【作者】 王磊；

【导师】 李根喜；

【作者基本信息】 南京大学 ， 生物学， 2021， 博士

【摘要】 外泌体是一种能被各种类型细胞分泌的细胞外囊泡,直径大小在30-200 nm之间。外泌体在生物体液中的分布范围很广,在血液、尿液、泪液、唾液、脊髓液、乳汁、精液、腹水和羊水中均能发现它的存在。越来越多的研究证实,外泌体与疾病的发生密切相关,且参与了肿瘤的发生转移。由于其来源广泛,取样相对简单,外泌体作为液体活检中一种很有前景的肿瘤标志物有望应用于临床中的疾病诊断和预后判断。当前,纳米粒子示踪技术对于检测纯化后的外泌体总体浓度和粒径分布具有重要作用,酶联免疫吸附技术（ELISA）和蛋白印迹技术（western blotting）是鉴别外泌体中所含蛋白的常用标准方法。然而,这些方法具有灵敏度低、样品用量大、特异性差和操作复杂的缺点,使得对外泌体进行精准和高灵敏分析遇到了重要挑战。鉴于外泌体是一个含有许多蛋白的膜结构,对于外泌体的分析检测可以转化成对外泌体膜和膜蛋白的检测。同时,借助适体与膜蛋白的特异性结合、外泌体膜的磷脂双分子层的负电荷性和疏水性等特点,将外泌体的检测转化成与之结合的核酸分子,并结合现有的许多信号扩增技术,比如纳米技术、酶介导的信号放大、核酸等温扩增等,以大幅度提高外泌体检测的灵敏度。在本论文工作中,我们在现有研究的基础上基于纳米材料的合成、酶介导的核酸信号扩增、以及多种信号扩增方法的联合使用,发展了多种新的信号放大方法,以最大程度提高对外泌体检测的灵敏性、特异性和简易性,并为外泌体在复杂生物样品中的检测提供技术支持。本论文的研究工作分为以下几个部分:1.基于磷脂膜间锆-磷酸配位键的信号放大锆离子(Zr⁴⁺)是一种多价阳离子,能通过配位作用强烈地与羧基、磷酸基团或磷酸酯等负电荷阴离子结合。基于外泌体和脂质体本身是由磷脂双分子层组成的囊泡结构,我们假设锆离子通过配位键能结合外泌体和脂质体,从而在外泌体表面引入许多带有信号分子的人工囊泡。基于磷酸-Zr⁴⁺-磷酸之间的配位相互作用,我们设计了一个无标记、低成本、简单的外泌体检测方法。该方法只需要简单易操作的磁分离步骤和一步信号释放过程,避免使用昂贵的仪器设备和设计复杂的信号扩增过程。此外,由于锆离子和磷脂膜的直接相互作用,不需要额外的化学修饰就能容易实现信号转化。引入脂质体作为优越的信号放大元件,检测灵敏度得到了提升,检测限为7.6×10³particles/μL。因此,我们构建了一种易操作、无修饰探针、灵敏的外泌体检测新方法,且在生物样品中也展现出优异的分析检测性能。此外,该方法第一次利用Zr⁴⁺与膜上磷酸根结合的特性来检测外泌体,为充分利用外泌体自身固有的特性进行外泌体分析提供了新的参考角度。2.基于无模板依赖的酶介导多重信号放大在本部分工作中,我们利用CD63抗体免疫磁珠特异性捕获外泌体,并用延长的Apt_MUC1适体与外泌体上的MUC1蛋白特异性结合。通过对外泌体的检测转换成对延长的Apt_MUC1适体检测,我们构建了三重不同的信号放大方法即HCR反应、DNase I酶切割、Td T酶扩增,用于外泌体的高灵敏检测。其设计思路是,与膜蛋白特异性结合的适体可引发大量的双链DNA（ds DNA）在外泌体表面组装,并使用核酸内切酶切割这些ds DNA形成短的核酸链,而这些短核酸链可作为Td T酶的引物被延长后产生大量信号。由于使用多重信号放大策略,该检测方法获得了对外泌体的高灵敏度分析,检测限为10 particles/μL。本方法中使用的酶不依赖于DNA模板序列,避免了复杂的核酸序列设计,而靶标外泌体的捕获和信号扩增需要两种膜蛋白同时存在于外泌体上,提高了方法的特异性。该方法中若将适体部分换成其它膜蛋白的特异性序列,就可以对外泌体上其它膜蛋白进行特异性分析,具有一定的通用性。此外,利用磁珠的简单易分离特点,该设计方法在复杂生物样品中也能灵敏地检测外泌体,显示出该方法优异的抗干扰能力,为其临床应用创造了条件。3.基于球形核酸的级联信号放大球形核酸（spherical nucleic acids,SNAs）是一种在纳米材料表面修饰的一层致密且定向的寡核苷酸。与游离核酸相比,球形核酸与其互补核酸链、蛋白受体或者酶的结合亲和力更高。许多材料如金纳米颗粒、配位聚合物颗粒和脂质体等能作为模板合成球形核酸。与脂质体类似,外泌体也是由脂质双层膜构成且能够在其表面进行功能化修饰。因此,我们提出了一种基于外泌体为模板合成球形核酸和酶介导的级联信号扩增策略在外泌体膜上进行核酸结合、生长、切割的循环信号放大方法并且进一步用于通用型、高灵敏外泌体的检测。具体来说,首先利用膜蛋白适体特异性捕获外泌体,然后,使疏水的磷脂双分子层与修饰胆固醇的单链DNA基于疏水作用力而结合,最后,结合上外泌体的单链引发信号放大反应。在我们提出的这个策略中存在三重信号放大设计:1)一个外泌体作为模板结合大量引物链形成球形核酸;2)一条引物链被Td T聚合形成poly T,并以此作为模板在Exo III作用下大量切割探针A;3)切割后形成的探针A短链作为新的引物不停地引发第二轮信号放大过程。该方法无需复杂的适体和探针链设计,仅需一种无模板引物链,在Td T、Exo III和探针A存在情况下,于同一反应液中进行三重信号放大过程,达到高灵敏检测外泌体的目的,检测限为44particles/μL。基于适体特异性捕获外泌体,以及胆固醇基团和外泌体膜间的疏水作用,我们设计的这个策略能够保证该方法具有高特异性和低背景信号。此外,无需通过高速离心分离外泌体,只需将临床血清样本简单离心去除细胞碎片,通过滤膜,稀释一定倍数后即可进行检测。该方法能够较好地区分临床样本中的结直肠肿瘤患者和健康受试者,为临床应用打下了基础。4.基于适体包裹脂质体和末端转移酶介导的信号放大在这部分工作中,我们提出了一种灵敏度高、成本低、易操作的方法来检测并分型外泌体。由于脂质体制备相对容易,且能在溶液中维持很长时间的悬浮状态而不聚集,并且在复杂生物环境中也具有很好的生物相容性,因此,我们先合成单体为1,2-dioleoyl-3-trimethylammonium-propane（DOTAP）的阳离子脂质体,然后加入带有大量负电荷的适体,适体基于静电相互作用吸附到脂质体表面,导致末端转移酶（terminal deoxynucleotidyl transferase Td T）难以识别、延长适体。然而,当溶液中存在外泌体时,适体和外泌体膜蛋白之间的强特异性相互作用取代适体和脂质体间较弱的静电相互作用,因此适体上的3’-OH能够被Td T酶识别并引发扩增反应。由于Td T酶的底物中富含d GTP,Td T扩增的底物就会形成G-四联体结构（G-quadruplex structure）,而G-四联体与硫磺素T（thioflavin T,Th T）结合并发出强烈的荧光。进一步研究表明,外泌体上膜蛋白的丰度与溶液的荧光强度成正相关。该策略利用负电荷适体与正电荷脂质体间的静电相互作用,而且二者形成的空间位阻可阻止Td T识别适体,因此,无需经过复杂的设计和适体标记,只需经过Td T酶一步信号放大方式,我们提出的检测方法就达到了对外泌体灵敏、低成本检测。所有步骤均在一锅法进行,无需分离过程和信号标记,有助于同时进行大量的样品分析,并降低操作难度和实验成本。此外,我们使用这个方法对不同肿瘤细胞分泌的外泌体和外泌体上不同的膜蛋白进行了分析,获得了不同外泌体上几种膜蛋白的分型图,进一步展现了不同外泌体的异质性。更多还原

