CN113278584B

CN113278584B - Method for extracting thrombus exosome of acute myocardial infarction patient and application

Info

Publication number: CN113278584B
Application number: CN202110554258.6A
Authority: CN
Inventors: 何尤夫; 吴强; 钱宇; 艾丽琼; 周瑜; 黄晶; 龙向淑; 蒙宣彤
Original assignee: Guizhou Provincial Peoples Hospital
Current assignee: Guizhou Provincial Peoples Hospital
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2023-05-09
Anticipated expiration: 2041-05-20
Also published as: CN113278584A

Abstract

The invention relates to a method for extracting thrombus exosomes of acute myocardial infarction patient and application thereof, which uses an ultracentrifugation method to successfully extract exosomes in thrombus after dissolving thrombus by using a collagenase mixing method, which belongs to the first example at home and abroad, and extracts tissue exosomes by comparing the existing collagenase method.

Description

Method for extracting thrombus exosome of acute myocardial infarction patient and application

Technical Field

The invention relates to the technical field of medicines, in particular to a method for extracting thrombus exosomes of acute myocardial infarction patients and application thereof.

Background

Myocardial infarction is also called myocardial infarction, and myocardial infarction (myocarpalinfunction) is a coronary occlusion, and the blood flow is interrupted, so that partial myocardial necrosis occurs due to severe persistent ischemia.

Clinically, there is severe and persistent poststernal pain, fever, leukocytosis, accelerated erythrocyte sedimentation rate, increased activity of serum myocardial enzymes and progressive electrocardiographic changes, and arrhythmia, shock or heart failure can occur.

Acute myocardial infarction is sudden, should be found early, treated early, and the pre-admission treatment is enhanced. The treatment principle is to save dying cardiac muscle, reduce infarct area, protect heart function and treat various complications in time.

At present, research at home and abroad is carried out based on exosomes of a specific cell source or exosomes in circulating blood, but local micro-environments may have certain differences with the circulating blood due to the special pathophysiological condition that thrombus blocks coronary arteries in local myocardial infarction, and the thrombus has complex components and is not a single cell line, so that the current conventional exosome research system is not applicable. In contrast, previous reports of Vella et al (Vella LJ, scicluna BJ, cheng L, et al A rigorous method to enrich for exosomes from brain tissue.J excel Vesicles.2017.6 (1): 1348885.) have demonstrated that brain tissue is solubilized by a mixture of collagenase and pancreatin, and tissue exosomes are proposed.

The study is based on the report and is improved, the exosomes in thrombus are successfully extracted by using an ultracentrifugation method after the thrombus is dissolved by using a collagenase mixed pancreatin method, which is the first example at home and abroad, and tissue exosomes are extracted by comparing the existing collagenase method.

Disclosure of Invention

The invention aims to provide a method for extracting thrombus exosomes of acute myocardial infarction patients.

The invention also aims to provide an application of the exosome in preparing a medicament for treating acute myocardial infarction diseases.

The method for extracting thrombus exosomes of acute myocardial infarction patients comprises the following steps:

s1, taking adult coronary thrombosis after myocardial infarction, and standing in a 15ml centrifuge tube filled with 5-10ml phosphate buffer solution (phosphate buffer saline, PBS) overnight;

s2, adding pancreatin and type II collagenase;

s3, after full shaking and uniform mixing, placing the mixture on a shaking table at a constant temperature of 37 ℃ for 4 hours until thrombus is completely dissolved;

s4, removing blood in a non-coagulated state, then sucking supernatant and discarding, and taking 90-110mg of precipitated thrombus into a 15ml centrifuge tube filled with 4ml PBS for standby;

s5, centrifuging the dissolved thrombus tissue dissolving solution in a high-speed centrifuge at 2-5 ℃ for 2000gX 7-13min, removing dead cells and cell fragments remained in the dissolving solution, and taking supernatant for later use;

s6, performing S6; adding the filtered and sterilized liquid into an ultracentrifuge tube for ultracentrifugation at the temperature of 2-5 ℃ and the speed of 120000gX for 60-80 min;

s7, discarding the supernatant, and re-suspending the sediment with 100ul of sterile PBS to obtain the exosomes.

Preferably, the aforementioned step S1 is left standing overnight for 6 hours or more.

Preferably, in the step S2, 10-200. Mu.g/ml of pancreatin is added.

Preferably, 20. Mu.g/ml pancreatin is added in step S2 above.

Preferably, 10-200U/ml type II collagenase is added in step S2.

