CN110343664B

CN110343664B - Method for extracting exosome and exosome protein

Info

Publication number: CN110343664B
Application number: CN201810287204.6A
Authority: CN
Inventors: 钱小红; 张养军; 高方园; 焦丰龙; 夏朝双; 吕雅瑶; 吴琼; 余谦
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2018-04-03
Filing date: 2018-04-03
Publication date: 2021-12-24
Anticipated expiration: 2038-04-03
Also published as: CN110343664A

Abstract

本发明公开了从人体体液或者细胞的培养液中提取外泌体及外泌体蛋白的方法。本发明提供的提取外泌体及外泌体蛋白的方法包括：向含有外泌体的待提取液中加入提取材料提取得到外泌体；提取材料为金属氧化物或修饰有所述金属氧化物的功能材料；金属氧化物能与磷酸根离子发生双配位作用；所述待提取液为来源于人体的体液、所述体液的稀释液或细胞的培养产物。实验证明，本发明的外泌体提取方法可以实现对人体体液或细胞的培养液中外泌体的高效提取，提取时间短，获得的外泌体纯度高，可以通过洗脱液洗脱获得完整的外泌体，并且所用提取材料可回收重复利用；另外提取所得外泌体蛋白可发现，所得外泌体蛋白种类多且与外泌体功能更密切。The invention discloses a method for extracting exosomes and exosome proteins from human body fluids or cell culture fluids. The method for extracting exosomes and exosome proteins provided by the present invention includes: adding an extraction material to a liquid to be extracted containing exosomes to extract exosomes; the extraction material is a metal oxide or modified with the metal oxide The metal oxide can have a double coordination effect with phosphate ions; the liquid to be extracted is a body fluid derived from the human body, a dilution of the body fluid or a culture product of cells. Experiments have shown that the exosome extraction method of the present invention can realize the efficient extraction of exosomes in human body fluids or cell culture fluids, the extraction time is short, the obtained exosomes have high purity, and complete exosomes can be obtained by elution with the eluent. Exosomes, and the extracted materials can be recycled and reused; in addition, the obtained exosomal proteins can be found in many types and are more closely related to the function of exosomes.

Description

Method for extracting exosome and exosome protein

Technical Field

The invention relates to a method for extracting exosome and exosome protein from human body fluid or culture solution of cells in the field of biotechnology.

Background

Exosomes are vesicular structures secreted by mammalian cells out of the cell by the process of "endocytosis-efflux" and are approximately 30-150nm in diameter. Exosomes are present not only in cell culture fluid, but also in body fluids such as blood, urine, amniotic fluid, breast milk, semen, saliva, pleural effusion and the like, and the composition of exosomes includes lipid bilayers and substances such as proteins, RNA, DNA and the like related to cell sources. Almost all cells produce exosomes, and exosomes from different cell sources contain both common components associated with cell formation, structure and material transport, and specific molecules associated with the biological function of the source cell. Exosomes are increasingly gaining attention as an important means for intercellular communication and the transfer of a large number of biologically functional molecules. Research has shown that exosomes play a crucial role in a range of physiological and pathological processes. The tumor-related exosomes can induce cancer, participate in tumor-targeted metastasis, improve the microenvironment for tumor growth, inhibit immune system immune supervision on tumors and mediate drug resistance of tumor cells, and the analysis and detection of the tumor-related exosomes are favorable for early diagnosis, prognosis analysis and curative effect evaluation of tumors.

Currently, the commonly used methods for exosome extraction include ultracentrifugation, density gradient centrifugation, polymer precipitation, ultrafiltration, size exclusion chromatography, and immunoaffinity. The first four methods realize extraction based on the unique physical parameters of exosome such as density, size and the like, lack specificity and cannot meet the requirements of tumor cell exosome specificity extraction and analysis in practical samples. Particularly, a human body fluid sample contains a large amount of particles such as low-density lipoprotein and chylomicron, the particle size and the density of the particles are close to those of exosome, and most methods cannot separate the particles from the exosome, so that the subsequent analysis of the exosome is interfered.

Disclosure of Invention

The technical problem to be solved by the invention is how to extract exosomes.

