Exosome derived from glioma internal cystic fluid and application of exosome in preparation of anti-glioma medicine

Abstract

The invention relates to an exosome derived from glioma internal cystic fluid and application of the exosome in preparation of anti-glioma drugs, and belongs to the technical field of biology. The glioma endocystic fluid source exosome is prepared to solve the treatment difficulty caused by the fact that a blood-brain barrier hinders drug delivery and glioma is high in invasiveness and drug resistance in the prior art. The particle size of the exosome is 60-130 nm, the exosome specifically expresses CD63, CD81 and TSG101 proteins, and a four-stage gradient centrifugation method (300 g to 3000g to 10000g to 100000g) is adopted for separation and extraction. Experiments prove that the exosome inhibits glioma cell invasion and proliferation by regulating and controlling tumor cell ribosome functions. Compared with a traditional treatment medicine, the exosome has the advantages of efficient targeting property, low toxicity and low cost, and further development of a new medicine is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to exosomes derived from cystic fluid inside gliomas and applications thereof in the preparation of anti-glioma drugs. Background Art

[0002] Glioma is a type of malignant tumor originating from glial cells in the central nervous system, accounting for 40%-60% of all primary intracranial tumors and is the most common primary intracranial tumor in adults. Its pathogenesis has not yet been fully clarified, but it is known to be closely related to genetic factors (such as chromosomal aberrations, oncogene amplification and inactivation of tumor suppressor genes), environmental carcinogens (such as high-dose ionizing radiation) and viral infections. The clinical manifestations of glioma are mainly determined by the space-occupying effect and the involvement of brain area functions. Common symptoms include headache, nausea and vomiting, epileptic seizures and neurological dysfunction (such as language disorders, movement disorders, etc.). Glioma is highly heterogeneous. According to the classification criteria of the World Health Organization (WHO2021 edition), the median survival time of glioblastoma with wild-type IDH1 molecules is only 12-18 months. In recent years, the incidence rate has been increasing year by year, and there is a trend of gradually decreasing age. Due to the invasive growth pattern of glioma cells along neurofibrosis and blood vessels, the boundary between them and the surrounding normal brain tissue is blurred, making complete surgical removal difficult. Almost 100% of patients experience recurrence after surgery. Furthermore, the presence of the blood-brain barrier limits the delivery and effectiveness of chemotherapy drugs, resulting in an extremely poor prognosis for patients. Although a series of advances have been made in the comprehensive treatment of gliomas, including maximal safe surgical resection, concurrent chemoradiotherapy plus adjuvant chemotherapy, electric field therapy, and immunotherapy, recent developments in molecular pathology have driven a shift in glioma classification from histological phenotypes to molecular phenotypes. For example, molecular markers such as IDH1 / 2 mutations, 1p / 19q co-deletion, and MGMT promoter methylation are used for prognostic assessment and personalized treatment. The exploration of targeted therapies (such as EGFR inhibitors and bevacizumab) and immunotherapies (such as CAR-T cell therapy) has brought new hope to patients, and precision radiotherapy technologies such as proton beam therapy have also shown the potential to prolong survival in clinical trials. Despite this, the complex biological characteristics of gliomas still require multidisciplinary collaboration and the continued development of new treatment strategies. The research on new and effective treatment strategies and therapeutic drugs will have a positive impact on the treatment of gliomas.

[0003] Glioma, especially glioblastoma multiforme (GBM), is highly susceptible to intratumoral cystic degeneration. Rapid tumor cell proliferation and delayed vascular development lead to severe localized ischemia, hypoxia, and necrosis, resulting in significant necrotic areas within the tumor. This necrotic area then gradually develops into an internal fluid component independent of the cerebrospinal fluid. Currently, there are no reports on the internal cystic fluid of gliomas, and the function of this fluid has not been investigated. Summary of the Invention

[0004] In view of this, one of the objects of the present invention is to provide exosomes derived from the internal cystic fluid of gliomas, and the second object is to provide an application of exosomes derived from the internal cystic fluid of gliomas in the preparation of anti-glioma drugs.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides exosomes derived from the internal cystic fluid of gliomas, wherein the exosomes are derived from the internal cystic fluid of gliomas, the exosomes have a particle size of 60-130 nm, and the exosomes specifically express CD63, CD81, and TSG101;

