CN109929795B - Improved extraction method of urine small extracellular vesicle - Google Patents
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Abstract
The invention discloses an improved extraction method of urine small extracellular vesicles, which comprises the following steps of: sequentially adding sucrose solutions with different concentration gradients to the bottom of the test tube from low concentration to high concentration to form gradient liquid with sequentially increasing concentration from top to bottom, slowly injecting coarse vesicle resuspension at the uppermost layer of the gradient liquid, and centrifuging; the method can obviously improve the extraction yield, reduce the sample consumption, greatly shorten the extraction time, obviously improve the extraction purity by combining with a differential centrifugation and ultrafiltration method, and is simpler, more reliable and faster in clinical use.
Description
Technical Field
The invention belongs to the technical field of biomedicine, relates to an outer vesicle separation and extraction technology, and particularly relates to an improved extraction method of urine small extracellular vesicles.
Background
With the discovery and study of urine extracellular vesicles, urine has been considered as a sample that reflects disease dynamics. Extracellular vesicles are nanoparticles of lipid membrane structure and can be divided into three classes: exosomes, microvesicles and apoptotic bodies. The formation mechanism is respectively as follows: the multivesicular bodies are fused with plasma membranes to release exosomes; cell membrane budding directly into microvesicles; vesicles shed from dying cells form apoptotic bodies. The diameter of the exosome is 40-100 nm, the diameter of the microvesicle is 100-1,000 nm, and the microvesicle is generally larger than the exosome, but the smaller microvesicles are similar to the exosome in size, the apoptotic bodies are different in size (the diameter is 800-5,000 nm), the appearance is different, and the microvesicle is generally larger than other types of extracellular vesicles. During their formation, urine extracellular vesicles can transfer various cell-specific components (proteins, lipids, and nucleic acids) to target cells. As methodology research becomes more stringent, and the optimization of isolation, purification and characterization methods has further advanced, extracellular vesicles have received great attention for the treatment of diseases, these studies are promoting the recognition that urine extracellular vesicles serve as important mediators in kidney pathology and physiology.
The extraction method of the extracellular vesicles comprises a reagent precipitation method, an ultra-separation method, an ultrafiltration method, a chromatographic exclusion method and a density gradient centrifugation method. The reagent precipitation method has been proved by many studies to have low purity, and the extract obtained by using the ultra-separation method, the ultra-filtration method or the chromatographic exclusion method only contains more hetero-proteins and non-vesicle nucleic acids. Meanwhile, urine of a patient suffering from nephropathy contains a large amount of soluble proteins, detection of extracellular vesicle proteins in the urine is interfered, and an additional purification step is required to remove the soluble proteins. At present, the ultra-separation method combined with density gradient centrifugation is still the most effective method for extracting extracellular vesicles, the vesicles extracted by the method contain high purity of exosome, and the extracellular vesicles extracted by the density gradient centrifugation method are generally called small extracellular vesicles. However, the density gradient centrifugation takes a long time, and the traditional density gradient centrifugation needs at least 14 hours, which hinders the clinical application of the traditional density gradient centrifugation.
Uromodulin (also known as Tamm-Horsfall uroglycoprotein (THP)) is a membrane protein synthesized only in the thick segment of the loop ascending branch, and is the protein most abundant in urine under physiological conditions. Besides interfering the detection of vesicle protein, the uromodulin can form a net to capture urine extracellular vesicles, and the density of the small extracellular vesicles is changed so that the small extracellular vesicles are not distributed and concentrated in a gradient liquid, thereby reducing the extraction amount and purity of the small extracellular vesicles.
Therefore, in order to further meet the needs of clinical application and scientific research, a density gradient centrifugation method for improving the extracellular vesicles in the urine specimen needs to be developed, which can extract the extracellular vesicles in the urine with high yield and high purity, shorten the extraction time, and provide a practical, simple, precise and reliable method for extracting the extracellular vesicles in the urine in clinical and scientific research.
