脑缺血后适应microRNA保护作用机制的研究

发布时间：2018-06-07 01:54

本文选题：缺血后适应 + 缺血/再灌注损伤　；参考：《昆明医科大学》2016年博士论文

【摘要】：目的：本研究旨在通过microRNA芯片技术探讨microRNAs在缺血／再灌注损伤及缺血后适应后小鼠脑组织中的表达变化,筛选出差异表达的microRNAs,进而进一步在体内及体外模型进行验证,深入研究和探讨miRNA参与脑缺血后适应的神经保护作用及其可能涉及的调控机制。方法：实验分为三部分,第一部分小鼠脑缺血后适应microRNA表达变化的研究：在小鼠脑缺血/再灌注损伤的模型基础上,通过microRNAs芯片筛查技术筛选出后适应后大脑皮层及海马组织内差异性表达的microRNAs,采用qRT-PCR技术对差异表达明显的miRNAs进行验证,并利用信息学软件进行靶基因分析及筛选。第二部分：对差异表达变化明显的miRNA-124进行进一步研究,探讨miRNA-124参与脑缺血后适应对小鼠缺血/再灌注损伤的神经保护作用的机制：采用小鼠侧脑室给药的方案,给予miRNA-124激动剂(agomir)及抑制剂(antagomir)处理,通过TTC染色法、TUNEL法、NeuN染色、Western blot、 qRT-PCR方法比较抑制及过表达miRNA-124后,脑梗死面积、细胞凋亡及存活情况、对PI3K/Akt2信号通路的影响、抗凋亡蛋白Bcl-2及凋亡相关蛋白Bax. Caspases 3的表达情况。第三部分miRNA-124调节PI3K/Akt2信号通路,减轻缺血再灌注损伤细胞凋亡的机制研究：采用PC12细胞体外实验,氧糖剥夺法模拟缺血/再灌注损伤模型,进一步验证miRNA-124通过调控PI3K/Akt2信号通路对抗缺血/再灌注损伤：建立PC12细胞氧糖剥夺/再灌注模型,niRNA-124agomir及antagomir进行细胞预处理,抑制及过表达miRNA-124,采用超氧化物阴离子探针、CCK8法、Annexin V FITC/PI流式双染、免疫荧光染色、Western blot、qRT-PCR等方法比较PC12细胞在缺血/灌注损伤后细胞缺氧情况、细胞活力及凋亡情况、PIK3/Akt2号通路、抗凋亡蛋白Bcl-2及凋亡相关蛋白Bax, Caspases 3的表达情况。结果：①microRNAs芯片筛查结果显示：与脑缺血/再灌注组相比,后适应处理后,小鼠皮层及海马区多种miRNAs表达水平发生显著变化,部分miRNA出现相同的变化,后适应下调了miRNA-1,let-7和miRNA-124的表达,上调了miRNA-19a的表达(p值均0.05)。通过qRT-PCI进一步验证了miRNAs的表达变化(p0.05)。②体内实验结果显示：抑制miRNA-124的表达后协同了后适应对缺血/再灌注损伤的脑保护作用,在后适应基础上进一步缩小了脑梗死体积、保护了神经细胞、减少了细胞凋亡,增强了PI3K/Akt2信号通路表达,抗凋亡蛋白Bcl-2表达上调、凋亡相关蛋白Bax、Caspases3表达下调(P值均0.05),过表达miRNA-124后抵消了后适应的脑保护效果。③体外实验结果显示：抑制PC12细胞中miRNA-124的表达后PI3K/Akt2信号通路表达增强,有效减少氧糖剥夺/再灌注导致的PC12细胞凋亡、增强细胞活力(P值均0.05),抗凋亡蛋白Bcl-2表达上调、凋亡相关蛋白Bax、Caspases3表达下调(P值均0.05),而过表达miRNA-124后加重了细胞缺血/再灌注损伤。结论：缺血后适应通过调控皮层及海马脑组织中miRNAs的表达水平对抗脑缺血/再灌注损伤。在小鼠I/R模型中,miRNA-1、let-7、miRNA19a和miRNA-124可能是与后适应保护机制相关的miRNAs。miRNA-124参与了脑缺血/再灌注损伤病理生理过程,并通过靶向负性调控PI3K/Akt2通路参缺血后适应在小鼠脑缺血/再灌注损伤中的抗凋亡作用。
[Abstract]:Objective: the purpose of this study was to investigate the changes in the expression of microRNAs in the cerebral tissue of mice after ischemia / reperfusion injury and post ischemic adaptation by microRNA chip technology, and to screen out the differential expression of microRNAs, and further verify in vivo and in vitro models, and further study and explore the neuroprotection of miRNA in the adaptation to cerebral ischemia. The effect and its possible regulatory mechanism. Methods: the experiment is divided into three parts. The first part is the study of the changes in the expression of microRNA after cerebral ischemia in mice: on the basis of the model of cerebral ischemia / reperfusion injury in mice, the differential expression of micro in the large cerebral cortex and hippocampus after adaptation is screened by microRNAs chip screening technique. RNAs, using qRT-PCR technology to verify the distinctly expressed miRNAs, and use the information software to carry out the target gene analysis and screening. The second part: to further study the miRNA-124 with distinct differential expression change, and explore the mechanism of miRNA-124 involved in the neuroprotective effect of miRNA-124 on the injury of blood / reperfusion injury in mice after cerebral ischemia Using the scheme of the mouse lateral ventricle administration, the miRNA-124 agonist (agomir) and the inhibitor (antagomir) were treated with TTC staining, TUNEL, NeuN staining, Western blot, and qRT-PCR