CN117586952B - Separation reagent and separation method for separating plasma exosomes - Google Patents
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- 210000001808 exosome Anatomy 0.000 title claims abstract description 84
- 238000000926 separation method Methods 0.000 title claims abstract description 64
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 40
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 28
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 20
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 20
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims description 21
- 238000005119 centrifugation Methods 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 11
- 108090000190 Thrombin Proteins 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 229960004072 thrombin Drugs 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 4
- 230000001376 precipitating effect Effects 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 10
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 4
- 229920001477 hydrophilic polymer Polymers 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 70
- 230000000052 comparative effect Effects 0.000 description 23
- 239000000243 solution Substances 0.000 description 17
- 239000002245 particle Substances 0.000 description 16
- 239000000523 sample Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000001166 ammonium sulphate Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
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- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
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- 238000005199 ultracentrifugation Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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Abstract
The invention relates to the technical field of biology, in particular to a separation reagent and a separation method for separating plasma exosomes. The separating agent comprises polyethylene glycol, ammonium sulfate and water. The separation reagent adopts the hydrophilic polymer polyethylene glycol to be matched with inorganic ammonium sulfate, so that exosomes are separated out from the plasma sample, the total exosomes in the purified plasma can be separated and separated in 1-2 hours, high-efficiency separation is realized, special centrifugal equipment is not needed, the separation reagent has the characteristics of high recovery rate, high purity and low price, can meet scientific research and clinical detection application, and has the same effect as the international common plasma exosome separation reagent.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a separation reagent and a separation method for separating plasma exosomes.
Background
The exosomes are generally 50-150nm in diameter and have a double-layer membrane structure, and are widely used in various body fluid samples such as blood plasma, cerebrospinal fluid and urine. The exosome has small volume, stable structure and rich content, and has wide application prospect in clinical diagnosis, scientific research and other aspects.
In the related art, an ultracentrifugation or kit separation method is generally adopted to separate the plasma exosome sample, the method generally adopts an ultrahigh rotating speed of more than 100000g, the centrifugal equipment is expensive, the separation time is more than 10 hours, and the kit Exoquick for separating the exosome of the SBI company is expensive and cannot be widely applied.
At present, the technology for separating the plasma exosomes is not mature, and the common separation method has the problems of long time consumption, high reagent cost and the like, thus preventing the exosomes from being applied to scientific research and clinical diagnosis.
Disclosure of Invention
In order to solve the technical problems, the invention provides a separating reagent and a separating method for separating plasma exosomes, wherein the separating reagent adopts polyethylene glycol (PEG) to be matched with inorganic ammonium sulfate salt, so that exosomes are separated out from a plasma sample, the total exosomes in the purified plasma can be separated in 1-2h, and special centrifugal equipment is not needed.
According to a first aspect of the present invention there is provided a separation reagent for separating plasma exosomes comprising polyethylene glycol, ammonium sulphate and water.
In the scheme, the separating agent for separating the plasma exosomes comprises polyethylene glycol, ammonium sulfate and water, wherein the polyethylene glycol belongs to hydrophilic macromolecular substances, can compete with exosome surface proteins for water molecules, and can further separate the exosomes out by the rejection action of exosomes and the salting-out action generated by the ammonium sulfate with higher concentration, so that the total exosomes in the purified plasma can be separated and separated within 1-2 hours, high-efficiency separation is realized, special centrifugal equipment is not needed, and the separating agent has the characteristics of high recovery rate, high purity and low price, can meet scientific research and clinical detection application, and has the effect equivalent to that of the international common separating agent for the plasma exosomes.
Further, the separation reagent comprises 10% -18% polyethylene glycol, 1% -4% ammonium sulfate and 80% -88% water by weight percent.
In the scheme, the dosage of polyethylene glycol, ammonium sulfate and water in the separation reagent is limited within a reasonable range value, so that each component can play a better synergistic effect, and a better separation effect is achieved. Particularly, when the amount of ammonium sulfate in the separating agent is less than 1%, the separating effect is poor, and when the amount of ammonium sulfate in the separating agent is more than 4%, the ammonium sulfate is not well dissolved in a solution formed by polyethylene glycol and water.
To achieve a better separation effect, further, the separation reagent comprises 15% polyethylene glycol, 2% ammonium sulfate and 83% water by weight.
Further, the polyethylene glycol has a molecular weight of 5000-8000 daltons.
Further, the polyethylene glycol has a molecular weight of 6000 daltons.
