【作者】 张锐；

【导师】 杜怡峰；

【作者基本信息】 山东大学 ， 神经病学（专业学位）， 2022， 博士

【摘要】 研究背景:细胞外囊泡(EVs)主要有三种类型:外泌体(30-150nm)、微囊泡(100-1000nm)和凋亡小体(1000nm-),其中外泌体是最受关注的研究对象,外泌体(Exosome)是细胞内的多泡体(Multivesicular bodies,MVB)与细胞质膜融合后主动分泌到细胞外的一种小囊泡。外泌体的直径在30-150nm之间,由脂质双分子层包绕,其中包含蛋白质、脂质、mRNA、miRNA等多种物质,存在于血液、母乳、唾液、尿液、脑脊液等多种体液中。其过程首先由细胞膜内陷形成早期核内体(Early endosome),早期核内体膜向内出芽并将胞质中的RNA、miRNA、蛋白包裹其中形成管腔内囊泡(ILVs),管腔内囊泡逐步积累增多,而早期核内体则生长成熟,形成晚期核内体(Late endosome),或者叫做多囊泡体(MVBs);晚期核内体的结局一般会出现两种情况:一是与溶酶体融合后囊泡内容物降解;二是与细胞膜融合后将其体内的囊泡释放到细胞外,也就是外泌体,外泌体在被发现的早期,它仅仅被当做是细胞向外排出代谢废物的载体,随着人们对外泌体内容物逐渐了解增多,尤其是功能性蛋白及mRNA,miRNA的发现,使得外泌体成为了近十年的研究热点之一。运动可以延长寿命,是一种可以降低与衰老相关的疾病的风险改善人类健康的非药理学方法。短期高强度运动是一种可以提高代谢功能和有氧能力的非常有效的方法,其特点是短时且剧烈。且已被证明比中等耐力运动更有效。通常认为外泌体至少通过两种机制,即依赖或在不依赖转运必需内体分选复合物(ESCRT)机制,不依赖ESCRT机制可能与神经酰胺有关,因而我们提出一种假设,即在小鼠中,高强度运动是通过神经酰胺的分泌介导了外泌体的生成和增加。经过查阅文献,我们尝试以C57BL/6小鼠为实验动物,探索上述假设的可能性。鉴于目前对细胞外囊泡的定义尚不完全明确,而且由于当前技术的限制,迄今为止的许多研究分析的是粗糙的细胞外囊泡制剂,国际胞外囊泡协会(ISEVs)发布的指南提出使用细胞外囊泡一词代替外泌体和(脱落)微泡。所以我们虽然以外泌体为研究对象,但是仍然使用细胞外囊泡来表达以更符合实际情况,这也是诸多文献中的常用说法。研究目的:1.EVs在不运动和高强度运动后的释放情况。2.EVs在外周和中枢神经系统中的释放。3.在小鼠中是否存在神经酰胺依赖性促进EVs释放的可能机制。研究方法:本课题以C57BL/6小鼠为研究对象,喂养至4-5个月龄。将其分为运动组及非运动组,运动组又分为不同的观察时间点,在高强度运动后,对脑组织释放的外泌体以及外周血中的外泌体进行提取,通过免疫荧光检测外泌体标记蛋白表达情况、western-blot检测蛋白定量,用Nanosight LM10检测外泌体的粒径分布和数量,检测神经酰胺,比较运动组和非运动组在表达上的差异,以及脑组织中的外泌体在高强度运动后不同时间段的释放情况进行统计学分析。观察小鼠在高强度运动下,脑组织和外周中的外泌体分泌情况,探索短期高强度的运动后是否通过神经酰胺机制促进了脑组织和血浆外泌体的释放。统计分析:定量数据采用均数±标准差表示,两组间比较采用T检验,多组间比较采用单因素方差分析,P值小于0.05被认为具有统计学差异。使用SPSS 22.0进行统计分析。研究结果:1.为研究血清中EVs释放与高强度锻炼之间的关系,运动组小鼠进行增量跑步机锻炼30分钟。从外周血血清EVs被纯化后,用免疫印迹分析EVs标记TSG101,CD63,HSC70表达。蛋白谱带显示HSC70水平在高强度运动(0分钟)后迅速升高(P<0.01),运动(90分钟)后迅速恢复到初始水平(P>0.05)。运动前后TSG101和CD63水平无显著差异(P>0.05)。2.采用脑组织免疫荧光染色研究短期运动对海马EVS释放及与Tuj1+神经元共定位的影响。本研究检测了神经元外泌体中选择性外泌体标志物HSC70和TSG101的表达。其中,不运动组与运动后即刻评估组海马区总Tuj1+细胞中TSG101+Tuj1+细胞和HSC70+Tuj1+细胞的比例存在显著差异(TSG101 P<0.05;HSC70 P<0.05)。这些结果提示运动后海马神经元中EVs较运动前是增加的。3.为了研究高强度运动与脑内EVs释放之间的关系,运动组小鼠进行30分钟递增跑步机运动。我们分别在运动前、运动后立即(0 min)和运动后90分钟(90min)采集小鼠脑组织。EVs蛋白的Western blot分析证实,运动后即刻0 min EVs释放量明显大于运动前。此外,短期和高强度运动对HSC70和TSG101阳性囊泡的影响更明显(P<0.05),C D63阳性小泡在运动后无明显变化(P>0.05)。Western blot分析还显示,运动后90分钟,TSG101和HSC70水平几乎恢复到基线水平(P>0.05)。此外,小鼠在运动前、运动后(0分钟)和运动后90分钟(90min)的细胞外囊泡的分泌也得到纳米颗粒追踪分析(N anoparticle Tracking Analysis NTA)的进一步证实。细胞外囊泡的的集中度(×108颗粒/ml)在运动后高于运动前(P<0.01),运动后90min恢复到基线水平(P>0.05),而E Vs的平均直径在运动前后没有显着变化(P>0.05)。4.为了确定EVs的分泌是否依赖神经酰胺的合成,我们通过薄层色谱(TLC)测量了脑组织中神经酰胺的分泌。结果显示,短期高强度运动后0 min神经酰胺释放量显著高于运动前(P<0.05),运动后90min神经酰胺释放量基本恢复至基线水平(P>0.05)。结论:短期高强度运动改变了大脑的EVs水平并增加C57BL/6小鼠脑组织EVs释放及血清的释放水平。通过western blot、免疫荧光和NTA检测均得到了证实,同时,短期高强度运动显著影响C57BL/6小鼠脑组织神经酰胺释放,神经酰胺的增加可能是促进外泌体释放增加的机理之一。更多还原

【Abstract】 Research Background:There are three main types of extracellular vesicles(EVs):exosomes(30-150nm),microvesicles(100-1000nm)and apoptotic bodies(1000nm-),of which exosomes are the most studied object,Exosomes are small vesicles that are actively secreted outside the cell after the fusion of intracellular multivesicular bodies(MVB)with the cytoplasmic membrane.The diameter of exosomes is between 30-150nm,surrounded by lipid bilayers,which contain proteins,lipids,mRNA,miRNA and other substances,and exist in blood,saliva,urine,cerebrospinal fluid and breast milk.in body fluids.The process is that first the cell membrane invaginates to form an early endosome,and then the early endosome membrane buds inward and encapsulates proteins,RNAs and miRNAs in the cytoplasm to form intraluminal vesicles(ILVs).With the gradual accumulation of intraluminal vesicles(ILVs),early endosomes