Disclosure of Invention
The method provided by the invention can obviously improve the extraction yield, reduce the sample consumption, greatly shorten the extraction time, obviously improve the extraction purity by combining a differential centrifugation method and an ultrafiltration method, and is simpler, more reliable and more rapid in clinical use.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an improved extraction method of urine small extracellular vesicles, which comprises the following steps of: sequentially adding sucrose solutions with different concentration gradients to the bottom of the test tube from low concentration to high concentration to form gradient liquid with sequentially increasing concentration from top to bottom, slowly injecting coarse vesicle resuspension at the uppermost layer of the gradient liquid, and centrifuging;
the sucrose solution is a 20mm Tris-HCl sucrose heavy water solution with the pH value of 8.4-8.8, and the crude vesicle heavy suspension is prepared by suspending crude vesicles in a 20mm Tris-HCl water solution with the pH value of 8.4-8.8.
The invention adopts 20mm Tris-HCL sucrose heavy water solution with specific pH range to carry out density gradient centrifugation on the crude vesicle, compared with the traditional extraction method, such as the sucrose heavy water solution cushion centrifugation with a certain concentration or the PBS sucrose heavy water solution density gradient centrifugation, the method can obviously improve the extraction yield of the small extracellular vesicle in urine, reduce the sample usage amount, and is more convenient for clinical preparation and use.
As a preferred embodiment of the invention, the sucrose solution is a 20mm Tris-HCl sucrose heavy aqueous solution with pH8.6, and the crude vesicle heavy suspension is prepared by suspending crude vesicles in a 20mm Tris-HCl aqueous solution with pH 8.6. Under the alkaline condition, the aggregation degree of TH protein can be reduced, thereby reducing the capture of the TH protein to extracellular vesicles, keeping the original density of the vesicles, and concentrating the small extracellular vesicles in a section of specific gradient layer to obtain the small extracellular vesicles with the maximum yield.
As a preferred embodiment of the invention, the concentration of the gradient liquid in the test tube from top to bottom is 5%, 10%, 20%, 40% and 60% in sequence, and the concentration gradient is favorable for the enrichment of the small extracellular vesicles in the middle layer and the subsequent extraction.
As a preferred embodiment of the present invention, the centrifugation is carried out at 25 ℃ for 5 hours. The difference between the method and the traditional method is that the traditional density gradient centrifugation needs at least 14 hours at 4 ℃ to obtain the small extracellular vesicles with a certain yield, and the method adopts 20mm Tris-HCL sucrose heavy water solution, so that the small extracellular vesicles with a larger yield can be obtained only by centrifugation for 5 hours at 25 ℃, and the extraction time is greatly shortened. It should be understood that the 25 ℃ described in the present invention allows for some tolerance in the accuracy of the instrument settings, typically + -2 ℃, and that temperatures within this range of variation are also within the scope of the present invention.
As a preferred embodiment of the present invention, the coarse vesicle is obtained by extracting a urine sample at normal temperature by differential centrifugation. The differential centrifugation method is to remove dead cells and large cell debris by low-speed centrifugation, and then remove soluble proteins, protein aggregates and other impurities co-precipitated with extracellular vesicles by high-speed centrifugation, thereby separating the microvesicles and exosomes, and the method is well known to those skilled in the art. The differential centrifugation method is generally carried out at the low temperature of 4 ℃, and when the differential centrifugation method is combined with the density gradient centrifugation of 20mm Tris-HCL sucrose heavy water solution, the differential centrifugation method at the normal temperature is more favorable for improving the extraction yield of the small extracellular vesicles.
The normal temperature of the invention is 18-28 ℃. The differential centrifugation method is used at normal temperature and is carried out at 18-28 ℃.
Preferably, the differential centrifugation method specifically comprises: at normal temperature, the urine sample is centrifuged at 500g, the precipitate is removed, the supernatant is centrifuged at 2,000g, the precipitate is removed, the supernatant is centrifuged at 16,000g, the precipitate is removed, and the supernatant is centrifuged at 200,000g for 1 hour by using an ultracentrifuge to obtain crude vesicles as the precipitate.