Further, the separating agent is obtained by mixing polyethylene glycol, ammonium sulfate and water at room temperature for 10min-15min and filtering with a 0.22 μm filter membrane.
According to a second aspect of the present invention, there is also provided a method of separating plasma exosomes, comprising the steps of:
step S1: centrifuging the plasma sample for the first time, collecting supernatant, and discarding precipitate;
step S2: adding thrombin into the supernatant obtained in the step S1, flicking and uniformly mixing, performing secondary centrifugation after room temperature treatment for a period of time, collecting the supernatant, and discarding the precipitate;
Step S3: adding the separating reagent into the supernatant obtained in the step S2, mixing the mixture upside down, precipitating the mixture on ice, centrifuging the mixture for the third time, and discarding the supernatant to retain the precipitate;
step S4: and (3) fully dissolving the precipitate obtained in the step (S3) by using PBS to obtain the total exosome solution of the plasma.
The room temperature is generally 10 to 30 ℃.
In the scheme, the separation method for separating the plasma exosome comprises the steps of firstly carrying out first centrifugation pretreatment on a plasma sample to obtain a plasma sample with cells and cell fragments removed, then adding thrombin to treat the plasma sample to remove fibrin in the plasma sample to obtain a treated plasma sample solution, then adding the separation reagent provided by the invention, mixing the mixture upside down, precipitating on ice, centrifuging, and removing other plasma components to obtain the plasma total exosome, wherein the total plasma exosome can be obtained in the whole process of 1-2 hours. The separation method provided by the invention does not need expensive treatment equipment, can efficiently extract the plasma exosome sample, has the characteristics of high recovery rate, high purity and low cost, can meet scientific research and clinical detection application, and is an ideal purification method.
Further, in the step S1, the first centrifugation is performed at 5000g-7000g for 15min-30min at 3-5 ℃.
In the scheme, the temperature, the centrifugal force and the centrifugal time of the first centrifugation are limited in a reasonable range value, so that cell fragments, apoptotic bodies and the like in the plasma sample can be separated more easily, and the separation effect can be improved.
Further, in the step S2, the volume ratio of the supernatant obtained in the step S1 to thrombin is (100-150): 1; preferably 125:1;
and/or thrombin at a concentration of 600U/mL-650U/mL; preferably 611U/mL;
and/or, the second centrifugation is performed for 3-8min at 10000g-15000g under the condition of 3-5 ℃.
In the above scheme, the volume ratio of the supernatant obtained in the step S1 to thrombin, the concentration of thrombin and the secondary centrifugation condition are limited in a reasonable range, so that the removal of fibrin in the blood plasma is facilitated, and the improvement of the separation effect is facilitated.
Further, in step S3, the volume ratio of the supernatant obtained in step S2 to the separation reagent is 1:1;
And/or the number of upside down is 5-10; preferably 8 times.
And/or the temperature of the ice precipitation is-4 ℃ to 4 ℃ and the time is 20min to 40min;
And/or, the third centrifugation is at 1000g-2000g for 3min-8min at 3-5 ℃.
In the above scheme, the volume ratio of the supernatant obtained in the step S2 to the separating reagent, the times of upside down, the temperature and time of ice precipitation and the third centrifugation condition are limited in a reasonable range, so that the removal of other plasma components and the improvement of the separation effect are facilitated.
The technical scheme provided by the invention has the following beneficial effects:
The separating agent for separating the plasma exosomes comprises polyethylene glycol, ammonium sulfate and water, and the hydrophilic polymer polyethylene glycol is matched with inorganic salt ammonium sulfate, so that the exosomes are separated out from a plasma sample, the total exosomes in the purified plasma can be separated and purified within 1-2 hours, high-efficiency separation is realized, special centrifugal equipment is not needed, and the separating agent has the characteristics of high recovery rate, high purity and low price, can meet scientific research and clinical detection application, and has the effect equivalent to that of the international common separating agent for the plasma exosomes.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart of a separation method for separating plasma exosomes according to example 4 of the present invention;
FIG. 2 is a graph showing the particle size distribution of the total plasma exosomes obtained in example 4 of the present invention;
FIG. 3 is a graph showing the particle size distribution of the total plasma exosomes obtained in example 5 of the present invention;
FIG. 4 is a graph showing the particle size distribution of the total plasma exosomes obtained in example 6 of the present invention;
FIG. 5 is a graph showing the particle size distribution of the total plasma exosomes obtained in comparative example 1 of the present invention;
FIG. 6 is a graph showing the particle size distribution of the total plasma exosomes obtained in comparative example 2 of the present invention;
FIG. 7 is a graph showing the particle size distribution of the total plasma exosomes obtained in comparative example 3 of the present invention;
FIG. 8 is a graph showing the comparison of the surface marker test results of the total plasma exosomes obtained in examples 4, 5, 6 and comparative example 1 of the present invention;
FIG. 9 is a graph showing the comparison of the surface marker test results of the total plasma exosomes obtained in example 4 and comparative examples 1 and 2 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
Example 1
The present example provides a separation reagent for separating plasma exosomes, consisting of 15g polyethylene glycol, 2g ammonium sulphate and 83g deionized water, the molecular weight of the polyethylene glycol being 6000 daltons. The separating reagent is obtained by mixing polyethylene glycol, ammonium sulfate and water at room temperature for 15min and filtering with a 0.22 μm filter membrane.
