【作者】 张蕾；

【导师】 贾友超；

【作者基本信息】 河北大学 ， 肿瘤学， 2021， 硕士

【摘要】 外泌体是一种磷脂双层膜包裹的生物纳米颗粒,直径为30-200 nm。近年来,由于外泌体携带大量遗传物质,在介导细胞-细胞间通信中发挥重要作用,已成为液体活检最理想的分析目标。然而,外泌体的低浓度使得其有效富集仍然是一个具有挑战性的任务。本论文设计开发了一种新型的Tim4@ILI-01免疫亲和薄片材料。基于Tim4与外泌体表面的磷脂酰丝氨酸结合的Ca²⁺依赖特性,捕获的外泌体在中性条件下可通过添加螯合剂轻松洗脱释放。该材料已成功用于富集肺癌患者血清中的外泌体。全文共分为四章:第一章:首先,主要对外泌体研究背景及分离方法进行概述;随后简述了金属有机框架的研究进展及相关应用;最后阐述了本论文的主要内容。第二章:设计开发了一种新型的金属有机框架（Metal-organic frameworks,MOFs）材料。首先,通过4-溴丁酸甲酯和1-（三甲基硅基）咪唑反应合成了溴化1,3-双（4-羧丁基）咪唑鎓（1,3-bis（4-carboxybutyl）-imidazolium bromide:ILI）离子液体。以ILI为有机配体,与六水合硝酸锌Zn（NO₃）₂·6H₂O制备了强亲水性ILI-01 MOFs材料。利用傅里叶变换红外光谱仪、X射线粉末衍射仪、扫描电子显微镜等技术证明了ILI-01 MOFs材料已成功制备。接触角测试表明ILI-01 MOFs材料具有强亲水特性。采用牛血清白蛋白溶液考察了ILI-01 MOFs材料的非特异性吸附,用洗涤缓冲液洗涤两次后,标准蛋白的非特异性吸附可降低到0.7%,基本消除了杂蛋白的非特异性吸附,降低外泌体分离时高丰度杂蛋白的干扰。第三章:基于ILI-01 MOFs材料上固有的丰富羧基,将Tim4抗体键合到到ILI-01MOF材料上。荧光倒置显微镜观察到Tim4@ILI-01免疫亲和材料已成功合成,BCA蛋白定量实验结果表明Tim4抗体的结合率达到82%。将制备的Tim4@ILI-01免疫亲和材料用于分离肺腺癌细胞系H1299培养上清中的外泌体,并通过透射电子显微镜,纳米颗粒示踪分析和Western Blotting进行鉴定。结果表明经Tim4@ILI-01免疫亲和材料分离后的外泌体大小和形态与之前的研究一致,富集后样品中标志性蛋白质含量显著提高。该方法捕获外泌体的效率可达到85.2%,比CD63@ILI-01和CD81@ILI-01等方法分别高出2.2倍和2.4倍。此外,进一步将所制备的Tim4@ILI-01免疫亲和材料与金标准“超速离心法”进行比较,该方法的捕获效率是超速离心法的5.2倍。利用Tim4抗体与外泌体表面磷脂酰丝氨酸结合的Ca²⁺依赖特性,Tim4@ILI-01免疫亲和材料捕获外泌体后通过加入EDTA缓冲液作为螯合剂,捕获的外泌体很容易释放。因此,利用细胞划痕实验和顺铂作用下的细胞毒性试验证明Tim4@ILI-01免疫亲和材料分离得到的外泌体具有生物学活性,可用于后续生物学分析和临床实验的应用。最后,将Tim4@ILI-01免疫亲和材料用于人血清样品中外泌体的分离富集,发现健康人和肺腺癌患者外泌体分泌之间的差异。通过实验比较不同时期的肺腺癌患者肿瘤干性基因CD44的表达水平,发现在蛋白水平上CD44基因在肺腺癌患者不同时期的表达发生了显著变化。这些结果表明Tim4@ILI-01免疫亲和材料是一种稳定的富集基质,具有良好的临床应用潜力。最后总结全文,本文制备了一种Tim4@ILI-01免疫亲和材料。该免疫亲和材料具有强亲水性,低非特异性吸附性能和无损释放外泌体的特点,可用于细胞培养基和血清中外泌体的有效分离和富集,具有广阔的应用前景。更多还原