As a preferred embodiment of the present invention, the supernatant obtained during the differential centrifugation is concentrated by ultrafiltration. Ultrafiltration is a method of separating exosomes relying on the pressure difference across an ultrafiltration membrane as a motive force, and methods thereof are well known to those skilled in the art. The applicant of the invention researches and discovers that a differential centrifugation method is combined with an ultrafiltration method, so that a large amount of foreign proteins can be removed, and the purity of the small extracellular vesicles is improved while the yield is ensured.
As a preferred embodiment of the invention, the ultrafiltration is ultrafiltration concentration in a 100kd ultrafiltration tube, and high-purity small extracellular vesicles can be obtained to the maximum extent.
The invention also provides a more specific improved extraction method of urine small extracellular vesicles, which comprises the following steps:
(1) extracting coarse vesicles by a differential centrifugation method: centrifuging a urine sample at the normal temperature by 500g, removing precipitates, centrifuging a supernatant by 2,000g, removing precipitates, adding the supernatant into a 100kd ultrafiltration tube, concentrating the supernatant by 10 times under the centrifugal force of 2,000, centrifuging a concentrated liquid by 16,000g, removing precipitates, centrifuging the supernatant by an ultracentrifuge at the centrifugal force of 200,000g for 1 hour to obtain precipitate crude vesicles, and suspending the crude vesicles in a 20mm Tris-HCL aqueous solution with the pH of 8.6 to obtain a crude vesicle heavy suspension;
(2) extracting small extracellular vesicles by density gradient centrifugation: sequentially filling 1ml of 20mm Tris-HCL sucrose heavy aqueous solution with the concentration of 5%, 10%, 20%, 40% and 60% and the pH value of 8.6 into the bottom of a test tube by using a flat-head needle injector, slowly adding the crude vesicle heavy suspension to the top layer of the prepared gradient liquid, centrifuging for 5 hours at 25 ℃ by 110,000g of centrifugal force, dividing the gradient liquid into 12 layers from top to bottom, taking out the 6, 7, 8 and 9 layers enriched with the small cell outer vesicle, respectively adding the layers into a super-separation tube, diluting to 8ml by using 20mm Tris-HCL aqueous solution with the pH value of 8.6, and centrifuging for 1 hour at 4 ℃ by 200,000g to obtain precipitates.
The improved extraction method adopts a differential centrifugation method combined with an ultrafiltration method to extract coarse vesicles, and adopts 20mm Tris-HCL sucrose heavy water solution to carry out density gradient centrifugation, so that the small extracellular vesicles with the maximum purity can be obtained.
In conclusion, compared with the prior art, the invention has the following beneficial effects:
the method improves the traditional density gradient centrifugation method, can eliminate the interference of urocortin and the like on the extraction of the urine small cell outer vesicles to the maximum extent, reduces the use amount of samples, obviously improves the extraction yield of the urine small cell outer vesicles, greatly shortens the extraction time, and can obtain the small cell outer vesicles with the maximum yield by carrying out the improved density gradient centrifugation extraction on the coarse vesicles obtained by differential centrifugation (without using salting-out and ultrafiltration methods) at normal temperature based on the improved method; the coarse vesicles obtained by combining the differential centrifugation at normal temperature with the 100kd ultrafiltration method are subjected to the improved density gradient centrifugation extraction of the invention, and then the small extracellular vesicles with the maximum purity can be obtained.
