Use of AML-12 exosomes in the preparation of a medicament for treating cholestatic liver injury

Abstract

The application discloses application of AML-12 exosomes in preparation of medicines for treating cholestatic liver injury, relates to the technical field of biological medicine, and discloses application of AML-12 cell-derived exosomes in preparation of medicines for preventing and treating ANIT-induced cholestatic liver injury; the treatment is manifested as reduction of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in serum. The application proves that the AML-12 cell-derived exosomes have a significant treatment effect on ANIT-induced cholestatic liver injury, successfully opens up the application of the biological preparation in a brand-new disease field, and fills the blank of the prior art; through in-vivo experiments, it is proved that AML-12 exosome treatment can significantly reduce liver cell injury markers and alkaline phosphatase, a key index of cholestasis, in serum of ANIT model mice, and improve pathological injury of liver tissue, thereby constituting a complete curative effect evidence chain from function to morphology.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of AML-12 exosomes in the preparation of drugs for treating cholestatic liver injury. Background Technology

[0002] Cholestatic liver injury is a common and serious liver disease that can be induced by various factors such as drugs and toxins. Alpha-naphthyl isothiocyanate (ANIT) is a classic hepatotoxic agent that specifically damages bile duct epithelial cells, causing bile duct obstruction and impaired bile excretion. This leads to the accumulation of bile acids in hepatocytes, triggering hepatocyte necrosis, apoptosis, and a strong inflammatory response. Therefore, the ANIT-induced mouse liver injury model is widely recognized as a classic experimental model for studying the pathogenesis of human cholestatic liver injury and screening therapeutic drugs.

[0003] In recent years, exosomes, as key mediators of intercellular communication, have shown great potential in the field of disease treatment. Exosomes are nanoscale (30-150 nm) vesicles actively secreted by cells, containing various bioactive molecules such as proteins, RNA, and DNA. They possess low immunogenicity, good biocompatibility, and natural targeted delivery capabilities. In existing technologies, the therapeutic potential of exosomes from different cell sources has been extensively explored. For example, Chinese patent literature (publication number: CN 117695311 A) discloses that exosomes derived from AML-12 cells can be used to alleviate liver ischemia-reperfusion injury.

[0004] However, the mechanisms of liver diseases are complex and diverse. The core of ischemia-reperfusion injury lies in the energy metabolism disorders and oxidative stress caused by hypoxia; while the core of ANIT-induced cholestatic liver injury lies in the direct toxicity of the biliary system and the resulting bile acid toxicity. The initiation points and core pathways of these two types of injury are fundamentally different. It is well known to those skilled in the art that the effectiveness of a treatment for a liver disease with a specific mechanism does not necessarily and reasonably imply its effectiveness for another liver disease with a completely different mechanism. To date, no research or patent has disclosed or suggested that exosomes derived from AML-12 cells can be used to treat ANIT-induced cholestatic liver injury; their roles and mechanisms in cholestasis and the regulation of the hepatic immune microenvironment remain unknown.

[0005] Therefore, the purpose of this invention is to fill this gap by proposing and fully validating a novel application of AML-12 exosomes in the treatment of ANIT cholestatic liver injury. Through systematic experimental studies, its unique mechanism of action in remodeling the immune microenvironment has been revealed, providing a solid scientific foundation for the translational application of this exosome. This provides a novel and effective candidate drug and solution for the treatment of this type of disease. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of AML-12 exosomes in the preparation of drugs for treating cholestatic liver injury.

[0007] The abbreviations used in this study are as follows: ALT, Alanine aminotransferase; AST, Aspartate Transaminase; ALP, Alkaline phosphatase; Alpha Mouse Liver12, AML-12 cells; Transmission Electron Microscopy (TEM); Nanoparticle Tracking Analysis (NTA); α-Naphthyl isothiocyanate (ANIT); Exosome; Neutrophils; Macrophages.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: Application of AML-12 cell-derived exosomes in the preparation of drugs for the prevention and treatment of ANIT-induced cholestatic liver injury.

[0009] Preferably, the treatment involves reducing the levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in the serum.

[0010] Preferably, the drug is a pharmaceutical composition comprising exosomes derived from AML-12 cells and a pharmaceutically acceptable carrier; the dosage form of the drug is an injection.

[0011] Preferably, the exosomes derived from AML-12 cells have one or more of the following characteristics: (a) Expression of marker proteins Alix, TSG101 and CD9; (b) Under transmission electron microscopy, it exhibits the morphology of nanoscale vesicles with a lipid bilayer membrane structure; (c) The main peak of the particle size, as determined by nanoparticle tracking analysis, is between 120 nm and 130 nm.