Preferably, 50U/ml type II collagenase is added in step S2.

Preferably, the precipitated thrombus is removed from step S4 to a 15ml centrifuge tube containing 4ml PBS for further use.

Preferably, the dissolved thrombus tissue lysate is centrifuged at 2000gX10min at 4 ℃ in a high-speed centrifuge to remove dead cells and cell debris remained in the lysate, and the supernatant is taken for later use.

Preferably, in the step S6, the liquid after the filtration and sterilization is added into an ultracentrifuge tube for ultracentrifugation at 4 ℃ and 120000gX70 min.

The exosome obtained by the method is applied to the preparation of the medicine for treating acute myocardial infarction diseases.

The beneficial effects are that:

compared with the prior art, the invention has the following beneficial effects:

problems of the prior art: at present, research at home and abroad is carried out based on exosomes of a specific cell source or exosomes in circulating blood, but local micro-environments may have certain differences with the circulating blood due to the special pathophysiological condition that thrombus blocks coronary arteries in local myocardial infarction, and the thrombus has complex components and is not a single cell line, so that the current conventional exosome research system is not applicable.

The invention has the beneficial effects that: the method of using collagenase to mix pancreatin to dissolve thrombus and then using ultracentrifugation method to successfully extract exosomes in thrombus is the first example at home and abroad, and tissue exosomes are extracted by comparing the existing collagenase method.

Drawings

Fig. 1: thrombolytic maps of each group: a: thrombus conditions of each group at 0min; b: each group of thrombus conditions at 15 min; c: each group of thrombus conditions at 30 min; d: each group of thrombus conditions at 45 min; e: thrombus condition of each group at 1h.

Fig. 2: statistical graphs of the residual mass of thrombolysis of each group (1).

Fig. 3: the white precipitate material shown by the circle is the exosome precipitated after ultracentrifugation.

Fig. 4: a: WB identified the exosome markers CD36 and internal reference GAPDH, B: and (5) statistics of WB gray scale values of each group.

Fig. 5: exosome electron microscopy images were extracted from each group of thrombolytic solutions: a: (1) group small dose pancreatin (10 μg/ml) +small dose collagenase type II (10U/ml) group, B: (2) group: small dose type II collagenase + large dose pancreatin group, C: (3) group: pancreatin+type II collagenase group, D: (4) group: large dose type II collagenase + large dose pancreatin group, E: (5) group: large dose type II collagenase + small dose pancreatin group. The exosomes in each group are seen to be in the form of a standard disc with a central depression, between 50 and 200nm in size.

Fig. 6: exosome NTA result graphs are extracted from various groups of thrombolytic liquid: a: (1) group small dose pancreatin (10 μg/ml) +small dose collagenase type II (10U/ml) group, with the result that the exosomes were seen to peak mainly at 123.6nm, 154.2nm, 191.2nm, followed by a peak centered at 226.9nm; b: (2) group: the small dose of collagenase type II + large dose of pancreatin groups, with the result that the exosomes were seen to peak mainly at 132.3nm, 161nm, 192nm, followed by a peak centered at 242.5nm; c: (3) group: pancreatin+type II collagenase group: as a result, it was found that the peak of exosomes was mainly 127.8nm, 174.2nm, 106.9nm, followed by a peak concentrated at 236.9nm; d: (4) group: large dose type II collagenase + large dose pancreatin group: as a result, the peak values of the exosomes are mainly 140.7nm, 167.2nm and 68nm, and the group has no peak value exceeding 200 nm; e: (5) group: large dose type II collagenase + small dose pancreatin group: as a result, it was found that the peak values of exosomes were mainly 135.5nm, 167.3nm, 114.4nm, and that the group had no peak value exceeding 200 nm.

Fig. 7: thrombolytic maps of each group: a: thrombus conditions of each group at 0min; b: each group of thrombus conditions at 15 min; c: each group of thrombus conditions at 30 min; d: thrombus condition of each group at 1h.

Fig. 8: statistical graphs of the residual mass of thrombolysis of each group (2).

Fig. 9: a: WB identified the exosome marker CD36 of each group and the internal reference GAPDH. B: and (5) statistics of WB gray scale values of each group.

Fig. 10: and (3) an exosome electron microscope detection result diagram of each group, wherein the diagram A is a collagenase group, the diagram B is an alteplase group, and the diagram C is a pancreatin+collagenase group. The exosomes in each group are seen to be in the form of a standard disc with a central depression, between 50 and 200nm in size.