[0007] Furthermore, the exosome preparation method comprises the following steps:

[0008] (1) Centrifuge the glioma intracystic fluid at 300 g and 4°C for 10 minutes, remove the precipitate, and retain the supernatant;

[0009] (2) centrifuging the supernatant obtained in step (1) at 3000 g and 4° C. for 10 minutes, removing the precipitate and retaining the supernatant;

[0010] (3) centrifuging the supernatant from step (2) at 10,000 g and 4° C. for 30 minutes, removing the precipitate and retaining the supernatant;

[0011] (4) centrifuging the supernatant of step (3) at a centrifugal force of 100,000 g and 4° C. for 70 minutes, removing the supernatant and retaining the precipitate, which is the exosomes derived from the cystic fluid inside the glioma;

[0012] Furthermore, the application of exosomes derived from cystic fluid inside gliomas in inhibiting the proliferation of glioma cells;

[0013] Furthermore, the use of exosomes derived from the cystic fluid of glioma in the preparation of anti-glioma drugs;

[0014] Preferably, the anti-glioma drug comprises the exosomes and pharmaceutically acceptable excipients;

[0015] Furthermore, the use of the exosomes derived from the cystic fluid inside the glioma in the preparation of anti-glioma drug delivery vectors;

[0016] The present invention provides a drug for treating brain glioma, which contains exosomes derived from the cystic fluid inside the glioma.

[0017] The beneficial effects of the present invention are:

[0018] The most prominent characteristics of glioma cells are uncontrolled proliferation and widespread, diffuse invasion. The exosomes derived from glioma cyst fluid prepared by the present invention have the ability to combat malignant gliomas, primarily by altering the translational function of glioma cell ribosomes, thereby inhibiting glioma cell proliferation and invasion. The glioma cyst fluid exosomes prepared by the present invention possess "autophagy inhibition" properties, exerting a therapeutic effect by regulating tumor cell ribosome function. Experiments have shown that after exosomes act on glioma cells, the number of ribosomes increases by 30.6%-81.2%, while the rate of ribosomal protein synthesis increases by 30.2%-102.1%, leading to abnormal protein accumulation and cell cycle arrest. This mechanism breaks through the traditional signaling pathway targeting approach and provides a new approach for the treatment of malignant gliomas.

[0019] This invention discloses a novel use of exosomes derived from glioma cystic fluid in drug preparation. This not only expands the application scope and enhances the value of exosomes derived from glioma cystic fluid, bringing new hope for the treatment of malignant gliomas, but also facilitates the development of new drugs. Using glioma cystic fluid-derived exosomes as lead compounds, structural modification or engineering is expected to further enhance their activity or reduce side effects. This lays a solid foundation for the development of highly effective, safe, and cost-effective anti-glioma drugs, and promotes the clinical translation of personalized precision treatment models.

[0020] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0022] Figure 1 To demonstrate the presence of cystic fluid within gliomas using preoperative cranial MRI results, T1, T2, T2flair, T1-enhanced images, and local magnification images of cranial MRI were presented. Postoperatively, tumor tissue was sectioned and stained with HE to confirm that the tumor was a glioma.

[0023] Figure 2 To use schematic diagrams to demonstrate the process of obtaining cystic fluid, including preoperative navigation, planning the puncture route, craniotomy puncture, cystic fluid extraction, and cystic fluid collection steps;

[0024] Figure 3The schematic diagram illustrates the collection process of exosomes from cyst fluid (differential centrifugation), which includes crude centrifugation (300g, 10 minutes), medium-speed centrifugation (3000g, 10 minutes), high-speed centrifugation (10000g, 30 minutes), and ultrahigh-speed centrifugation (100000g, 70 minutes). The resulting exosomes are relatively pure.