In order to solve the above technical problems, the present invention first provides a method for extracting exosomes, comprising: adding an extraction material into an extract to be extracted containing exosomes to extract the exosomes to obtain exosomes; the extraction material is metal oxide or a functional material modified with the metal oxide;

the metal oxide can generate double coordination with phosphate ions.

In the above method, the metal oxide may be TiO₂、ZrO₂、Al(OH)₃、Ga₂O₃、Fe₃O₄、Fe₂O₃、Nb₂O₃、SnO₂、HfO₂And/or Ta₂O₅；

The functional material modified with the metal oxide can be specifically graphene oxide, magnetic nanoparticles or silicon spheres modified with the metal oxide.

The particle size of the material can be 1-50 μm.

In the above method, the liquid to be extracted may be a body fluid derived from a human body, a dilution of the body fluid, or a culture product of cells.

The dilution liquid of the body fluid can be obtained by diluting the body fluid with a buffer solution. The buffer may be a solution having a pH buffering capacity and capable of maintaining the osmotic pressure of exosomes. The buffer may specifically be DMEM buffer (Gibico, cat. No. 31053-. When the body fluid is diluted by using a buffer solution, the volume ratio of the body fluid to the buffer solution can be 1: 1-1: 5. The volume ratio of the body fluid and the buffer solution when the body fluid is diluted with the buffer solution may further be 1: 1.

In the present invention, when the viscosity of the body fluid is high (such as plasma and saliva), the exosome can be extracted by using the diluent of the body fluid. When the viscosity of the body fluid is low (such as urine), the exosome can be extracted by directly using the body fluid.

The culture product of the cells can be obtained by culturing the cells for 24-48 hours by using a culture solution prepared by exosome-removing serum. The exosome-removing serum can be supernatant obtained by centrifuging fetal calf serum for 12-16 hours at 110000 g.

The culture product may be a product obtained by culturing the cells, or may be a non-cellular part of the culture product. The non-cellular fraction can be obtained by subjecting the culture product to centrifugation.

In the above method, the body fluid may be blood, serum, urine or saliva; the cell is a tumor cell. The tumor cell can be cervical cancer cell, liver cancer cell or pancreatic cancer cell.

In the above method, adding an extraction material to the solution to be extracted containing exosomes to extract exosomes may include: adding the extraction material to the exosome-containing to-be-extracted solution to obtain an extraction mixture; and centrifuging the extraction mixture to enable the exosomes to enter a precipitate, collecting the precipitate, and removing the extraction material in the precipitate to obtain the exosomes.

The above method may further comprise the step of removing dead cells and/or cell debris from the liquid to be extracted before adding the extraction material to the liquid to be extracted. The removal of dead cells and/or cell debris from the extract can be achieved by subjecting the extract to gradient low speed centrifugation and/or filtration membranes. The gradient low-speed centrifugation can be carried out in sequence according to the following steps: centrifuge 300g for 10 min, 2000g for 10 min, 10000g for 30 min. The filter may be a 0.2 μm filter.

The above method may further comprise the step of incubating the extraction mixture prior to centrifuging the extraction mixture. The incubation time can be 1-10 minutes. The incubation time may be 2 to 5 minutes (e.g., 5 minutes). The incubation can be carried out at 1000-2500 rpm. The incubation can be carried out in particular at 1500 rpm. The incubation can be performed at room temperature (25 ℃) or at low temperature. The low temperature may be 4 ℃.

In the above method, the centrifugation may be carried out under conditions of 2000 g. The time for centrifugation may be 30 seconds.