grow and mature to form late endosomes,also known as multivesicular bodies(MVBs).There are two fates of late endosomes:1.The vesicle contents are degraded after fusion with lysosomes;2.After fusion with the cell membrane,these vesicles are released outside the cell,that is,exosomes.For a long time after it was discovered,exosomes were only regarded as garbage carriers for cells to excrete waste,but as the contents of exosomes gradually became known,especially functional proteins and mRNAs,miRNAs The discovery of exosomes has made exosomes one of the research hotspots in the past decade.Exercise can prolong life and is a non pharmacological method that can reduce the risk of aging related diseases and improve human health.Short term high-intensity exercise is a very effective method that can improve metabolic function and aerobic capacity.It is characterized by short-term and intense exercise.It has been proved to be more effective than moderate endurance exercise.It is generally believed that exosomes pass through at least two mechanisms,i.e.dependent or independent transport essential endosomal sorting complex(eccrt)mechanism,and the independent ESCRT mechanism may be related to ceramide.Therefore,we propose a hypothesis that high-intensity exercise mediates the formation and increase of exosomes through the secretion of ceramide in mice.After consulting the literature,we tried to take C57BL/6 mice as experimental animals to explore the possibility of the above hypothesis.Given that the current definition of extracellular vesicles is not fully defined,and due to the limitations of current technology,many studies to date have analyzed crude extracellular vesicle preparations,guidelines issued by the International Society for Extracellular Vesicles(ISEVs)propose Use the term extracellular vesicles instead of exosomes and(exfoliated)microvesicles.Therefore,although we use exosomes as the research object,we still use extracellular vesicles to express more in line with the actual situation,which is also a common saying in many literatures.Research purposes:1.EVs are released after inactivity and high-intensity exercise.2.Release of EVs in the peripheral and central nervous system.3.Possible mechanisms to promote the release of EVs.Research methods:In this project,C57BL/6 mice were used as the research objects and were fed to 4-5 months old.They were divided into exercise group and non-exercise group,and exercise group was divided into different observation time points.After high-intensity exercise,exosomes released from brain tissue and exosomes in peripheral blood were extracted and analyzed by immunofluorescence.Detection of exosome marker protein expression,western-blot detection of protein quantification,Nanosight LM10 to detect the particle size distribution and number of exosomes,detection of ceramides,comparison of the difference in expression between exercise and non-exercise groups,and brain tissue The release of exosomes in different time periods after high-intensity exercise was statistically analyzed.To observe the secretion of exosomes in the brain tissue and the periphery of mice under high-intensity exercise,and explore whether short-term high-intensity exercise promotes the release of exosomes in brain tissue and plasma through a ceramide mechanism.Statistical analysis:Quantitative data were expressed as mean ± standard deviation,t-test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups,and