Method for extracting small extracellular vesicles in urine

Abstract

The invention discloses a method for extracting small extracellular vesicles in urine. The method comprises the following steps: carrying out first-time differential centrifugal treatment, second-time differential centrifugal treatment and third-time differential centrifugal treatment on a urine sample to obtain supernate 1; treating the precipitate by using a surfactant, and carrying out centrifugal treatment on the treated solution to obtain supernate 2; respectively carrying out ultracentrifugation treatment on the two kinds of supernate to obtain a first crude vesicle and a second crude vesicle, mixing the first crude vesicle and the second crude vesicle, and resuspending by using a Tris-HCL aqueous solution; tris-HCL sucrose heavy water solutions with different concentrations are sequentially added to the bottom of an ultracentrifuge tube to form a gradient solution, the uppermost layer of the gradient solution is filled with a proper proportion of coarse vesicle resuspension, ultracentrifugation treatment is performed by using a specific centrifugal force and a specific centrifugation time, and a small extracellular vesicle enrichment layer which is more intensively distributed can be obtained. According to the method, the extraction yield and purity of the small extracellular vesicles in the urine can be remarkably improved, and meanwhile, the extraction time is greatly shortened.

Description

Technical Field

[0001] The invention relates to the field of purification of small extracellular vesicles, and in particular to a method for extracting small extracellular vesicles from urine. Background Art

[0002] Exosomes (EVs) are a heterogeneous group of lipid-encapsulated structures secreted by cells and widely present in biological fluids. EVs act as mediators of intercellular communication under physiological and pathological conditions, transmitting biological information between cells. EVs play an important role in the early diagnosis, stratification, treatment and prognosis of diseases.

[0003] Based on the mechanism of formation, EVs can be divided into three overlapping size and density categories: exosomes, microvesicles, and apoptotic bodies. However, many studies on EVs have used low-specificity separation methods, which may lead to erroneous experimental conclusions. According to the statement of the International Exosome Society, density gradient centrifugation remains the most widely used high-purity separation method for recovering EVs. EVs separated by this method are smaller in diameter and contain more exosomes, which more and more scholars call small extracellular vesicles (sEVs).

[0004] Urine is an ideal non-invasive diagnostic specimen. Urine, as a waste product excreted by the body, also contains a certain amount of EVs. Uromodulin (UMOD), also known as Tamm-Horsfall urine glycoprotein (THP), is a membrane protein synthesized only in the ascending segment of the medulla and is the most abundant protein in urine under physiological conditions. It is precisely because THP is present in large quantities in urine that it is often difficult to achieve both high yield and high purity in urine sEV extraction. THP can capture EVs in urine so that they are precipitated and removed after three-step differential gradient centrifugation. In addition, EVs can be adsorbed on THP, affecting the sedimentation coefficient of the vesicles and changing their distribution in the density gradient, thereby reducing the yield and purity of sEVs. Another problem is that the relative centrifugal force and temperature in the urine EV separation method are currently not unified, and centrifugation takes a long time. It is necessary to develop an extraction method that improves yield and purity.

[0005] At the same time, the Chinese patent application (application number 201910221861.5) discloses an improved extraction method for urine small extracellular vesicles, but due to the low concentration of EVs in urine and the inability to completely eliminate THP interference, the yield and purity of urine sEV extraction still have room for improvement; secondly, the time consumption of the improved method can be further shortened. Therefore, it is necessary to further improve the density gradient centrifugation method for urine sEV extraction, including the differential centrifugation step, to obtain a higher yield, higher purity and more convenient sEV separation method. Summary of the invention

[0006] In view of the problems of the existing method for extracting small extracellular vesicles from urine, the present invention proposes a method for extracting small extracellular vesicles from urine to solve the problems raised in the above background technology.