[0012] A method for preparing exosomes derived from AML-12 cells, the method comprising the following steps: S1: Culture AML-12 cells and collect the cell culture supernatant; S2: Pre-treat the cell culture supernatant to remove cells, cell debris and large particulate impurities; S3: Add polyethylene glycol solution to the pretreated supernatant to induce precipitation; S4: Resuspend and purify the precipitate to obtain the exosomes derived from the AML-12 cells.

[0013] Preferably, in S1, the AML-12 cells are cultured using DMEM basal medium containing 4.5 g / L L-glucose, L-glutamine, and sodium pyruvate, with the addition of 1% penicillin-streptomycin antibiotic solution and 10% fetal bovine serum.

[0014] Preferably, in step S1, the fetal bovine serum used for cell culture is pre-treated by ultra-high speed centrifugation to remove exosomes from the serum; the conditions for ultra-high speed centrifugation are: 120,000g, 4°C, centrifugation for 15 hours.

[0015] Preferably, the pretreatment in S2 includes: centrifuging the cell culture supernatant sequentially at 500g for 5 minutes and collecting the supernatant; centrifuging the supernatant at 3000g for 20 minutes and collecting the supernatant; and finally filtering the supernatant through a 0.22-micron filter. In S3, the PEG solution is a PEG6000 solution with a final concentration of 10%. The addition involves adding an equal volume of the PEG solution to the pretreated supernatant and precipitating at 4°C for at least 12 hours.

[0016] Preferably, S4 includes: S41: Centrifuge the mixture precipitated by PEG at 4°C and 6000g for 1 hour, discard the supernatant, and obtain the preliminary precipitate; S42: Resuspend the preliminary precipitate with phosphate buffer and centrifuge at 4°C and 10,000g for 1 hour, then collect the supernatant; S43: Centrifuge the supernatant obtained in S42 at ultra-high speed, discard the supernatant, and resuspend the precipitate with phosphate buffer to obtain the exosomes derived from AML-12 cells.

[0017] The beneficial effects of this invention are as follows: 1. This invention confirms that exosomes derived from AML-12 cells have a significant therapeutic effect on ANIT-induced cholestatic liver injury, successfully opening up the application of this biological agent in a completely new disease field and filling the gap in the existing technology.

[0018] 2. This invention has demonstrated through rigorous in vivo experiments that AML-12 exosome therapy can significantly reduce hepatocyte damage markers (ALT, AST) and the key cholestasis indicator alkaline phosphatase (ALP) in the serum of ANIT model mice, and significantly improve pathological damage to liver tissue, thus forming a complete chain of efficacy evidence from function to morphology. Attached Figure Description

[0019] Figure 1 Characterization diagrams of exosomes derived from AML-12 cells in this invention (A: Schematic diagram of extraction of exosomes derived from AML-12 cells; B: Western blotting identification of exosomes derived from AML-12 cells, showing positive expression of Alix, TSG101, and CD9; C: Transmission electron microscopy (TEM) image, showing the classic "teacup-shaped" concave morphology of exosomes, with a clearly visible lipid bilayer membrane structure at the edges; D: Nanoparticle tracking analysis (NTA), showing particle size distribution). Figure 2 The images shown are for validation of the ANIT-induced acute liver injury model of this invention (A: Schematic diagram of ANIT-induced acute liver injury model establishment; BC: Comparison of serum ALT and AST levels; D: Representative image of liver tissue H&E staining (40×); E: Statistical analysis of necrosis area of ​​liver tissue H&E staining; F: Statistical analysis of inflammatory infiltration area of ​​liver tissue H&E staining; *p<0.05, **p<0.01, ***p<0.001, ns indicates no statistical difference). Figure 3 The images show a comparison of serum biochemical indicators in mice treated with AML-12 exosomes according to this invention, as well as pathological images of liver tissue H&E staining (A: Schematic diagram of the ANIT-induced acute liver injury model treated with AML-12 exosomes; BD: Serum ALT, AST, and ALP levels; E: Representative images of liver tissue H&E staining; F: Statistics of necrotic area stained with H&E staining in liver tissue; G: Statistics of inflammatory infiltration area stained with H&E staining in liver tissue, *p<0.05, **p<0.01, ns indicates no statistical difference). Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0021] Example 1: Extraction and characterization of exosomes derived from AML-12 cells Cell culture and exosome extraction: AML-12 cells were cultured in DMEM basic (1X), Gibco medium containing 4.5 g / L D-glucose, L-glutamine, and 110 mg / L sodium pyruvate, supplemented with 1% Penicilin-Streptomycin (100X) (NCM-Biotech) and 10% FBS (G8002-500ML, Sewell) at 37°C in a 5% CO2 incubator. To collect exosomes, when cell confluence reached 80%-90%, the medium was replaced with exosome-free serum-free medium, and the cells were cultured for another 48 hours. The cell culture supernatant was then collected. Subsequently, the cell culture supernatant underwent a series of centrifugations, filtrations, and PEG precipitation, followed by ultracentrifugation to precipitate exosomes, and resuspending in sterile PBS.

