CN110218693B

CN110218693B - Reagent combination, kit and method for extracting exosomes

Info

Publication number: CN110218693B
Application number: CN201910592769.XA
Authority: CN
Inventors: 王家亮
Original assignee: Shanghai Chengran Biology Technology Co ltd
Current assignee: Shanghai Chengran Biology Technology Co ltd
Priority date: 2019-07-01
Filing date: 2019-07-01
Publication date: 2021-02-26
Anticipated expiration: 2039-07-01
Also published as: CN110218693A

Abstract

The invention discloses a reagent combination, a kit and a method for extracting exosomes, and relates to the field of biomedicine. The invention discloses a reagent combination for extracting exosome, which comprises the following components: polybrene, polyethylene glycol and dextran. Adopt this reagent combination to extract exosome, exosome's extraction efficiency and purity are higher, and one set of reagent can be applicable to the extraction of serum or plasma simultaneously, and easy operation does not rely on expensive equipment or reagent consumptive material.

Description

Reagent combination, kit and method for extracting exosomes

Technical Field

The invention relates to the field of biomedicine, in particular to a reagent combination, a kit and a method for extracting exosomes.

Background

Exosomes (exosomes) are a class of extracellular vesicles 30-150nm in diameter with intact membrane structures, and are involved in a variety of physiological and pathological processes. Exosomes are thought to be a new mechanism of intercellular communication, allowing cells to exchange proteins, lipids and genetic material. In the past, exosomes secreted by cells were once thought to be involved only in waste excretion; with the development of high-throughput proteomics and genomics, exosomes are widely regarded as highly conserved pathways for short-distance and long-distance communication between cells in organisms, and such cell-cell communication is very important for both normal cells and tumor cells.

All eukaryotic cell types secrete exosomes under culture conditions. Exosomes can be isolated from all body fluids in the body, including blood, saliva, cerebrospinal fluid, ascites, and even urine. These exosomes transmit a variety of signal molecules, including proteins, mRNA, LncRNA, MicroRNA, DNA, etc., which carry molecules that can be taken up by other cells. Thus, exosomes may transfer biological information to neighboring cells. This intercellular communication is not only related to physiological functions, but also to the pathogenesis of several diseases, including tumors, cardiovascular diseases and neurodegenerative diseases. It is now found in disease models that exosomes play an important role through molecular messaging. Exosomes are also increasingly considered as biomarkers and prognostic important indicators of disease, with important clinical diagnostic and therapeutic implications.

In recent years, the application of exosomes in the field of disease research has been widely developed. The exosome samples are also diversified, but the samples for clinical detection mainly comprise plasma, serum and the like. Plasma and serum are the most commonly used sample sources for clinical diagnosis, and because of their abundant exosome content, research on clinical application of blood exosomes has been intensively carried out. Serum, plasma samples can be studied for exosomes of various pathological states.

At present, an exosome extraction method mainly comprises an ultra-high-speed centrifugation method, the obtained exosome has high purity, but the extraction efficiency is too low, the separation time is long, and the requirement on equipment is high; the immunomagnetic bead method can specifically capture exosome with a protein label, and has high cost and higher extraction efficiency than the ultra-high speed centrifugation method due to the need of a large amount of antibodies, magnetic beads and the like; the kit precipitation method, such as related serum exosome extraction reagents of Thermofish company and SBI company, has the highest exosome extraction efficiency compared with the other two methods, but the exosome purity is relatively low, and high abundance proteins in body fluid can be precipitated while the exosome is captured. Because the exosome research requires a rapid and simple extraction method, the demand of a kit method for separating exosomes gradually increases in the market, the quality of exosomes directly influences the capture and detection of subsequent exosome nucleic acids and proteins, and particularly, the purity, impurities and recovery rate of exosomes directly influence the research on exosome nucleic acids and proteins.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a reagent combination for extracting exosome. The reagent combination is adopted to extract exosome, and the extraction efficiency and purity of exosome are higher.

It is another object of the present invention to provide a kit for extracting exosomes. The exosome is extracted by the kit, so that the extraction efficiency and purity can be improved, the operation is simple, the practicability is high, and the kit is suitable for extracting exosomes of plasma or serum samples.