a P value less than 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 22.0.Research result:1.To study the relationship between the release of EVs in serum and high-intensity exercise,mice in the exercise group performed incremental treadmill exercise for 30 minutes.Serum EVs were purified from peripheral blood,and the expression of EVs markers TSG101,CD63,and HSC70 were analyzed by western blot.The protein spectrum showed that the level of HSC70 increased rapidly(P<0.01)after high-intensity exercise(0 min),and returned to the initial level rapidly(P>0.05)after exercise(90 min).There was no significant difference in the levels of TSG101 and CD63 before and after exercise(P>0.05).2.The effect of short-term exercise on hippocampal EVS release and colocalization with Tuj 1+neurons was investigated by immunofluorescence staining of brain tissue.This study examined the expression of selective exosomal markers HSC70 and TSG101 in neuronal exosomes.Among them,the proportions of TSG101+Tuj1+cells and HSC70+Tuj1+cells in the total Tuj1+cells in the hippocampus of the non-exercise group and the immediate post-exercise assessment group were significantly different(TSG101 P<0.05;HSC70 P<0.05).These results suggest that EVs in hippocampal neurons are increased after exercise compared to before exercise.3.To investigate the relationship between high-intensity exercise and the release of EVs in the brain,mice in the exercise group performed 30-min incremental treadmill exercise.We collected mouse brain tissue before exercise,immediately after exercise(0 min),and 90 min after exercise(90 min).Western blot analysis of EVs protein confirmed that the amount of EVs released at 0 min immediately after exercise was significantly greater than that before exercise.In addition,short-term and high-intensity exercise had more significant effects on HSC70 and TSG101-positive vesicles(P<0.05),while CD63-positive vesicles had no significant change after exercise(P>0.05).Western blot analysis also showed that TSG101 and HSC70 levels almost returned to baseline levels 90 minutes after exercise(P>0.05).In addition,the secretion of extracellular vesicles in mice before exercise,after exercise(0 min)and 90 minutes after exercise(90 min)was further confirmed by NTA.The concentration of extracellular vesicles(×108 particles/ml)was higher after exercise than before exercise(P<0.01),and returned to the baseline level 90 min after exercise(P>0.05),while there was no significant change(P>0.05)of the average diameter of EVs before and after exercise.4.To determine whether EVs secretion is dependent on ceramide synthesis,we measured ceramide secretion in brain tissue by thin-layer chromatography(TLC).The results showed that the release of ceramide at 0 min after short-term high-intensity exercise was significantly higher than that before exercise(P<0.05),and the release of ceramide basically returned to the baseline level at 90 minutes after exercise(P>0.05).In conclusion:Short-term high-intensity exercise altered the level of EVs in the brain and increased the release of EVs in the brain tissue and serum of C57BL/6 mice.It was confirmed by western blot,immunofluorescence and NTA detection.At the same time,short-term high-intensity exercise significantly affected the release of ceramide in the brain tissue of C57BL/6 mice,and the release of ceramide may be one of the mechanisms that promote the increased release of exosomes.更多还原