[0022] Experimental methods: 1.1 Extraction of exosome-free serum Prepare six 70ml ultracentrifuge tubes (BECKMAN). Aliquot the purchased fetal bovine serum (G8002-500ml, Seville) into the ultracentrifuge tubes, filling them completely and ensuring no air bubbles are formed. Balance the tubes (weight difference accurate to ±0.05). Then, centrifuge the balanced tubes in an ultracentrifuge (BECKMAN) at 120,000g for 15 hours at 4°C. After centrifugation, remove the ultracentrifuge tubes and transfer the FBS to clean, sterile 50ml centrifuge tubes for future use in preparing fresh exo-free medium.

[0023] 1.2 Preparation of 20% PEG PEG stock solution (final concentration 10%): Dissolve 200g PEG6000 and 58.44g sodium chloride in water, bring the volume to 1000ml, and filter through a 0.45 μm filter for later use.

[0024] 1.3 Gradient centrifugation for exosome extraction The cell culture supernatant collected after 48 hours of culture was centrifuged at 500g for 5 minutes at 4°C, the precipitate was discarded, and the supernatant was retained. Cell debris was removed from the supernatant at 4℃, 3000g, for 20 minutes. The precipitate was discarded, and the supernatant was retained.

[0025] The supernatant is filtered through a 0.22-micron filter.

[0026] Add an equal volume of filtered 20% PEG stock solution to the filtered supernatant and incubate overnight (at least 12 hours) at 4°C.

[0027] The benchtop centrifuge was used at 4°C and centrifuged at 6000g for 1 hour (Eppendorf, model 5810 R, 3214g).

[0028] Discard the supernatant and pour it away. Resuspend the suspension in 10 ml of PBS. Centrifuge at 10000g for 1 hour at 4°C using a benchtop centrifuge (Eppendorf, model 5810 R, 3214g), collect the supernatant and discard the precipitate.

[0029] Transfer the supernatant to a cleaned and autoclaved 70ml ultracentrifuge tube (BECKMAN), filling it completely to prevent air bubbles from forming, and balance it (weight difference accurate to ±0.05). Then place the balanced ultracentrifuge tube in an ultracentrifuge (BECKMAN) and centrifuge at 120,000g for 70 minutes at 4℃.

[0030] After centrifugation, discard the supernatant in the centrifugation tube and resuspend the precipitate in 700 / 750 μL of PBS. Then, take 25 μL of the suspension, add 25 μL of RIPA lysis buffer containing 1% PMSF, lyse on ice for 30 min, and store the remaining suspension at -80℃ for later use.

[0031] After the ice cracking was complete, 25 μL was aspirated and the exosomal protein concentration was measured using the BCA protein quantification kit.

[0032] Identification of exosomes Each time, 10 μL of exosomal protein sample was taken for Western blotting identification, mixed with 5× loading buffer, and boiled at 95°C for 3 min to denature the protein.

[0033] Western Blot 1.1 Electrophoresis Electrophoretic separation was performed using a 10% SDS-polyacrylamide gel. After sample loading, the power was turned on initially at 80V for 20 minutes. Once the sample entered the separating gel, the voltage was adjusted to 110V for approximately 40 minutes, stopping when bromophenol blue appeared (reaching the green line at the bottom). The power was then turned off, the connecting wires were disconnected, the electrophoresis buffer was discarded, and the gel interlayer, along with the upper tank, was removed to recover the electrophoresis solution.