[0007] A method for extracting small extracellular vesicles from urine, the method comprising the following steps: S1. Obtain a urine sample, perform a first differential centrifugation on the urine sample to obtain a primary supernatant, and perform a second differential centrifugation on the primary supernatant to obtain a secondary supernatant; S2, performing a third differential centrifugation on the secondary supernatant to obtain a precipitate and a supernatant 1; S3, treating the precipitate with a surfactant, and centrifuging the treated solution to obtain a supernatant 2; S4, performing ultracentrifugation treatment on the supernatant 1 and the supernatant 2 respectively to obtain a first crude vesicle and a second crude vesicle, mixing the first crude vesicle and the second crude vesicle and resuspending them in a Tris-HCl aqueous solution to obtain a crude vesicle resuspension; S5. Take an ultracentrifuge tube, add Tris-HCL sucrose heavy water solutions of different concentration gradients to the bottom of the ultracentrifuge tube in order from low to high concentration to form a gradient solution with increasing concentration from top to bottom, slowly add the crude vesicle resuspension solution to the top layer of the gradient solution, perform ultracentrifugation, take out the gradient solution from top to bottom, take the specific component solution of the layered solution to obtain small extracellular vesicles.

[0008] According to the method for extracting urine small extracellular vesicles proposed by the present invention, the beneficial effects of the present invention are: By treating the precipitate after the third differential centrifugation with 0.05% surfactant ethylphenyl polyethylene glycol, the small extracellular vesicles in the precipitate can be released into supernatant 2; by obtaining two crude vesicles from the two supernatants separately after ultracentrifugation, instead of mixing the two supernatants together to extract the crude vesicles, the influence of the surfactant ethylphenyl polyethylene glycol in supernatant 2 on the extraction of crude vesicles in supernatant 1 was avoided; by remixing the two crude vesicles obtained and resuspending them with 20mm Tris-HCL aqueous solution, the change of THP on the sedimentation coefficient of small extracellular vesicles can be reduced, which can effectively eliminate the interference of urea modulin on the extraction of urine small extracellular vesicles, and significantly improve the extraction yield and purity of urine small extracellular vesicles.

[0009] In addition, the method for extracting urine small extracellular vesicles provided by the present invention may also have the following additional technical features: Preferably, after step S1 and before step S2, the method comprises: using an ultrafiltration element to perform ultrafiltration concentration treatment on the secondary supernatant.

[0010] Preferably, the ultrafiltration element is a 100kd ultrafiltration tube.

[0011] Preferably, S3 is specifically as follows: adding a surfactant to the precipitate, placing it in a water bath at 37° C. for 5 minutes, rotating and mixing for 10 seconds every 1 minute during the water bath, and centrifuging it at 16000 g for 20 minutes after the water bath to obtain supernatant 2.

[0012] Preferably, the surfactant is a 20 mm Tris-HCL aqueous solution with a pH of 8.4 to 8.8 containing 0.05% ethylphenyl polyethylene glycol.

[0013] Preferably, the step S1, the step S2, and the step S3 are all centrifuged at room temperature.

[0014] Preferably, the Tris-HCL aqueous solution is 2.5 ml of a 20 mm Tris-HCL aqueous solution with a pH of 8.4 to 8.8.

[0015] Preferably, in step S5, the ultracentrifugal force is 200000 g, and the centrifugation time is 2 hours.

[0016] Preferably, the step S5 is specifically as follows: Take an ultracentrifuge tube, insert a flat-head needle syringe into the bottom of the tube, and add 1 ml of 20 mm Tris-HCL sucrose heavy aqueous solution with a pH of 8.4 to 8.8 at a concentration of 5%, 10%, 20%, 40%, and 60% to the bottom of the ultracentrifuge tube in sequence to form a gradient solution with increasing concentration from top to bottom, slowly add the crude vesicle resuspension solution to the top layer of the gradient solution with a pipette, centrifuge at 200,000 g for 2 h at 24° C. to 26° C., and then take out the gradient solution from top to bottom to obtain 5 layers of component solutions, take out the 3rd and 4th layer component solutions in the layered solution, centrifuge at 200,000 g for 1 h at 4° C., and take out the precipitate after centrifugation to obtain small extracellular vesicles.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Transmission electron microscopy images of small extracellular vesicles extracted from the supernatant after differential centrifugation using three different precipitation treatment agents provided in the embodiments of the present invention; Figure 2 Western Blot identification diagrams (A, B) of crude vesicles and small extracellular vesicle markers extracted from the supernatant after differential centrifugation using three different precipitation treatment agents, and TRPS nanoparticle counting results (C) provided in the embodiments of the present invention; Figure 3 A design diagram of a supercentrifuge tube before density gradient centrifugation (A) and a schematic diagram of the internal component stratification of the supercentrifuge tube after centrifugation (B) provided in an embodiment of the present invention; Figure 4 Western Blot identification of small extracellular vesicle markers extracted from each component of the improved density gradient method and the original density gradient centrifugation method; Figure 5 Western Blot identification diagram (A) of small extracellular vesicle markers, nanoparticle TRPS counting results (B) and small extracellular vesicle purity results (C) obtained in test examples a to f provided in the embodiments of the present invention.

