CN114480257A - Method for identifying and enriching exosomes - Google Patents
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a method for identifying and enriching exosomes, which adopts nucleic acid aptamers to identify and enrich exosomes. Mixing the magnetic beads modified by the nucleic acid aptamers with a culture solution, and specifically recognizing the nucleic acid aptamers and combining with exosomes in the culture solution; the magnetic bead-aptamer-exosome compound is retained in the magnetic field through the magnetic field, and the rest components are washed away, so that the exosome can be separated and purified; adding the antisense complementary strand of the aptamer, and competitively binding with the aptamer, thereby competitively releasing the previously captured exosomes for exosome enrichment. The method has the advantages of low preparation cost, good stability and high separation efficiency; in the elution link, the antisense strand is used for competitively releasing the exosome, so that the damage to the structure and the function of the exosome possibly caused in an immune separation method using an antibody is avoided, the integrity of the exosome is kept to the maximum extent, and the yield and the purity of the exosome are improved.
Description
Technical Field
The invention relates to a method for identifying and enriching exosomes.
Background
Exosomes, 40-100 nm in diameter, are signaling organelles secreted by normal and diseased cells through the endolysosomal pathway. Exosomes have an intact lipid bilayer membrane structure coating, contain a sub-proteome of the cell, and are present in many body fluids. They carry lipids, nucleic acids, proteins and other molecules from the mother cell that reflect its function. The research on exosomes from cell sources has become one of the most popular research fields at present, and exosomes obtained by separation and purification from a cell culture medium are used for disease diagnosis, drug delivery or therapeutic action and have wide application value.
As the exosome enters blood, tissue fluid or cell culture medium after being secreted from cells and is mixed with other components, an efficient exosome separation and purification means has important significance for research and application of exosomes. So far, no method can simultaneously ensure the content, purity and biological activity of the exosome.
The exosomes commonly used in the prior art are extracted by the following techniques:
a centrifugal method: at present, the most common method for extracting the exosome is used, the obtained exosome is large in amount, but the purity is insufficient, and the exosome is found to be aggregated into blocks during electron microscope identification, so that the subsequent experiment is not facilitated.
Density gradient centrifugation: usually sucrose density gradient centrifugation is used, the sample is ultracentrifuged together with 2 sucrose solutions and the different components in the sample settle into their respective isopycnic regions. The exosome obtained by the method has high purity, but the preliminary preparation work is complicated, time-consuming and less in amount.
Filtration and centrifugation: the relative molecular mass of the exosome is larger than that of the general protein, and ultrafiltration membranes with different sizes for intercepting the relative molecular mass can be selected to separate the exosome from other macromolecular substances. The method is simple and time-saving to operate, does not influence the biological activity of the exosome, but has the defect that the purity of the extracted exosome is insufficient.
A rotary ultrafiltration method: similar to the principle of filtration and centrifugation. The method has the advantages that the pressure generated by the nitrogen is utilized to filter the supernatant, and the rupture of exosome caused by excessive pressure can be reduced. Meanwhile, the required time is short, and the reduction of the amount of the exosome caused by over-long exposure of the exosome in an open system is avoided.
Continuous adaptation serum cell culture coupled with modified ultracentrifugation: the cells are gradually reduced in dependence on serum in the process of decreasing the serum gradient and finally adapt to serum-free culture. The collected supernatant was centrifuged 2 times, and the 2 nd centrifugation was centrifuged at least 3 times.
Chromatography: and (3) separating the solute by utilizing the relative relation between the pore size of the gel pores and the molecular size of the sample to extract exosomes. Modified size exclusion chromatography: morphologically intact functional exosomes can be reliably and easily recovered in small volumes (1mL) of plasma from cancer patients. The disadvantage is that the impurities of similar size to the exosomes are difficult to separate and the final purity obtained is not high enough.
Immunomagnetic bead method: the magnetic beads coated with the antibody of the exosome-associated antigen (such as CD9 and CD63) are combined with exosomes for separation, ultracentrifugation is not needed, but washing liquid can affect the bioactivity of the exosomes, and the obtained exosomes are difficult to perform subsequent research.
Among the above methods, the immunomagnetic bead method is a technique closer to the one that we have applied for patent. Antibodies are used in immunomagnetic bead methods to recognize and enrich exosomes. The antibody as a protein has the characteristics of difficult preparation, easy inactivation, difficult modification and the like, and particularly has great challenges when being fixed on other support surfaces and high cost.
Disclosure of Invention
The invention aims to solve the technical problems that the content, purity and biological activity of exosomes cannot be ensured simultaneously in the prior art, and the aptamer probe is used for recognizing and enriching the exosomes instead of an antibody so as to solve the problems of poor stability, difficult preparation and narrow application range of the antibody probe.