It is another object of the present invention to provide a method for extracting exosomes. The method has simple operation, high extraction efficiency, and high purity of the extracted exosome.

The invention is realized by the following steps:

in a first aspect, the present invention provides a combination of reagents for the extraction of exosomes, comprising: polybrene, polyethylene glycol and dextran.

Preferably, the reagent combination comprises the following components:

a first component comprising polybrene;

a second component comprising polyethylene glycol and dextran;

an optional third component comprising thrombin;

preferably, the molecular weight of the polyethylene glycol is 7000 9000;

preferably, the dextran has a molecular weight of 4000-6000.

Further, in some embodiments of the invention, the polybrene is solid polybrene or polybrene solution with concentration of more than or equal to 1mg/mL, preferably powder polybrene with purity of more than or equal to 94%;

preferably, the molecular weight of the polyethylene glycol is 8000;

preferably, the dextran has a molecular weight of 4000;

preferably, the thrombin has a specific activity of 1000U/mg.

The reagent combination provided by the invention can be used for extracting exosome, can be suitable for extracting serum or plasma samples, and polybrene can be used for removing macromolecular impurities such as protein in the samples; the polyethylene glycol and the glucan are mixed for precipitating the exosome in the sample and separating the exosome from the sample. The exosome extracted by adopting the reagent combination has higher extraction efficiency, and the extracted exosome purity can be improved.

In a second aspect, the present invention provides a kit for the extraction of exosomes comprising a combination of reagents as described above;

preferably, the kit further comprises a filter;

preferably, the kit further comprises at least one of a 0.22 μm filter and a 0.1 μm filter;

preferably, the extracted sample of the kit is serum or plasma.

The exosome is extracted in the reagent combination, the extraction efficiency and the purity of the exosome are higher, the operation is simple, the practicability is high, and the reagent combination can be simultaneously suitable for extracting plasma or serum samples.

In a third aspect, the present invention provides a method for extracting exosomes, comprising the steps of:

adding polybrene into a sample from which exosomes are to be extracted to obtain a first solution;

and simultaneously adding polyethylene glycol and glucan into the first solution to obtain a second solution.

Preferably, the method is performed using a combination of reagents as described above or a kit as described above.

Further, in some embodiments of the present invention, the final concentration of polyethylene glycol in the second solution is 6% to 10% (mass concentration), the final concentration of dextran is 8 to 40 mg/mL;

preferably, the final concentration of polyethylene glycol in the second solution is 8% and the final concentration of dextran is 8-32 mg/mL.

Preferably, the final concentration of dextran in the second solution is 8-32mg/mL, more preferably 16-32mg/mL, and even more preferably 32 mg/mL.

The research of the invention finds that the final concentration of the polyethylene glycol is controlled to be 6-10%, the final concentration of the glucan is controlled to be 8-32mg/mL, the extraction rate of the exosome can be improved, preferably, the final concentration of the polyethylene glycol is controlled to be 8%, the final concentration of the glucan is controlled to be 16-32mg/mL, and the extraction efficiency of the exosome is obviously improved.

Further, in some embodiments of the invention, the method further comprises: after adding polyethylene glycol and dextran, the second solution is placed at 2-8 ℃ for reaction for 30-90 min.

Further, in some embodiments of the invention, the method further comprises: after the reaction is finished, centrifuging the second solution, taking the precipitate, and carrying out heavy suspension to obtain a first heavy suspension;

preferably, the conditions of centrifugation are as follows: 10000-14000g, and centrifuging for 1-2 minutes;

preferably, the pellet is resuspended using phosphate buffer.

Further, in some embodiments of the invention, the method further comprises: and filtering the first resuspension solution, simultaneously adding polyethylene glycol and glucan into the filtrate, uniformly mixing, centrifuging, taking the precipitate, and carrying out resuspension to obtain a second resuspension solution containing the exosome.

