Method for separating and purifying various exosome subgroups

A technology of exosomes and subpopulations, applied in artificial cell constructs, tumor/cancer cells, vertebrate cells, etc., can solve the problems of lengthy, low yield, time-consuming and expensive, and achieve simple operation and great application prospects Effect

Active Publication Date: 2017-12-08
SOUTH CHINA AGRI UNIV
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Problems solved by technology

There are also only two ways to try to isolate the different subclasses of exosomes, one is based on density gradient ultracentrifugation, which is a long, laborious and low-yielding procedure; the other is immunoprecipitation, wh

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  • Method for separating and purifying various exosome subgroups
  • Method for separating and purifying various exosome subgroups

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[0056] Example 1 Methods for isolating and purifying different exosome subsets

[0057] 1. The present invention constructs a method for separating and purifying different exosome subsets, which can also be used to specifically enrich the exosomes secreted by cancer cells, including the following specific steps:

[0058] (1) A method for isolating and purifying total exosomes from culture medium (cell culture fluid):

[0059] Centrifuge 100-500ml of cell culture solution at 500g at 4°C for 5 minutes to remove suspended cells, and then centrifuge the supernatant at 2500g at 4°C for 15 minutes to remove cell debris, and then filter through a 0.22-micron pore filter to remove larger microvesicles; the supernatant The solution was concentrated using an ultrafiltration centrifugal filter tube ultravel-100 membrane. This step not only concentrates exosomes, but also eliminates a large portion of impurity proteins with molecular weights less than 100 kDa. The concentrated supernata...

Example Embodiment

[0072] Example 2 Analysis of isolated and purified exosome subsets

[0073] 1. The protein concentration of the isolated and purified exosome subsets in Example 1 was determined by the absorbance at 280 nm of the Nanodrop device, and the number and particle size distribution of exosomes were detected by a nanoparticle tracking analyzer. Western blot and Dotblot analysis were used to analyze the specific proteins contained in the obtained exosome subsets.

[0074] Additionally, total RNA and DNA of each exosome subset were extracted and assessed for RNA and DNA using an Agilent 2100 Bioanalyser. The proteomic analysis of exosome subsets refers to the method reported in the literature (Shevchenko, A et al. 2006, Nature protocols 1, 2856-2860) and the Singapore Experimental Therapeutics Centre Sample Preparation Manual for Proteomic Analysis.

[0075] 2. We observed that exosomes can be eluted at different phosphate concentrations, and the elution curves of exosomes released ...

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Abstract

The invention discloses a hydroxyapatite-based method for separating and purifying various exosome subgroups. The method comprises the following steps: firstly, separating and purifying a total exosome suspension from a medium or a biological fluid sample; and then, on the basis of hydroxyapatite, implementing chromatographic separation, so that the various exosome subgroups are obtained, wherein exactly, the step comprises the following operations: mixing the total exosome suspension with the hydroxyapatite, implementing incubation under a shaking condition, transferring an incubated material to an affinity chromatographic column, washing the column by virtue of a low-concentration sodium phosphate solution, then sequentially eluting the column by virtue of sodium phosphate solutions of which concentrations are gradually increased, so that various eluents are obtained, and removing impurities, so that the various exosome subgroups are obtained. The method is simple and rapid; the obtained various exosome subgroups can nearly avoid overlap in distribution of protein molecules, RNA molecules, DNA molecules and the like; moreover, an exosome desorption process is mild and obtained exosome can be further applied; and the method can be also used for specifically enriching exosome excreted by cancer cells. The method has a good clinical application foundation; and the method is great in application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological detection. More specifically, it relates to a method for isolating and purifying different exosome subpopulations. Background technique [0002] Exosomes (exosomes) are microvesicles with a double-layer plasma membrane structure, about 30-150nm in diameter, which are released by cells into the intercellular space or body fluid through exocytosis. Active biomolecules such as proteins, lipids and nucleic acids. [0003] Exosomes play a role in the pathogenesis of cancer, metastasis of cancer cells and some degenerative diseases. Their abundance in circulation increases with disease including cancer. For example, the level of secreted exosomes in stage II to IV ovarian cancer patients was significantly higher than that in normal controls and early stage cancer patients. Patients with colorectal cancer and lung cancer also produced higher amounts of exosomes than healthy controls. Breast cance...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0602C12N5/0693
Inventor 王俊霞徐领会张炼辉
Owner SOUTH CHINA AGRI UNIV
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