外泌体分离与纯化技术研究进展

外泌体是所有真核细胞分泌到细胞外的直径介于30～150 nm的一种膜性纳米囊泡,参与细胞间生物信号的传递。大量实验证据表明,外泌体参与多种生物功能并发挥重要作用,包括蛋白质、RNA和脂质等生物分子的转移及多种疾病生理和病理过程的调节,被认为是疾病诊断、治疗和预后的重要的生物标志物和药物载体,因此发展简单、高效、经济的外泌体分离与纯化技术将有助于疾病的早期诊断和精准治疗。目前,利用外泌体的物理化学和生物化学特性已开发出多种分离外泌体的技术,但仍缺乏标准化和规模化临床级外泌体的分离方法,从而限制了其临床应用。另外,对分离出的外泌体的特征、纯度和数量的鉴定是判断外泌体分离纯化方法优劣的重要指标。本文综述了外泌体分离与纯化技术以及鉴定方法的研究进展,主要讨论分离技术的机制、性能、挑战和前景以及外泌体的鉴定方法,以期为外泌体的分离纯化提供新的思路和解决策略。     

甲基丙烯酰胺基明胶水凝胶研究进展

甲基丙烯酰胺基明胶(GelMA)水凝胶的制备及其在生物医学领域的应用是最近十几年的研究热点。GelMA水凝胶因其独特的光致交联特性,可以加工成不同形貌的水凝胶支架材料,同时,因其具有可控的力学性能、降解性能,以及优秀的生物相容性,已成为具有广泛应用前景的生物高分子聚合物材料。本文主要介绍了GelMA水凝胶在止血材料、创伤敷料、组织工程支架、药物控释、骨缺损修复等领域的研究进展。     

Exosome based miRNA delivery strategy for disease treatment

miRNA, which is a common non-coding RNA, can target various m RNAs to regulate their physiological activities. Therefore, mi RNAs play an important role in various physiological and pathological processes,and so they have been proposed as a powerful tool to treat different diseases efficiently. However, the characteristic of mi RNA degradation in vivo limits its further clinical application. Exosomes have the advantage of crossing the biological barrier and achieving long-distance communication ...     

外泌体分离技术及其临床应用研究进展

外泌体是细胞通过胞吐过程分泌的一类粒径为30~200 nm的囊泡，其组成包括脂质双分子层以及其内部包裹的细胞来源的蛋白质、核糖核苷酸（RNA）和脱氧核糖核苷酸（DNA）等生物分子。作为一种细胞间交流的重要方式，外泌体在一系列生理和病理过程中起着至关重要的作用。由于体液环境复杂，加之自身体积小、密度低，外泌体的富集与分离对于其后续分析和功能研究至关重要。该文介绍了外泌体的研究策略、表征手段及生物学功能和临床应用研究进展，特别对外泌体的提取方法进行了详细介绍，并加以系统评述。     

Plasma Exosomes of Patients with Breast and Ovarian Tumors Contain an Inactive 20S Proteasome

Exosomes are directly involved in governing of physiological and pathological conditions of an organism through the transfer of information from producing to receiving cells. It can be assumed that exosomes are one of the key players of tumor dissemination since they are very stable and small enough to penetrate from various tissues into biological fluids and then back, thus interacting with tissue target cells. We evaluated the enzymatic activity and the level of 20S proteasome in tissue and exosomes of healthy females (n = 39) and patients with ovarian (n = 50) and breast (n = 108) tumors to reveal the critical role of exosomal cargo in the mediation of different types of metastases. Exosomes from plasma and ascites were isolated and characterized in according to International Society for Extracellular Vesicles guidelines. The level of 20S proteasome in tissue and exosomes was determined using Western blot analysis. Chymotrypsin- and caspase-like (ChTL and CL, respectively) peptidase activities of the proteasomes were determined using fluorogenic Suc-LLVY-AMC and Cbz-LLG-AMC substrates, respectively. We observed increased levels of 20S proteasome in ovarian cancer tissue and luminal B subtype breast cancer tissue as well as in plasma exosomes from cancer patients. Moreover, the level of the 20S proteasome in plasma exosomes and ascites exosomes in patients with ovarian tumors is comparable and higher in ovarian cancer patients with low volume ascites than in patients with moderate and high-volume ascites. We also found increased ChTL and CL activities in breast cancer and ovarian cancer tissues, as well as in peritoneal metastases in ovarian cancer, while proteasomal activity in exosomes from plasma of healthy females and all patients, as well as from ascites of ovarian tumor patients were lower than detection limit of assay. Thus, regardless of the type of tumor metastasis (lymphogenous or peritoneal), the exosomes of cancer patients were characterized by an increased level of 20S proteasome, which do not exhibit enzymatic activity.     