[0020] The embodiments of the present invention will be further described below with reference to the accompanying drawings. DETAILED DESCRIPTION

[0021] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the examples. Several embodiments of the present invention are given in the examples. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0022] Example 1 In Example 1 of the present invention, a method for extracting small extracellular vesicles from urine is provided, the method comprising the following steps: S1. Obtain a urine sample, perform a first differential centrifugation on the urine sample to obtain a primary supernatant, and perform a second differential centrifugation on the primary supernatant to obtain a secondary supernatant; Among them, the centrifugal force of the first differential centrifugation is 500g, and the centrifugation time is 10min.

[0023] The centrifugal force of the second differential centrifugation was 2000 g, and the centrifugation time was 20 min.

[0024] S2, performing a third differential centrifugation on the secondary supernatant to obtain a precipitate and a supernatant 1; It should be noted that step S1 and step S2 are both performed at room temperature, that is, the three differential centrifugation processes are all performed at room temperature, so as to reduce THP aggregation, thereby reducing its capture of small extracellular vesicles, so that the small extracellular vesicles are retained in the supernatant.

[0025] S3, treating the precipitate with a surfactant, and centrifuging the treated solution to obtain a supernatant 2; Specifically, S3 is as follows: adding a surfactant to the precipitate, placing the precipitate in a water bath at 37° C. for 5 minutes, rotating and mixing for 10 seconds every 1 minute during the water bath, centrifuging at 16000 g centrifugal force for 20 minutes after the water bath, removing the bottom precipitate, to obtain supernatant 2; Preferably, the surfactant is a 0.05% ethylphenyl polyethylene glycol solution in Tris-HCL, wherein the Tris-HCL solution is a 20 mm Tris-HCL solution with a pH of 8.4 to 8.8; It should be noted that the ethylphenyl polyethylene glycol is specifically NP40, and the small extracellular vesicles in the precipitate can be released into the supernatant 2 through NP40. The small extracellular vesicles in the supernatant 2 are precipitated by ultracentrifugation to obtain the second crude vesicles.

[0026] S4, performing ultracentrifugation treatment on the supernatant 1 and the supernatant 2 respectively to obtain a first crude vesicle and a second crude vesicle, mixing the first crude vesicle and the second crude vesicle and resuspending them in a Tris-HCl aqueous solution to obtain a crude vesicle resuspension; Specifically, in the present invention, the two supernatants are subjected to ultracentrifugation respectively to obtain crude vesicles and then the crude vesicles are mixed, rather than mixing the two supernatants and then extracting the crude vesicles together. Since the crude vesicles are extracted by ultracentrifuging the supernatant to obtain the precipitate, NP40 reduces the aggregation of urine protein and reduces the precipitation of small extracellular vesicles. The present invention avoids the influence of NP40 in supernatant 2 on the extraction of crude vesicles in supernatant 1. The two crude vesicles obtained were mixed again and resuspended in 2.5 ml of a 20 mm Tris-HCL aqueous solution with a pH of 8.4 to 8.8. The resuspension solution is alkaline, which can reduce the aggregation of THP protein and its interference with the sedimentation coefficient of small extracellular vesicles, thereby facilitating the subsequent density gradient centrifugation to separate small extracellular vesicles.

[0027] S5. Take an ultracentrifuge tube, add Tris-HCL sucrose heavy water solutions of different concentration gradients to the bottom of the ultracentrifuge tube in order from low to high concentration to form a gradient solution with increasing concentration from top to bottom, slowly add a suitable proportion of the crude vesicle resuspension solution to the top layer of the gradient solution, use a specific centrifugal force and centrifugation time for ultracentrifugation, and take out the gradient solution in layers to obtain a more concentrated small extracellular vesicle enriched layer, take the specific component solution of the layered solution to obtain small extracellular vesicles.

