缺血预处理后不同间隔时间对大鼠脑缺血再灌注自噬的影响

发布时间：2018-04-28 10:09

本文选题：自噬 + 缺血预处理　；参考：《河北联合大学》2014年硕士论文

【摘要】：目的研究自噬相关蛋白Beclin-1、LC3-II在缺血预处理后不同时间间隔大鼠脑缺血再灌注海马中的表达，探讨自噬在脑缺血预处理神经保护作用的可能机制。方法1.健康雄性SD大鼠50只随机分为3组：假手术组、缺血再灌注组、缺血预处理组，缺血预处理组再按不同缺血预处理间隔时间分为24h、72h、5d3个亚组。2.参照Zea-Longa线栓法制作大鼠大脑中动脉缺血预处理和缺血再灌注模型。3.利用TTC染色、photoshop图像分析法、HE染色、免疫组织化学染色、观察脑梗死体积、海马神经细胞病理变化、海马神经细胞自噬相关蛋白Beclin-1及LC3-II表达的情况。4.透射电镜观察神经细胞中自噬超微结构的变化。5.比较各组神经功能缺失、脑梗死体积、海马神经细胞病理变化、海马神经细胞自噬相关蛋白Beclin-1及LC3-II表达、自噬体和各级溶酶体数量。6.以Excel数据库整理后采用SPSS17.0统计学软件进行数据分析，所得数据均以x±s表示，P0.05认为差异有统计学意义。采用Microsoft Excel软件作图。结果1.缺血预处理组神经功能缺失症状较缺血再灌注组有所改善，差异具有统计学意义（P0.05）。2.与缺血再灌注组相比，缺血预处理组脑梗死体积缩小，差异具有统计学意义（P0.05）。3.HE染色可见假手术组神经细胞无损伤；与缺血再灌注组相比，缺血预处理各亚组海马神经细胞损伤减轻。4.与假手术组相比，缺血再灌注组、缺血预处理组Beclin-1的表达均明显增强，差异具有统计学意义（P0.05）；缺血预处理组与缺血再灌注组比较，可见Beclin-1阳性细胞数减少，差异具有统计学意义（P0.05）。5.假手术组只见极少量LC3-II阳性细胞表达，而在缺血再灌注组及缺血预处理组均可见大量LC3-II阳性细胞，差异有统计学意义（P0.05）。缺血预处理各亚组LC3-II阳性细胞表达显著低于缺血再灌注组，差异有统计学意义（P0.05）。6.透射电镜下缺血预处理组及缺血再灌注组均可见数量不等、处于不同期间的自噬体和自噬溶酶体，其数量在缺血预处理组明显减少。7.缺血预处理72h亚组相比较24h亚组和5d亚组脑梗死体积缩小（P0.05），，海马神经细胞损伤减轻，Beclin-1、LC3-II阳性细胞数不同程度减少（P0.05），自噬体的形成和溶酶体的激活减少。结论1.采用改良后Zea-Longa线栓法制作的大鼠大脑中动脉缺血预处理和缺血再灌注模型方法简便、安全、数据可靠。2.缺血预处理可减低缺血再灌注24小时后大鼠神经功能障碍评分、脑梗死体积、海马神经细胞损伤，对缺血再灌注后神经细胞损伤具有保护作用。3.缺血预处理可能通过下调缺血再灌注24小时后自噬相关蛋白Beclin-1、LC3-II的表达，以减少缺血再灌注损伤神经细胞的自噬性死亡，减轻神经功能缺损症状，缩小脑梗死体积，从而发挥其脑保护作用。4.缺血预处理72h亚组脑梗死体积低于缺血预处理24h、5d亚组，Beclin-1、LC3-II阳性细胞数低于缺血预处理24h、5d亚组，偶见自噬体，各级溶酶体数量相对较少，提示最佳预处理诱导时间间隔为72h。
[Abstract]:Objective to study the expression of autophagy related protein Beclin-1 and LC3-II in hippocampus of ischemic reperfusion rats at different time intervals after ischemic preconditioning, and to explore the possible mechanism of autophagy in ischemic preconditioning. Method 1. 50 healthy male SD rats were randomly divided into 3 groups: sham operation group, ischemia reperfusion group, ischemic preconditioning group, The ischemic preconditioning group was then divided into 24h, 72h, 5d3 subgroup and 5d3 subgroup,.2. referred to Zea-Longa line embolus to make the rat middle cerebral artery ischemic preconditioning and ischemia reperfusion model.3. using TTC staining, Photoshop Image analysis, HE staining, immunohistochemical staining, observation of cerebral infarction volume and hippocampal neurocytopathic disease. Changes in the expression of autophagy related protein Beclin-1 and LC3-II in hippocampal neurons.4. transmission electron microscope observation of the ultrastructure changes of autophagy in the nerve cells.5. compared with the absence of nerve function, the volume of cerebral infarction, the pathological changes of hippocampal neurons, the expression of autophagic related protein Beclin-1 and LC3-II in the hippocampal neurons, autophago and each The number.6. of the grade lysosome was analyzed by the SPSS17.0 statistics software after the Excel database. The data were all expressed in X + s, and P0.05 thought the difference was statistically significant. The Microsoft Excel software was used to map.
