Let-7a通过MKP1介导缺血再灌注后神经细胞损伤的机制研究

发布时间：2018-07-31 10:16

【摘要】：本研究分为3部分： 1.let-7a在脑缺血再灌注后的表达及其作用在本部分研究中，我们通过对正常大鼠、缺血再灌注大鼠脑内let-7a的表达的检测，发现let-7a在脑缺血再灌注后，在脑组织内高表达。为进一步研究let-7a在体内的作用，我们构建过表达let-7a的AAV9质粒，通过尾静脉注射的方式使之在大鼠体内过表达。与普通大鼠相比，过表达let-7a的大鼠在缺血再灌注后脑组织病理改变严重、神经功能缺损评分增高，并且脑组织的内炎症因子IL-6和TNF-α含量高于普通大鼠，细胞凋亡程度也明显重于普通大鼠。此结果提示let-7a在脑缺血再灌注后表达上调，并促进缺血再灌注后的炎症反应和细胞凋亡，进而加重神经损伤。 2.MKP1在脑缺血再灌注后的表达及其作用 MKP1是一种重要的抗炎、抗凋亡蛋白，通过数据库比对可以发现MKP1可能是let-7a的下游作用靶点。在本部分研究中，我们对MKP1在脑缺血再灌注后的作用进行研究。将大鼠分为假手术（Sham）组、对照组（Control）、MKP1抑制剂组（MKP1抑制剂-Sanguinarine chloride，血根碱）。其中，Sham组不做任何处理，Control组大鼠经尾静脉注射生理盐水，抑制组经大鼠尾静脉注射MKP1抑制剂，Sham组大鼠在脑缺血再灌注模型制备时线栓未阻断血流。与对照组大鼠相比，MKP1抑制剂组的大鼠在缺血再灌注后出现脑组织病理改变严重、梗死面积大、神经功能缺损评分高。同时，MKP1抑制剂组大鼠脑组织的炎症因子TNF-α和IL-6含量高于对照组大鼠，Tunel和染色阳性细胞数以及小胶质细胞增生也明显多于对照组大鼠。结果提示MKP1有抑制炎症反应、细胞凋亡、神经保护的作用。 3.let-7a对MKP1的调节在本部分研究中，我们对let-7a与MKP1两者之间的具体作用进行研究。通过western blot实验结果证实，let-7a mimic可下调神经元细胞系PC12细胞中MKP1蛋白的表达，而let-7a抑制剂let-7a inhibitor可促进其蛋白表达，但两者均不影响MKP1mRNA的水平。Luciferase assay显示，转染let-7a mimic后，含野生型MKP1的3’-UTR段的荧光报告质粒的荧光表达强度明显降低，将MKP13’-UTR突变后，let-7a不能降低荧光报告质粒的荧光表达强度。此结果表明，let-7a可通过结合MKP13’-UTR端在转录后水平下调MKP1的表达。本研究还发现，let-7a可上调缺氧状态下PC12细胞的凋亡，，过表达MKP1的PC12细胞可抑制let-7a促PC12细胞凋亡的作用。进一步研究说明，let-7a可直接靶向作用于重要抗炎、抗凋亡因子MKP1，通过对MAPK信号通路的调控从而加促进神经细胞损伤。
[Abstract]:This study is divided into three parts: the expression of 1.let-7a after cerebral ischemia-reperfusion and its effects in this part of the study, we measured the expression of let-7a in the brain of normal rats and ischemia-reperfusion rats. It was found that let-7a was highly expressed in brain tissue after cerebral ischemia reperfusion. To further study the role of let-7a in vivo, we constructed a AAV9 plasmid expressing let-7a, which was overexpressed in rats by tail vein injection. Compared with the normal rats, the brain tissue pathological changes and neurological deficit score of the rats with overexpression of let-7a were serious, and the contents of IL-6 and TNF- 伪 in the brain tissues were higher than those in the normal rats. The degree of apoptosis was also more severe than that of normal rats. These results suggest that the expression of let-7a is up-regulated after cerebral ischemia-reperfusion, and it also promotes the inflammatory response and apoptosis after ischemia-reperfusion. The expression and effect of 2.MKP1 after cerebral ischemia-reperfusion is an important anti-inflammatory and anti-apoptotic protein. It can be found that MKP1 may be the downstream target of let-7a by database comparison. In this part of the study, we studied the role of MKP1 after cerebral ischemia reperfusion. Rats were divided into sham-operated (Sham) group and control (Control) MKP1 inhibitor group (MKP1 inhibitor -Sanguinarine chloride, root alkaloid). The rats in the control group were injected with normal saline via caudal vein, while the rats in the control group were injected with MKP1 inhibitor, MKP1 inhibitor, through the tail vein of the control group, and the blood flow was not blocked in the model of cerebral ischemia-reperfusion. Compared with the control group, the rats in the MKP1 inhibitor group had severe pathological changes of brain tissue, large infarct area and high neurological deficit score after ischemia-reperfusion. At the same time, the contents of TNF- 伪 and IL-6 in brain tissue of MKP1 inhibitor group were higher than those of control group. The number of Tunel and staining positive cells and the proliferation of microglia in MKP1 inhibitor group were significantly higher than those in control group. The results suggest that MKP1 can inhibit inflammation, apoptosis and neuroprotection. The regulation of MKP1 by 3.let-7a in this part of the study, we study the specific role between let-7a and MKP1. The results of western blot assay confirmed that mimic could down-regulate the expression of MKP1 protein in the neuronal cell line PC12 cells, while let-7a inhibitor, the inhibitor of let-7a, could promote the expression of MKP1 protein, but neither of them could affect the level of MKP1mRNA. Luciferase assay showed that the expression of MKP1 protein was decreased after let-7a mimic transfection. The fluorescence expression intensity of the fluorescent reporter plasmid containing the 3'-UTR segment of wild type MKP1 was significantly decreased, but the fluorescence expression intensity of the fluorescent reporter plasmid could not be reduced after the mutation of MKP13'-UTR. These results suggest that let-7a can down-regulate the expression of MKP1 at post-transcriptional level by binding to the MKP13'-UTR terminal. It was also found that let-7a could up-regulate the apoptosis of PC12 cells under hypoxia, and that PC12 cells over-expressing MKP1 could inhibit the apoptosis of PC12 cells induced by let-7a. It is further demonstrated that let-7a can directly target the important anti-inflammatory and anti-apoptotic factor MKP1 and promote neuronal injury by regulating the MAPK signaling pathway.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.3

【参考文献】