methods to suppress and overexpress miRNA-124, the area of cerebral infarction, the cell withering and survival, the effect on the PI3K/Akt2 signaling pathway, and the anti apoptotic protein B The expression of Cl-2 and apoptosis related protein Bax. Caspases 3. Third part miRNA-124 regulates the PI3K/Akt2 signaling pathway to reduce the mechanism of apoptosis induced by ischemia-reperfusion injury: in vitro experiment of PC12 cells, oxygen glucose deprivation is used to simulate ischemia / reperfusion injury model, and one step is to verify that miRNA-124 is regulated by the PI3K/Akt2 signaling pathway. Against ischemia / reperfusion injury: PC12 cell oxygen glucose deprivation / reperfusion model, niRNA-124agomir and antagomir cell preconditioning, inhibition and overexpression of miRNA-124, using superoxide anion probe, CCK8, Annexin V FITC/PI flow double staining, immunofluorescence staining, Western blot, qRT-PCR and other methods to compare PC12 cells in ischemia / The condition of cell hypoxia, cell vitality and apoptosis, PIK3/Akt2 pathway, anti apoptotic protein Bcl-2 and apoptosis related protein Bax, Caspases 3 expression. Results: 1. The results of microRNAs chip screening showed that after the cerebral ischemia / reperfusion group, after adaptation, there were a variety of miRNAs expression water in the cortex and hippocampus of mice. There was a significant change in the level of miRNA, and the expression of miRNA-1, let-7 and miRNA-124 was down regulated, and the expression of miRNA-19a was up-regulated (P value was 0.05). The expression of miRNAs was further verified by qRT-PCI (P0.05). The cerebral protective effect of perfusion injury further narrowed the volume of cerebral infarction on the basis of post adaptation, protected the nerve cells, reduced the apoptosis, enhanced the expression of PI3K/Akt2 signaling pathway, increased the expression of anti apoptotic protein Bcl-2, Bax, Caspases3 expression (all P 0.05), and counteracted the postadaptation after overexpression of miRNA-124. The results of brain protection in vitro showed that the expression of PI3K/Akt2 signaling pathway was enhanced after the inhibition of miRNA-124 expression in PC12 cells, effectively reducing the apoptosis of PC12 cells induced by oxygen deprivation / reperfusion, enhanced cell viability (P value 0.05), up regulation of anti apoptotic protein Bcl-2 expression, and down regulation of apoptosis related protein Bax, Caspases3 expression (P value of 0). 5), after overexpression of miRNA-124, it aggravates cell ischemia / reperfusion injury. Conclusion: adaptation to ischemic / reperfusion injury by regulating the expression level of miRNAs in the cortex and hippocampal brain tissue after ischemia. In the mouse I/R model, miRNA-1, let-7, miRNA19a and miRNA-124 may be associated with the postadaptation mechanism of miRNAs.miRNA-124. The pathophysiological process of cerebral ischemia / reperfusion injury and the anti apoptosis effect of PI3K/Akt2 pathway on cerebral ischemia / reperfusion injury in mice are regulated by target negative regulation.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.3

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