CN115109742A - Clinical grade high-purity exosome separation and purification kit in blood or urine - Google Patents
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Abstract
The invention relates to a clinical-grade separation and purification kit for exosomes in high-purity blood or urine, which combines differential centrifugation and ultrafiltration centrifugation, and adds the steps of dilution and ultrafiltration concentration to separate and purify exosomes so as to greatly improve the purity of exosomes and reduce the loss of exosomes. Also comprises the application of the kit in gastric cancer diagnosis. Compared with the prior art, the invention obtains a large amount of high-purity stable exosomes with the average size of 30-150 nm, complete teacup saucer-like structure and uniform particle size distribution, and carries the marker proteins CD9, CD81 and CD63 on the surface, thereby overcoming the defects of low purity, low yield, low expression quantity of characterization proteins and the like in the prior art.
Description
Technical Field
The invention belongs to the technical field of nano biology, and particularly relates to a clinical grade separation and purification kit for exosomes in high-purity blood or urine.
Background
Exosomes (exosomes) are 30-150 nm 'saucer-shaped' phospholipid bilayer vesicles secreted by many types of cells, have the density of 1.13-1.19 g/mL, carry various proteins, lipids and nucleic acids, have complex functions, and participate in immune regulation, regulation of extracellular matrix reconstruction, activation of signal pathways of cells and the like. The separated exosome is used for further detecting tumor markers, such as miRNA, and can be used for screening and early diagnosis of tumors. The method for separating the exosome has been reported so far, but has some problems, the purity of the exosome obtained by the traditional ultracentrifugation is still acceptable, but the yield is not high; the methods of ultrafiltration, dialysis and the like designed according to the exosome size principle cannot well remove the foreign protein with the same size as the exosome; meanwhile, exogenous substances which are not suitable for clinical treatment are introduced to different degrees by adopting a polyethylene glycol (PEG) precipitation method and an immunomagnetic bead method. The research and development of a novel high-purity exosome separation kit have great clinical requirements.
Disclosure of Invention
The present invention aims at overcoming the defects of the prior art and providing a clinical grade high purity exosome separating and purifying kit in blood or urine so as to generate a large amount of clinical grade high purity exosomes.
Further, the invention also aims to provide application of the kit.
The purpose of the invention is realized by the following technical scheme: a clinical grade high-purity exosome separation purification kit in blood or urine chooses differential centrifugation and ultrafiltration centrifugation to combine together, increases to dilute and ultrafiltration concentration step separation purification exosome to improve its purity and reduce exosome's loss by a wide margin, includes the following steps:
(1) collecting at least 2ml of blood sample of a patient, centrifugally separating out supernatant, transferring the supernatant into another centrifuge tube, and diluting the collected supernatant with phosphate buffer solution at 4 ℃ to obtain diluent; and/or
Collecting at least 2ml of urine sample of a patient, centrifugally separating out supernatant, transferring the supernatant into another centrifugal tube, and diluting the collected supernatant by adopting phosphate buffer solution at 4 ℃ to obtain diluent;
(2) centrifuging the diluent prepared in the step (1) at the rotating speed of 3000 rpm at the temperature of 4 ℃, reserving the supernatant, and discarding the precipitate;
(3) centrifuging the supernatant obtained in the step (2) at the temperature of 4 ℃ at the rotating speed of 8000-;
(4) diluting the supernatant obtained in the step (3) by using a phosphate buffer solution at the temperature of 4 ℃, continuously centrifuging for 10min by using a 100 kDa-150 kDa ultrafiltration tube at the speed of 3000-4000 rpm, removing small molecular impurities and simultaneously obtaining a concentrated supernatant so as to reduce the workload and the cost of the subsequent ultracentrifugation;
(5) centrifuging the concentrated supernatant obtained in the step (4) at the rotating speed of 8000 g at the temperature of 4 ℃ for 60 min, discarding the supernatant after centrifugation is finished, and keeping precipitate;
(6) and (4) resuspending the precipitate obtained in the step (5) by using a phosphate buffer solution, transferring the precipitate into a dialysis bag, and dialyzing for 3 hours to obtain the target liquid of the exosome in the required high-purity serum or urine.
Wherein, the formula of the phosphate buffer solution is as follows: KCL 200 mg/L; NaCL 8 g/L; KH (Perkin Elmer) 2 PO 4 240 mg/L；Na 2 HPO 4 1.44 g/L。
On the basis of the scheme, the dilution volume ratio of the phosphate buffer solution to the supernatant in the step (1) is 1: 1.
further, the centrifugation time in the step (2) is 8-10 min; the centrifugation time in the step (3) is 15-25 min.