Drawings
FIG. 1 is a schematic diagram showing the layering of the components in the test tube before centrifugation in a density gradient (modified method) of a 20mm Tris-HCL sucrose heavy aqueous solution in example 1 of the present invention;
FIG. 2 is a schematic diagram showing the layering of the components in the tube after centrifugation in a 20mm Tris-HCl sucrose heavy aqueous solution density gradient (modified method) in example 1 of the present invention;
FIG. 3 is a graph showing the difference in the distribution of extracellular vesicle markers in each layer of gradient fluid after centrifugation by the conventional method and the improved method in example 1 of the present invention;
FIG. 4 is a flow chart of the scheme for maximum yield extraction of extracellular vesicles of test example d and a flow chart of the scheme for maximum purity extraction of extracellular vesicles of test example h in example 2 of the present invention (A);
FIG. 5 is a transmission electron microscope image of the small extracellular vesicles obtained in test examples a to h of example 2 of the present invention;
FIG. 6 shows the Western Blot identification chart (A) of the small extracellular vesicle markers, the TRPS counting result (B) of the nanoparticles and the purity result (C) of the small extracellular vesicles obtained in test examples B to h of example 2 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It should be understood that the scope of the above-described subject matter of the present invention is not limited to the following examples, and any technique realized based on the contents of the present invention is within the scope of the present invention. The technical means and procedures used in the following examples are conventional means and procedures well known to those skilled in the art, and the raw materials used are commercially available, unless otherwise specified.
Example 1
Comparison of different sucrose heavy aqueous solution Density gradient centrifugation
1. Preparation of sucrose heavy water solution
(1) Preparation of PBS sucrose heavy water solution
Weighing 0.03g of PBS powder and 1.5g of sucrose in a test tube, adding heavy water to fix the volume to 30ml, adjusting the pH to 7.4, and uniformly mixing and dissolving to obtain 5% PBS sucrose heavy water solution. Weighing 0.02g of PBS powder and 12g of sucrose in a test tube, adding heavy water to a constant volume of 20ml, adjusting the pH value to 7.4, and uniformly mixing and dissolving to obtain 60% PBS sucrose heavy water solution. The 10%, 20% and 40% sucrose heavy water solution is prepared by mixing the two solutions according to the proportion.
(2) Preparation of 20mm Tris-HCL sucrose heavy water solution
Weighing 0.07g of Tris powder and 1.5g of sucrose in a test tube, adding heavy water to a constant volume of 30ml, adding concentrated hydrochloric acid to adjust the pH value to 8.6, and uniformly mixing and dissolving to obtain a 5% 20mm Tris-HCL sucrose heavy water solution. Weighing 0.05g of Tris powder and 12g of sucrose in a test tube, adding heavy water to a constant volume of 20ml, adding concentrated hydrochloric acid to a pH value of 8.6, and uniformly mixing and dissolving to obtain a 60% 20mm Tris-HCL sucrose heavy water solution. The 10%, 20% and 40% sucrose heavy water solution is prepared by mixing the two solutions according to the proportion.
2. Differential centrifugation method for extracting coarse vesicles
The urine sample was centrifuged at 500g for 10 minutes, the precipitate was removed, the supernatant was centrifuged at 2,000g for 20 minutes, the precipitate was removed, and then the supernatant after 2,000g centrifugation was centrifuged at 16,000g for 20 minutes in a fixed angle rotor. The crude vesicle pellet was obtained by centrifuging 16,000g of supernatant at 200,000g centrifugal force for 1 hour in a SW41Ti horizontal rotor at maximum acceleration and lower deceleration using a Beckman XL-80 ultracentrifuge (Beckman Coulter).
3. Extraction of urine small cell outer vesicle
The precipitates (crude vesicles) obtained by differential centrifugation were resuspended in 3ml of PBS (conventional method) water (H) at pH 7.4 2 O) solution or 20mm Tris-HCl (modified) water (H) at pH8.6 2 O) solution, using a flat-head needle injector to sequentially inject 5%, 10%, 20%, 40%, 60% of PBS (traditional method) sucrose heavy water solution with pH 7.4 or 20mm Tris-HCL (improved method) sucrose heavy water solution with pH8.6 into the bottom of a test tube by 1ml respectively, slowly adding the coarse vesicle resuspension solution to the top layer of the prepared gradient solution by using an injection gun (the components in the test tube are layered and shown in figure 1 before the centrifugation by the improved method), respectively at 4 ℃ and 25 ℃, centrifuging at 110,000g centrifugal force for 5 hr, taking out the gradient solution from top to bottom into 12 layers (the contents in the tube are shown in figure 2 after centrifugation by modified method), adding into a centrifuge tube, diluting to 8ml with PBS aqueous solution (conventional method) with pH 7.4 or 20mm Tris-HCl aqueous solution (modified method) with pH8.6, centrifuging at 4 deg.C for 1 hr at 200,000g, the pellet was examined using Western Blot for the extracellular vesicle markers Alix and TSG101 protein in the 12 layer fraction to determine the extracellular vesicle layer. And identifying and recording the layering of the small extracellular vesicles, and extracting the small extracellular vesicles to take out the layering containing the small extracellular vesicles together without re-identification.
