Background:
Since winning the Nobel Prize in 2013, exosomes have been a research hotspot in the international scientific community. In the past ten years, its popularity has remained high, and now it still sits firmly in the fifth place in the national natural hotspot list! Let's go ahead and unravel the mystery of this extracellular vesicle!
Exosome Personal Business Card:
Name:exosomes ;
Species: extracellular vesicles;
Big, small: diameter 30-150nm;
Density：1.1-1.18g/ml；
Shape: "saucer-shaped";
Source:: Secreted by all living cells;
exists: Present in almost all tissues, intercellular spaces, body fluids;
Quantity: There are about 1014 of them in the human body, which is almost 1000-10000 per cell on average;
Functions: Regulate cell metabolism, control inflammation, promote angiogenesis, promote tissue repair, immune regulation, cancer occurrence and development, antigen presentation, etc.
Honor:Winner of the 2013 Nobel Prize in Physiology or Medicine.
Carrying substances:lipids, proteins, DNA, and RNA (miRNA, LNCRNA, circular RNA, mRNA);
Outstanding features: Heterogeneity, i.e., exosomes secreted by the same cell may have great functional differences;
Figure Exosome structure and characteristics.
Properties of exosomes and related applications:
1. Heterogeneity, which can be used as a biomarker for disease diagnosis
The exosomes secreted by different cells are different in number, cell membrane and content composition, and biological function, and are heterogeneous. The size and content of exosomes can reflect the type and state of secretory cells, and can accurately regulate various physiological activities in the body, which can be used as biomarkers for disease diagnosis. By isolating and purifying exosomes in body fluids, analyzing the characteristic mirNAs or proteins contained in them, and then comparing them with disease characteristic markers, the type of disease can be determined.
Present, can be used for immune regulation
Exosomes generally complete the process of antigen presentation, immune activation inhibition, and immune tolerance through the delivery of a variety of biomolecules. Among them, exosomes secreted by antigen-presenting cells can carry and present related complexes to achieve the purpose of immunity by weakening or enhancing the immune response. In addition, exosomes are loaded with many immune-related molecules, including lysosome-associated membrane proteins 1 and 2, immunoglobulin superfamily members8, and MHC, which can specifically bind to related peptide chains and induce immune responses.
3. Deimmunity, which can be used as a drug delivery carrier
Exosomes are characterized by low immunogenicity and can evade recognition and attack by most of the immune system. Compared with the disadvantages of traditional chemical drugs, such as poor biocompatibility, easy removal by the human body, poor solubility and permeability, exosomes have many advantages, such as nanoscale volume, low cytotoxicity, good biofilm permeability and biocompatibility, and their lipid bilayer membrane structure can protect the contents from degradation and maintain biological activity. Therefore, exosomes can be used as carriers to deliver drugs, helping drugs escape the immune system, cross tissue barriers, and improve the efficiency of drug delivery and absorption.
4. Tumorigenic, can be applied to tumors**
The tumorigen-promoting nature of tumor cell ** exosomes is manifested in the regulation of the body's immune function while also transmitting harmful messages to the recipient cells, resulting in cell lesions; In addition, tumor cells will secrete exosomes to excrete a part of the drug out of the cell or bind to functional drug antibodies, reducing drug efficacy and drug resistance. Many studies have shown that exosomes secreted by advanced tumor cells can inhibit the anti-tumor immune response and promote the growth and spread of tumors. In recent years, exosomes have given rise to many new interventions in the application of tumors, such as blocking exosome production, secretion, and blocking exosome-mediated cellular communication.
5. Inducible, can be applied to regenerative medicine
Exosomes also have the function of inducing cells** and differentiation, which can promote the differentiation of mesenchymal stem cells (MSCs) into different cells to replace damaged tissue cells, which is used in the field of regenerative medicine. For example, exosomes extracted from the conditional culture medium of human umbilical cord mesenchymal stem cells can promote the proliferation and migration of cells, inhibit the mitochondrial apoptosis signaling pathway, and thus promote the repair of the first wound. Many companies have developed exosome-based regenerative medicine products, covering wound regeneration, cosmeceutical products, cosmetology, and other fields.
Exosome Research Holistic Approach:
Figure Flow chart of the overall exosome research protocol.
Exosome identification:
In the last issue of the article "National Natural Research Hotspot "Evergreen" - Introduction to Exosomes", I gave you a detailed introduction to various methods of exosome extraction and purification. After exosome isolation and purification, characterization is required to determine the purification effect and evaluate whether the obtained exosomes meet the needs of downstream experiments. There are many methods for the characterization and identification of exosomes, and the indicators analyzed are also multifacetedSize, morphology, surface markersThese three aspects of detection are the most important and are the methods recommended by the International Society for Extracellular Vesicles (ISEV) for the characterization of isolated exosomes.