In the above method, the extraction material may be the metal oxide; the proportion of the extraction material to the liquid to be extracted in the extraction mixture meets the following requirements of a1), a2), a3) or a 4):

a1) the extract to be extracted is the culture solution of the cells, and the ratio of the extraction material to the extract to be extracted in the extraction mixture is a11) or a 12):

a11)5mg:(10～300)mL；

a12)5mg:10mL；

a2) the extracting solution is serum or serum diluent, and the proportion of the extracting material to the extracting solution in the extracting mixture is a21) or a 22):

a21)5mg:(30～500)μL；

a22)5mg:100μL；

a3) the extracting solution is saliva or a dilution of the saliva, and the proportion of the extracting material to the extracting solution in the extracting mixture is a31) or a 32):

a31)5mg:(1～125)μL；

a32)5mg:100μL；

a4) the extracting solution is urine, and the proportion of the extracting material to the extracting solution in the extracting mixture is a41) or a 42):

a41)5mg:(10～75)mL；

a42)5mg:20mL。

in the above method, the removing the extraction material in the precipitate may comprise: adding an eluent into the precipitate to obtain an elution mixture, wherein the eluent is (3-20) g/100mL of ammonia water (such as 15g/100mL of ammonia water); and centrifuging the elution mixture to enable the exosome to enter a supernatant, collecting the supernatant, and removing impurities in the supernatant to obtain the exosome.

The centrifugation conditions may be 2000g for 30 seconds.

The method may further comprise the step of washing the precipitate before adding the eluent to the precipitate. The wash buffer used for the wash may be PBS.

The removing of the impurities in the supernatant may include: and centrifuging the supernatant to enable the exosome to enter a precipitate, and collecting the precipitate to obtain the exosome. The centrifugation conditions can be 2000g for 30 s.

The following X1) or X2), also belong to the scope of protection of the invention:

x1) an extraction method of exosome proteins, comprising: extracting to obtain exosome by using the exosome extraction method, and cracking the exosome to obtain exosome cracking solution; extracting exosome protein from the exosome lysate;

x2) an assay method for exosome proteins comprising: obtaining the exosome protein by using the method of X1), and then analyzing the exosome protein to finish the analysis of the exosome protein.

The lysis of the exosomes may be performed using a lysis solution. The lysate can be 4g/100mL SDS aqueous solution, 8M urea aqueous solution, dithiothreitol and/or RIPA lysate. The lysis may also be assisted by ultrasound. The ultrasonic condition may be 100W ultrasonic for 20 minutes.

The analysis of the exosome protein may be performed using SDS-PAGE or LC-MS/MS.

An exosome extraction kit or an exosome protein extraction kit comprising the extraction material also belongs to the scope of protection of the present invention.

The kit may contain the extraction material alone or may consist of the extraction material together with other reagents required for the extraction of exosomes.

The further reagent may be the wash buffer and/or the eluent.

Any one of the following Y1) -Y4), also belongs to the protection scope of the invention:

y1) use of the extraction material for exosome protein extraction;

y2) use of the extraction material in exosome protein analysis or identification;

y3) the use of the kit for exosome protein extraction;

y4) in the analysis or identification of exosome proteins.

Experiments prove that the exosome extraction method can realize the high-efficiency extraction of exosomes in human body fluid or cell culture solution, has short extraction time and high purity of the obtained exosomes, can obtain complete exosomes by elution of eluent, and can recycle the used extraction materials; in addition, the obtained exosome protein is extracted, so that the obtained exosome protein has multiple types, and compared with the conventional method, the obtained exosome protein has more close functions with exosome. The invention has the following beneficial effects:

1. through utilizing the chemical action combination through two coordination between the naked phosphate radical in exosome membrane phospholipid bilayer surface and the metal oxide to realize the high specificity of external secretion and extract, easy operation, extraction rate is fast, and the rate of recovery is high.

2. By changing the conditions of the eluent, the desorption of the extracted exosomes is realized, the exosomes with complete vesicle structures are obtained, and the eluted materials can be recycled.

3. By directly cracking exosomes, the extracted exosome components can be directly subjected to subsequent analysis, such as protein, RNA and the like.

Drawings

FIG. 1 shows the results of detection of marker proteins TSG101 and CD9 in exosomes.

FIG. 2 shows the results of mass spectrometric identification of exosome proteins extracted by three methods. Wherein, the metal oxide method is the extraction method of the exosome of the invention; u1, U2 and U3 respectively represent three parallel experiments for extracting exosomes by an ultracentrifugation method, K1, K2 and K3 respectively represent three parallel experiments for extracting exosomes by a commercial kit method, and T1, T2 and T3 respectively represent three repeated experiments for extracting exosomes by a metal oxide method.