The results of the distribution difference of the markers in each extracellular vesicle obtained by PBS (traditional method) 4 ℃ density gradient centrifugation, PBS (traditional method) 25 ℃ density gradient centrifugation, 20mm Tris-HCL (improved method) 4 ℃ density gradient centrifugation and 20mm Tris-HCL (improved method) 25 ℃ density gradient centrifugation (shown in figure 3) show that the improved method sucrose gradient solution can obtain more concentrated extracellular vesicle distribution layers at 25 ℃ than the other three schemes by ultracentrifugation, and the distribution layers are mainly concentrated in the 6 th, 7 th, 8 th and 9 th layers.
Example 2
Comparison of different differential centrifugation protocols in combination with Tris-HCL (modified method) sucrose heavy aqueous solution density gradient centrifugation
1. Extraction of urine small extracellular vesicles
Test example a: 30% sucrose cushion method
Crude vesicles were obtained by differential centrifugation as described in example 1 at ambient temperature, and resuspended in 6ml of 20mm Tris-HCl water (H) 2 O) solution (pH 8.6), the bottom of the test tube was filled with 1ml each of a coarse vesicle heavy suspension, 30% and 60% 20mm Tris-HCl sucrose heavy aqueous solutions (pH 8.6) in this order by using a flat-head needle syringe, and after centrifugation at 25 ℃ for 5 hours at a centrifugal force of 110,000g, 1ml of the 30% sucrose heavy aqueous solution layer was taken out and added to an ultracentrifuge tube, diluted to 8ml with 20mm Tris-HCl aqueous solution (pH 8.6), and centrifuged at 4 ℃ for 1 hour at 200,000g to precipitate a sample rich in extracellular vesicles.
Test example b: differential centrifugation at room temperature combined salting out and improved density gradient centrifugation
(1) Differential centrifugation combined with salting-out for extracting coarse vesicles
Crude vesicles were extracted by differential centrifugation as described in example 1 at room temperature, except that after centrifugation at 2,000g, the supernatant was added with sodium chloride to give a concentration of 0.58M, the mixture was allowed to stand at room temperature for 2 hours, and after centrifugation at 2,000g for 20 minutes, the supernatant was centrifuged at 16,000 g.
(2) Extraction of urine small extracellular vesicles
Extracting urine small extracellular vesicles from the crude vesicles obtained in the step (1) according to the following method: the crude vesicles were resuspended in 3ml of 20mm Tris-HCl (modified) water (H) pH8.6 2 O) solution, using a flat-head needle injector to sequentially inject 1ml of 5%, 10%, 20%, 40%, 60% of 20mm Tris-HCL (modified method) sucrose heavy aqueous solution with pH8.6 into the bottom of a test tube, slowly adding the coarse vesicle heavy suspension to the uppermost layer of the prepared gradient liquid (shown in figure 1) by using an adding gun, centrifuging at 25 ℃ for 5 hours under a centrifugal force of 110,000g, taking out the vesicle enrichment layer outside the small cell, adding the vesicle enrichment layer into a super-separation tube, diluting to 8ml by using 20mm Tris-HCL aqueous solution with pH8.6, and centrifuging at 4 ℃ for 1 hour of 200,000g respectively to obtain precipitates.
Test example c: differential centrifugation at 4 ℃ combined with improved density gradient centrifugation
Crude vesicles were extracted by differential centrifugation as described in example 1 at 4 ℃ and small extracellular vesicles were extracted as described in step (2) of test example b.
Test example d: differential centrifugation combined with modified density gradient centrifugation at room temperature (see figure 4A)
Crude vesicles were extracted at ambient temperature by differential centrifugation as described in example 1, and small extracellular vesicles were extracted as described in step (2) of test example b.