Fig. 11: graphs of the results of the detection of NTA in exosomes of each group. Graph a: collagenase group, it was seen that the peak of exosomes appeared at 127.1nm, but still remained peak after 210.8nm, suggesting a relatively low purity. B: the alteplase group showed peak peaks at 136.7nm and 177nm for the exosomes, but peaks at 243nm and 202.6nm, suggesting relatively low purity. C: the pancreatin + collagenase group, with visible exosome peaks within 200nm, highest peaks at 127.8nm and only one peak at 236.9nm, suggests a significant improvement in purity over the first two groups.

Detailed Description

The technical scheme of the invention is further specifically described by the following specific examples.

Example 1 extraction method of thrombus exosome from acute myocardial infarction patient

S1, taking adult coronary thrombosis after myocardial infarction, placing the coronary thrombosis into a 15ml centrifuge tube filled with 5-10ml phosphate buffer solution, and standing for more than 6 hours;

s2, adding 20 mug/ml pancreatin and 50U/ml type II collagenase;

s4, removing blood in a non-coagulated state, then sucking supernatant and discarding, and taking 100mg of precipitated thrombus into a 15ml centrifuge tube filled with 4ml of PBS for standby;

s5, centrifuging the dissolved thrombus tissue dissolving solution at a high-speed centrifuge at 4 ℃ for 2000gX10min, removing dead cells and cell fragments remained in the dissolving solution, and taking supernatant for later use;

s6, performing S6; adding the filtered and sterilized liquid into an ultracentrifuge tube, and ultracentrifugating at 4 ℃ for 120000gX70 min;

Example 2 extraction method of thrombus exosome from acute myocardial infarction patient

S1, taking adult coronary thrombosis after myocardial infarction, and standing in a 15ml centrifuge tube filled with 5-10ml phosphate buffer solution overnight;

s2, adding 10 mug/ml pancreatin and 10U/ml type II collagenase;

s4, removing blood in a non-coagulated state, then sucking supernatant and discarding, and taking 90mg of precipitated thrombus into a 15ml centrifuge tube filled with 4ml of PBS for later use;

s5, centrifuging the dissolved thrombus tissue dissolving solution at a high-speed centrifuge at 2 ℃ for 2000gX 7min, removing dead cells and cell fragments remained in the dissolving solution, and taking supernatant for later use;

s6, performing S6; adding the filtered and sterilized liquid into an ultracentrifuge tube for ultracentrifugation at the temperature of 2 ℃ and 120000gX for 60 min;

Example 3 extraction method of thrombus exosome from acute myocardial infarction patient

s2, adding 200 mug/ml pancreatin and 200U/ml type II collagenase;

s4, removing blood in a non-coagulated state, then sucking supernatant and discarding, and taking 110mg of precipitated thrombus into a 15ml centrifuge tube filled with 4ml of PBS for later use;

s5, centrifuging the dissolved thrombus tissue dissolving solution at a high-speed centrifuge at a temperature of 5 ℃ for 2000gX 13min, removing dead cells and cell fragments remained in the dissolving solution, and taking supernatant for later use;

s6, performing S6; adding the filtered and sterilized liquid into an ultracentrifuge tube for ultracentrifugation at 5 ℃ for 120000gX 80 min;

Experimental example:

1. thrombolysis

1.1 use of formulations: pancreatin (0.05% edta, purchased from GIBCO, cat No. 25300054), type II collagenase (type II. Collagen is mainly present in tissues such as the intima of blood vessels, heart and intestinal tract, thrombus may contain some blood vessels and heart tissue, so type II. Purchased from GIBCO, cat No. 17101015), and heparin (drug name: low molecular heparin calcium injection with the specification of 1ml:5000IU, the specification of You Nishu, the manufacturer of Hainan general alliance pharmaceutical Co., ltd.), urokinase (pharmaceutical Ming: recombinant human prourokinase for injection with the specification of 5 mg/expenditure, the trade name of Prtime gram, the manufacturer of Shanghai Tianshi Liu pharmaceutical Co., ltd.), alteplase (pharmaceutical name: atteplase for injection with the specification of 20 mg/branch, the trade name of Aishi Lily, the manufacturer of Boehringer Ingelh eim Pharma GmbH & Co.KG), streptokinase (pharmaceutical name: recombinant streptokinase for injection with the specification of 10 ten thousand units/branch, the trade name of Sikatong, the manufacturer of Beijing tetracyclic biopharmaceutical Co., ltd.), phosphate buffer (phosphate buffer saline, PBS, purchased from GBICO)

1.2 use of the instrument: constant temperature shaking table (Shanghai shanzhi, model: TS-2102)

1.3 steps: coronary thrombus was taken after adult myocardial infarction and allowed to stand overnight (> 6 h) in a 15ml centrifuge tube containing 5-10ml phosphate buffer (phosphate buffer saline, PBS) for removal of non-coagulated blood, after which the supernatant was carefully aspirated and discarded, and the precipitated thrombus was taken to be about 100mg in a 15ml centrifuge tube containing 4ml PBS for use.