[0028] Wherein, the step S5 is specifically as follows: Take an ultracentrifuge tube, insert a flat-head needle syringe into the bottom of the tube, and add 1 ml of 20 mm Tris-HCL sucrose heavy aqueous solution with a pH of 8.4 to 8.8 at a concentration of 5%, 10%, 20%, 40%, and 60% to the bottom of the ultracentrifuge tube in sequence to form a gradient solution with increasing concentration from top to bottom, slowly add the crude vesicle resuspension solution to the top layer of the gradient solution with a pipette, centrifuge at 200,000 g for 2 h at 24° C. to 26° C., and then take out the gradient solution from top to bottom to obtain 5 layers of component solutions, take out the 3rd and 4th layer component solutions in the layered solution, centrifuge at 200,000 g for 1 h at 4° C., and take out the precipitate after centrifugation to obtain small extracellular vesicles; Specifically, the gradient solution is a 20 mm Tris-HCL sucrose heavy water gradient solution with increasing concentration from top to bottom. After configuration, a crude vesicle resuspension solution is added to the top layer of the gradient solution. After centrifugation, a 5-layer component solution is formed. Then, a specific component solution, i.e., the 3rd and 4th layer component solutions, is extracted. After ultracentrifugation, the precipitate is taken to obtain small extracellular vesicles.

[0029] Example 2 In Example 2 of the present invention, a method for extracting small extracellular vesicles from urine is provided. The extraction method provided in Example 2 is basically the same as the extraction method provided in Example 1, except that: after step S1 and before step S2 in Example 1, the method provided in Example 2 also includes: using an ultrafiltration element to ultrafilter and concentrate the secondary supernatant, and the ultrafiltration element is a 100kd ultrafiltration tube.

[0030] Specifically, a 100 kd ultrafiltration tube is used for concentration, thereby reducing the amount of urine samples, reducing the time required for the third differential centrifugation, and removing a large amount of impurities, thereby maximizing the acquisition of high-purity small extracellular vesicles.

[0031] Furthermore, in order to further verify the effect of the present invention, the present invention conducted the following experiments: 1. Verification of the effect of differential centrifugation combined with NP40 extraction of crude vesicles and small extracellular vesicles: A urine sample was centrifuged at 500 g for 10 minutes, and the precipitate was removed to obtain a first supernatant. The first supernatant was centrifuged at 2000 g for 20 minutes to remove the precipitate to obtain a second supernatant. The second supernatant was then centrifuged at 16000 g in a fixed angle rotor for 20 minutes, and the supernatant was retained to obtain supernatant 1. The precipitate after centrifugation was resuspended with 0.5 ml PBS (control group), 20 mM Tris-HCl (pH 8.4-8.8), 0.05% NP40 solution, and 100 mg / ml DTT Tris-HCl solution, respectively, and the suspension was incubated at 37°C for 5 minutes, shaking for 10 seconds every minute. After incubation, centrifuge at 16000 g for 20 minutes at room temperature. Supernatant 2 in the test tube was aspirated with a disposable pipette, and supernatant 1 was mixed with supernatant 2. Another sample of the same amount of urine was operated according to the above method to obtain supernatant 2, which was not mixed with supernatant 1. After dilution to 8 ml, crude vesicles and small extracellular vesicles were extracted separately. The crude vesicles were obtained by centrifugation at 200000g centrifugal force in a SW41Ti horizontal rotor at maximum acceleration and minimum deceleration for 1 hour using a Beckman XL-80 ultracentrifuge. Western Blot was used to detect crude vesicles and small extracellular vesicles extracted from supernatant 1 and supernatant 2 alone, and crude vesicles and small extracellular vesicle protein markers extracted from the two supernatants. TRPS particle counting was used to count the number of particles in the small extracellular vesicle samples extracted from the three supernatants. The results showed that the small extracellular vesicle samples extracted from supernatant 2 after 16000g precipitation treated with the three reagents and PBS (control group) contained small extracellular vesicle markers TSG101 and Alix, and the typical cup-shaped vesicle morphology of about 100nm was observed under transmission electron microscopy (such as Figure 1 ), proving that small extracellular vesicles were extracted from supernatant 2. Compared with Tris-HCl (pH 8.4-8.8) and 100 mg / ml DTT, more small extracellular vesicle markers TSG101 and Alix were obtained by combining NP40 treatment. Only the samples extracted from the NP40 group showed weak small extracellular vesicle marker syntenin-1 (such as Figure 2 However, the extraction of mixed supernatants 1 and 2 did not significantly enhance the expression of TSG101, Alix, and syntenin-1 in crude and minicellular vesicle samples (e.g. Figure 2 A and B in the figure show that mixed extraction cannot increase the yield of small extracellular vesicles. TRPS particle counting also verified the above conclusions and also proved that more vesicle particles (such as Figure 2C in the above). Since the extraction of crude vesicles by mixing supernatant 1 and supernatant 2 together cannot increase the yield of small extracellular vesicles, the present invention uses differential centrifugation combined with NP40 treatment to extract crude vesicles from supernatant 2 and supernatant 1 separately and then mix the crude vesicles to extract small extracellular vesicles, and the beneficial effect of the method is demonstrated in the comparison of the experimental examples.