On the basis of the scheme, the phosphate buffer solution adopted in the step (6) is 1 mL-4 mL, the aperture of the dialysis bag is 30 nm-35 nm, and the dialysate is the phosphate buffer solution.
Preferably, 1 mL-4 mL of phosphate buffer solution is adopted in the step (6) for heavy suspension and is transferred into a dialysis bag, the aperture of the dialysis bag is 30 nm-35 nm, the phosphate buffer solution is used as dialysate, and the required target liquid of exosomes in high-purity serum or urine can be obtained after dialysis for 3-8 h.
Filtering the target solution obtained in step (6) with a 0.22 μm sterile filter.
The invention also provides application of the kit in detection of gastric cancer related markers, which adopts a flow cytometer to detect the markers of CD9, CD81 and CD63 and is used for detecting gastric cancer related markers such as miRNA markers.
Specifically, 5 mul of liquid is taken out from the prepared exosome, PI dye is added into the liquid and mixed evenly, deionized water is added into the liquid to reach the total volume of 100 mul, a flow cytometer is added into the liquid for analysis, and the CD9, CD81 and CD63 antigen markers are confirmed to exist on the surface of the exosome.
Compared with the prior art, the preparation method obtains a large amount of high-purity stable exosomes, the average size is 30-150 nm, the exosomes have complete teacup saucer-like structures, the particle size distribution is uniform, and the surface of the exosomes carries the marker proteins CD9, CD81 and CD63, so that the defects of low purity, low yield, low expression quantity of the characterization proteins and the like in the prior art are overcome.
Drawings
Fig. 1, Transmission Electron Microscope (TEM) photograph of the exosomes obtained.
FIG. 2, Western Blot detection, high expression of exosome marker proteins CD9, CD81 and CD 63.
FIG. 3, Nanoparticle Tracking Analysis (NTA) detection, NTA detection exosome size Analysis, obtained exosome size AnalysisThe average particle size of the particles is 113nm +/-20 nm, and the number of exosomes is up to 6 x 10 7 。
Detailed Description
Example 1: separation and purification of exosome in serum
The utility model provides a clinical grade high-purity exosome separation purification kit in blood or urine, includes the box body, chooses differential centrifugation and ultrafiltration centrifugation to combine together for use, increases to dilute and ultrafiltration concentration step separation purification exosome to improve its purity and reduce the loss of exosome by a wide margin, according to following step:
(1) collecting 2ml of blood sample of a gastric cancer patient, centrifugally separating out supernatant, transferring the supernatant into another centrifugal tube, and diluting the collected supernatant with phosphate buffer solution at 4 ℃ to obtain diluent;
(2) centrifuging the diluent prepared in the step (1) at 4 ℃ at the rotating speed of 3000 rpm for 8 min, precipitating to obtain cells, reserving supernatant, and discarding the precipitate;
(3) centrifuging the supernatant obtained in the step (2) at 4 ℃ at 9000 rpm for 15min, collecting the supernatant, and removing the precipitate;
(4) diluting the supernatant obtained in the step (3) by using a phosphate buffer solution at the temperature of 4 ℃, continuously centrifuging for 5min by using a 110 kDa ultrafiltration tube at the speed of 3000-;
(5) centrifuging the concentrated supernatant obtained in the step (4) at the rotating speed of 100,000g at the temperature of 4 ℃ for 60 min, discarding the supernatant after the centrifugation is finished, and keeping the precipitate;
(6) and (4) resuspending the precipitate obtained in the step (5) by using a phosphate buffer solution, transferring the precipitate into a dialysis bag, and dialyzing for 8 hours to obtain the target solution of a large amount of exosomes with high purity.
The obtained exosomes are characterized by using a Transmission Electron Microscope (TEM), a Western Blot (Western Blot) and a Nanoparticle Tracking Analysis (Nanoparticle Tracking Analysis), and the specific characterization results are as follows:
FIG. 1: transmission Electron Microscope (TEM) photograph: the characterization means of the exosome purity is mainly a TEM (transmission electron microscope), if the exosome purity is insufficient, a lot of impurities exist, and the exosome cannot be seen clearly. A TEM picture of the exosome obtained in this example is taken, and as shown in fig. 1, the exosome is clear and complete in the visual field range, which indicates that the exosome obtained by the method has high purity and complete exosome structure.
FIG. 2: western Blot detection: the main characterization means of the biological activity of the exosomes obtained in the present example is Western Blot detection, and as can be seen from fig. 2, the exosomes highly express the exosome marker proteins CD9, CD81 and CD 63.