A／W生物活性微晶玻璃的制备

生物活性玻璃陶瓷材料由于能够与活体组织自发产生紧密的化学键合而成为骨修复材料中的重要分支,其中A／W生物微晶玻璃更是由于在保持良好生物活性的基础上,力学性能接近甚至超过自然骨而作为承重的生物活性材料应用于临床。采用正硅酸乙酯和磷酸三乙酯,利用溶胶-凝胶法制备玻璃粉体,这种玻璃粉体经过一定的热处理工艺可以得到含有磷灰石和硅灰石的A／W生物微晶玻璃。     

Exosome isolation and purification via hydrophobic interaction chromatography using a polyester,capillary‐channeled polymer fiber phase

Extracellular vesicles, including microvesicles and exosomes, are lipidic membrane‐derived vesicles that are secreted by most cell types. Exosomes, one class of these vesicles that are 30–100 nm in diameter, hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. For exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Most current exosome isolation methods have practical problems including being too time‐consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary‐channeled polymer (C‐CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from diverse matrices of practical concern. Initial results demonstrate the ability to isolate extracellular vesicles enriched in exosomes with comparable yields and size distributions on a much faster time scale when compared to traditional isolation methods. As a demonstration of the potential analytical utility of the approach, extracellular vesicle recoveries from cell culture milieu and a mock urine matrix are presented. The potential for scalable separations covering submilliliter spin‐down columns to the preparative scale is anticipated.     

Difference gel electrophoresis analysis of Ras-transformed fibroblast cell-derived exosomes

Exosomes are membrane vesicles of endocytic origin released by many cell types. The molecular composition of exosomes reflects the specialised functions of their original cells. For example, these vesicles can mediate communication through their ability to bind to target cells, facilitating processes such as vascular homeostasis and antigen presentation. Although the proteomes of exosomes from several cell types are known, exploration of exosomes from additional cell types may improve our understanding of their potential physiological roles. Here, we describe the isolation and characterisation of exosomes isolated from the culture medium of murine fibroblast NIH3T3 cells and Ras-transformed NIH3T3 cells. The vesicular nature and size (30-100 nm) of the purified fibroblast exosomes was confirmed by electron microscopy. 2-D difference gel electrophoresis (DIGE) was used to compare protein profiles of exosomes secreted from NIH3T3 cells and Ras-transformed NIH3T3 cells. LC-MS/MS sequencing identified proteins in 188 protein spots in the exosomes from the two cell lines, many of which have been previously identified in exosomes from other cell types. However, some proteins identified are novel for fibroblast exosomes, such as Serpin B6. Over 34 proteins, including milk fat globule EGF factor 8 (lactadherin), collagen alpha-1 (VI), 14-3-3 isoforms, guanine nucleotide-binding proteins (G proteins), the eukaryotic translation initiation factors elF-3 gamma and elF-5A accumulated (>2-fold) in exosomes upon Ras-induced oncogenic transformation. Significantly, the 10.4-fold increase in v-Ha-Ras p21 protein in exosomes derived from Ras-transformed NIH3T3 cells suggests that exosome secretion may be implicated in eradication of obsolete proteins.     