FIG. 3 shows the results of Gene Ontology analysis of exosome proteins extracted by the exosome extraction method (a), the ultracentrifugation method (b) and the commercial kit (c) of the present invention.

FIG. 4 is the extraction of exosomes from cell culture fluid.

FIG. 5 shows the results of a reversible elution experiment.

FIG. 6 shows the result of directly extracting exosomes from cell culture fluid using metal oxide. The arrows indicate exosomes.

FIG. 7 is exosome extraction in urine.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The DMEM buffer solution used in the examples described below was Gibico, cat # 31053-.

The PBS in the following examples is available from Corning Inc., cat #: 21-040-CVR.

The invention provides an exosome extraction method, which comprises the following steps: adding an extraction material into an extract to be extracted containing exosomes to extract the exosomes to obtain exosomes; the extraction material is metal oxide or functional material modified with the metal oxide; the metal oxide can generate double coordination with phosphate radical ions.

The following metal oxide is TiO₂The method for extracting exosome is specifically explained by taking human serum, cell culture solution and urine as examples of the extract to be extracted. The centrifugation in the following examples was carried out at 4 ℃ unless otherwise specified.

Example 1 exosome extraction

Extraction of exosomes in human serum and LC-MS/MS analysis of protein thereof

Step 1: serum sample pretreatment

Taking 100 mu L of human serum (informed by the consent of the person), and adding DMEM buffer solution into the serum according to the volume ratio of 1:1 for dilution to obtain serum diluent; and (3) carrying out gradient density centrifugation on the serum diluent to remove dead cells and/or cell residues, wherein the centrifugation conditions are as follows: centrifuging for 10 minutes at 300g, centrifuging for 10 minutes at 2000g and centrifuging for 30 minutes at 10000g, taking supernatant, filtering the supernatant by a filter membrane of 0.2 mu m, and collecting filtrate, namely the pretreated serum sample.

Step 2: exosome extraction in serum samples

Weighing 5mg TiO₂Rinsing with pure water and DMEM buffer solution for 2 times to obtain washed TiO₂(ii) a To the washed TiO₂Adding 200 mu L of the pretreated serum sample obtained in the step 1 to obtain an extraction mixture; mixing the extractsAfter incubating the mixture at 4 ℃ for 5 minutes with shaking (1500rpm), the mixture was centrifuged at 2000g for 30 seconds to allow exosomes to enter the pellet, and the pellet was collected; adding a washing buffer solution (the buffer solution is PBS) into the precipitate, washing the precipitate by shaking (the washing is completed by centrifuging at 2000g for 30 seconds to collect the precipitate), and obtaining a washed precipitate; adding an eluent (the eluent is 10g/100mL of ammonia water) into the washed precipitate to obtain an elution mixture; centrifuging the elution mixture at 2000g for 30 seconds to allow exosomes to enter a supernatant, and collecting the supernatant to obtain an exosome extracting solution; and centrifuging the obtained exosome extracting solution for 30 seconds at 2000g to enable exosomes to enter precipitates, and collecting the precipitates to obtain the exosomes.

The exosome proteins are extracted, and the exosome marker proteins TSG101 and CD9 are detected by using western-blot, and the result is shown in figure 1, and the obtained exosome contains TSG101 and CD 9.