The addition of polyethylene glycol and dextran can re-precipitate the exosomes from the solution, further improving the purity of the exosomes.

preferably, the pellet is resuspended using phosphate buffer;

preferably, the first resuspension is filtered using a 0.1 μm filter. The size of the exosome is usually in the range of 30-150nm, and large-particle impurities can be filtered by filtering through a 0.1-micron filter, so that the purity of the obtained exosome is improved.

Further, in some embodiments of the invention, the method further comprises:

before adding polyethylene glycol and glucan, placing the first solution at the temperature of 2-8 ℃ for reaction for 30-90 min;

preferably, the final concentration of polybrene in the first solution is 1-4 mg/mL;

more preferably, the final concentration of polybrene in the first solution is 2 mg/mL.

Further, in some embodiments of the invention, the method further comprises: filtering the sample prior to adding polybrene;

preferably, the sample is filtered using a 0.22 μm filter; the large-particle impurities can be filtered by a filter with the diameter of 0.22 mu m, which is beneficial to improving the purity of the obtained exosome. Preferably, before filtering the sample, the method further comprises: diluting the sample with physiological saline;

preferably, the sample is serum or plasma;

preferably, when the sample is plasma, the method further comprises: after filtering the sample, adding thrombin into the filtered sample, uniformly mixing, and standing for 25-35 min;

preferably, thrombin is added in an amount of: 18-22U thrombin was added per ml of the sample.

The method for extracting the exosome provided by the invention uses the reagent combination of polybrene, polyethylene glycol and glucan to extract the exosome, the polybrene promotes the polymerization of the exosome, the polyethylene glycol and the glucan are mixed to promote the exosome to be separated out from a sample, and large-particle impurities in an exosome solution are removed by combining with the treatment of 0.22 mu m and 0.1 mu m filters, so that the extraction efficiency and the purity of the exosome are greatly improved.

Compared with the existing ultracentrifugation method or exosome extraction kit and the like, the kit and the method have higher extraction efficiency and purity, one set of reagent can be simultaneously suitable for extracting serum or plasma, the operation is simple, and expensive equipment or reagent consumables are not relied on.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Fig. 1A shows exosome concentration and diameter as detected by nanoparticle tracking analyzer NS 300. The working concentration of glucan is 8mg/mL, and the working concentration of polyethylene glycol is 8% by mass. The X axis represents the range of vesicles detected, from 0 to 1000nm, and the Y axis represents the concentration of vesicles of a certain diameter detected in the sample detected, in units of number/mL, i.e. particles/mL.

Fig. 1B shows exosome concentration and diameter as detected by nanoparticle tracking analyzer NS 300. The working concentration of glucan is 16mg/mL, and the working concentration of polyethylene glycol is 8% by mass.

Fig. 1C shows exosome concentration and diameter as detected by nanoparticle tracking analyzer NS 300. The working concentration of the glucan is 24mg/mL respectively, and the working concentration of the polyethylene glycol is 8 percent by mass.

Fig. 1D shows exosome concentration and diameter as detected by nanoparticle tracking analyzer NS 300. The working concentration of glucan is 32mg/mL and the working concentration of polyethylene glycol is 8% by mass, and the results in fig. 1A-D show that the number of exosomes increases with the increase of the working concentration of glucan.

FIG. 2 is a comparison of the concentration of exosomes by ultracentrifugation and the method of the present invention. When the working concentration of glucan in the method is less than 16mg/mL, the concentration of exosome in the ultracentrifugation method is similar to that in the method, and when the working concentration of glucan is 24-32mg/mL, the concentration of exosome extracted by the method is far higher than that in the ultracentrifugation method.

FIG. 3 is a comparison of the results of total RNA distribution measurements obtained by Agilent2100 bioanalyzer for different exosome extraction methods. Compared with the method of the present invention, the SBI and QIAGEN kit method has uniform distribution of nucleic acid fragments, which are all located at 25-200nt, but the total amount of nucleic acid is different. The total amount of nucleic acid of the method is the highest (FU is more than 8), the SBI kit method is the lowest (FU is less than 6), and the QIAGEN kit method is intermediate (FU is more than 6 and less than 8).