Example 2
This example provides a separation reagent for separating plasma exosomes, which differs from example 1 in that it consists of 15g polyethylene glycol, 1g ammonium sulphate and 84g deionized water.
Example 3
This example provides a separation reagent for separating plasma exosomes, which differs from example 1 in that it consists of 15g polyethylene glycol, 4g ammonium sulphate and 81g deionized water.
Example 4
The embodiment provides a separation method for separating plasma exosomes, the flow chart of which is shown in fig. 1, comprising the following steps:
Step S1: collecting peripheral blood samples of 1 volunteer by using EDTA anticoagulation blood collection tube, and preserving at 4 ℃; after removal, the human plasma sample was centrifuged at 6000g for 30min at 4℃to leave a supernatant, and the pellet was discarded.
Step S2: adding 2ul thrombin with concentration of 611U/mL into 250ul supernatant obtained in step S1, flicking, mixing, treating at room temperature for 5min, centrifuging at 12000g at 4deg.C for 5min, collecting supernatant, and discarding precipitate.
Step S3: adding the separating reagent of the example 1 into the supernatant obtained in the step S2 according to the volume ratio of 1:1, uniformly mixing the mixture upside down for 8 times, precipitating the mixture on ice at the temperature of 0 ℃ for 30min, centrifuging the mixture at 1500g for 5min at the temperature of 4 ℃, and discarding the supernatant to keep the precipitate.
Step S4: and (3) fully dissolving all the precipitates obtained in the step (S3) by using a 1mLPBS buffer solution to obtain a plasma total exosome solution.
Example 5
The present example provides a separation method for separating plasma exosomes, which differs from example 4 in that the separation reagent of example 2 is used in step S3.
Example 6
The present example provides a separation method for separating plasma exosomes, which differs from example 4 in that the separation reagent of example 3 is used in step S3.
Comparative example 1
This comparative example provides a separation method for separating plasma exosomes, which differs from example 4 in that in step S3 a separation reagent of SBI company model Exoquick is used.
Comparative example 2
This comparative example provides a separation method for separating plasma exosomes, which is different from example 4 in that the separation reagent used in step S3, specifically, the separation reagent comprises 15g polyethylene glycol and 85g deionized water, and the molecular weight of the polyethylene glycol is 6000 daltons.
Comparative example 3
This comparative example provides a separation method for separating plasma exosomes, which is different from example 4 in that the salt ion in the solution is sodium chloride, and the separation reagent used in step S3 is different from that in the embodiment, specifically, the separation reagent includes 4g sodium chloride, 15g polyethylene glycol and 81g deionized water, and the molecular weight of the polyethylene glycol is 6000 daltons.
The total plasma exosome solutions obtained in examples 4 to 6 and comparative examples 1 to 3 were subjected to exosome particle size distribution testing by nanoparticle tracking analysis (Nanoparticle TRACKING ANALYSIS, NTA).