[0034] 1.2 Transfer of film Cut a PVDF membrane according to the size of the gel (cut off a corner for marking), activate it in methanol for 30 seconds to 1 minute, then equilibrate it in transfer buffer for 2-3 minutes. Next, place the sandwiches, black side down, in an iron pan, and add transfer buffer to completely cover the sandwiches. Use a gel remover to trim off excess gel. Place the sandwiches in the following order: black gel, white membrane, sponge, 3 layers of filter paper, gel, membrane, 3 layers of filter paper, sponge. After each layer is placed, use a test tube to remove air bubbles. Secure the two plates together, ensuring alignment. Place the assembled sandwiches in the electroporation cell (black side corresponding to the black side of the cell, white side to the red side). Add transfer buffer, add ice to raise the liquid level until the sandwiches are completely covered, insert the electrodes, and determine the transfer time based on the protein molecule size (larger molecular weight requires a longer transfer time).

[0035] Using ice transfer: 300mA current, 2 hours. Immerse the entire outer tank in crushed ice. After the transfer is complete, turn off the power, disassemble the transfer device from top to bottom, and peel off each layer one by one.

[0036] Closed After completing the transfer, transfer the membrane to a petri dish or other suitable container (incubator). Depending on the membrane size, add an appropriate amount of rapid blocking buffer (NcmBlot Blocking Buffer, New Semiconductor), ensuring the membrane is completely submerged. Block with 5% skim milk powder for 1 hour. After blocking, discard the blocking buffer (it is recyclable; simply place the container in the refrigerator). Add 1*TBST and wash the membrane three times, 10 minutes each time, while rapidly shaking on a horizontal shaker.

[0037] Incubation of primary antibody Dilute the antibody according to the ratio of 1:5000 (antibody: diluent = 1:5000), approximately 5 ml per membrane, which is 1 μL antibody to 5 ml diluent. Prepare the diluted solution for later use. Use an internal control at 1:5000 and a general antibody at 1:1000. Add the primary antibody (rabbit anti-TSG101 polyclonal antibody; rabbit anti-ALIX polyclonal antibody; rabbit anti-CD9 polyclonal antibody) diluted with the diluent and incubate overnight at 4°C. The next day, recover the primary antibody, wash the membrane three times with TBST for 5 minutes each time, and gently shake on a horizontal shaker.

[0038] Incubation of secondary antibodies Western blot (WB) secondary antibody is diluted with 5% skim milk: 5mL 5% skim milk: 0.25g skim milk powder + 5mL TBST Dilute the antibody according to the ratio of antibody to diluent = 1:5000, approximately 5 ml (1 μL) per membrane. Discard the TBST, add the diluted secondary antibody to the incubation chamber (note: mouse-to-mouse and rabbit-to-rabbit ratios), cover the membrane surface, and incubate at room temperature for 1 hour. Discard the secondary antibody, wash the membrane three times with TBST for 5 minutes each time, and shake rapidly on a horizontal shaker. After the final wash, do not discard the TBST; keep the membrane moist.

[0039] Prepare the developer: Mix solution A and solution B in a 1:1 ratio before use. The developer should cover the membrane during application. Acquire signals using a chemiluminescence imaging system.

[0040] Western Blot results like Figure 1 As shown in Figure B, the extracted sample exhibited clear and specific bands at the molecular weight positions corresponding to ALIX, TSG101, and CD9. This confirms the successful acquisition of high-purity, intact, functional exosomes derived from AML-12 cells, providing a suitable material basis for subsequent in vivo efficacy experiments.

[0041] TEM results Transmission electron microscopy (TEM) observation: 10 μL of exosome suspension was dropped onto a copper grid, allowed to settle and precipitate, then negatively stained with 2% phosphotungstic acid, dried at room temperature, and observed under a TEM. Figure 1 As shown in Figure C, transmission electron microscopy (TEM) observations revealed typical nanoscale vesicle structures in the extracted samples, exhibiting the classic "teacup-shaped" concave morphology of exosomes, with clearly visible lipid bilayer membrane structures at the edges, thus morphologically confirming the successful isolation of exosomes.

[0042] NTA results Nanoparticle tracking analysis (NTA): For exosome samples, particle size distribution and concentration are determined: First, the sample cell is rinsed with deionized water; then, the instrument is calibrated with polystyrene microspheres (particle size: 100 nm, catalog number: 3100A, brand: ThermoFisher); next, the sample cell is rinsed with 1X PBS buffer (Biological Industries, Israel); finally, the sample is diluted with 1X PBS buffer and injected for analysis. Figure 1 As shown in Figure D, the sample's peak diameter is 128.8 nm, and the median diameter is 144.9 nm, consistent with the typical particle size distribution characteristics of exosomes. Furthermore, the sample exhibits a single-peak distribution with a sharp peak shape, indicating the absence of impurity proteins and cell debris, suggesting high exosome purity.