[0025] Figure 4 The extracted exosomes were identified by scanning electron microscopy, flow cytometry, and Western blot, respectively. A is an electron microscopy image of cyst fluid-derived exosomes, B is a flow cytometry image of cyst fluid-derived exosomes, and C is a Western blot image of protein markers (CD63, CD81, and TSG101) detected in cyst fluid-derived exosomes.

[0026] Figure 5 This study examined changes in glioma cell invasion and proliferation after treatment with cyst fluid-derived exosomes, as well as their effects on intracranial growth in orthotopic glioma-bearing mice. A shows the results and statistical graph of a Transwell chamber perforation assay, B shows the results and statistical graph of colony formation, C shows the CCK-8 growth curve, and D shows an image of a cranial MRI scan of orthotopic tumor-bearing mice (**p < 0.01 vs. control group).

[0027] Figure 6 To detect changes in ribosome number and ribosomal protein synthesis efficiency in glioma cells after treating them with cyst fluid-derived exosomes. (A) is the electron microscopy scanning result of the cells, and (B) is the result of the ribosomal protein synthesis efficiency test of the cells. DETAILED DESCRIPTION

[0028] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0029] Among them, the accompanying drawings are only for illustrative purposes and represent only schematic diagrams rather than actual pictures, and should not be understood as limiting the present invention. In order to better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged or reduced, and do not represent the dimensions of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the accompanying drawings.

[0030] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "back", etc. indicating directions or positional relationships, they are based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operate in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0031] Example 1 Preparation of exosomes from glioma cyst fluid

[0032] 1. Preoperative evaluation of intratumoral cystic fluid in glioma patients

[0033] The presence of glioma and glioma cystic fluid was initially confirmed by preoperative MRI examination of the patient. Figure 1 As shown: T1-weighted images show iso- or hypointense signals in the solid portion of the tumor, with marked hypointense signals in the central necrotic or cystic areas, and patchy hyperintense signals in the presence of hemorrhage. T2-weighted images and FLAIR sequences show slightly hyperintense signals in the solid portion, with marked hyperintense signals in the necrotic and cystic areas. The surrounding edema is hyperintense, widespread, and disproportionate to the tumor volume. T1-enhanced sequences show markedly heterogeneous enhancement, with irregular, thick-walled rings or nodules (garland-like enhancement) in the solid portion of the tumor, no enhancement in the necrotic areas, and unevenly thickened walls. Postoperative HE staining reveals highly heterogeneous tumor cell morphology, including cells of varying sizes, increased nuclear-cytoplasmic ratios, and some mononuclear or multinuclear giant cells. Mitotic figures are frequent, and typical pseudo-palisading necrosis is present.

[0034] 2. Collection of glioma intracystic fluid

[0035] like Figure 2 As shown: Preoperative high-resolution thin-slice MRI scans of the patient's skull are burned to a disc and then imported into the neurosurgery preoperative magnetic bead navigation system for data import, registration, data extraction, and modeling. After fixation, skull bony positioning registration is then performed, and an appropriate puncture path is planned. After disinfection, draping, and craniotomy, the predetermined path is navigated again. Using a 20mL syringe connected to a puncture needle, negative pressure puncture is performed according to the navigation path. Cyst fluid is collected until it enters the syringe. After complete extraction, the puncture needle is slowly withdrawn. The cystic fluid in the syringe is sealed and sterilely aliquoted, labeled, and stored (frozen at -80°C).

