微球-磁珠-纳米孔筛分离纯化富集检测肿瘤外泌体新技术及骨肉瘤临床运用研究

Tumor derived exosome has the potential huge value and usage in tumor diagnosis biomarker, metastasis mechanism and treatment researches. However, the traditional exosome separation, purification and enrichment techniques have some critical defects such as protein contamination, extracellular vesicles and nucleic acid impurity, and time consuming and expensive et al. These critical defects may result in inaccuracy, error, and inefficiency of the protein and nucleic acid extracted from exosome. Therefore, it is essential and important to improve the exosome separation, purification enrichment and detection techniques. Bases on the preliminary work, the established micro-nano hydroxyapatite surface exosome enrichment technique, combined with the other techniques, nano hole griddle and antibody coating polystyrene magnetic bead, the research try to resolve the protein contamination, extracellular vesicles impurity, and time consuming and expensive problems in exosome separation, purification enrichment and detection procedure. Matrix array will be designed to make the novel exosome efficient high throughput sifting sort system to screen out the osteosarcoma exosome biomarkers related to metastasis to prove its potential clinical usage. The novel exosome separation, purification enrichment and detection technique above provide the essential and important technique for tumor biomarker sorting, metastasis mechanism and treatment researches or clinical experience. It has wide potential clinical usage and provide the tumor derived exosome related researches core technique.

外泌体在肿瘤诊断标志物、肿瘤转移及治疗等研究中具有极大运用前景，但传统外泌体分离、纯化、富集技术存在胞外囊泡混杂、核酸和蛋白质污染、耗时昂贵等问题，导致基于外泌体蛋白质、核酸检测的肿瘤标志物和治疗等研究存在误差及低效问题。所以研发新技术解决传统外泌体分离、纯化、富集、检测方法的缺陷和问题是关键科学问题之一。本课题将基于前期研究基础，结合纳米孔筛分离外泌体、精准调控表面微纳米羟基磷灰石结构的微球富集外泌体和单抗聚苯乙烯磁珠纯化技术，构建微球-磁珠-纳米孔筛新技术用于外泌体分离、纯化、富集、检测和筛选，解决外泌体分离纯化中胞外囊泡混杂、核酸和蛋白质污染、耗时昂贵等问题。建立高效高通量矩阵的微球-磁珠-纳米孔筛新技术，筛选骨肉瘤转移相关潜在外泌体诊断标志物。研究将为肿瘤外泌体的标志物筛选、肿瘤转移机制、治疗策略等研究和临床运用提供核心技术基础和平台，新技术具有广泛的临床运用。

外泌体在肿瘤诊断标志物、肿瘤转移及治疗等研究中具有极大运用前景，但传统外泌体分离、纯化、富集技术存在胞外囊泡混杂、核酸和蛋白质污染、耗时昂贵等问题，导致基于外泌体肿瘤标志物筛查和治疗等误差及低效问题。. 为解决外泌体分离纯化和检测关键科学问题，提出本研究目标（1） 建立外泌体分离纯化的新技术用于解决外泌体纯化中胞外囊泡混杂、外泌体核酸纯度、蛋白质污染问题。（2）以骨肉瘤外泌体为研究对象，采用新技术检测筛选骨肉瘤肺转移外泌体标志物并验证及运用。. 本研究通过骨肉瘤肺转移患者外泌体、肺转移标本、细胞株外泌体样本等mRNA测序等研究，初步获得了骨肉瘤转移特性的mRNA特征生物信息，并初步证实外泌体可作为骨肉瘤肺转移的潜在诊断方式。为评估现有外泌体分离纯化技术，对外泌体商业化试剂盒进行比较研究，研究结果明确商业外泌体分离纯化试剂盒获得外泌体生物信息存在较大差异和缺陷，临床运用此类试剂盒分离纯化外泌体存在潜在临床风险。. 研发微流控分离纯化技术去除外泌体蛋白质杂质。基于重新认知外泌体复合体的整体性检测分析特征谱思路，本项目研究确认外泌体质谱检测分析方法可行，开展质谱检测分析外泌体生物特征谱，获得外泌体骨肉瘤肺转移诊断特征谱。该技术具备快速、简便、稳定临床诊断运用价值，满足本研究核心目标，即建立外泌体分离纯化的新技术用于解决外泌体纯化中胞外囊泡混杂、蛋白质污染问题，检测筛选骨肉瘤肺转移外泌体标志物。.研究进一步采用氧化锌纳米材料结合微流控技术研发外泌体芯片，可捕获定量外泌体且检测分析外泌体膜标志物，通过外泌体标志物与骨肉瘤肺转移临床数据间映射关系，检测筛选骨肉瘤肺转移外泌体标志物。 . 本研究将促进外泌体分离纯化和检测分析技术发展，对骨肉瘤肺转移外泌体标志物临床运用具有价值，新技术对外泌体诊断肿瘤疾病具有广泛的临床运用价值。