【作者】 李艳；

【导师】 施福东；

【作者基本信息】 天津医科大学 ， 临床医学（专业学位）， 2022， 硕士

【摘要】 背景和目的:在全世界,缺血性卒中是人类致残和死亡的主要原因之一。使用重组组织型纤溶酶原激活剂静脉溶栓和机械取栓是目前治疗缺血性脑卒中的有效方法,但由于严格的时间窗的限制和纳入标准限制,只有不到15%的患者可以获益。除此之外,缺血性脑损伤可重塑外周免疫,使外周免疫系统功能由正常状态转为抑制状态,进而增加感染的风险,主要包括肺感染以及泌尿道感染。自然杀伤细胞（natural killer cell,NK）是固有免疫系统的重要组分,卒中急性发作后,外周NK细胞数目减少、功能受损,引起与NK细胞相关的免疫缺陷以及卒中并发症的发展。Micro RNAs（miRNAs）参与免疫细胞转录后的调控活动。在我们前期的研究中,我们对来源于缺血性卒中病人外周血的NK细胞进行miRNA转录组学测序,发现miR-451a表达显著变化,但其对免疫应答具体的作用及潜在机制尚不明确。在本研究中,我们利用免疫缺陷小鼠,探讨缺血性卒中后miR-451a对NK细胞功能的影响并进而阐明相应的作用机制。方法:我们通过磁珠分选技术从野生型小鼠C57BL/6脾脏中分选NK细胞,在体外转染miR-451a模拟物（mimics）、抑制剂（inhibitor）及阴性对照（negative control）,后通过尾静脉被动转输至T,B淋巴细胞缺失,NK细胞失活的NOD/Prkdc^scid/IL-^null（NPG）小鼠体内,并对其进行大脑中动脉闭塞模型（middle cerebral artery occlusion,MCAO）诱导。评估模型诱导后第1、3天的行为学评分及梗死体积比较临床结局;采用肺部病理及肺部菌落培养评估卒中后肺部自发感染情况;采用流式细胞术评估卒中后第1、3天脑内和脾脏内NK细胞表型及功能改变;同时运用实时荧光定量PCR以及mTOR抑制剂AZD8055探索miR-451a调控NK细胞功能的机制。结果:首先我们应用实时荧光定量PCR验证了MCAO模型诱导后第3天脑内以及脾脏NK细胞中miR-451a的表达,证明缺血后第3天脑中及脾脏miR-451a显著升高。随后我们发现诱导MCAO模型后,miR-451a mimics抑制了脑内及外周的NK细胞的表型和功能,表现在抑制性受体KLRG1表达增加,活化标志CD69表达减少,以及效应分子IFN-以及perforin表达减少。而miR-451a inhibitor保留了NK细胞的活化和细胞毒性反应。进一步我们发miR-451a在脑内抑制NK细胞免疫应答,但对神经缺损无影响,但miR-451a在外周抑制NK功能,伴随着严重的肺部感染,包括菌落形成增多以及炎性细胞浸润增多。同时miR-451a inhibitor减轻肺部感染。药物抑制PI3K-Akt-mTOR通路阻断了miR-451a对NK细胞功能的影响。结论:在本研究中,我们证明了miR-451a通过PI3K-Akt-mTOR通路抑制脑缺血后外周和中枢NK细胞的功能,进一步抑制肺部菌落清除能力,为缺血性卒中后NK细胞的器官特异性靶向miRNA提供了新的见解,为卒中后恢复免疫防御提供了新的靶点。更多还原

【Abstract】 Background and purpose Stroke is a devastating and debilitating disease and a leading cause of death worldwide.Intravenous thrombolysis with recombinant tissue plasminogen activator（rt-PA）and mechanical thrombectomy are currently effective treatments for ischemic stroke.But due to the short therapeutic window and restrictive inclusion criteria,fewer than 15%patients benefit from these treatments.Moreover the injured brain can reshape peripheral immunity and transition the functional status of the peripheral immune system from competence to suppression,which increased the susceptibility to infections,predominantly pneumonia and urinary tract infection.Micro RNAs（miRNAs）act as the post-transcriptional regulator of immune cells.Our previous study had revealed that miR-451a was significantly altered in peripheral blood NK cells from patients with ischemic stroke,but the effects and the exact mechanism on immune defense remain elusive.In this study,using immunodeficient mice,we studied the effect and specific pathway of miR-451a on NK cells after ischemic stroke.Method Cell suspensions from spleen of wild type mice C57BL/6 were enriched for NK cells by using a magnetic-bead sorting system.Sorted NK cells were treated with miR-451a mimics,negative control or inhibitor.After transfected for 24 h,NK cells were transferred intravenously into NOD/Prkdc^scid/IL-^null（NPG）mice prior to middle cerebral artery occlusion（MCAO）.Neurological assessment and infarct volume at day 1 and day3 after MCAO were used to evaluate the stroke outcome.Lung tissues were collected for histological staining and bacteriological analysis to assess the spontaneous pulmonary infection.The phenotype and function of NK cells in spleen and brain were quantified by using flow cytometry at day 1 and day 3after ischemic stroke.Meanwhile,we used real-time PCR and mTOR inhibitor AZD8055 to explore the mechanism of miR-451a on NK cell function.Results Firstly,we verified the increased expression of miR-451a in NK cells in brain and spleen at day 3 after MCAO by using PCR.Secondly,miR-451a mimics suppressed NK cells activation and cytotoxicity in the ischemic brain and spleen,including over-expressed inhibitory receptor KLRG1,down-regulated activation marker CD69,and reduced production of effector molecules IFN-miR-451a inhibitor preserved NK cell activation and cytotoxicity responses.Additionally,miR-451a mediated brain-infiltrated NK cell dysfunction had no significant effect on neurological deficits.Importantly,miR-451a impaired NK cell function in the periphery and exacerbated the lung infection,which were indicated by the colony-forming units and inflammatory cells.And miR-451a inhibitor attenuated the bacterial burden of lung.Finally,pharmacological inhibition of PI3K-Akt-mTOR pathway blocked the miR-451a effects on NK cell functions.Conclusion In this study,we demonstrated that miR-451a suppressed the phenotype and function of NK cells in brain and periphery via PI3K-Akt-mTOR pathway after ischemic stroke.These effects of miR-451a may dampen the capacity of bacteria clearance.This study provides new insight for organ-specific targeting miRNA in NK cells and helps us to find the new target to boost periphery immunity after ischemic stroke.更多还原