【Abstract】 Exosomes with a diameter of 30-200 nm are extracellular vesicles that can be secreted by various types of cells.Exosomes are widely distributed in biological fluids in the range of blood,urine,tears,saliva,spinal fluid,milk,semen,ascites and amniotic fluid.More and more studies have confirmed that exosomes are closely related to the process of diseases and are even involved in the origin and metastasis of tumors.Benefitting from the diverse sources and relatively simple sampling,exosomes,as a promising tumor marker for liquid biopsy,are expected to be applied in clinical disease diagnosis and prognosis.At present,nanoparticle tracking analysis（NTA）plays an important role in detecting the total concentration and particle size of purified exosomes.Enzyme labeled immunosorbent assay（ELISA）and Western blotting are common standard methods for the identification of proteins existing in exosomes.However,these methods suffer from low sensitivity,large sample consumption,poor specificity,and tedious operation,making accurate and highly sensitive analysis of exosomes a huge challenge.Since exosome is a vesicle containing all kinds of proteins,the analysis of exosome can be converted to the examination of the membrane and membrane protein of exosomes.By taking advantage of the specific combination between aptamers and membrane proteins,negative charge and hydrophobicity of the phospholipid bilayer of exosome membranes,the detection of exosomes can be converted into nucleic acids which bind to exosomes.The sensitivity of exosome detection can be greatly improved by the signal amplification techniques,such as nanomaterial technology,enzyme-mediated signal amplification,isothermal nucleic acid amplification,and the combination of these technologies.In this paper,in order to maximumly improve the sensitivity,specificity and simplicity of exosome detection,we have developed a variety of new signal amplification methods based on the synthesis of nanomaterials,enzyme-mediated nucleic acids signal amplification,and the combined utilization of multiple signal amplification strategy,providing the foundation for the detection of exosomes in complex clinical samples.The research work of this paper is present in the following parts:1.Signal amplification based on zirconium phosphate coordination chemistry between phospholipid membranesZr⁴⁺ion can bind strongly to phosphate group of the phospholipid membranes via electrostatic interactions.Considering that exosomes and liposomes are actually vesicles composed of phospholipid bilayer,we hypothesize that Zr⁴⁺can bind with both exosomes and liposomes through coordination chemistry.Thus,artificial vesicles encapsulating with many signaling molecules are combined on the surface of the exosomes,and a sensitive,label-free and cost-effective method for detection of exosomes has been proposed by making use of phosphate-Zr⁴⁺-phosphate interaction.This method takes advantage of magnetic bead for easy separation and liposomes for one-step signal release process,which avoids the involvement of expensive equipment and complicated signal amplification designs.Due to the direct interaction between zirconium ions and phospholipid membranes,the signal transformation of the biosensor is easily achieved without extra chemical modification.With liposomes as superior signal amplification elements in this method,the detection sensitivity for exosomes is improved,and the limit of detection is 7.6×10³particles/μL.Hence,a new method with advantages