The following grouping is: (1) group: small dose pancreatin (10 μg/ml) +small dose collagenase type II (10U/ml) group, (2) group: small dose type II collagenase (10U/ml) +large dose pancreatin group (200 μg/ml), (3) group: pancreatin+type II collagenase group (20 μg/ml pancreatin+50U/ml collagenase (preferred group)), (4) group: large dose collagenase type II (200U/ml) +large dose pancreatin (200 μg/ml) group, (5) group: the large dose of collagenase type II (200U/ml) +the small dose of pancreatin (10 μg/ml) was taken out from 5 groups, and the thrombus was weighed with forceps for 0min, 15min, 30min, 45min, and 1h, respectively, and photographed.

1.4 results (see fig. 1, fig. 2, table 1):

table 1: the residual thrombolysis mass statistics (the results are represented by mean value + -standard deviation)

2. Exosome extraction

2.1 use reagents and consumables: ultracentrifuge tubes (cat No. 355644, beckmann), phosphate buffer (phosphate buffer saline, PBS, purchased from GBICO), 5ml syringe, 0.22 μm needle filter (Millipore)

2.2 use of the instrument: ultracentrifuge (Beckmann, optima XPN-100, using TYPE45TI rotor), high speed cryocentrifuge (Ai Bende, eppendorf 5810R)

3. The steps are as follows:

1) Centrifuging the dissolved thrombus tissue dissolving solution at a high-speed centrifuge at 4 ℃ for 2000gX10min to remove dead cells and cell fragments remained in the dissolving solution, and taking supernatant for later use;

2) Adding the filtered and sterilized liquid into an ultracentrifuge tube, and ultracentrifugating at 4 ℃ for 120000gX70 min;

3) The supernatant was discarded and the pellet was resuspended in sterile PBS 100ul to give the exosomes (see FIG. 3).

3. Exosome identification

3.1 using respectively:

1) And (5) identifying the size and the shape of the exosomes by using an electron microscope.

2) Nanoparticle tracking analyzers (Nanoparticle Tracking Analysis, NTA) identify the particle size of exosomes.

3) Western Blot identified exosome marker CD63 and CD9 expression.

3.2 instruments required: zetaView PMX 110 (Particle Metrix, meerbusch, germany), small vertical electrophoresis transfer device (Bio-Rad, 1658033, U.S. Pat. No. 5), gel imager (Bio-Rad, gel Doc XR, U.S. Pat. No. 5), microplate reader (Sieimer's fly, varioskan LUX)

The required reagents and consumables: formvar-carbon sample-carrying copper mesh, PBS, uranyl oxalate, methylcellulose, BCA protein quantification reagent (Thermo Scientific), 5 Xloading buffer (Beyotime), SDS-PAGE gel preparation kit (Beyotime), PVDF membrane (0.45 μm, millpore, U.S.A.), CD36 rabbit anti-human antibody (Abcam, ab133625, U.S.A.), HBR sheep anti-rabbit IgG antibody (Abcam, ab6721, paraformaldehyde (Beyotime), glutaraldehyde (Beyotime), ECL developer (Millpore, U.S.A.)

3.3 BCA protein quantification

3.3.1 sample treatment: adding a proper amount of pyrolysis liquid into the extracted and purified exosomes, and carrying out ice pyrolysis for 30min to be detected;

balancing the BCA protein quantitative kit to room temperature, and observing whether each reagent has sediment, and waiting for dissolution if the sediment exists;

3.3.2 preparing working solution:

3.3.3 well count calculation: total number of wells of 8 standards + samples;

3.3.4 total volume calculation: 200ul of working fluid per well;

3.3.5 working solution preparation: reagent A in total volume: reagent b=50: 1.