FIG. 4 shows the comparison of Ct values obtained by detecting actb gene by fluorescent PCR of nucleic acid of exosome extracted by the method of the present invention and commercial kit, the average Ct value of the method of the present invention is 23.86, the average Ct value of SBI kit is 27.62, and the average Ct value of QIAGEN kit is 26.28.

FIG. 5 is a Western blot detecting the exosome-specific proteins CD63, TSG101 and CD 9. The total protein loading of the exosomes extracted by the ultracentrifugation method, the SBI kit and the QIAGEN kit is 20mg, and the loading of the method of the invention is 10 mg. From the result of the quantitative imprinting signal intensity, the total amount of the exosome-specific protein enriched by the method is higher than that of the other three exosome separation and extraction methods.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

The reagents and materials used in the examples were as follows:

and (3) glucan: molecular weight 5000, purchased from Sigma-Aldrich;

polyethylene glycol: molecular weight 8000, available from Sigma-Aldrich;

thrombin powder: specific activity 1000U/mg, purchased from Sigma-Aldrich;

polybrene powder: the purity is more than or equal to 94 percent.

Example 1

The method for extracting exosomes provided by the embodiment operates as follows:

1. mixing dextran with polyethylene glycol, dissolving in PBS phosphate buffer solution, and making into mixed solution; wherein the mass concentration of the polyethylene glycol in the mixed solution is controlled to be 40%, and the concentration of the glucan is controlled to be 40 mg/mL.

2. A200. mu.L plasma sample from which exosomes are to be extracted is made up to 1mL with 0.9% physiological saline.

3. 1mL of a constant volume plasma sample was filtered through a 0.22 μm filter.

4. The filtered plasma samples were supplemented with 20U thrombin (using ddH)₂Dissolving thrombin powder by O to prepare a thrombin solution with the concentration of 1U/. mu.L), and standing at room temperature for 30min after uniformly mixing if the thrombin is not required to be added into the serum sample.

5. The sample after standing was added with polybrene solution (using ddH)₂Dissolving polybrene powder with O to obtain stock solution with concentration of 0.2g/mL), mixing to give final concentration of 2mg/mL, shaking, and reacting at 4 deg.C for 60 min.

6. After the reaction is finished, adding a mixed solution of glucan and polyethylene glycol, wherein the volume ratio of the sample to the mixed solution is 4:1, the final concentration of the polyethylene glycol is 8%, and the final concentration of the glucan is 8 mg/mL; the reaction was carried out at 4 ℃ for 60 min.

7. After the reaction was completed, 12000g was centrifuged for 1 minute, and the supernatant was removed.

8. The resulting pellet was resuspended in 1mL phosphate buffered PBS.

9. The resulting solution was filtered with a 0.1 μm filter, and the filtered solution was added again to the mixed solution of dextran and polyethylene glycol, sufficiently mixed, 12000g, centrifuged for 1 minute, and the supernatant was removed.

10. The resulting pellet was resuspended in 200. mu.L phosphate buffered PBS, which contains exosomes.

11. Detection of

The exosomes obtained from the above operation were analyzed using a nanoparticle tracking analysis system NS300, and the results are shown in fig. 1A and fig. 2.

Example 2

The extraction method of this example is substantially the same as that of example 1, except that: the dextran concentration in step 1 was 80mg/ml and the final dextran concentration in step 6 was 16 mg/ml. The results are shown in FIG. 1B and FIG. 2.

Example 3

The extraction method of this example is substantially the same as that of example 1, except that: the dextran concentration in step 1 was 120mg/ml and the final dextran concentration in step 6 was 24 mg/ml. The results are shown in FIG. 1C and FIG. 2.

Example 4

The extraction method of this example is substantially the same as that of example 1, except that: the dextran concentration in step 1 was 160mg/ml and the final dextran concentration in step 6 was 32 mg/ml. The results are shown in FIGS. 1D and 2.

Comparative example 1

The exosomes were extracted by ultracentrifugation and the results are shown in figure 2.

The ultracentrifugation method comprises the following steps: take a 200 microliter serum sample as an example.