of sensitive,easy to operate,no modification probe has been developed for the detection of exosomes.This biosensor also shows good performance in the complex biological samples for the detection of exosomes.Moreover,it is the first time to detect exosomes by linking the phospholipids in both exosomes and liposomes with Zr⁴⁺,which may bring about inspiration in the construction of biosensor for biomedical research by the rational utilization of the intrinsic phospholipid of exosomes.2.Template-free enzyme-mediated multiple signal amplificationIn this work,exosomes are specifically captured by immunomagnetic beads modified with CD63 antibody and another MUC1 protein on the surface of exosomes is combined with the extended MUC1 aptamer(Apt _MUC1).Thus,the concentration of exosomes is positively correlated with the content of elongated Apt _MUC1,and we have designed triple signal amplification methods based on HCR reaction,DNase I cleavage,and TDT mediated amplification for the highly sensitive detection of exosomes.The scheme of design is that a large number of ds DNA stemed from HCR reaction triggered by aptamers are digested by endonuclease to form short nucleic acid chains,and then these short nucleic acid chains acting as primers are extended by Td T enzyme to generate signals.Due to the strategy of multiple signal amplification methods,this assay has achieved a highly sensitive analysis of exosomes with limit of detection as 10 particles/μL.The enzymes used in this method are independent of DNA template sequence,avoiding the complex nucleic acid sequence design.The processes of exosomes capture and signal amplification require the simultaneous presence of two kinds of membrane proteins on exosomes,which improves the specificity of the method.In addition,by taking advantage of the simple and easy separation of magnetic beads,the assay holds the capacity to detect exosomes in complex biological samples with high sensitivity and effectiveness,which indicates that the proposed method has excellent anti-interference ability and provides potentials for the clinical application of exosomes.3.Spherical nucleic acids-based cascade signal amplificationSpherical nucleic acids（SNAs）are a type of nanomaterial with a small spherical core functionalized with dense and highly oriented oligonucleotides.Compared with free nucleic acids,SNAs have higher binding affinity with their complementary nucleic acid chains,protein receptors or enzymes.SNAs have been synthesized by many kinds of templates,such as gold particles,coordination polymer particles,and liposomes.Similar to liposomes,exosomes are also composed of lipid bilayer membrane and can be remolded by membrane surface engineering.Therefore,we have proposed a strategy of exosomes-templated SNAs and enzyme-mediated cyclic signal amplification based on the binding,growth,and incision of DNA for universal and highly sensitive detection of exosomes.Namely,aptamers are used to specifically capture exosomes,and then the hydrophobic phospholipid bilayer of exosomes binds to the cholesterol-modified single-strand DNA based on the hydrophobic interaction,which can initiate the single-strand DNA-based the signal amplification reaction.In this proposed strategy,there is a triple signal amplification:1)one exosome can bind with a number of primer