【作者】 张新华；

【导师】 刘国权；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2017， 硕士

【摘要】 炎症反应是一种常见的有机体对外来刺激做出防御性反应的病理过程。通常,炎症反应作为机体的一种自动防御系统是有益于机体的,但是过度性的、失控性的炎症反应会对机体造成极大的伤害。猪繁殖与呼吸综合征病毒(PRRSV)感染猪后引起的猪肺损伤主要是因肺部过度的炎症反应所致。目前,对于这种肺损伤尚无有效的治疗方法。在炎症反应过程中,大量的中性粒细胞被肺血管内皮捕获,黏附于内皮上,并最终进行跨内皮迁移、渗出,破坏内皮的完整性,导致其渗透性增加而造成肺水肿。细胞间粘附分子1(ICAM-1)在中性粒细胞被内皮捕获、迁移、活化并渗出的过程中扮演了十分重要的角色。黏着斑激酶(FAK)能够介导细胞黏附和迁移,调节黏着斑的翻转,并且还参与了内皮被破坏后的修复过程。有研究表明,内皮受到伤害时,内皮细胞的迁移明显增强,同时FAK的磷酸化程度显著提高;当阻止FAK的活化后,内皮细胞的迁移也随之被阻断。本研究探究了FAK的表达与炎症的关系以及ICAM-1对FAK表达的调控,期望为缓解PRRSV感染所致猪肺损伤等过度性炎症反应提供一定的理论基础。本研究首先检测了四种炎症条件下FAK的表达变化,结果显示其显著增加。然后检测ICAM-1基因敲除对FAK表达的影响,发现FAK的表达显著降低。同时,我们发现ICAM-1基因敲除能够促进miR-15b-5p的表达,且利用生物信息学软件预测发现miR-15b-5p能够靶向FAK的3’UTR区。随后我们利用双荧光素酶实验证明了miR-15b-5p能够靶向FAK的3’UTR区。最后,在细胞水平超表达miR-15b-5p,结果表明超表达miR-15b-5p能够抑制FAK的表达。因此,本研究试验结果表明炎症能够促进FAK的表达以及ICAM-1能够通过miR-15b-5p调控FAK的表达。更多还原

【Abstract】 Inflammatory response is a common pathology process that organism makes a defensive response to external stimuli.Usually,the inflammatory response as an organism’s automatic defense system is beneficial to the body,but the excessive,uncontrolled inflammatory response to the organism is a great harm.The main reason for pig lung injury caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)infection is excessive inflammatory response.At present,there is no effective treatment for this lung injury.In the inflammation process,a large number of neutrophils is captured by pulmonary vascular endothelial,then adheres to endothelium,and eventually migrates across the endothelium,and exudates resulting in pulmonary edema by destructing the integrity and increasing permeability of the endothelium.Intercellular adhesion molecule-1(ICAM-1)plays a very important role in the process of endothelial capture,migration,activation and exudation of neutrophil.Focal adhesion kinase(FAK)can mediate cell adhesion and migration,and regulate the turnover of focal adhesion,and is also involved in the repair process after endothelial destroyed.Studies have shown that in the injured endothelium,the migration of endothelial cells significantly increased,while the degree of FAK phosphorylation significantly increased.When the activation of FAK is prevented,endothelial cell migration is subsequently blocked.This study explored the relationship between the expression of FAK and inflammation,the regulation of ICAM-1 on FAK expression,and it is expected to provide a theoretical basis for lung injury caused by PRRSV infection and other excessive inflammatory response.This study first examined the expression change of FAK in four inflammatory conditions,and the results showed a significant increase.The effect of ICAM-1 knockout on FAK expression was then examined and the expression of FAK was found to be significantly reduced.Simultaneously,we found that ICAM-1 knockout can promote the expression of miR-15b-5p,and the bioinformatics software predicts that miR-15b-5p can target the 3’UTR region of FAK.We demonstrate that miR-15b-5p is capable of targeting the 3’UTR region of FAK by dual-luciferase reporter assay.At last,miR-15b-5p is overexpressed at the cellular level,and the results show that overexpression of miR-15b-5p inhibites the expression of FAK.Therefore,the results of this study show that inflammation can promote the expression of FAK and ICAM-1 can regulate the expression of FAK through miR-15b-5p.更多还原