【Abstract】 Exosomes are bio-nanoparticles wrapped in a phospholipid double-layer membrane with a diameter of 30-200 nm.In recent years,because exosomes carry a large amount of genetic material and play an important role in mediating cell-cell communication,they have become the most ideal analysis target for liquid biopsy.However,the low concentration of exosomes makes their effective enrichment still a challenging task.This paper designed and developed a new type of Tim4@ILI-01 immunoaffinity flake material.Based on the Ca²⁺-dependent properties of Tim4 binding to phosphatidylserine on the surface of exosomes,the captured exosomes can be easily eluted and released by adding a chelating agent under neutral conditions.The material has been successfully used to enrich the exosomes in the serum of lung cancer patients.The full text is divided into four chapters:Chapter 1:Firstly,the research background and separation methods of exosomes were summarized.Then,the research progress and related applications of metal-organic framework are summarized.At last,the main content of this thesis is expoundedChapter 2:Designed and developed a new type of metal-organic frameworks（MOFs）material.First,1,3-bis（4-carboxybutyl）-imidazolium bromide（ILI）ionic liquid was synthesized by the reaction of methyl 4-bromobutyrate and 1-（trimethylsilyl）imidazole.The strongly hydrophilic ILI-01 MOFs were prepared with ILI as the organic ligand and zinc nitrate hexahydrate Zn（NO₃）₂·6H₂O.The use of fourier transform infrared spectra,X-ray diffraction signals,scanning electron microscopic and other techniques have proved that the ILI-01 MOFs material has been successfully prepared.Water contact angle shows that the ILI-01 MOFs material has strongly hydrophilic properties.The non-specific adsorption of ILI-01 MOFs material was investigated with bovine serum albumin solution.After washing twice with washing buffer,the non-specific adsorption of standard protein can be reduced to0.7%,which basically eliminates the non-specific adsorption of impurity proteins.The interference of high abundance contaminants during exosomes separation is eliminated.Chapter 3:Based on the abundant carboxyl groups inherent in the ILI-01 MOFs material,the Tim4 antibody was successfully bonded to the ILI-01 MOF material.Fluorescence inverted microscope observed that Tim4@ILI-01 immunoaffinity material has been successfully synthesized,and the results of BCA protein quantitative experiment showed that the binding rate of Tim4 antibody reached 82%.The prepared Tim4@ILI-01 immunoaffinity material was used to separate exosomes in the culture supernatant of lung adenocarcinoma cell line H1299.Exosomes were furture identified by transmission electron microscopy,nanoparticle trace analysis and Western Blotting.The results showed that the size and morphology of exosomes separated by Tim4@ILI-01 immunoaffinity material were consistent with previous studies,and the content of marker proteins in the samples was significantly increased after enrichment.The efficiency of this method to capture exosomes can reach 85.2%,which is 2.2 times and 2.4 times higher than that of CD63@ILI-01 and CD81@ILI-01,respectively.In addition,the prepared Tim4@ILI-01 immunoaffinity material was further compared with the gold standard"ultracentrifugation method".The capture efficiency of this method is 5.2 times that of the ultracentrifugation method.Taking advantage of the Ca²⁺-dependent properties of Tim4 antibody binding to phosphatidylserine on the surface of exosomes,the Tim4@ILI-01 immunoaffinity material captures exosomes by adding EDTA buffer as a chelating agent,and the captured exosomes are easily released.Therefore,the cell wound-healing assay and the cytotoxicity test under the action of cisplatin proved that the exosomes isolated from the Tim4@ILI-01 immunoaffinity material have biological activity and can be used for subsequent biological analysis and clinical experiments.Finally,the Tim4@ILI-01 immunoaffinity material was used for the separation and enrichment of exosomes in human serum samples,and the difference in exosomes secretion between healthy people and patients with lung adenocarcinoma was found.Through experiments to compare the expression level of tumor stem gene CD44 in patients with lung adenocarcinoma at different stages,it was found that the expression of CD44 gene in lung adenocarcinoma patients at different stages changed significantly at the protein level.These results indicate that Tim4@ILI-01 immunoaffinity material is a stable enrichment matrix and has good clinical application potential.Finally,the full text is summarized.In this work,the Tim4@ILI-01 immunoaffinity material with strongly hydrophilicity,low non-specific adsorption performance and non-destructive release of exosomes was developed.It can be used for the effective separation and enrichment of exosomes in cell culture media and serum,and has broad application prospects.更多还原