The first step is as follows: centrifuging at 300g for 10min at 4 ℃.

The second step is that: the supernatant layer in the first step is pipetted about 190 microliters to a new centrifuge tube and centrifuged at 2000g for 10min at 4 degrees.

The third step: and sucking the supernatant layer in the second step by a pipette about 180 microliters to a new centrifugal tube, and centrifuging for 30min at 10000g under the condition of 4 ℃.

The fourth step: and (4) sucking the supernatant layer in the third step by a pipette about 170 microliters to a new centrifuge tube, and centrifuging for 120min at 100000g under the condition of 4 degrees.

The fifth step: about 160. mu.l of the supernatant layer in the fourth step was removed by pipette and the exosomes were resuspended by adding 90. mu.l of PBS solution, yielding about 100. mu.l of exosome solution.

The results in FIG. 1 show the sizes of the exosomes obtained in examples 1-4:

the exosomes of examples 1-4 were analyzed by NS300 to obtain exosome concentrations of 8-9 orders of magnitude of 10, where a is 0.7 x 10⁹B is 1.1X 10⁹C is 3.8X 10⁹D is 6.3X 10⁹The method is characterized in that the number of exosomes separated and enriched from a sample is remarkably increased along with the increase of working concentration of glucan, meanwhile, the average value of the diameters of the exosomes A is 131nm, the average value of the diameters of the exosomes B is 89nm, the average value of the diameters of the exosomes C is 108nm, and the average value of the diameters of the exosomes D is 108nm, so that the mixture of polyethylene glycol and glucan can effectively separate and enrich the exosomes from the sample.

The results in fig. 2 show that the methods of examples 1-4 extract exosome concentrations much higher than those of comparative example 1, especially the exosome contents extracted in examples 3 and 4, compared to the existing ultracentrifugation method. The exosome contains nucleic acid, protein and other biological macromolecules, the number of the exosome is more, the content of the contained biological macromolecules is richer, the subsequent analysis on the content of the exosome is facilitated, the total amount of the exosome obtained by an ultracentrifugation method is lower, the content of the biological macromolecules in the exosome is low, the existing scientific research instrument and equipment is frequently large in error and inaccurate in measurement in detection on the nucleic acid and the protein with lower contents, and the biological significance of the exosome sample is facilitated to be effectively analyzed by improving the total amount of the exosome and the total amount of the nucleic acid and the protein in the exosome.

Experimental example 1

Comparing the effect of the commercially available exosome nucleic acid extraction kit (SBI, QIAGEN) with the method of example 4 above on exosome nucleic acid extraction:

1.5mL of plasma samples were divided into three 500. mu.L aliquots and separately treated with Exo from SBI

Exosome Isolation and RNA Purification kit(for Serum&Plasma), exoRNeasy Serum/Plasma Mi from QIAGENdi Kit, and the method of example 4, and combine with miRNeasy Micro Kit of QIAGEN company to extract the nucleic acid of plasma exosome, and detect the content and distribution of the exosome nucleic acid obtained by the three methods through Agilent2100 bioanalyzer. The results are shown in FIG. 3.

From FIG. 3, it can be seen that the nucleic acids extracted from exosomes by either the method of the present invention or the SBI, QIAGEN kit method have a significant distribution in the range of 25-500 nt. However, the higher peak value (FU > 8) compared to SBI (FU > 4) or QIAGEN (FU > 6) kits in the present invention was analyzed on the ordinate, indicating that the total amount of nucleic acid that can be captured by the present invention was higher than in the two commercial kits. FU is called fluoroescence unit and represents fluorescence intensity, and in the detection of the Agilent2100 bioanalyzer, nucleic acid is labeled by fluorescent dye in advance, so that the larger the value of FU in the detection result, the higher the total amount of nucleic acid representing the sample.

