【作者】 邓芸；

【导师】 罗阳；

【作者基本信息】 重庆大学 ， 生物学， 2022， 硕士

【摘要】 近年来,液体活检因其微创取样广受临床研究关注,其样本来源于循环系统,可以规避组织活检取样的局部限制性,更容易获得全面的“诊断信息”。作为液体活检的重要标志物,肿瘤来源sEVs(cancer-derived sEVs,csEVs)在肿瘤发展进程中具有介导免疫刺激和免疫抑制的功能,灵敏检测生物样本中csEVs并分析其相对丰度对肿瘤发展进程的追踪评估具有重要意义。然而,实现复杂临床样本中csEVs的识别和鉴定仍存在一定的挑战。一方面,csEVs粒径小、丰度低,难以实现特异性捕获;另一方面,细胞来源的异质性以及分离手段带来的异质性可能掩盖csEVs在肿瘤发生发展中的真实作用,造成误诊。基于物理特性的定量方式以及传统生化分析手段难以满足现行临床实践中对于高精度分析肿瘤来源sEVs的迫切需求。因此,发展一种新型的精准、灵敏的肿瘤来源sEVs检测手段既是临床研究的目的,也是亟待解决的科学问题。凭借着优异的信号放大能力以及一定的环境耐受性,催化发夹组装反应(catalytic hairpin assembly,CHA)已经在分子诊断领域取得了突破性研究进展,同时也为csEVs的量化技术研究带来了良好的发展契机。基于sEVs相关研究,本文以乳腺癌细胞MCF-7来源的csEVs和乳腺上皮细胞MCF-10A来源的sEVs为模型,联合功能化磁珠富集和CHA信号放大技术,有效实现对血浆样本中csEVs灵敏检测并进一步分析其相对丰度,以期揭示csEVs与肿瘤发生发展的关系。主要研究内容与结果如下:(1)具有识别功能的催化发夹组装体系的构建。为解决常规生化分析技术灵敏度低的问题,引入CHA作为核心元件,围绕其循环检测的特点设计和构建相关体系。CHA具有高灵敏、无酶参与、对环境依赖性低、可实现指数级信号扩增等优势,已被广泛应用于新型生物传感器的开发。本实验以sEVs膜表面标志性CD63蛋白和肿瘤特异性EpCAM蛋白为检测靶标,使用核酸适配体序列搭载催化发夹组装反应的引发链,构建具有识别功能的适配体-引发链,通过NUPACK软件进行结构模拟,发现增加引发链序列不会对适配体特殊结构产生影响。同时,CHA信号放大元件展示出稳定的茎环结构,为该反应低背景提供保证。利用凝胶电泳实验及荧光检测手段验证功能化CHA体系信号放大效果,设置相互干扰实验证明两组独立的CHA反应程序可以在同一体系下相互协作。以上结果表明,具有识别功能的催化发夹组装体系的构建成功。(2)基于催化发夹组装反应的csEVs检测策略研究。为了攻克csEVs检测以及分离问题,本课题以功能化CD9磁珠为富集单元,csEVs标志性CD63蛋白和肿瘤特异性EpCAM蛋白为靶标元素,结合具有识别功能的CHA信号放大元件,实现了临床样本中csEVs的灵敏检测,且最低检测限为420 particles/μL。同时引入csEVs相对丰度指标,一定程度上规避了临床样本提取得到的sEVs总量差异,进一步验证csEVs相对丰度指标与乳腺癌患者病理分期之间的潜在关联,结果表明疾病严重程度与病人血浆中csEVs相对丰度呈正相关,后续将扩大临床样本进一步验证这一相关性。综上,为解决csEVs粒径小、丰度低的问题,本论文提出了一种新型csEVs检测策略,利用核酸适配体赋予CHA反应体系独立识别EpCAM信号和CD63信号的能力,便于同一检测样本中EpCAM和CD63信号的读取,可有效实现对细胞系来源和临床血浆样本来源的csEVs灵敏检测,并引入csEVs相对丰度指标,发现其与乳腺癌患者病理分期之间存在一定关联,为揭露csEVs在肿瘤发展进程中的真实作用奠定基础。更多还原