3.3.6 dilution of standards at different rates

25ul of standard and a target sample with proper concentration (25 ul can be directly loaded if the target sample has a low pre-judging concentration, 25ul can be loaded after dilution with a diluent if the pre-judging concentration is high, for example, 5 times of dilution, namely 5ul of sample plus 20ul of diluent) are respectively added into the microwells of the 96-well plate. 200ul BCA working solution is added to each hole, and the mixture is gently shaken and mixed. The 96-well plate cover is covered, the mixture is incubated for 30 minutes at 37 ℃ (if the concentration is lower, the color is lighter, the incubation temperature can be properly increased or the incubation time can be prolonged), the microplate reader is opened in advance, and the measurement procedure is selected. Cooled to room temperature, and the protein concentration (the final protein concentration of the sample is multiplied by the corresponding dilution times) is calculated according to a standard curve by measuring A562 by an enzyme-labeled instrument.

3.4 Western Blot

3.4.1 sample treatment

The BCA-quantified exosome protein sample is taken and added with 1/4 volume of 5 Xloading buffer solution in proper quantity, and the mixture is boiled for 10min after uniform mixing.

3.4.2 preparation of SDS-PAGE

Washing and airing a glass plate, putting the glass plate into a manufacturing tool according to requirements, preparing SDS-PAGE glue according to requirements, firstly preparing separating glue, adding 7ml of separating glue into the glass plate, adding 2ml of absolute ethyl alcohol, fully solidifying the separating glue after 30min, preparing concentrated glue, discarding absolute ethyl alcohol in the glass plate, adding 2ml of concentrated glue, and inserting comb teeth.

3.4.3 electrophoresis on samples

After the gel is well solidified, placing the gel into an electrophoresis tank, filling electrophoresis buffer solution, pulling out a comb, washing a sample loading hole by the electrophoresis buffer solution, loading the prepared samples, taking the same total protein amount from each sample, slowly adding the samples into the sample loading hole, and carrying out 120mA electrophoresis for 90min.

3.4.4 immunoblotting (Wet transfer)

After electrophoresis, using a transfer electrophoresis device, and electrotransferring under 300mA constant current condition for 60min in ice bath, transferring protein onto PVDF film: pouring a proper amount of electrotransfer buffer solution into a tray, taking out the gel from the electrophoresis device, and sequentially placing from a negative electrode to a positive electrode: filter paper-glue-PVDF film-filter paper (no bubble is generated between the glue and the PVDF film in the placing process), placing the jig into a transfer electrophoresis device, and adding a proper amount of electrotransfer buffer solution to transfer the film.

3.4.5 Immunochromatographic

Closing: the PVDF membrane was blocked with a blocking solution (TBST solution containing 10% skimmed milk) at room temperature for 1h.

Incubation resistance: the primary antibody is diluted by the sealing liquid, then incubated with the sealed PVDF membrane at 4 ℃ for overnight, and the membrane is washed: PBST was washed 4 times for 8min each.

Secondary antibody incubation: diluting the secondary antibody with a blocking solution 1:5000, and incubating the PVDF membrane for 1h at room temperature

Washing the film: PBST was washed 4 times for 8min each.

Color development was performed using Immobilon Western HRP luminescence reagent (millipore Corp.)

And (3) X-ray development: the film for obtaining the display strip is carried out in a darkroom, and the specific steps of ECL adding, exposure, development and fixation are as follows:

placing PVDF film on a tiled preservative film, and mixing with 1:1, mixing the solution A and the solution B in proportion, uniformly dripping the mixed solution on the PVDF film, and reacting in a dark place.

Taking out the film, sucking up the redundant ECL substrate reaction liquid, putting into a cassette, paving a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding moving the X-ray film), closing the cassette, and exposing.

Taking out the X-ray film, putting into developing solution, taking out after about 1min, rinsing in clear water for a few seconds, and putting into fixing solution for at least 2min.

Taking out the X-ray film, airing and analyzing.

And (3) placing the dried X-ray film into a cassette, marking the position of a protein pre-dyeing marker and the name of a sample on the X-ray film by using a marker, and then analyzing the result.

3.5 electron microscope:

(1) resuspending exosomes into 50-100 μl2% paraformaldehyde;

(2) applying the 5 mu lexosome suspension to a Formvar-carbon-loaded copper mesh;

(3) mu.l of PBS was added to the sealing film. Placing a copper mesh (Formvar membrane face down) on the PBS droplets with tweezers for cleaning;

(4) the copper mesh was placed on 50 μl of 1% glutaraldehyde droplets for 5min;

(5) the copper mesh was placed in 100 μl ddH2O 2min (washed 8 times);

(6) the copper mesh was placed on 50 μl uranium oxalate drops at pH 75min;

(7) placing the copper mesh on methyl cellulose liquid drops for 10min, and operating on ice;

(8) placing the copper net on a stainless steel ring at the top end of the sample table, and sucking redundant liquid on the filter paper;

(9) drying in air for 5-10 min;

the copper mesh is placed in a box, and an electron microscope photograph is taken at 80 kV.