FIG. 3: particle size analysis of NTA detection exosome: the cell number of the exosomes obtained in this example is characterized by NTA, and it can be seen from FIG. 3 that the exosomes obtained in the present invention have an average particle size of 113nm + -20 nm and an exosome number as high as 6 x 10 7 。
Example 2
The utility model provides a clinical grade high-purity exosome separation purification kit in blood or urine, includes the box body, chooses differential centrifugation and ultrafiltration centrifugation to combine together for use, increases to dilute and ultrafiltration concentration step separation purification exosome to improve its purity and reduce the loss of exosome by a wide margin, according to following step:
(1) collecting at least 2ml of urine sample of a patient, centrifugally separating out supernatant, transferring the supernatant into another centrifugal tube, and diluting the collected supernatant by adopting phosphate buffer solution at 4 ℃ to obtain diluent;
(2) centrifuging the phosphate buffer solution prepared in the step (1) at 4 ℃ at the rotating speed of 3000 rpm for 9min, reserving supernatant, and removing precipitate;
(3) centrifuging the supernatant obtained in the step (2) at 4 ℃ at 10000 rpm for 20min, reserving the supernatant, and removing the precipitate;
(4) diluting the supernatant obtained in the step (3) with a phosphate buffer solution at 4 ℃, continuously centrifuging for 5min by using a 110 kDa ultrafiltration tube at a speed of 3500 rpm, removing small molecular impurities and simultaneously obtaining a concentrated supernatant so as to reduce the workload and cost of the subsequent ultracentrifugation;
(5) centrifuging the concentrated supernatant obtained in the step (4) at the rotating speed of 100,000g at the temperature of 4 ℃ for 60 min, discarding the supernatant after the centrifugation is finished, and keeping the precipitate;
(6) and (4) resuspending the precipitate obtained in the step (5) by using a phosphate buffer solution, transferring the precipitate into a dialysis bag, and dialyzing for 7 hours to obtain the target solution of a large amount of exosomes with high purity.
And (3) taking 5 mu l of target liquid from the prepared exosome, adding monoclonal antibodies of anti-CD 9, CD81 and CD63 marked by PI dyes, uniformly mixing, adding deionized water to a total volume of 100 mu l, adding a flow cytometer, and analyzing to confirm that the CD9, CD81 and CD63 antigen markers exist on the surface of the exosome.
The above detailed description of the two embodiments of the present invention is provided only for illustrating the technical concept and features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. The utility model provides a clinical grade is exosome separation purification kit in high-purity blood or urine, which characterized in that chooses differential centrifugation and ultrafiltration centrifugation to combine together, increases the purification exosome of dilution and ultrafiltration concentration step separation to improve its purity and reduce the loss of exosome by a wide margin, includes the following step:
(1) collecting at least 2ml of blood sample of a patient, centrifugally separating out supernatant, transferring the supernatant into another centrifuge tube, and diluting the collected supernatant with phosphate buffer solution at 4 ℃ to obtain diluent; and/or
Collecting at least 2ml of urine sample of a patient, centrifuging to separate out supernatant, transferring to another centrifuge tube, and diluting the collected supernatant by adopting phosphate buffer solution at 4 ℃ to obtain diluent;
(2) centrifuging the diluent prepared in the step (1) at the rotating speed of 3000 rpm at the temperature of 4 ℃, reserving the supernatant, and discarding the precipitate;
(3) centrifuging the supernatant obtained in the step (2) at the rotation speed of 8000-12000 rpm at the temperature of 4 ℃, reserving the supernatant, and removing the precipitate;
(4) diluting the supernatant obtained in the step (3) by using a phosphate buffer solution at the temperature of 4 ℃, continuously centrifuging for 5-10min by using a 100 kDa-150 kDa ultrafiltration tube at the speed of 3000-4000 rpm, removing small molecular impurities and simultaneously obtaining a concentrated supernatant so as to reduce the workload and the cost of the subsequent ultracentrifugation;
(5) centrifuging the concentrated supernatant obtained in the step (4) for 60 min at the rotating speed of 8000-;
(6) and (4) resuspending the precipitate obtained in the step (5) by using a phosphate buffer solution, transferring the precipitate into a dialysis bag, and dialyzing for 3 hours to obtain the required target liquid of the exosome in the high-purity serum or urine.
2. The kit for the separation and purification of exosomes in clinical grade high-purity blood or urine according to claim 1, wherein the phosphate buffer formulation is: KCL 200 mg/L; 8g/L of NaCL; KH (Perkin Elmer) 2 PO 4 240 mg/L；Na 2 HPO 4 1.44 g/L。
3. The clinical grade high purity blood or urine exosome separation and purification kit according to claim 1 or 2, wherein the dilution volume ratio of phosphate buffer solution to supernatant in step (1) is 1: 1.