不同金属/适配体双功能复合磁性纳米材料的制备及其对外泌体的富集性能

外泌体作为一种细胞外囊泡,其内容物可以反映亲代细胞的重要信息,而自身也具有独特的结构,能够执行特征的生物学功能。基于外泌体的表面化学和生物学特征,制备了不同类型的金属/适配体(Apt)双功能复合磁性纳米材料,并将其应用于外泌体的富集纯化。将适配体和外泌体表面目标膜蛋白的特异性结合性能与以钛、锆为代表的金属氧化物和外泌体磷脂双层膜的特异性亲和作用结合,可极大地提高分离材料对外泌体的分离选择性和富集容量。分别以Fe₃O₄@Zr-MOFs、Fe₃O₄@Zr-Ti-MOFs和Fe₃O₄@TiO₂等金属有机框架(MOFs)/金属氧化物磁性纳米材料为基底,制备对应的双功能MOFs/金属氧化物-适配体复合磁性纳米材料Fe₃O₄@Zr-MOFs-Apt、Fe₃O₄@Zr-Ti-MOFs-Apt和Fe₃O₄@TiO₂-Apt,并进一步对不同材料的外泌体富集性能加以评价。以超速离心法提取的模型外泌体以及尿液为样品,对修饰相同质量适配体和不同含量金属氧化物的双功能材料的富集性能加以对比。将3种双功能磁性纳米材料应用于尿液外泌体的富集,得到的外泌体裂解后经质谱鉴定,分别得到233、343和832个外泌体蛋白。这一结果也表明双功能磁性纳米材料可以充分结合核酸适配体亲和的高选择性和金属氧化物的高富集容量优势,对于复杂生物样品中外泌体的快速、高效分离纯化具有潜在的应用价值,而针对材料制备和分离纯化方法的设计也为新型外泌体富集材料的设计提供了一条可行的新思路。     

Chemotactic ion-releasing hydrogel for synergistic antibacterial and bone regeneration

The repair of critical-sized bone defects remains a major concern in clinical care. Herein, a multifunctional hydrogel is rationally designed to synergistically photothermal antibacterial and potentiate bone regeneration via adding magnesium oxide nanoparticle and black phosphorus nanosheet (BPNS) into poly(vinyl alcohol)/chitosan hydrogel (PVA/CS-MgO-BPNS). Under the dual effect of near-infrared irradiation and CS intrinsic antibacterial properties, PVA/CS-MgO-BPNS hydrogel can kill more than 99.9% of Staphylococcus aureus and Escherichia coli. The released Mg ions stimulate the migration of mesenchymal stem cells (MSCs) to hydrogels and synergize with released phosphate to promote osteogenic differentiation. The PVA/CS-MgO-BPNS hydrogel also promotes calcium phosphate particle formation and therefore improves the biomineralization ability. Furthermore, the potential molecular mechanism of PVA/CS-MgO-BPNS to regulate MSCs migration and differentiation is through activating phosphoinositide 3-kinase (PI3K)-Akt signaling pathways through RNA-seq analysis. Finally, the PVA/CS-MgO-BPNS hydrogel could significantly promote endogenous bone tissue regeneration in the rat skull defect model. Taken together, this easy fabricated multifunctional hydrogel has good clinical applicability for the repair of large-scale bone defects.     

Modulation of material properties of a decellularized myocardial matrix scaffold

Injectable materials offer the potential for minimally invasive therapy for myocardial infarction (MI), either as an acellular scaffold or as a cell delivery vehicle. A recently developed myocardial matrix hydrogel, derived from decellularized porcine ventricular tissue, has the potential to aid in cardiac repair following an MI. Herein, we set out to study the effects of cross-linking on the cardiac hydrogel stiffness, degradation properties, cellular migration, and catheter injectability in vitro. Cross-linking increased stiffness, while slowing degradation and cellular migration through the gels. Additionally, the cross-linked material was pushed through a clinically relevant catheter. These results demonstrate that the material properties of myocardial matrix can be tuned via cross-linking, while maintaining appropriate viscosity for catheter injectability.     