3.6 NTA：

(1) Washing the sample cell with deionized water;

(2) the instrument was calibrated with polystyrene microspheres (110 nm);

(3) washing the cuvette with 1X PBS;

samples were diluted with 1 XPBS and assayed by sample injection.

3.7, results (see fig. 4, 5, 6):

the results show that: the CD36 in three groups of large-dose collagenase, small-dose collagenase, pancreatin, small-dose collagenase and small-dose pancreatin all express strong positives, while the other two groups express weak positives. We considered to be related to damage to the exosome membrane by overdosing with pancreatin.

Finally, combining the thrombolysis efficiency, CD36 expression condition and NTA and electron microscopy results, we found that the number of large-particle exosomes was significantly reduced with the increase of the pancreatin amount, but the exosomes injury was possibly increased, and the extraction efficiency was significantly inhibited, so we finally selected the group of pancreatin+type II collagenases (20 μg/ml pancreatin+50U/ml collagenase).

Subsequently, for comparison with several anticoagulants commonly seen in clinic, we further grouped into: pancreatin group (50 μg/ml, quoted: PMID: 32489530), collagenase type II group (75U/ml, quoted: PMID: 28804598), heparin group (4000U, calculated as 50-70U/Kg recommended by national Wei Jian Committee for acute ST elevation myocardial infarction thrombolysis therapy (2 nd edition) 2019), alteplase group (15 mg, 15mg recommended first dose by national Wei Jian Committee for acute ST elevation myocardial infarction thrombolysis therapy (2 nd edition) 2019), urokinase group (150 ten thousand units, reasonable drug therapy by national Wei Jian Committee for acute ST elevation myocardial infarction thrombolysis therapy (2 nd edition), streptokinase group (2 ten thousand IU, indicated by single bolus in AMI coronary artery with reference to the pharmaceutical specification), pancreatin+collagenase group (20 μg/ml pancreatin+50U/ml collagenase), total 7 groups.

The groups were placed on a thermostatically controlled rocking bed, incubated at 37℃and the thrombi were then weighed with forceps and photographed at 0min, 15min, 30min, 1h, respectively.

Results (see fig. 7, 8, table 2):

table 2: the residual thrombolysis mass statistics (the results are represented by mean value + -standard deviation)

4. Exosomes extracted from seven sets of thrombi were quantified by BCA and multiplied by volume (200 ul) with the final results: pancreatin group (100 μg), collagenase type II group (80 μg), heparin group (18 μg), alteplase group (220 μg), urokinase group (130 μg), streptokinase group (110 μg), pancreatin+collagenase group (220 μg), and the results are shown in fig. 9, fig. 10, and fig. 11.

The results show that: the alteplase group, the collagenase group and the pancreatin plus collagenase group are all strong positive for CD36, the urokinase, the collagenase and the streptokinase are expressed in weak positive, and the heparin group is almost free of CD36 expression, which is considered to be related to the damage of large doses of pancreatin, urokinase and streptokinase to the exosome membrane.

The invention extracts thrombus exosome for the first time at home and abroad, and extracts tissue exosome by comparing the existing collagenase method, and the experiment reduces the consumption of collagenase and improves the purity of exosome at the same time after adding pancreatin.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A method for extracting thrombus exosomes of acute myocardial infarction patients, which is characterized by comprising the following steps:

s2, adding 20 mug/ml pancreatin and 50U/ml type II collagenase;

s6, adding the filtered and sterilized liquid into an ultracentrifuge tube for ultracentrifugation at the temperature of 2-5 ℃ and 120000gX for 60-80 min;

2. The method of claim 1, wherein step S1 is left to stand overnight for more than 6 hours.

3. The method of claim 1, wherein step S4 takes 100mg of precipitated thrombus to a 15ml centrifuge tube containing 4ml of PBS for use.

4. The method according to claim 1, wherein in step S5, the lysed thrombus tissue lysate is centrifuged at 2000gX10min at 4℃in a high-speed centrifuge to remove dead cells and cell debris remaining in the lysate, and the supernatant is taken for use.

5. The method of claim 1, wherein step S6 is performed by adding the filtered sterilized liquid to an ultracentrifuge tube at 4 ℃ and an ultracentrifuge tube at 120,000 gx70 min.