And step 3: exosome lysis and LC-MS/MS analysis of its proteins

Putting the material containing the exosomes obtained in the step 2 (namely the washed precipitate) on ice, adding 18 mu L of 4g/100mL of SDS aqueous solution, and then carrying out ultrasonic (ultrasonic conditions are carried out at 100W) lysis for 20 minutes to obtain an ultrasonic product; centrifuging the sonicated product at 16000g for 5 minutes, and collecting the whole supernatant; adding 82 mu L of 8M urea aqueous solution and dithiothreitol into the supernatant to obtain lysate, wherein the concentration of the dithiothreitol in the lysate is 20 mmol/L; incubating the lysate for 4 hours at 37 ℃ to obtain a lysate; transferring the lysate to a FASP tube, and washing with 8M urea aqueous solution (FASP tube is a tube with molecular weight cut-off, and the washing process is completed by centrifugation under 14000g for 10 min, after which most of the solution and small molecule substances are centrifuged into the lower receiving tube and the exosomes are cut off in the upper FASP tube) 2 times; adding iodoacetamide (the final concentration is 50mmol/L), reacting for 1 hour at room temperature in the dark, washing for 3 times by 50mM ammonium bicarbonate aqueous solution, adding 1 mu g of trypsin, carrying out enzyme digestion for 12 hours at 37 ℃, carrying out heat drying on the obtained solution at 45 ℃, carrying out constant volume by 0.1% (v/v) formic acid aqueous solution to obtain an exosome protein extracting solution, and loading the exosome protein extracting solution for LC-MS/MS analysis, wherein the loading amount is 1 mu g.

Mass spectral data were collected and then retrieved using a MAXQUANT search target database. The target database used was RefSeq human database. The search parameter is set as trypsin full digestion, 2 protease cleavage missing sites are set, the protein fixing modification is Carbammidomethyl (C), and the variable modification is oxidation (M). The mass spectrum primary mass error is 15ppm, the secondary mass error is 0.6Da, and the false positive rate is 1%.

The results showed that the number of proteins identified by the serum exosome protein obtained by the above method was 384, which is significantly superior to that of the ultracentrifugation method (identification of proteins by the same method, the number of proteins identified was 228) and the commercial kit (Minute)^TMHi-Efficiency Exosome Precipitation Reagent, Inc.: invent Biotechnologies, Inc, cat #: CAT # EI-O27) (identification of protein by the same method, and the number of identified proteins obtained was 252) (fig. 2). Gene Ontology analyzes the cell components of the exosome protein extracted by the invention, and the result shows that the protein obtained by the method has more close functions with the exosome (figure 3). The method for extracting the exosome has a better extraction effect.

Wherein, the step of extracting the serum exosome by using the ultracentrifugation method comprises the following steps: taking 200 microliters of serum, adding PBS for dilution in the same volume, centrifuging for 30 minutes at 2000g at 4 ℃, taking the supernatant, centrifuging for 45 minutes again at 12000g, filtering the supernatant by using a 0.2 mu m filter membrane, centrifuging for 70 minutes at 110000g, discarding the supernatant, cleaning a bottom layer precipitate by using PBS, centrifuging for 70 minutes at 110000g, discarding the supernatant, and obtaining a precipitate, namely the serum exosome obtained by an ultracentrifugation method.

2. Exosome extraction in cell culture fluids

2.1 ultracentrifugation method for the extraction of exosomes

Culturing HeLa cells with a culture solution prepared from exosome-free fetal calf serum for 24-48 hours, and collecting the culture solution; centrifuging the culture solution at 4 ℃ for 10 minutes by 300g to remove cells, centrifuging for 10 minutes by 2000g to remove dead cells, centrifuging for 30 minutes by 10000g to remove organelles and the like, collecting supernatant, centrifuging 110000g of supernatant for 70 minutes, wherein the precipitate contains exosomes, cleaning the precipitate with PBS, centrifuging for 70 minutes by 110000g, collecting precipitate, centrifuging for 70 minutes by 110000g, and collecting precipitate to obtain exosomes in the cell culture solution.

2.2 evaluation of Metal oxide enrichment Effect by Standard exosomes

And (3) dividing the exosome obtained in the step 2.1 into a plurality of portions serving as standard exosomes, wherein each portion comprises exosomes with the corresponding protein content of 2 mu g. 2mg of TiO are taken₂Resuspending in 100 μ L DMEM buffer solution, adding 1 part of standard exosome, incubating at 4 deg.C under shaking at 1500rpm for 5 min, centrifuging 2000g suspension obtained after incubation for 30 s, washing precipitate with PBS 3 times, centrifuging 2000g for 30 s, collecting precipitate, and collecting precipitate, i.e. TiO enriched with exosome₂。

Carrying out scanning electron microscope and transmission electron microscope on the TiO enriched with exosome₂The characterization was performed, and the results are shown in FIG. 4, wherein a is TiO not enriched with exosomes₂The characterization picture of scanning electron microscope, b is TiO enriched with exosome₂The characterization of scanning electron microscope shows that c and d are TiO enriched with exosome₂D is a partial enlarged view of c. The results show that TiO₂Can be successfully enriched to exosomes.