In order to solve the technical problems, the invention provides the following technical scheme:
the aptamer as a single-stranded oligonucleotide molecule can specifically recognize marker proteins on the surface of exosomes, such as CD9, CD63 and CD81, and has similar affinity with antibodies. Because of easy modification, the nucleic acid aptamer is very easy to fix on the surface of the magnetic bead, and the stability of the nucleic acid aptamer is greatly improved compared with that of an antibody. The magnetic beads modified by the aptamer are mixed with a cell culture solution, and the aptamer can specifically recognize and bind with exosomes in the culture solution. The magnetic bead-aptamer-exosome compound can be retained in a magnetic field by passing the magnetic field in the cell culture added with the aptamer-magnetic bead, and the rest components are washed away, so that the separation and purification of exosome can be realized. In the elution step, we add the antisense complementary strand of the aptamer, which can competitively bind with the aptamer, thereby competitively releasing the previously captured exosomes to achieve the enrichment effect.
Preferably, besides using magnetic beads and magnetic field, the aptamer can be immobilized on other surfaces to prepare other separation methods such as affinity chromatography, and the core is still the function of utilizing the affinity and specific recognition of the aptamer. The core part of this patent is the use of aptamers instead of antibodies as affinity probes to recognize and capture exosomes in cell culture broth.
In addition, the method is also suitable for separating exosomes in blood, tissue fluid or other body fluids, and is not limited to the separation of exosomes in cell culture fluid
The invention has the following beneficial effects: the separation and purification method for preparing the aptamer has the characteristics of low preparation cost, good stability and high separation efficiency. In the elution link, the antisense strand is used for competitively releasing the exosome, so that the damage to the structure and the function of the exosome possibly caused in an immune separation method using an antibody is avoided, the integrity of the exosome is kept to the maximum extent, and the yield and the purity of the exosome are improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram of the principles of the present invention;
FIG. 2 is a diagram of data detected by the ultra-separation method in the example;
FIG. 3 is a diagram showing data of detection by the magnetic bead method of aptamer;
FIG. 4 is a comparison of the electrophoresis of three samples of CD81 obtained by the aptamer magnetic bead method, and the expression of CD81 is shown in the electrophoresis;
FIG. 5 shows a comparison of the electrophoresis of CD9 in three samples obtained by the aptamer magnetic bead method, and shows that CD9 is expressed in the electrophoresis.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Examples
A method of identifying and enriching exosomes, comprising the steps of:
s1, mixing the magnetic beads modified by the nucleic acid aptamers with a cell culture solution, and specifically recognizing the nucleic acid aptamers and combining with exosomes in the culture solution;
s2, the magnetic bead-aptamer-exosome compound is retained in the magnetic field through the magnetic field, and the rest components are washed away, so that the exosome can be separated and purified;
s3, adding the antisense complementary strand of the aptamer, and competitively binding with the aptamer, thereby competitively releasing the previously captured exosome for exosome enrichment.
Nucleic acid aptamer TAACACGACAGACGTTCGGAGGTCGAACCCTGACAGCGTGGGC recognizing CD63
The cell culture medium is a serum-free complete culture medium, the source of the culture medium is placenta stem cell culture solution, and the cell concentration is as follows: 1.2*106Per ml
CD63 aptamer antisense sequence ATTGTGCTGTCTGCAAGCCTCCAGCTTGGGACTGTCGCACCCG
The data for the ultracentrifuge measurements are shown in FIG. 2.
Median:64.75nm
Mean:66.36nm
Std Dev:10.43nm
Sample Concentration:3.19*1012Particles/ml
The data of aptamer magnetic bead detection are shown in FIG. 3: in FIG. 3, 1, 2 and 3 are samples obtained by the aptamer magnetic bead method, and 4, 5 and 6 are samples obtained by the ultra-separation method, and it can be seen from the electrophoretogram that the aptamer magnetic bead method has higher purity.
Median:64.25nm
Mean:69.89nm
Std Dev:13.84nm
Sample Concentration:3.88*1012Particles/ml。
As can be seen from the detection results of FIGS. 1 and 2, the number obtained by the aptamer magnetic bead method is better than that obtained by the ultraseparation method.
Human CD63-WB (placental stem cell exosomes) data are shown in fig. 4 and 5:
FIG. 4 is a comparison of the electrophoresis of three samples of CD81 obtained by the aptamer magnetic bead method, and the expression of CD81 is shown in the electrophoresis; FIG. 5 is a comparison of CD9 electrophoresis of three samples obtained by the aptamer magnetic bead method, and the expression CD9 is shown in the electrophoresis chart
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A method for identifying and enriching exosomes, characterized in that exosomes are identified and enriched using nucleic acid aptamers.
2. A method of identifying and enriching exosomes according to claim 1, comprising the steps of:
s1, mixing the magnetic beads modified by the nucleic acid aptamers with the culture solution, and specifically recognizing the nucleic acid aptamers and combining with the exosomes in the culture solution;
s2, the magnetic bead-aptamer-exosome compound is retained in the magnetic field through the magnetic field, and the rest components are washed away, so that the exosome can be separated and purified;
s3, adding the antisense complementary strand of the aptamer, and competitively binding with the aptamer, thereby competitively releasing the previously captured exosome for exosome enrichment.
3. The method for identifying and enriching exosomes according to claim 1, wherein the culture fluid is blood, tissue fluid or cell culture fluid.
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