Results there was a significant difference between the 1. ischemic preconditioning group and the ischemia reperfusion group. The difference was statistically significant (P0.05) compared with the ischemia reperfusion group, the volume of cerebral infarction in the ischemic preconditioning group was reduced, and the difference was statistically significant (P0.05).3.HE staining showed that the nerve cells in the sham operation group were not injured; and the ischemia reperfusion group was in the ischemic reperfusion group. Compared with the ischemic preconditioning group, the damage of the hippocampal neurons in the ischemic preconditioning group was significantly enhanced, compared with the sham group. The expression of Beclin-1 in the ischemic preconditioning group was significantly enhanced, and the difference was statistically significant (P0.05). Compared with the ischemic reperfusion group, the number of Beclin-1 positive cells decreased and the difference was statistically significant compared with the ischemic reperfusion group. The difference was statistically significant (P0.05). The expression of a very small number of LC3-II positive cells was found in the sham operation group of P0.05.5., while a large number of LC3-II positive cells were found in the ischemic reperfusion group and the ischemic preconditioning group. The difference was statistically significant (P0.05). The expression of LC3-II positive cells in each subgroup of ischemic preconditioning was significantly lower than that of the ischemia reperfusion group, and the difference was statistically significant (P0.05).6. transmission electricity The number of autophagosomes and autophagic lysosomes in the ischemic preconditioning group and ischemia reperfusion group were in different periods. The number of the ischemic preconditioning group was significantly reduced by the.7. ischemic preconditioning 72h subgroup, compared with the 24h subgroup and the 5D subgroup, the cerebral infarction volume decreased (P0.05), the hippocampal neuronal damage was reduced, Beclin-1, LC3-II positive were fine. The number of cells decreased (P0.05), autophagosome formation and lysosome activation decreased.
Conclusion 1. the model of ischemic preconditioning and ischemia-reperfusion model of middle cerebral artery made by modified Zea-Longa thread embolus method is simple and safe. The data reliable.2. ischemic preconditioning can reduce the neurological dysfunction score of rats after 24 hours of ischemia reperfusion, the volume of cerebral infarction, the damage of the hippocampal nerve cells, and the fine nerve after ischemia-reperfusion. .3. ischemic preconditioning may reduce the autophagic related protein Beclin-1, LC3-II expression after 24 hours of ischemia-reperfusion, reduce the autophagic death of ischemic reperfusion injury, reduce the symptoms of nerve function defect, reduce the volume of cerebral infarction, and thus play its protective role of.4. ischemic preconditioning 72h The volume of cerebral infarction in subgroup was lower than that of ischemic preconditioning (24h), 5D subgroup, Beclin-1, LC3-II positive cells were lower than that of ischemic preconditioning 24h, 5D subgroup and autophagosome, and the number of lysosomes at all levels was relatively small, suggesting that the best preconditioning time interval was 72h..

【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

【参考文献】