【作者】 杨莉；

【导师】 姚红红；

【作者基本信息】 东南大学 ， 免疫学， 2021， 博士

【摘要】 目的:脑卒中是全球成年人残疾的主要原因,严重影响患者生活质量,但目前尚无有效的改善患者预后的药物。新近研究表明,环状RNA（circular RNA,circ RNA）在缺血性脑卒中发生发展中发挥重要作用,筛选有治疗前景的circ RNA具有重要意义。同时,如何安全有效地将效应circ RNA靶向递送至作用部位也是目前仍待解决的科学问题。基于此,本研究拟筛选与脑卒中密切相关的circ RNA,进一步设计和验证基于细胞外囊泡（extracellular vesicle,EV）的靶向治疗方案,旨在探寻可以促进缺血后脑修复的、具有临床转化价值的有效治疗策略。方法:基于临床样本,通过急性缺血性脑卒中（acute ischemic stroke,AIS）患者血浆circ RNA芯片和定量聚合酶链反应（quantitative polymerase chain reaction,q PCR）技术,筛选出AIS患者血浆中和光化学诱导（photothrombotic,PT）卒中模型鼠血浆及组织中差异表达circ RNA;采用统计学分析circ SCMH1与AIS严重程度的相关性,及对患者诊断和预后的价值。基于狂犬病病毒糖蛋白（rabies virus glycoprotein）RVG-Lamp2b-脑靶向修饰的技术构建RVG-circ SCMH1-EV,通过纳米粒径跟踪分析（nanoparticle tracking analysis,NTA）、透射电子显微镜（transmission electron microscope,TEM）、western blot、q PCR等表征RVG-circ SCMH1-EV;采用HE染色和流式细胞术评估RVG-circ SCMH1-EV的安全性。通过行为学实验评估RVG-circ SCMH1-EV对卒中模型鼠感觉运动功能的影响;通过尼氏染色和核磁成像评估RVG-circ SCMH1-EV对卒中模型鼠脑梗死体积的影响。采用高尔基体染色结合Sholl分析、病毒转染、免疫荧光染色和流式细胞分选术等技术,探究circ SCMH1促进缺血后脑修复的细胞机制。基于蛋白质组学和转录组学,结合生物信息学和分子生物学实验,探究circ SCMH1促进缺血后脑修复的分子机制。结果:circ SCMH1在AIS患者血浆中显著降低,该现象在PT模型鼠中得以证实;统计分析显示卒中1 d时的circ SCMH1水平与AIS严重程度弱相关,对诊断评估的曲线下面积为0.7896;在预后较好的AIS患者血浆中,卒中1 d时的circ SCMH1水平较高,且随患者恢复而回升;预后评估的曲线下面积为0.6864。RVG-circ SCMH1-EV粒径集中分布在117 nm左右;脑靶向信号在EV表面表达;circ SCMH1在EV中高表达;RVG-circ SCMH1-EV可安全高效地将circ SCMH1靶向递送至梗死周围区并被神经细胞摄取。RVG-circ SCMH1-EV显著促进卒中模型鼠感觉运动功能恢复;其对PT模型鼠梗死体积无显著影响,但可显著减小远端大脑中动脉闭塞（distal middle cerebral artery occlusion,d MCAO）和暂时性大脑中动脉闭塞（transient middle cerebral artery occlusion,t MCAO）模型鼠梗死体积,可能有神经保护作用。细胞机制研究表明,RVG-circ SCMH1-EV可逆转缺血引起的神经元树突棘、树突分支、树突长度和树突复杂度损伤的现象,增强神经元可塑性;抑制梗死周围区星形胶质细胞和小胶质细胞活化;抑制外周免疫细胞CD4⁺T细胞、CD8⁺T细胞、B220⁺B细胞和Ly6G^-Ly6C^lo细胞浸润梗死周围区。分子机制研究表明,circ SCMH1与转录因子甲基Cp G结合蛋白2（methyl-Cp G binding protein 2,Me CP2）结合,解除Me CP2对靶基因的转录抑制作用,从而促进卒中后脑修复。结论:本研究立足临床数据,基于RVG-Lamp2b-EV递载系统,从多个层面证实了RVG-circ SCMH1-EV可在缺血后促进脑修复,解析了核心效应分子-circ SCMH1作用的细胞及分子机制。本研究将为开发有效促进缺血性脑卒中患者预后的治疗方案提供基础。更多还原