Test example e: differential centrifugation at 4 ℃ combined with 30kd ultrafiltration and improved density gradient centrifugation
(1) Extraction of coarse vesicle by differential centrifugation combined with ultrafiltration
Crude vesicles were extracted by differential centrifugation as described in example 1 at 4 ℃ except that after centrifugation at 2,000g, the supernatant was taken and added to a 30kd ultrafiltration tube, the supernatant was concentrated 10-fold at 2,000g centrifugation force, and centrifugation at 16,000g was performed.
(2) Extraction of urine small cell outer vesicle
The extraction of small extracellular vesicles was performed as described in step (2) of test example b.
Test example f: differential centrifugation combined with 30kd ultrafiltration and improved density gradient centrifugation at room temperature
(1) Differential centrifugation method combined with ultrafiltration method for extracting coarse vesicles
Crude vesicles were extracted at room temperature by differential centrifugation as described in example 1, except that after centrifugation at 2,000g, the supernatant was taken and added to a 30kd ultrafiltration tube, the supernatant was concentrated 10-fold at 2,000g centrifugation force, and centrifugation at 16,000g was performed.
(2) Extraction of urine small cell outer vesicle
The extraction of small extracellular vesicles was performed as described in step (2) of test example b.
Test example g: differential centrifugation at 4 ℃ combined with 100kd ultrafiltration and improved density gradient centrifugation
(1) Differential centrifugation method combined with ultrafiltration method for extracting coarse vesicles
Crude vesicles were extracted at 4 ℃ by differential centrifugation as described in example 1, except that after centrifugation at 2,000g, the supernatant was taken and added to a 100kd ultrafiltration tube, the supernatant was concentrated 10-fold at 2,000g centrifugation, and centrifugation at 16,000g was performed.
(2) Extraction of urine small extracellular vesicles
The extraction of small extracellular vesicles was performed as described in step (2) of test example b.
Test example h: differential centrifugation at room temperature in combination with 100kd ultrafiltration and modified density gradient centrifugation (see FIG. 4B)
(1) Differential centrifugation method combined with ultrafiltration method for extracting coarse vesicles
Crude vesicles were extracted at room temperature by differential centrifugation as described in example 1, except that after centrifugation at 2,000g, the supernatant was taken and added to an ultrafiltration tube of 100kd, and the supernatant was concentrated 10-fold at 2,000g of centrifugal force, followed by centrifugation at 16,000 g.
(2) Extraction of urine small extracellular vesicles
The extraction of small extracellular vesicles was performed as described in step (2) of test example b.
The test contents and the operating conditions of the above test examples a to h are shown in the following table.
2. Examination of urine small extracellular vesicles
The small extracellular vesicles obtained in each experimental group were observed by Transmission Electron Microscopy (TEM) (see fig. 5), in which solid arrows indicate TH protein and open arrows indicate extracellular vesicles adhered to TH protein. The results of the study showed that Tris-HCl discontinuous density gradient centrifugation (runs b-h) removed more TH protein than centrifugation using a 30% Tris sucrose pad alone (run a).
The total protein content of the small extracellular vesicles obtained in each test example was measured by Coomassie method, Western Blot identification chart (see FIG. 6A) compares the differences of the exosome marker proteins in each test example, and the number difference of nanoparticles in each test example was counted by adjustable resistance pulse sensing (TRPS), and the results were calculated as the number of nanoparticles extracted per ml of urine (see FIG. 6B). The purity of the small extracellular vesicles was calculated from the number of nanoparticles extracted from the sample of small extracellular vesicles and the ratio of the total protein amount (see FIG. 6C). The results show that TEM (see figure 5) and Western Blot identification (see figure 6A) show that test examples B and d can extract more extracellular vesicles and vesicle markers Alix, TSG101 and Syntenin-1 without ultrafiltration at normal temperature by differential centrifugation, and TRPS (see figure 6B) shows that the maximum number of nanoparticles can be obtained under the condition that test example d does not combine ultrafiltration and salting-out methods by differential centrifugation at normal temperature, so that test example d is a scheme for extracting the small extracellular vesicles with the maximum yield (the flow chart is shown in figure 4A); the result of calculating the purity (see figure 6C) by the number of nanoparticles/total protein shows that the test example h can obtain the extracellular vesicles with the maximum purity by combining differential centrifugation at normal temperature and 100kd ultrafiltration, and is a scheme for extracting the extracellular vesicles with the maximum purity (see figure 4B for a flow chart).