[0043] Example 2: Establishment and evaluation of an ANIT-induced mouse model of acute cholestatic liver injury 1. Experimental animals and grouping: SPF-grade healthy male C57BL / 6 mice (6-8 weeks old, weighing 20-22g) were randomly divided into two groups after one week of acclimatization: normal control group (control group) and ANIT model group.

[0044] 2. Modeling method: Mice in the ANIT model group were administered ANIT (purchased from Aladdin Company, with corn oil as the solvent, at a dose of 70 mg / kg body weight) by gavage once. The normal control group was administered an equal volume of corn oil by gavage only.

[0045] 3. Sample Collection: Forty-eight hours after ANIT gavage, mice were anesthetized, and blood was collected from the eyeballs. After the blood was allowed to clot, it was centrifuged at 3500 rpm for 5 minutes at 4°C to separate the serum. This centrifugation was repeated twice to reduce the number of red blood cells in the serum, and then stored at -80°C for later use. Subsequently, the mice were dissected, and tissue from the same location in the right lobe of the liver was taken and fixed with 4% paraformaldehyde solution for pathological analysis.

[0046] 4. Detection Indicators and Methods Serum biochemical analysis: The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected strictly in accordance with the operating procedures of the kit (purchased from Nanjing Jiancheng Biotechnology Institute).

[0047] Histopathological analysis: Liver tissue fixed with paraformaldehyde was dehydrated with graded alcohol, cleared with xylene, embedded in paraffin, and then sectioned into 4 μm thick sections. After dewaxing to water, the sections were stained with hematoxylin and eosin (H&E) and mounted with neutral resin. Liver tissue damage was observed and assessed under an optical microscope by a senior pathologist whose grouping was unknown.

[0048] 5. Results: Serum biochemistry results: as shown in the figure Figure 2 As shown in BC, compared with the normal control group, the serum ALT and AST levels in the ANIT model group mice were significantly elevated (p<0.01), indicating that hepatocellular damage and cholestasis were successfully induced. These results demonstrate that ANIT successfully induced severe hepatocellular damage and bile excretion disorders.

[0049] Histopathological results: such as Figure 2 As shown in Figure D, the normal control group (Control group) exhibited intact liver lobule structure, neatly arranged hepatocyte cords, and normal cell morphology. In contrast, the ANIT model group (ANIT group) displayed typical pathological features of acute liver injury: extensive hydropic degeneration and focal necrosis of hepatocytes (black arrows), and abundant inflammatory cell infiltration, predominantly neutrophils, within the hepatic sinusoids (blue arrows). Figure 2 As shown in E and 2F, the inflammatory infiltration area and necrosis area in the model group were significantly higher than those in the control group.

[0050] This embodiment fully demonstrates the successful replication of the ANIT acute liver injury mouse model using two gold standard indicators: serum biochemistry and histopathology. This model exhibits significant liver parenchymal damage and cholestasis, providing a stable and reliable disease model for subsequent evaluation of the therapeutic effects of AML-12 exosomes.

[0051] Example 3: Evaluation of the therapeutic effect of AML-12 exosomes on ANIT-induced acute cholestatic liver injury 1. Experimental animals and grouping: SPF-grade healthy male C57BL / 6 mice were randomly divided into three groups: control group, ANIT model group, and AML-12 exosome treatment group. All mice were administered ANIT (70 mg / kg) by gavage according to the method in Example 2 to establish an acute liver injury model.

[0052] 2. Treatment regimen: The AML-12 exosome treatment group received three intraperitoneal injections of AML-12 exosomes prepared in Example 1 (each dose was 10 µg / g body weight, dissolved in 700 μL PBS) 24 hours before ANIT gavage (lead administration), during gavage (initial treatment), and 24 hours after gavage (maintenance treatment). The ANIT model group received an equal volume of PBS at the same time points as a negative control.

[0053] 3. Sample collection and testing: 24 hours after the last administration (i.e. 48 hours after ANIT gavage), serum and liver tissue were collected according to the method described in Example 2, and the levels of ALT, AST, and ALP were detected and H&E staining was performed.