1. Exosome size and morphological identification—electron microscopy
Electron microscopy such as scanning electron microscopy (SEM) or transmission electron microscopy (TEM) can visualize the morphology of particles and can therefore be used to identify the presence and integrity of exosomes. The operation steps are roughly as follows: resuspend the extracted exosomes with PBS, drop them on a sample copper mesh with a pore size of 2nm, stand at room temperature for 2min, use filter paper to absorb the liquid from the side of the filter screen, use 3% phosphotungstic acid solution to negative stain at room temperature for 5min, filter paper to absorb the negative staining solution, dry it at room temperature, and observe and take pictures with electron microscope. Exosomes have very obvious membrane boundaries under electron microscopy, and are saucers or cup-shaped structures of different sizes of 30 to 150 nm (there are also exosomes of other shapes below 200 nm that are difficult to identify).
Figure Electron microscopy for detection of exosome morphology.
Note: **On the Internet, if there is any infringement, please contact to delete.
2. Detection of exosome particle size concentration - nanoparticle tracer analysis (NTA).
NTA technology can detect the particle size and number of exosomes, and the operation steps are: the collected exosomes are diluted to 106 ml with PBS and injected into the nanoparticle tracking analyzer with a 1ml syringe; The laser beam passes through the specimen particles outside the sample room, the particles are visualized through a microscope equipped with a camera, the Brownian motion of the exosomes is captured, and finally the concentration and hydrodynamic diameter are calculated based on their motion using equations. Compared with other exosome identification methods, NTA technology has simpler sample processing, better guarantees the original state of exosomes, and faster detection speed.
Figure NTA detects exosome particle size concentration.
3. Identification of surface markers of exosomes - WB detection
The analysis and identification of exosome biochemistry is mainly the detection of specific protein markers carried by them, and WB detection is a common analysis technique for exosome-specific proteins (such as tetratransmembrane proteins, tumor susceptibility gene 101 protein, lipid raft labeling protein, apoptosis-inducing factor 6-interacting protein, etc.). The specific operation refers to the standard WB operation, and the antibody of exosome-specific protein is used for labeling, so as to qualitatively detect and analyze exosomes.
Figure WB detects exosomal signature proteins.
There are other methods that can be used to identify exosomes, such as ELISA, flow cytometry, and mass spectrometry, which are also commonly used to detect the number of exosomes and the expression of surface marker proteins such as CD9, CD63, and CD81.
Introduction of Exosome Quantitative Detection Kit:
The ABSIN ELISA Sandwich Detection Kit (ABS50054) uses high-performance anti-CD9 capture and anti-CD81 detection antibodies to detect human body fluids or cell culture supernatants, sensitively and quantitatively detect targets that co-express exosome surface markers CD9 and CD81, and achieve rapid and accurate quantification of intact exosomes in samples.
Figure Principle of exosome quantitative detection kit.
Product Components:
Protocol Procedure:
1. Reagent preparation
1) All reagents should be balanced at room temperature for at least 15min before use;
2) Take the concentrated lotion solution, dilute 20x concentrated wash solution into 1x working solution with distilled water or deionized water, mix well and set aside;
3) The chromogenic substrate solution should be mixed 1:1 between A and B before detection.
2. Experimental process
1) Prepare the slats and shake the reagents well;
2) 100 L of standard SO-S5 was taken with a pipette, and the samples to be tested were added to the microplate.
3) Seal the plate hole with a sealing film, 37, 60min;
4) Wash the working liquid wash 3 times, 300-350 l holes each time, and pat the residual liquid in the plate holes on absorbent paper;
5) Add 100 l of enzyme conjugate per well;
6) Seal the plate hole with sealing film, 37, 60min;
7) Wash the working solution 3 times, 300-350 l wells per well, and pat dry it on absorbent paper;
8) Add 100 L of chromogenic substrate solution working solution to the reaction well, 37 Avoid light for 30min, add 50 L of stop solution, read at 450nm on the microplate reader, and plot the XY linear curve.
3. Calibration procedure
xy linear fit, with a linear correlation coefficient r 099. Do a full set of calibrators for each experiment and draw the curve.
References
1] Shi Yuxiao, Lu Xiaohong, Lu Wangding, et al. Overview of the biological properties and applications of exosomes[J].Chinese Journal of Pharmaceutical Industry,2023,54(07):1008-1019doi:10.16522/j.cnki.cjph.2023.07.004.
2] wu y, deng w, klinke dj 2nd. exosomes: improved methods to characterize their morphology, rna content, and surface protein biomarkers. analyst. 2015 oct 7;140(19):6631-42. doi: 10.1039/c5an00688k. pmid: 26332016; pmcid: pmc4986832.
3] wang y, zhang l, li y, chen l, wang x, guo w, zhang x, qin g, he sh, zimmerman a, liu y, kim im, weintraub nl, tang y. exosomes/microvesicles from induced pluripotent stem cells deliver cardioprotective mirnas and prevent cardiomyocyte apoptosis in the ischemic myocardium. int j cardiol. 2015 aug 1;192:61-9. doi: 10.1016/j.ijcard.2015.05.020. epub 2015 may 8. pmid: 26000464; pmcid: pmc4469495.
4] Nande. Isolation and identification of exosomes of adipose stem cells and in vitro anti-liver cancer study of curcumin delivery[D].Dalian University of Technology, 2021doi:10.26991/d.cnki.gdllu.2020.001248.