2.3 reversible elution experiment (materials reusable)

The TiO enriched with exosome obtained in the step 2.2₂Washing with 100 μ L10 g/100mL ammonia, centrifuging at 2000g for 30 s, collecting supernatant, placing into FASP tube, washing with 100 μ L PBS for 2 times, and respectively characterizing the washed material and the eluted supernatant, with the result shown in FIG. 5, wherein a is TiO after washing with ammonia₂And b is a transmission electron microscope characterization image of the eluted supernatant. Indicating that the TiO enriched with exosome₂The exosomes on the above can be eluted to obtain TiO₂Can be repeatedly used.

2.3 direct extraction of exosomes from cell culture fluids using metal oxides

Adding 5mg of TiO₂The cells were added to 10mL of the culture obtained in step 2.1, incubated for 30 minutes with shaking by an oscillating machine, centrifuged at 2000g for 30 seconds, and the pellet was washed 2 times with PBS and characterized by scanning electron microscopy, as shown in FIG. 6. The results show that metal oxides can enrich exosomes in cell culture fluid.

3. Exosome extraction in urine

Collecting 20mL of morning urine, centrifuging at 2000g for 10 min, centrifuging at 10000g for 30 min, filtering the supernatant with 0.2 μm filter membrane, and adding 5mg TiO₂The material, after 30 minutes of shaking incubation with shaking machine, was centrifuged at 2000g for 30 seconds, the pellet was washed 2 times with PBS and characterized by scanning electron microscopy, the results are shown in figure 7. The results show that the metal oxide can enrich exosomes in urine.

Claims

1. A method for extracting exosomes, comprising: adding TiO ₂ to a solution to be extracted containing exosomes to obtain an extraction mixture; centrifuging the extraction mixture at 2000 g to allow exosomes to enter into a precipitate and collect the precipitate; Add an eluent to the precipitate to obtain an elution mixture, where the eluent is (3-20) g/100mL ammonia water; centrifuge the elution mixture to make the exosomes enter the supernatant , collect the supernatant, and remove the impurities in the supernatant to obtain exosomes;

The ratio of TiO ₂ in the extraction mixture to the liquid to be extracted satisfies the following a1), a2), a3) or a4):

a1) The to-be-extracted solution is the culture solution of the cells, and the ratio of TiO ₂ to the to-be-extracted solution in the extraction mixture is 5 mg:(10-300) mL;

a2) The extraction solution is serum or serum dilution, and the ratio of TiO ₂ in the extraction mixture to the to-be-extracted solution is 5 mg:(30-500) μL;

a3) The extraction solution is saliva or a dilution of saliva, and the ratio of TiO ₂ in the extraction mixture to the to-be-extracted solution is 5 mg:(1-125) μL;

a4) The extraction solution is urine, and the ratio of TiO ₂ in the extraction mixture to the to-be-extracted solution is 5 mg:(10-75) mL.

2. The method according to claim 1, characterized in that: the ratio of TiO ₂ to the liquid to be extracted in the extraction mixture satisfies the following a12), a22), a32) or a42):

a12) The to-be-extracted solution is the culture solution of the cells, and the ratio of TiO ₂ to the to-be-extracted solution in the extraction mixture is 5 mg: 10 mL;

a22) The extraction solution is serum or serum dilution, and the ratio of TiO ₂ in the extraction mixture to the to-be-extracted solution is 5 mg: 100 μL;

a32) The extraction solution is saliva or a dilution of saliva, and the ratio of TiO ₂ in the extraction mixture to the to-be-extracted solution is 5 mg:100 μL;

a42) The extraction solution is urine, and the ratio of TiO ₂ in the extraction mixture to the to-be-extracted solution is 5 mg: 20 mL.