【Abstract】 In recent years,liquid biopsies have been given serials attention in clinical application due to their minimally invasive sampling.Moreover,the samples are derived from the circulatory system,which can circumvent the limitations of conventional tissue biopsy and make it easier to obtain comprehensive "diagnostic information".As a crucial marker of liquid biopsy,cancer-derived sEVs(csEVs)have functions in signal mediation in both immune suppression and stimulation during tumor progression.In addition,sensitive detection of csEVs and determination of their relative abundance in biological samples are cruical for monitoring tumor progression.Identification and determination of csEVs in complex clinical samples are still challenging.On the one hand,csEVs are difficult to achieve specificity of capturation.On the other hand,heterogeneity of sEVs from different cell lines and isolation techniques may obscure assessment of the true role of sEVs.Therefore,conventional quantitative methods based on the physical and biochemical properties are difficult to deal with the urgent need for high precision analysis of csEVs in clinical practice.Due to the excellent signal amplification ability and certain thresholds of environment tolerance,catalytic hairpin assembly(CHA)has made a breakthrough in the field of molecular diagnosis.Meanwhile,this method supports potential to further development of quantitative techniques for deep csEVs research.Based on previous studies of sEVs,MCF-7 and MCF-10 A cell derived sEVs are investigated in this thesis.Associated with magnetic beads and CHA signal amplification,sensitive detection of csEVs in plasma samples are achieved and further to analysis their relative abundance.The relative abundance of csEVs and potential relationship between csEVs and tumorigenesis are explored.The contents and achievements are as follows:(1)Construction of a catalytic hairpin assembly circuit with recognition function.In order to break through low sensitivity of conventional biochemical techniques,CHA is employed to indentify target single csEVs.CHA is a highly sensitive,enzymefree and environment-insensitive isothermal amplification of nucleic acids,which can generate exponential signal amplification and has been widely used in biosensors.In this experiment,sEVs’ marker CD63 protein and tumor-specific EpCAM protein on the membrane surface of sEVs as targets,integrating aptamer sequence and initiator of CHA as novel trigger(Aptamer-trigger),conducting structural simulations via NUPACK software,showed that the addition of trigger sequence would not affect the specific structure of aptamer,and these CHA signal amplification elements exhibited a stable stem-loop structure,which provided low-background.Gel electrophoresis and fluorescence detection were carried out to verify the signal amplification,an interference experiment was set up to demonstrate that two independent CHA reaction programs can collaborate with each other.The above results indicate that successful construction of the catalytic hairpin assembly system with recognition function.(2)Catalytic hairpin assembly-based fluorescence sensing strategy for csEVs.In order to overcome difficulties caused by small particle size and low abundance of csEVs,the thesis focused on solving the heterogeneity brought by isolation techquies.This project developed,CD63 protein and cancer-relative EpCAM protein as the target elements,combined with CHA signal amplifier,and further to detect csEVs in biological sample.Introduction of the relative abundance of csEVs to resolve discrepancies of total sEVs obtained from different samples to a certain extent,next to investigate potential correlation between this index and pathological stage of breast cancer.The increasing relative abundance of csEVs in plasma of patients with higher disease severity.This correlation will be evaluated in the large-scale samples.In summary,to solve the problem of small size and low abundance of csEVs,this thesis proposed a novel csEVs detection strategy,using aptamers to endow the CHA reaction system with the ability to independently recognize EpCAM signals and CD63 signals,facilitating the readout of EpCAM and CD63 signals in a sample,which can realize the sensitive detection of csEVs from cell line and clinical samples,and introduction of the relative abundance index of csEVs revealed a certain correlation between them and the pathological stage of breast cancer,laying the foundation for uncovering the real role of csEVs in the tumor progression.更多还原