[0054] 4. Results: 1. Serum biochemical therapeutic effects: such as Figure 3 As shown in BD, serum ALT, AST, and ALP levels in the ANIT model group were significantly higher than those in the normal control group (p < 0.001), further confirming the success of the model. In contrast, ALT and AST activities in the AML-12 exosome treatment group were significantly reduced (*p<0.05, **p<0.01, ***p<0.001). This indicates that AML-12 exosome treatment demonstrates that exosome therapy can effectively reduce hepatocellular damage and alleviate cholestasis.

[0055] Histopathological improvement: such as Figure 3 As shown in Figure E, the ANIT model group exhibited extensive hepatocellular necrosis and severe inflammatory infiltration, with disruption of normal liver structure. In contrast, the treatment group showed significant repair of liver tissue structure, with a marked reduction in the extent of hepatocellular necrosis and the degree of inflammatory cell infiltration, and a more regular arrangement of hepatic cords. Figure 3Both F and G showed that, compared with the normal group and the treatment group, the necrotic area and inflammatory infiltration area in the model group were significantly higher than those in the treatment group and the normal group. The liver tissue structure of the AML-12 exosome treatment group (EXO group) was significantly repaired, with regular arrangement of hepatocyte cords and a significant reduction in necrotic areas and inflammatory cell infiltration, and was morphologically close to that of the normal control group.

[0056] The above results confirm that intraperitoneal injection of AML-12 exosomes can significantly improve ANIT-induced acute cholestatic liver injury, demonstrating a clear therapeutic effect.

[0057] This invention provides a novel candidate biological agent for the treatment of complex cholestatic liver injury, which has both therapeutic efficacy and the potential for multi-target synergistic effects, and has significant scientific value and clear prospects for clinical translation.

[0058] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Application of AML-12 cell-derived exosomes in the preparation of drugs for the prevention and treatment of ANIT-induced cholestatic liver injury.

2. The application according to claim 1, characterized in that, The treatment is characterized by reducing serum levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase.

3. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition comprising exosomes derived from the AML-12 cells and a pharmaceutically acceptable carrier; the dosage form of the drug is an injection.

4. The application according to claim 1, characterized in that, The AML-12 cell-derived exosomes have one or more of the following characteristics: (a) Expression of marker proteins Alix, TSG101 and CD9; (b) Under transmission electron microscopy, it exhibits the morphology of nanoscale vesicles with a lipid bilayer membrane structure; (c) The main peak of the particle size, as determined by nanoparticle tracking analysis, is between 120 nm and 130 nm.

5. A method for preparing exosomes derived from AML-12 cells, characterized in that, The method includes the following steps: S1: Culture AML-12 cells and collect the cell culture supernatant; S2: Pre-treat the cell culture supernatant to remove cells, cell debris and large particulate impurities; S3: Add polyethylene glycol solution to the pretreated supernatant to induce precipitation; S4: Resuspend and purify the precipitate to obtain the exosomes derived from the AML-12 cells.

6. The preparation method according to claim 5, characterized in that, In S1, the AML-12 cells are cultured using DMEM basal medium containing 4.5 g / L L-glucose, L-glutamine, and sodium pyruvate, with the addition of 1% penicillin-streptomycin antibiotic solution and 10% fetal bovine serum.

7. The preparation method according to claim 6, characterized in that, In S1, the fetal bovine serum used for cell culture is pre-treated by ultra-high speed centrifugation to remove exosomes from the serum; the conditions for ultra-high speed centrifugation are: 120,000g, 4°C, centrifugation for 15 hours.

8. The preparation method according to claim 5, characterized in that, The pretreatment of S2 includes: centrifuging the cell culture supernatant sequentially at 500g for 5 minutes and collecting the supernatant; centrifuging the supernatant at 3000g for 20 minutes and collecting the supernatant; and finally filtering the supernatant through a 0.22-micron filter. In S3, the PEG solution is a PEG6000 solution with a final concentration of 10%. The addition involves adding an equal volume of the PEG solution to the pretreated supernatant and precipitating at 4°C for at least 12 hours.

9. The preparation method according to claim 6, characterized in that, S4 includes: S41: Centrifuge the mixture precipitated by PEG at 4°C and 6000g for 1 hour, discard the supernatant, and obtain the preliminary precipitate; S42: Resuspend the preliminary precipitate with phosphate buffer and centrifuge at 4°C and 10,000g for 1 hour, then collect the supernatant; S43: Centrifuge the supernatant obtained in S42 at ultra-high speed, discard the supernatant, and resuspend the precipitate with phosphate buffer to obtain the exosomes derived from AML-12 cells.

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