4. the clinical-grade high-purity kit for separating and purifying exosomes in blood or urine according to claim 1, wherein the centrifugation time in the step (2) is 8-10 min; the centrifugation time in the step (3) is 15-25 min.
5. The kit for separating and purifying exosomes in clinical grade high-purity blood or urine according to claim 1 or 2, characterized in that 1 mL-4 mL of phosphate buffer is adopted in step (6) for resuspension and transfer into a dialysis bag, the aperture of the dialysis bag is 30 nm-35 nm, the phosphate buffer is used as dialysate, and the required target liquid of exosomes in high-purity serum or urine can be obtained after dialysis for 3-8 h.
6. The clinical grade high-purity blood or urine exosome separation and purification kit according to claim 1 or 2, characterized by comprising the following steps:
(1) collecting 2ml of blood sample of a gastric cancer patient, centrifugally separating out supernatant, transferring the supernatant into another centrifugal tube, and diluting the collected supernatant with phosphate buffer solution at 4 ℃ to obtain diluent;
(2) centrifuging the diluent prepared in the step (1) at 4 ℃ at the rotating speed of 3000 rpm for 8 min, precipitating to obtain cells, reserving supernatant, and discarding the precipitate;
(3) centrifuging the supernatant obtained in the step (2) at 4 ℃ at 9000 rpm for 15min, collecting the supernatant, and removing the precipitate;
(4) diluting the supernatant obtained in the step (3) by using a phosphate buffer solution at the temperature of 4 ℃, continuously centrifuging for 5min by using a 110 kDa ultrafiltration tube at the speed of 3000-;
(5) centrifuging the concentrated supernatant obtained in the step (4) at the rotating speed of 100,000g at the temperature of 4 ℃ for 60 min, discarding the supernatant after the centrifugation is finished, and keeping the precipitate;
(6) and (4) resuspending the precipitate obtained in the step (5) by using a phosphate buffer solution, transferring the precipitate into a dialysis bag, and dialyzing for 8 hours to obtain the target solution of a large amount of exosomes with high purity.
7. The clinical grade high-purity blood or urine exosome separation and purification kit according to claim 1 or 2, characterized by comprising the following steps:
(1) collecting at least 2ml of urine sample of a patient, centrifuging to separate out supernatant, transferring to another centrifuge tube, and diluting the collected supernatant by adopting phosphate buffer solution at 4 ℃ to obtain diluent;
(2) centrifuging the phosphate buffer solution prepared in the step (1) at 4 ℃ at the rotating speed of 3000 rpm for 9min, reserving supernatant, and removing precipitate;
(3) centrifuging the supernatant obtained in the step (2) at 4 ℃ at 10000 rpm for 20min, reserving the supernatant, and removing the precipitate;
(4) diluting the supernatant obtained in the step (3) with a phosphate buffer solution at 4 ℃, continuously centrifuging for 5min by using a 110 kDa ultrafiltration tube at a speed of 3500 rpm, removing small molecular impurities and simultaneously obtaining a concentrated supernatant so as to reduce the workload and cost of the subsequent ultracentrifugation;
(5) centrifuging the concentrated supernatant obtained in the step (4) at the rotating speed of 100,000g at the temperature of 4 ℃ for 60 min, discarding the supernatant after the centrifugation is finished, and keeping the precipitate;
(6) and (4) resuspending the precipitate obtained in the step (5) by using a phosphate buffer solution, transferring the precipitate into a dialysis bag, and dialyzing for 7 hours to obtain the target solution of a large amount of exosomes with high purity.
8. The kit for separating and purifying exosomes in clinical grade high-purity blood or urine according to claim 1, wherein the target solution obtained in the step (6) is filtered by a 0.22 μm sterile filter.
9. Use of the kit according to any one of claims 1 to 8 for detecting gastric cancer-associated markers, wherein the detection of the markers CD9, CD81 and CD63 is performed by flow cytometry, and the kit is used for detecting gastric cancer-associated markers such as miRNA markers.
10. Use according to claim 9, characterized in that: the detection of the markers of CD9, CD81 and CD63 by using a flow cytometer comprises the following steps: and (3) taking 5 mu l of target liquid from the prepared exosomes, adding monoclonal antibodies of anti-CD 9, CD81 and CD63 marked by PI dyes, uniformly mixing, adding deionized water to a total volume of 100 mu l, adding a flow cytometer, analyzing, and confirming whether the CD9, CD81 and CD63 antigen markers exist on the surface of the exosomes.
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Cited By (1)
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