基于核酸适体的外泌体分子识别研究进展

自研究者证实外泌体承担了细胞外RNA等物质的运输功能以来, 关于外泌体来源与功能的研究一直备受关注. 近年来外泌体被发现具有作为疾病生物标志物的潜力, 使得拥有特定表面蛋白以及特定装载物的外泌体成为分析化学领域有价值的检测对象. 从化学本质角度来说, 外泌体的获取与分析需要依赖特异性的分子识别过程. 核酸适体作为分子识别单元, 因其特异性强、 亲和力高、 生物活性稳定、 易于合成和保存、 而且其序列和结构上具有可编程性, 易于设计和修饰, 已成功地用在外泌体相关的生物传感体系中. 本文从外泌体的化学组成及其具有生理、 病理意义的组分出发, 从外泌体通用生物标志物识别、癌细胞来源外泌体的检测及外泌体蛋白谱的分析这3个方面综述了以核酸适体作为分子识别单元在外泌体分析领域的代表性工作, 总结了现有的靶向外泌体的核酸适体序列信息以及应用场景, 阐述了利用化学合成与修饰以及DNA自组装等化学调控手段增强核酸适体分子识别性能的最新进展, 并从适用于外泌体分子识别的核酸适体的筛选以及化学修饰的角度, 对未来的研究方向进行了展望.     

A fast on-demand preparation of injectable self-healing nanocomposite hydrogels for efficient osteoinduction

Injectable hydrogels have been considered as promising materials for bone regeneration,but their osteoinduction and mechanical performance are yet to be improved.In this study,a novel biocompatible injectable and self-healing nano hybrid hydrogel was on-demand prepared via a fast(within 30 s) and easy gelation approach by reversible Schiff base formed between-CH=O of oxidized sodium alginate(OSA) and-NH_2 of glycol chitosan(GCS) mixed with calcium phosphate nanoparticles(CaP NPs).Its raw materials can be ready in large quantities by a simple synthesis process.The mechanical strength,degradation and swelling behavior of the hydrogel can be readily controlled by simply controlling the molar ratio of-CH=O and-NH_2.This hydrogel exhibits pH responsiveness,good degradability and biocompatibility.The hydrogel used as the matrix for mesenchymal stem cells can significantly induce the proliferation,differentiation and osteoinduction in vitro.These results showed this novel hydrogel is an ideal candidate for applications in bone tissue regeneration and drug delivery.     

Advances in Exosomes‐Based Drug Delivery Systems

Exosomes, a subgroup of extracellular vesicles, are important mediators of long‐distance intercellular communication and are involved in a diverse range of biological processes such as the transport of lipids, proteins, and nucleic acids. Researchers, seeing the problems caused by the toxic effects and clearance of synthetic nanoparticles, consider exosomes as an interesting alternative to such nanoparticles in the specific and controlled transport of drugs. In recent years, there have been remarkable advances in the use of exosomes in cancer therapeutics or for treating neurological diseases, among other applications. The objective of this work is to analyze studies focused on exosomes used in drug delivery system, present and future applications in this field of research are discussed based on the results obtained.     

血清外泌体的蛋白质组分析及其在骨质疏松中的应用

外泌体是细胞分泌的微小具膜囊泡，作为重要媒介参与细胞间的信号传递，已在疾病的诊断和治疗中发挥独特作用。骨质疏松症是全身性骨代谢疾病，容易引起骨密度减少并导致骨折，在老年人群中发病率很高，目前急需发展特异性体液诊断技术。本论文采用超速离心的方法，对血清中的外泌体进行了分离富集和表征，并采用液相色谱-质谱进行了外泌体蛋白质组学分析，共鉴定到了179个外泌体蛋白质，主要参与防御响应和免疫应答等生物过程。针对来自正常对照组、骨量减少组和骨质疏松组的血清样本，分离富集其中的外泌体，通过免标记定量蛋白质组学分析，分别鉴定到188、224和185个蛋白质。定量分析显示17个蛋白质的表达量在骨质疏松组和骨量减少组有显著改变（p<0.05），包括Integrin β 3、Integrin α 2 β 1、Talin 1和Gelsolin等，说明人体骨质在衰变过程中发生了系统性变化，并体现在血清外泌体中。该研究可为骨质疏松研究提供潜在的分子标志物，有助于阐明其病变机制。     

基于超高效液相色谱-串联质谱的股骨头坏死组织外泌体脂质代谢组学分析

股骨头坏死(ONFH)是一种可导致股骨头塌陷进而需要接受全髋关节置换的疾病.外泌体作为一种细胞间交流的方式,在一系列生理和病理过程中起着至关重要的作用,已在疾病的诊断和治疗中发挥独特作用.该研究利用非靶向代谢组学方法,探讨股骨头坏死组织外泌体内的脂质代谢特征,阐释股骨头坏死时机体发生的脂质代谢变化.该研究采用超速离心的...     