【Abstract】 Aims:Stroke is a leading cause of adult disability that can severely compromise patients’quality of life in the worldwide,yet no effective medication currently exists to accelerate rehabilitation.A variety of circular RNA（circ RNA）molecules are known to function in ischemic brain injury,and screening of circ RNA with diagnostic and therapeutic prospects is of great significance.However,how to safely and effectively deliver the circ RNA to the targeted site remains to be resolved.Thus,the study sought to search the related circ RNA in the pathogenesis of stroke,and then exlpore the effective therapeutic strategies for brain repair after ischemia stroke based on extracellular vesicles（EVs）.Methods:circ RNA was screened from the plasma of acute ischemic stroke（AIS）patients using circ RNA microarray and valided in AIS and photothrombotic（PT）mice combined with quantitative polymerase chain reaction（q PCR）.Clinical statistic was used to analyze the relationship between circ SCMH1 and the severity of AIS,the potential in accessing the diagnosis and prognosis of AIS.Engineered rabies virus glycoprotein（RVG）-circ SCMH1-EVs based on modified RVG-lamp2b-EVs targeting brain were generated to deliver circ SCMH1.Nanoparticle tracking analysis（NTA）,transmission electron microscope（TEM）,western blot and q PCR were used to characterize RVG-circ SCMH1-EVs.HE staining and flow cytometry were used to examine the toxicity and immunogenicity,respectively.Behavioral tasks were performed to evaluate the effect of RVG-circ SCMH1-EVs on sensorimotor functions in rodent models.Nissl staining and magnetic resonance imaging（MRI）were used to examine the effect of RVG-circ SCMH1-EVs on infarct size.Golgi staining combined with Sholl analysis,lentivirus transduction,immunostaining,and flow cytometry were used to explore the cellular mechanism（s）of brain recovery.Proteomic assay and RNA-seq combined with transcriptional profiling and molecular profiling were used to identify the molecular mechanism（s）of brain recovery.Results:circ RNA SCMH1（circ SCMH1）levels were significantly decreased in plasma of AIS patients and PT mice,as well as in the peri-infarct zone of mice.Statistical analysis revealed that the baseline circ SCMH1 levels on the 1^st day were low related to AIS with a significant area under the curve（AUC）of 0.7896 in diagnosing AIS.Furthermore,there was a higher level of the baseline circ SCMH1 on the 1^st day between patients that eventually achieved good outcomes versus those with poor outcomes,and the level rose along with stroke recovery.ROC curve analysis was performed to calculate the predictive power of baseline circ SCMH1 levels for stroke outcome and the AUC was 0.6864.The engineered EVs were physically homogenous,with a size distribution peaking at 117 nm in diameter.Brain targeting signals were expressed on the surface of EVs,and circ SCMH1 was overexpressed in the EVs.circ SCMH1 was safely and effectively delivered to the peri-infarct zone via the engineered RVG-circ SCMH1-EVs.The study identified that RVG-circ SCMH1-EVs improved functional recovery of PT mice with no significant difference in infarct volume between RVG-circ SCMH1-EV group and RVG-Vector-EV group.However,the protective effect could not be ruled out as the infarct volume was decreased in distal middle cerebral artery occlusion（d MCAO）and transient middle cerebral artery occlusion（t MCAO）model mice.Furthermore,the study discovered that RVG-circ SCMH1-EVs relieved the damage to spines,branches,denderic lengenth and intersections,enhanced neuronal plasticity;and also inhibited astrocytes and microglia activation in the peri-infact area,as well as peripheral immune cell infiltration in PT mice,including CD4⁺T cells、CD8⁺T cells、B220⁺B cells and Ly6G^-Ly6C^lo cells.Elucidating a molecular mechanism that circ SCMH1bound to the transcription factor methyl-Cp G binding protein 2（Me CP2）,releasing repression of Me CP2 target gene transcription,thereby promoting brain repair after stroke.Conclusions:Based on data obtained from the plasma of AIS patients,using the RVG-Lamp2b-EV delivery system,this study reveals that RVG-circ SCMH1-EVs afford multiple advantages to promote functional recovery in the rodent stroke models,and demonstrates the basic mechanism of how circ SCMH1 can be therapeutically exploited to improve post-stroke outcomes.This study will provide the basis for the development of treatment that effectively promote the recovery of ischemic patients.更多还原