strands;2)one primer strand of SNAs can be extended by Td T and then act as template strands to digest numerous probe（probe A）;3)truncated probe A as a new primer triggers the circle of the second signal amplification progress.Therefore,under the assistance of Td T,Exo III,probe A,and primer strand,the highly sensitive detection of exosomes can be achieved without complicated nucleic acids sequence design,and the limit detection is 44 particles/μL.Meanwhile,exosomes are captured based on the specific recognition between aptamers and membrane proteins,and the signal is amplified based on the hydrophobic interaction between cholesterol group and exosomes membrane,which synergistically endow the biosensor with high specificity and low signal background.With simple centrifugation,filtration,and dilution of clinical serum samples to remove cell debris and other large extracellular vesicles,the proposed method can distinguish malignancy colorectal cancer patients from the control groups without the high-speed centrifugation,which offers an alternative solution in the diagnosis of cancer in clinical samples.4.Fabrication of an aptamer-coated liposome complex and terminal deoxynucleotidyl transferase-mediated signal amplificationIn this work,we have proposed a new and efficient method for the detection and profiling of exosomes with high sensitivity,cost-effectiveness,and easy operation process in small samples.We have first fabricated cationic1,2-dioleoyl-3-trimethylammoniumpropane（DOTAP）liposomes with aptamers absorbed via self-assembly for targeting exosomal membrane proteins.Aptamers with large numbers of negative charges will combine with DOTAP liposomes due to electrostatic interaction,resulting in no recognition or extension by terminal deoxynucleotidyl transferase（Td T）because of the steric hindrance between Td T and liposomes.However,in the presence of exosomes,the weaker and nonspecific combination between aptamers and liposomes is superseded by the stronger and specific binding between aptamers and exosomes,making the free 3′-OH of aptamers to be available to Td T,which will trigger the polymerization of aptamers to form multiple G-quadruplex structures under the condition of a d GTP-rich pool.The G-quadruplex can give rise to strong fluorescence in the presence of thioflavin T（Th T）,and the fluorescence intensity is relevant to the abundance of exosomal membrane proteins.The proposed strategy takes advantage of the electrostatic interaction between negative-charge DNA and positive-charge liposomes,and the resulting steric hindrance renders the Td T enzyme inaccessible to the aptamers.Therefore,without complex design and labels on aptamers,the proposed method achieves highly sensitive and cost-effective detection for exosomes with one step of Td T-mediated signal enlargement.In addition,all experiments are conducted in one-pot due to the rational design without separation procedures,so this method is beneficial to mass sample analysis simultaneously.The proposed method has been employed to interrogate various membrane proteins of different cancer cell-derived exosomes by using a panel of corresponding aptamers and obtained a heat map to describe the profiling of exosomes.Thus,patterns that differentiate the heterogeneity of different exosomes can be eventually obtained.更多还原