[0032] 2. Extraction of small extracellular vesicles by modified density gradient centrifugation The present invention further improves the original extraction method (the improved extraction method in the Chinese patent application with application number 201910221861.5). Specific implementation steps: resuspend the precipitate (the first crude vesicle and the second crude vesicle) obtained by differential centrifugation in 2.5 ml of a 20 mm Tris-HCL aqueous solution with a pH of 8.4 to 8.8, insert a flat-head needle syringe into the bottom of the tube, and add 1 ml of 20 mm Tris-HCL sucrose heavy aqueous solution with concentrations of 5%, 10%, 20%, 40%, and 60% to the bottom of the ultracentrifuge tube in turn, and add the crude vesicle resuspension very slowly to the top layer of the prepared gradient solution (such as 1 ml) using a pipetting gun. Figure 3 A in the figure), centrifuge at 200,000 g for 2 hours at 24°C to 26°C, and separate the gradient solution into 5 layers from top to bottom (e.g. Figure 3 B in the figure), take out each layer of fractions and add them to the ultracentrifuge tube, dilute to 8 ml with 20 mm Tris-HCL aqueous solution at pH 8.4-8.8, centrifuge at 200,000 g for 1 hour at 4 ° C, and use Western Blot to detect small extracellular vesicle markers Alix and TSG101 proteins in the 5-layer fractions to determine the small extracellular vesicle layer. Compared with the original method, the improved method for extracting small extracellular vesicles obtained small extracellular vesicle markers Alix and TSG101 that were more concentrated in two specific fractions (the third and fourth), and contained fewer markers in other fractions (such as Figure 4 ). At the same time, this method significantly shortens the extraction time compared with the original method.

[0033] Comparison of different differential centrifugation schemes combined with modified sucrose-deoxycholic acid density gradient centrifugation: 1. Test Example a: Differential centrifugation combined with density gradient centrifugation at room temperature (i.e., the improved extraction method in the Chinese patent application with application number 201910221861.5) (1) Extraction of crude vesicles by differential centrifugation The urine samples were centrifuged at 500 g for 10 min to remove the precipitate, the supernatant was centrifuged at 2000 g for 20 min to remove the precipitate, and the supernatant after 2000 g centrifugation was subsequently centrifuged at 16000 g for 20 min in a fixed-angle rotor. The 16000 g supernatant was centrifuged at 200000 g for 1 h in a Beckman XL-80 ultracentrifuge (Beckman Coulter) in a SW41Ti swing-out rotor at maximum acceleration and low deceleration to obtain a crude vesicle pellet; (2) Extraction of small extracellular vesicles from urine: Use a flat-tip syringe to add 1 ml of 20 mm Tris-HCL sucrose heavy aqueous solution with a concentration of 5%, 10%, 20%, 40%, and 60% at pH 8.6 to the bottom of the test tube in turn, and slowly add the crude vesicle resuspension to the top layer of the prepared gradient solution. After centrifugation at 110,000 g for 5 hours at 25°C, the gradient solution is divided into 12 layers from top to bottom. Take out the layers enriched with small extracellular vesicles, add them to ultracentrifuge tubes respectively, dilute to 8 ml with 20 mm Tris-HCL aqueous solution with a pH of 8.6, and centrifuge at 200,000 g for 1 hour at 4°C to obtain precipitates.

[0034] 2. Experimental Example b: Improved differential centrifugation combined with NP40 and improved density gradient centrifugation at room temperature (1) Improved differential centrifugation combined with NP40 to extract crude vesicles Crude vesicles were extracted at room temperature using the differential centrifugation method described in Example 1.

[0035] (2) Extraction of small extracellular vesicles from urine Extract according to the extraction method described in Example 1.