外泌体分离及其蛋白质组学分析的研究进展

外泌体是一种由细胞分泌的膜性囊泡小体,直径通常为30~100 nm,密度为1.10~1.18 kg/L。外泌体广泛存在于各种体液中,可携带脂类、蛋白质、信使RNAs（mRNAs）、microRNAs（miRNAs）、非编码RNAs（ncRNAs）等多种重要的生物功能分子。许多证据表明,外泌体形成了一种特殊的细胞间信息传递系统,不仅影响细胞的生理状态,而且与多种疾病的发生与发展密切相关。该文简要综述了近年来在外泌体的分离和纯化方面取得的研究进展,并对蛋白质组学技术在外泌体分析中的应用进行了概述。此外,还对外泌体蛋白质组学研究的发展前景进行了展望。     

强韧型自愈合水凝胶的研究进展

水凝胶作为一种由大量水和与众不同的三维网状结构构成的智能软材料,已经广泛应用于许多领域,如药物输送、软骨修复、废物处理及电子设备等。然而,水凝胶不良的机械性能及自愈合性极大地限制了它们的潜在应用。目前已报道的韧性水凝胶通常不具有或只有很弱的自修复性,而自修复水凝胶通常机械性能非常弱。因此,研发具有高效自修复性能和优异机械性能的水凝胶材料,无论是从学术角度还是工业角度都是非常重要的。本文总结了近些年来强韧型自愈合水凝胶的最新研究进展,从其制备方法、性能等方面进行了简要介绍,并对未来的发展前景进行了展望。     

A Polysaccharide-based Hydrogel and PLGA Microspheres for Sustained P24 Peptide Delivery: An In vitro and In vivo Study Based on Osteogenic Capability

A bone morphogenetic protein-2(BMP-2) derived synthetic oligopeptide, S [PO4]KIPKASSVPTELSAISTLYLDDD(P24), has shown great potential for facilitating bone regeneration. However, P24 cannot be directly used onto bone defects, while a continuous sustained delivery of P24 may lead to a better formation of bone tissue. Based on this issue, we have developed a sustained delivery system incorporating P24-loaded poly(lactide-co-glycolide)(PLGA) microspheres and nano-hydroxyapatite(n-HA) into the composite hydrogel. The P24-contained compound material was characterized with NMR, FTIR and SEM to demonstrate the fomiation of compound structure containing P24, PLGA and n-HA. A continuous drug release of P24 was observed for over 60 d that evidently enhanced the efficiency in promoting the proliferation of MC3T3-E1 cells and the secrete of alkaline phosphatase(ALP) in vitro. Moreover, the osteoinduction eflect of the hydrogel system with P24 peptide niicrospheres was demonstrated in vivo and manifested by the result of immunohistochemistry. This novel injectable composite hydrogel is expected to be applied to improving the bone defect treatment in bone tissue engineering.     

Efficient exosome extraction through the conjugation of superparamagnetic iron oxide nanoparticles for the targeted delivery in rat brain

Exosomes possess endogenous attributes and distinct biological functions, and thereby, their uses as drug nanocarriers have attracted increasing attention for biomedical practices. However, to achieve targeted therapeutic purposes, complicated extractions, as well as modifications of exosomes, are involved. Here, based on the use of superparamagnetic iron oxide nanoparticles conjugated exosome (Ex-SPIONs), a facile exosome extraction through magnetism was established. The produced Ex-SPIONs exhibited a uniform size distribution and desirable biocompatibility. Moreover, taking advantage of the magnetic properties of SPIONs, the targeted delivery of Ex-SPIONs was demonstrated in the rat brain. Therefore, the constructed SPIONs functionalized exosome shows promising therapeutic potentials, including the treatment of brain diseases.