 15 min before ICH, mice lateral ventricle were given autophagy inhibitor 3-MA or activator rapamycin (rap), and the same amount of preparation solvent (DMSO and normal saline 1:1 mixture) was injected into the lateral ventricle as control group. The motor test was used to evaluate the motor function of the experimental animals after ICH at 1 d ~ 7 d after ICH, and the whole brain was killed at 24 h and 72 h after ICH. The water content in the striatum, cortex and cerebellum of cerebral hemorrhage side and contralateral side were measured by dry wet weight method, 24 and 72 hours after ICH were taken as time points, and the whole brain was taken after intraperitoneal injection of (propidium iodide, Pi 1 hour before death. The number of Pi positive cells in central and peripheral areas of cerebral hemorrhage in mice was observed by stereological microscope to study the effect of different levels of autophagy activation on the neuronal death induced by ICH. In order to further explore the mechanism of different autophagy levels on brain injury after ICH, the mice were killed at 24 and 72 hours after ICH. The expression of autophagy active-associated protein (Beclin-1) LC3-IIP62 and apoptosis-related protein (Bcl-2AXA cleavedcaspase-3) at 24 and 72 hours after ICH were detected by Western blotting. Results: (1) the protein expression levels of Beclin-1 and LC3-II were significantly decreased after 3-MA preconditioning. The impairment of motor function was also significantly recovered. In contrast, rapamycin pretreatment up-regulated the expression of Beclin-1 and LC3-II, reduced the protein level of p62, but increased the number of cell death after intracerebral hemorrhage. (2) 3-MA pretreatment significantly decreased the up-regulated expression of Bax and cleaved caspase-3 protein, but reversed the down-regulation of Bcl-2, on the contrary, the protein expression of Bax and cleaved caspase-3 increased after rapamycin preconditioning. At the same time, the expression of Bcl-2 was further decreased. Conclusion: (1) ICH can induce autophagy enhancement and 3- MA pretreatment can decrease the level of autophagy expression after ICH. Rap pretreatment can further increase the level of autophagy expression after ICH, (2) inhibition of autophagy reduces the brain injury induced by ICH. In mouse ICH model autophagy can regulate the neuronal damage induced by ICH by activating apoptosis pathway.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.34

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