Experimental example 2

Comparing the detection difference of the invention and SBI and QIAGEN kits on the specific nucleic acid expression quantity of exosomes

By using a reverse transcription kit (Takara Co., PrimeScript)^TMII 1st Strand and cDNA Synthesis Kit), the exosome nucleic acid extracted by the present invention in experimental example 1 and the exosome nucleic acid extracted by SBI, QIAGEN Kit were reverse-transcribed into cDNA. And detecting the expression level of actb in the plasma exosome nucleic acid, wherein the reaction system is as follows:

the reaction procedure is as follows: step 1: 5min at 37 ℃; step 2: 5 ℃ for 10 min; and step 3: 95 ℃ for 10 sec; 60 deg.C for 1 min. Wherein the step 3: the reaction was carried out in 40 cycles.

After the reaction was completed, Ct values of each reaction well in the fluorescent PCR instrument ABI7500 were derived as shown in table 1 below:

TABLE 1

From Table 2 and FIG. 4, the average Ct value obtained by detecting actb gene according to the present invention is smaller than the Ct values obtained by the other two kit methods, and the standard deviation of Ct is also lower than the detection results of SBI kit and QIAGEN kit. According to the principle of fluorescence PCR, on the premise that the total amount of cDNA is consistent, the smaller the Ct value is, the more the copy number of the gene contained in the sample is, thus indicating that the exosome extracted by the method contains more nucleic acid. Indicating that the number of exosomes was positively correlated with the amount of nucleic acid therein.

Experimental example 3

Comparing the method of example 4 with gold standard ultracentrifugation, SBI and QIAGEN exosome extraction kits to detect differences in exosome-specific protein expression levels:

exosomes have a variety of specific marker proteins most commonly used, such as CD9, CD63, CD81, TSG101, and the like. The method of example 4 of the present invention was selected and compared with ultracentrifugation and two commercial kits, total protein extraction was performed after serum exosomes were extracted, and expression levels of CD9, CD63 and TSG101 were detected using western blot after protein BCA quantification was completed.

The method specifically comprises the following steps: according to the instructions of an SBI Exosome extraction kit (ExoQuick-TC) and a QIAGEN Exosome extraction kit (mircurY Exosome Kits), 200 mu L of serum total exosomes are respectively extracted by combining the method of the embodiment 4 of the invention according to the ultracentrifugation operation. Each exosome was resuspended in 50. mu.L of exosome lysate, and exosome was lysed in 50. mu.L of protein lysate RIPA, total protein was extracted, ice-washed for 60min, then centrifuged at 12000g at 4 ℃ for 30min, about 100. mu.L of supernatant was taken into a new EP tube, and exosome protein was quantified using the BCA kit, and the total protein concentration of exosomes was detected using a microplate reader. The quantified total protein was added to a loading buffer and denatured by boiling, and 10. mu.g of protein was added for immunoblotting.

The results are shown in FIG. 5: in comparison with other methods in the method of example 4 of the present invention, the tag protein of exosome can be detected by immunoblotting experiment. However, in comparison of different methodologies, the total amount of each tagged protein is different, and the result of measuring the content of the total amount of tagged protein by using the imager is as follows: the total amount of exosomes CD9 and CD63 extracted by the method is higher than that of the ultracentrifugation and SBI kit and is similar to that of the QIAGEN kit in result (Table 2, figure 5-B and figure 5-D), but in the result of the blotting experiment, the total protein of the exosomes extracted by the QIAGEN kit has an obvious nonspecific strip (figure 5-A), which indicates that the exosomes extracted by the kit are polluted, while the total protein extracted by the method is single in strip and has higher purity. CD9 and CD63 are exosome-specific proteins, exosomes separated and extracted by a QIAGEN kit method are subjected to protein extraction and immunoblotting, and CD9 and CD63 antibodies still have non-specific bands at positions where bands should appear, so that the vesicles extracted by the QIAGEN kit method possibly contain non-exosome hybrid proteins, and the purity of the vesicles is lower than that of the exosome extracted by the extraction method.

In addition, the total amount of exosome TSG101 extracted by the present invention was higher than the ultracentrifugation, SBI and QIAGEN kit (fig. 5-C). When each method is used for carrying out an immunoblotting experiment with the same total amount of protein, the more total amount of the exosome-tagged protein indicates that the number of the extracted exosomes is more, and the method has the optimal exosome protein enrichment capacity and higher extraction efficiency under the experimental conditions.