As shown in FIG. 2, the results of the examination of the total plasma exosome solution obtained in example 4 showed that the exosome particle size range was between 60 and 300nm, with a peak at about 97nm at about 100 nm. As shown in FIG. 3, the results of the examination of the total plasma exosome solution obtained in example 5 showed that the exosome particle size ranges between 30 and 300nm, and that a plurality of peaks occurred at 90nm, 112nm, 166nm, etc. As shown in FIG. 4, the results of the examination of the total plasma exosome solution obtained in example 6 revealed that the exosome particle size ranges between 30 and 400nm, and that a plurality of peaks occurred at 31nm, 97nm, 140nm, 194nm, etc. As shown in FIG. 5, the results of the measurement of the total plasma exosome solution obtained in comparative example 1 showed that the exosome particle size range was between 60 and 320nm, and that a plurality of peaks appeared at 103nm, 186nm, etc., with the highest peak concentrated around 103 nm. As shown in FIG. 6, the results of the detection of the total plasma exosome solution obtained in comparative example 2 showed that the exosome particle size ranges between 29 and 250nm, a plurality of peaks at 86nm, 114nm, 158nm, etc., and the exosome concentration was low. As shown in FIG. 7, the results of the examination of the total plasma exosome solution obtained in comparative example 3 revealed that the exosome particle size range was between 60 and 450nm, and that a plurality of peaks appeared at 87nm, 135nm, 194nm, etc., with the highest peak concentrated around 87 nm.
As can be seen from the results of fig. 2 to 7, the particle size distribution of the total plasma exosomes obtained by combining the separation reagent according to the present invention with the separation method according to the present invention is uniform, concentrated, and highly uniform, and the purity of the exosomes obtained by separation is high, and especially the detection result of the total plasma exosomes solution according to example 4 of the present invention is close to the detection result of the total plasma exosomes solution according to comparative example 1, and the proportion of particles is high around 100nm, which indicates that the particle size distribution of the total plasma exosomes obtained by combining the separation reagent according to the present invention with the separation method according to the present invention is comparable to the international commonly used plasma exosomes separation reagent.
The total plasma exosome solutions obtained in examples 4 to 6 and comparative examples 1 to 2 were used for detecting the content of the exosome marker protein by Western blot. Table 1 shows the comparison of the gray scale analysis values of the surface markers of the total plasma exosomes obtained in examples 4, 5, 6 and comparative example 1, and Table 2 shows the comparison of the gray scale analysis values of the surface markers of the total plasma exosomes obtained in example 4 of the present invention and comparative examples 1, 2.
TABLE 1
TABLE 2
As can be seen from the results of FIG. 8 and Table 1, the expression level of the total plasma exosome markers obtained by the combination of the separation reagent of the present invention with the separation method of the present invention was similar to that of comparative example 1, indicating that the purity of the exosome obtained by the separation was high.
As can be seen from the results of FIG. 9 and Table 2, the expression level of the total plasma exosome markers obtained by the separation method of the present invention using the separation reagent of the present invention was close to that of comparative example 1 and higher than that of comparative example 2, indicating that the exosome obtained by the separation of the present invention was higher in purity.
The results from FIGS. 5, 6 and 2 show that the concentration of total plasma exosomes isolated by the separation reagent of the present invention in combination with the separation method of the present invention is higher than that of comparative examples 1 and 2, indicating that the separation effect of the present invention is better.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (1)
1. A separation method for separating plasma exosomes, comprising the steps of:
step S1: centrifuging the plasma sample for the first time, collecting supernatant, and discarding precipitate;
step S2: adding thrombin into the supernatant obtained in the step S1, flicking and uniformly mixing, performing secondary centrifugation after room temperature treatment for a period of time, collecting the supernatant, and discarding the precipitate;
Step S3: adding a separating reagent into the supernatant obtained in the step S2, mixing the mixture upside down, precipitating on ice, centrifuging for the third time, and discarding the supernatant to retain the precipitate; the separation reagent comprises 15% polyethylene glycol, 2% ammonium sulfate and 83% water; the separating agent is obtained by mixing polyethylene glycol, ammonium sulfate and water at room temperature for 10min-15min, and filtering with 0.22 μm filter membrane; the molecular weight of the polyethylene glycol is 6000 daltons;
Step S4: fully dissolving the precipitate obtained in the step S3 by using PBS to obtain a total exosome solution of plasma;
in the step S1, the first centrifugation is performed for 30min at 6000g under the condition of 4 ℃;
In the step S2, the volume ratio of the supernatant obtained in the step S1 to thrombin is 125:1;
the concentration of thrombin was 611U/mL;
The second centrifugation is carried out at 12000g for 5min at 4 ℃;
In the step S3, the volume ratio of the supernatant obtained in the step S2 to the separating reagent is 1:1;
the number of times of upside down is 8;
The temperature of the precipitation on ice is 0 ℃ and the time is 3min;
The third centrifugation was performed at 1500g for 8min at 4 ℃.
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