Claims (8)
1. An improved extraction method of urine small extracellular vesicles is characterized in that density gradient centrifugation is carried out according to the following method: sequentially filling sucrose solutions with different concentration gradients from low concentration to high concentration into the bottom of the test tube to form gradient liquid with sequentially increasing concentration from top to bottom, slowly filling coarse vesicle heavy suspension into the uppermost layer of the gradient liquid, and centrifuging at 25 ℃;
the sucrose solution is 20mm Tris-HCL sucrose heavy water solution with the pH value of 8.4-8.8, and the crude vesicle heavy suspension is prepared by suspending crude vesicles in 20mm Tris-HCL water solution with the pH value of 8.4-8.8.
2. The improved extraction method of urine small extracellular vesicles according to claim 1, wherein the sucrose solution is 20mm Tris-HCl sucrose heavy aqueous solution with pH8.6, and the crude vesicle heavy suspension is prepared by resuspending crude vesicles in 20mm Tris-HCl aqueous solution with pH 8.6.
3. The improved extraction method of urine small extracellular vesicles as claimed in claim 1, wherein the gradient solution has a concentration of 5%, 10%, 20%, 40% and 60% from top to bottom in the test tube.
4. The improved process of claim 3, wherein the centrifugation is at 25 ℃ for 5 hours.
5. The improved extraction method of urine small extracellular vesicles according to any one of claims 1 to 4, wherein the coarse vesicles are obtained by extracting a urine sample at room temperature by differential centrifugation.
6. The improved extraction method of urine small extracellular vesicles as claimed in claim 5, wherein the supernatant obtained during the differential centrifugation is concentrated by ultrafiltration.
7. The improved extraction method of urine small extracellular vesicles as claimed in claim 6, wherein the ultrafiltration is ultrafiltration concentration in a 100kd ultrafiltration tube.
8. The improved extraction process of urine small extracellular vesicles as claimed in claim 7, wherein the process comprises the following steps:
(1) extracting coarse vesicles by a differential centrifugation method: centrifuging a urine sample at the normal temperature by 500g, removing precipitates, centrifuging a supernatant by 2,000g, removing precipitates, adding the supernatant into a 100kd ultrafiltration tube, concentrating the supernatant by 10 times under the centrifugal force of 2,000, centrifuging a concentrated liquid by 16,000g, removing precipitates, centrifuging the supernatant by an ultracentrifuge at the centrifugal force of 200,000g for 1 hour to obtain precipitate crude vesicles, and suspending the crude vesicles in a 20mm Tris-HCL aqueous solution with the pH of 8.6 to obtain a crude vesicle heavy suspension;
(2) extracting small extracellular vesicles by density gradient centrifugation: sequentially filling 1ml of 20mm Tris-HCl sucrose heavy water solution with the concentrations of 5%, 10%, 20%, 40% and 60% and the pH value of 8.6 to the bottom of the test tube by using a flat-head needle injector, slowly adding the coarse vesicle heavy suspension to the top layer of the prepared gradient liquid, centrifuging for 5 hours at the temperature of 25 ℃ by using a centrifugal force of 110,000g, dividing the gradient liquid into a plurality of layers from top to bottom, taking out the layers enriched in the vesicles outside of the small cells, respectively adding the layers into an ultra-separation tube, diluting to 8ml by using 20mm Tris-HCl water solution with the concentration of 8.6, and centrifuging for 1 hour at the temperature of 4 ℃ by using 200,000g of Tris-HCl water solution with the concentration of 8.6 to obtain precipitates.
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