TABLE 2

When the total amount of the extracted exosome protein and the immunoblotting loading protein in the example 4 of the present invention is half of those in the ultracentrifugation method, the SBI kit method and the QIAGEN kit method, the signal intensities of CD63 and CD9 are close to those in the QIAGEN kit, and the signal intensity of TSG101 is higher than those in the other three exosome extraction methods.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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Claims

1. A reagent combination for exosome extraction, characterized in that it comprises the following components:

a first component comprising polybrene;

a second component comprising polyethylene glycol and dextran;

a third component comprising thrombin;

the molecular weight of the polyethylene glycol is 7000-9000;

the molecular weight of the glucan is 4000-6000.

2. A reagent combination for exosome extraction according to claim 1, characterized in that the polybrene is solid polybrene or a polybrene solution with concentration not less than 1 mg/mL.

3. A reagent combination for exosome extraction according to claim 1, characterized in that polybrene is powdered polybrene with purity > 94%.

4. The reagent combination for exosome extraction according to claim 1, characterized in that the molecular weight of the polyethylene glycol is 8000, the molecular weight of the dextran is 4000, the specific activity of the thrombin is 1000U/mg.

5. A kit for the extraction of exosomes, characterized in that it comprises a combination of reagents according to any one of claims 1-4.

6. The kit of claim 5, further comprising a filter.

7. The kit of claim 5, further comprising at least one of a 0.22 μ ι η filter and a 0.1 μ ι η filter.

8. The kit of claim 5, wherein the extracted sample of the kit is serum or plasma.

9. A method for the extraction of exosomes, characterized in that it is carried out using the combination of reagents according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 8, comprising the following steps:

10. The method of claim 9, wherein the final concentration of polyethylene glycol in the second solution is 6% to 10% and the final concentration of dextran is 8 to 32 mg/mL.

11. The method of claim 10, wherein the final concentration of polyethylene glycol in the second solution is 8%.

12. The method of claim 10, wherein the final concentration of dextran in the second solution is 8-32 mg/mL.

13. The method of claim 12, wherein the final concentration of dextran in the second solution is 16-32 mg/mL.

14. The method of claim 12, wherein the final concentration of dextran in the second solution is 32 mg/mL.

15. The method of claim 10, further comprising: after adding polyethylene glycol and dextran, the second solution is placed at 2-8 ℃ for reaction for 30-90 min.

16. The method of claim 15, further comprising: after the reaction is finished, centrifuging the second solution, taking the precipitate, and carrying out heavy suspension to obtain a first heavy suspension; wherein, the centrifugation conditions are as follows: 10000-14000g, and centrifuging for 1-2 minutes; the pellet was resuspended using phosphate buffer.

17. The method of claim 16, further comprising: filtering the first heavy suspension, adding polyethylene glycol and glucan into the filtrate at the same time, mixing uniformly, centrifuging, taking the precipitate, and carrying out heavy suspension to obtain a second heavy suspension containing exosomes; wherein, the centrifugation conditions are as follows: 10000-14000g, and centrifuging for 1-2 minutes; resuspending the pellet using phosphate buffer; the first resuspension was filtered using a 0.1 μm filter.

18. The method of claim 9, further comprising:

and before adding the polyethylene glycol and the glucan, placing the first solution at the temperature of 2-8 ℃ for reaction for 30-90 min.

19. The method of claim 18, wherein the final polybrene concentration in the first solution is 1-4 mg/mL.

20. The method of claim 19, wherein the final concentration of polybrene in the first solution is 2 mg/mL.

21. The method of claim 9, further comprising: the sample was diluted with physiological saline before adding polybrene, and the diluted sample was filtered using a 0.22 μm filter.

22. The method of claim 21, wherein the sample is serum or plasma.

23. The method of claim 22, wherein when the sample is plasma, the method further comprises: and after filtering the sample, adding thrombin into the filtered sample, uniformly mixing, and standing for 25-35 min.

24. The method of claim 23, wherein the thrombin is added in an amount of: 18-22U thrombin was added per ml of the sample.