[0036] 3. Test Example c: Differential centrifugation combined with 30 kd ultrafiltration and improved density gradient centrifugation at room temperature (1) Extraction of crude vesicles by differential centrifugation combined with ultrafiltration The crude vesicles were extracted at room temperature according to the method described in Experimental Example a, except that the supernatant after the second differential centrifugation was ultrafiltered and concentrated 10 times using an ultrafiltration element, wherein the ultrafiltration element was a 30 kd ultrafiltration tube.

[0037] (2) Extraction of small extracellular vesicles from urine Urinary small extracellular vesicles were extracted according to the extraction method described in Example 1.

[0038] 4. Test Example d: Improved differential centrifugation combined with NP40 and 30kd ultrafiltration and improved density gradient centrifugation at room temperature (1) Extraction of crude vesicles by modified differential centrifugation combined with NP40 and ultrafiltration The crude vesicles were extracted at room temperature according to the differential centrifugation method described in Example 2, except that the molecular weight cutoff of the ultrafiltration element was 30 kd.

[0039] (2) Extraction of small extracellular vesicles from urine Urinary small extracellular vesicles were extracted according to the extraction method described in Example 1.

[0040] 5. Test Example e: Differential centrifugation combined with 100 kd ultrafiltration and modified density gradient centrifugation at room temperature (1) Extraction of crude vesicles by differential centrifugation combined with ultrafiltration The crude vesicles were extracted at room temperature according to the differential centrifugation method described in Experimental Example a, except that the supernatant after the second differential centrifugation was ultrafiltered and concentrated 10 times using an ultrafiltration element, wherein the ultrafiltration element was a 100 kd ultrafiltration tube.

[0041] (2) Extraction of small extracellular vesicles from urine Urinary small extracellular vesicles were extracted according to the extraction method described in Example 1.

[0042] 6. Test Example f: Improved differential centrifugation combined with NP40 and 100 kd ultrafiltration and improved density gradient centrifugation at room temperature (1) Extraction of crude vesicles by modified differential centrifugation combined with NP40 and ultrafiltration Crude vesicles were extracted at room temperature using the differential centrifugation method described in Example 2.

[0043] (2) Extraction of small extracellular vesicles from urine Urinary small extracellular vesicles were extracted according to the extraction method described in Example 1.

[0044] The test contents and operating conditions of the above test examples a to f are shown in the following table:

[0045] Identification and Yield and Purity Comparison of Urine Small Extracellular Vesicles: Western Blot was used to compare the differences in small extracellular vesicle marker proteins in each experimental case (e.g. Figure 5 The total protein content of the small extracellular vesicles obtained in each test case was determined by the Coomassie brilliant blue method, and the difference in the number of nanoparticles in each group was counted by tunable resistive pulse sensing (TRPS). The results were calculated as the number of nanoparticles extracted per milliliter of urine (e.g. Figure 5 The purity of SVs was calculated by the ratio of the number of nanoparticles and the total protein in the extracted SVs sample (e.g. Figure 5The results showed that Western Blot analysis showed that more extracellular vesicles and vesicle markers Alix, TSG101 and Syntenin-1 were extracted from the six experimental cases (e.g. Figure 5 A in the figure), the TRPS nanoparticle counting results showed that the modified differential centrifugation method at room temperature combined with NP40 in the test example b can obtain the maximum number of nanoparticles (such as Figure 5 B in the figure), therefore, Example b is the maximum yield extraction scheme for urine small extracellular vesicles, and its extraction yield is significantly higher than that of other examples; the purity is calculated by the number of nanoparticles / total protein amount. It is found that the improved differential centrifugation method combined with NP40 and 100kd ultrafiltration at room temperature in Example f can obtain small extracellular vesicles with the maximum purity (such as Figure 5 C) is the maximum purity extraction scheme for extracting urine small extracellular vesicles, and its extraction purity is significantly improved compared with other test examples. The total extraction time of the present invention is significantly shorter than that of test example a, and the total extraction time of test example b is the shortest.

