表面修饰磁珠提取外泌体的研究

REASEARCH ON MAGNETIC BEADS MODIFIED ON THE SURFACE USING FOR EXTRATION OF EXOSOMES

周锐

11849110

硕士

生物工程领域工程

葛东亮

2020-06-02

2020-07-20

哈尔滨工业大学

深圳

液体活检技术是指非入侵方式对受检者进行检查，通过分析来源于机体的体液，包括血液、尿液、唾液、脑脊液等，来实现对机体生理状态和病例状态的评估和监测。与传统活检技术一样，液体活检技术主要用于癌症等疾病的诊断和监测以及早期筛查和临床预后，优点是及时迅速而且基本无创。目前液体活检的研究主要集中于CTC（循环肿瘤细胞）和ctDNA（循环肿瘤DNA），对于外泌体的研究还并不多。随着研究的深入，外泌体及其在液体活检和靶向治疗领域的巨大潜力越来越被人们关注。然而，由于外泌体直径在20-200nm之间，并且细胞分泌产生的细胞外囊泡不仅包括外泌体，还包括粒径稍大的微囊泡（microvesicles）,分离起来非常困难。这目前常用的分离手段包括超速离心，化学沉淀，免疫分离，但是由于超速离心需要价格昂贵的超速离心机，而且效率低，化学沉淀会引入蛋白质杂质，免疫分离会破坏外泌体的膜结构，影响外泌体的生物活性，因此对现有广泛应用于外泌体分离提取方法存在一定的局限性。研究发现，包括外泌体在内的细胞外囊泡的膜结构表面上含有磷脂酰丝氨酸，后者可以在Ca2+充足的条件下与Tim4蛋白结合，当Ca2+耗尽时则分离。由于二者的结合是可逆的，不会影响外泌体活性，可以应用于高活性外泌体的分离富集。本实验尝试在前人基础上研究利用表面修饰的磁性纳米微球提取人体外周血中高纯度和活性的外泌体的方法。研究将Tim4蛋白特异性接枝在修饰改性过的纳米磁珠表面上，在富含CaCl2的条件下结合包含外泌体在内的细胞外囊泡，再用Ca2+螯合剂去除Ca2+分离出囊泡。同时对获取的细胞外囊泡内的miRNA进行分析，并与血浆背景中RNA做比对。通过一系列表征，本研究证实了通过Tim4磁珠亲和的方法能够得到丰富的高活性包含外泌体在内的细胞外囊泡，该方法成本低，操作简单，相比较传统的提取方法更方便广泛应用，为外泌体研究提供了新的技术支持。获得的细胞外囊泡活性和含量都很高，为应用于液体活检和临床治疗提供了支持。而且这种磁珠与囊泡的结合是可逆的，可以用于同一样本的多次提取。并且我们研究了提取细胞外囊泡内相关miRNA的含量，证实了获得的细胞外囊泡中含有相当数量的外泌体。将来的研究可以根据获得的miRNA含量通过反卷积算法推算出相应的外泌体所占比例，为临床诊断和治疗提供进一步的支持。

With the deepening of research, the great potential of exosomes in the fields of liquid biopsy and targeted therapy has attracted more and more attention. However, due to the size of exosomes ranging from 20 to 200nm, and the extracellular vesicles secreted by cells include not only exosomes, but also microvesicles with larger size, separation of exosomes is very difficult. At present commonly used methods include overspeed centrifugal separation, chemical precipitation and immuno separation. However, overspeed centrifugal need expensive ultracentrifuge and the efficiency is low. Chemical precipitation will introduce protein impurities.  immuno separation can secrete body outside the membrane structure, impact secrete biological activity of the body, so on a secrete body outside are widely used in the separation and extraction has certain limitation. Studies have shown that the membrane structure surface of extracellular vesicles, including exosomes, contains phosphatidylserine, which can bind to Tim4 protein in sufficient Ca2+ conditions and separate when Ca2+ is depleted. Since the combination of the two is reversible and will not affect the exosome activity, it can be applied to the separation and enrichment of highly active exosomes. In this study, we tried to study the method of extracting exosomes with high purity and activity from human peripheral blood by using magnetic nano-spheres modified on the basis of previous studies. In this study, Tim4 protein-specific grafting was performed on the surface of modified nanometer magnetic beads, combined with extracellular vesicles containing exosomes in the condition of rich in CaCl2, and then Ca2+ chelating agent was used to remove Ca2+ and isolate vesicles. Meanwhile, miRNA in extracellular vesicles was analyzed and compared with RNA in plasma background. Through a series of characterization, this study confirmed that the method of Tim4 magnetic bead affinity can obtain abundant extracellular vesicles with high activity including exosomes. This method has low cost and simple operation, and is more convenient and widely used than the traditional extraction method, which provides new technical support for the exosome research. The activity and content of extracellular vesicles obtained are pretty high, which provide practical significance for liquid biopsy and clinical treatment. Moreover, the combination of the magnetic beads and vesicles is reversible and may be used for  repeatedly extraction of the same kind of sample. Moreover, we studied the contents of several types of miRNA extracted from extracellular vesicles and confirmed that the obtained extracellular vesicles contained a considerable number of miRNAs that could be used for gene analysis. Future studies can calculate the proportion of exosomes based on the obtained miRNA content through deconvolution algorithm, providing further support for clinical diagnosis and treatment.

外泌体 细胞外囊泡 分离 纳米磁球 Tim4蛋白

exosomes extracellular vesicles isolation megnetic beads Tim4

中文

联合培养

南方科技大学

周锐. 表面修饰磁珠提取外泌体的研究[D]. 深圳. 哈尔滨工业大学,2020.