[0046] The extraction time of schemes a to e in test examples is as follows:

[0047] In summary, the present invention uses 0.05% NP40 to treat the precipitate after the third differential centrifugation, so that the small extracellular vesicles in the precipitate can be released into the supernatant 2; by subjecting the supernatant 1 and the supernatant 2 to ultracentrifugation respectively to obtain crude vesicles and then mixing the crude vesicles to extract the small extracellular vesicles, the influence of NP40 in the supernatant 2 on the extraction of crude vesicles in the supernatant 1 is avoided; by performing differential centrifugation at room temperature and resuspending and mixing the two obtained crude vesicles with a 20mm Tris-HCL alkaline aqueous solution, the change of THP on the sedimentation coefficient of small extracellular vesicles can be reduced, and the interference of urea modulin on the extraction of urine small extracellular vesicles can be effectively eliminated; by using the improved density gradient centrifugation method, more concentrated small extracellular vesicles can be obtained, and the extraction time is shortened; the present invention significantly improves the extraction yield and purity of urine small extracellular vesicles, and greatly shortens the extraction time.

[0048] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0049] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for extracting small extracellular vesicles from urine, characterized in that: The method comprises the following steps: S1. Obtain a urine sample, perform a first differential centrifugation on the urine sample to obtain a primary supernatant, and perform a second differential centrifugation on the primary supernatant to obtain a secondary supernatant; S2, performing a third differential centrifugation on the secondary supernatant to obtain a precipitate and a supernatant 1; S3, treating the precipitate with a surfactant, and centrifuging the treated solution to obtain a supernatant 2; S4, performing ultracentrifugation treatment on the supernatant 1 and the supernatant 2 respectively to obtain a first crude vesicle and a second crude vesicle, mixing the first crude vesicle and the second crude vesicle and resuspending them in a Tris-HCl aqueous solution to obtain a crude vesicle resuspension; S5. Take an ultracentrifuge tube, add Tris-HCL sucrose heavy water solutions of different concentration gradients to the bottom of the ultracentrifuge tube in order from low to high concentration to form a gradient solution with increasing concentration from top to bottom, slowly add the crude vesicle resuspension solution to the top layer of the gradient solution, perform ultracentrifugation, take out the gradient solution from top to bottom, take the specific component solution of the layered solution to obtain small extracellular vesicles.

2. The method for extracting urine small extracellular vesicles according to claim 1, characterized in that: After step S1 and before step S2, the method includes: using an ultrafiltration element to perform ultrafiltration concentration treatment on the secondary supernatant.

3. The method for extracting urine small extracellular vesicles according to claim 1, characterized in that: The ultrafiltration element is a 100kd ultrafiltration tube.

4. The method for extracting urine small extracellular vesicles according to claim 1, characterized in that: The S3 is specifically as follows: adding a surfactant to the precipitate, placing it in a water bath at 37° C. for 5 minutes, rotating and mixing for 10 seconds every 1 minute during the water bath, centrifuging it at 16000 g for 20 minutes after the water bath, removing the bottom precipitate, and obtaining supernatant 2.

5. The method for extracting urine small extracellular vesicles according to claim 1, characterized in that: The surfactant is specifically a 20 mm Tris-HCL aqueous solution with a pH of 8.4 to 8.8 containing 0.05% ethylphenyl polyethylene glycol.

6. The method for extracting urine small extracellular vesicles according to claim 1, characterized in that: The step S1 and the step S2 are both performed at room temperature.

7. The method for extracting urine small extracellular vesicles according to claim 1, characterized in that: The Tris-HCL aqueous solution is 2.5 ml of a 20 mm Tris-HCL aqueous solution with a pH of 8.4 to 8.

8.

8. The method for extracting urine small extracellular vesicles according to claim 1, characterized in that: In step S5, the centrifugal force of ultracentrifugation is 200000 g, and the centrifugation time is 2 hours.

9. The method for extracting urine small extracellular vesicles according to claim 1, characterized in that: The step S5 is specifically as follows: Take an ultracentrifuge tube, insert a flat-head needle syringe into the bottom of the tube, and add 1 ml of 20 mm Tris-HCL sucrose heavy aqueous solution with a pH of 8.4 to 8.8 at a concentration of 5%, 10%, 20%, 40%, and 60% to the bottom of the ultracentrifuge tube in sequence to form a gradient solution with increasing concentration from top to bottom, slowly add the crude vesicle resuspension solution to the top layer of the gradient solution with a pipette, centrifuge at 200,000 g for 2 h at 24° C. to 26° C., and then take out the gradient solution from top to bottom to obtain 5 layers of component solutions, take out the 3rd and 4th layer component solutions in the layered solution, centrifuge at 200,000 g for 1 h at 4° C., and take out the precipitate after centrifugation to obtain small extracellular vesicles.

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