[0036] 3. Extraction of exosomes from glioma tumors

[0037] like Figure 3 As shown, after the cyst fluid collected in step 2 is completely thawed, it is roughly centrifuged using a low-speed centrifuge at a centrifugal force of 300g, 4°C, and centrifugation for 10 minutes; after completion, the precipitate (cells and large debris) is removed and the supernatant is retained, which is transferred to a new centrifuge tube for medium-speed centrifugation at a centrifugal force of 3000g, 4°C, and centrifugation for 10 minutes; after completion, the precipitate (small cell fragments) is removed and the supernatant is retained, which is transferred to a new centrifuge tube again, and after complete balancing, high-speed centrifugation is performed at a centrifugal force of 10000g, 4°C, and centrifugation for 30 minutes; after completion, the precipitate (apoptotic small fragments) is removed and the supernatant is retained, which is transferred to a new centrifuge tube again, and after complete balancing, high-speed centrifugation is performed at a centrifugal force of 10000g, 4°C, and centrifugation for 30 minutes. The supernatant was retained and transferred to an ultracentrifuge tube again. After being completely balanced, ultracentrifugation was performed at a centrifugal force of 100,000 g at 4°C for 70 minutes. After completion, the supernatant was removed and the precipitate was retained. Sterile cold PBS was added to the ultracentrifuge tube again, and after being completely balanced, ultracentrifugation was performed again at a centrifugal force of 100,000 g at 4°C for 70 minutes. After centrifugation, the supernatant was removed, the precipitate was retained and slightly dried, and then the precipitate was resuspended with 100 uL of sterile pre-cooled PBS solution, which is the exosomes derived from the glioma cyst fluid.

[0038] Example 2 Identification of Exosomes from Glioma Cyst Fluid

[0039] 1. Scanning electron microscope: The exosomes extracted in Example 1 were fixed with sufficient glutaraldehyde (2.5% concentration) at 4°C for 2 hours to maintain structural integrity. The fixed exosomes were aspirated for negative staining. The exosome sample was dropped onto a carbon film copper grid and stained with phosphotungstic acid (5%) and uranyl acetate for 1.5 minutes in sequence. The excess stain was aspirated to enhance contrast. The samples were then observed and photographed under a scanning electron microscope. The results are shown in the figure below. Figure 4 A;

[0040] 2. Flow cytometry exosome particle size detection: dilute the above-extracted exosome sample 50 times with DPBS to ensure that the particle concentration is within the range of 20-100 particles / field of view. Instrument calibration: start the Nanosight NS300 device, clean the sample pool and connect the laser module, and flush the pipeline with DPBS until no particles are displayed in the background. Parameter setting: adjust the Screen Gain (screen gain) and Camera Level (camera sensitivity) to ensure that the particles are clearly visible. Set the number of measurements, single measurement time and dilution multiple. Data acquisition: inject the diluted sample into the sample pool with a syringe, track the Brownian motion of the particles with a high-speed camera, and generate a particle size distribution diagram and concentration data. Analysis and saving: export .avi (motion trajectory), .csv (raw data) and .pdf (report) files, and use GraphPad to redraw the map. The results are as follows: Figure 4 B.

[0041] 3. Western-Blot detection: Pipette 20uL of the extracted exosomes, add 20uL of RIPA lysis buffer, add 4X loading buffer at a ratio of 1:3 after sufficient lysis, mix thoroughly, and boil at 100 degrees Celsius for 10 minutes to denature the protein. Add the prepared protein sample to the electrophoresis gel loading well, run electrophoresis at 95V until the end, transfer the protein on the gel to the PVDF membrane, block with 5% milk powder solution at room temperature for 1 hour, incubate with antibodies (CD63, CD81, TSG101) at 4°C overnight, remove the primary antibody working solution the next day, wash the membrane three times with PBST solution, and apply the corresponding primary antibody-derived HRP-labeled secondary antibody working solution. Incubate at room temperature for 1 hour, wash the membrane three times with PBST solution, and develop it under a developer. The results are as follows: Figure 4 As shown in C.

[0042] Example 3 Glioma intracystic fluid-derived exosomes effectively inhibit the migration of glioma cells

[0043] The invasive ability of the glioma cell line U251 was tested by the Transwell chamber perforation assay to understand the effect of exosomes derived from glioma cyst fluid on the invasion of glioma cells. For the Transwell perforation assay, Matrigel was diluted at a ratio of 1:8 and then coated on the membrane inside the Transwell chamber (pore size 8μm). U251 cells were plated at 2x10 per well. 4 The cells were divided into two groups and respectively plated on the bottom of the chamber, and then placed in a 24-well cell culture plate and cultured in serum-free DMEM / F12 medium. 5uL of cyst fluid-derived exosomes were added to the exosome-treated group, and 5uL of PBS solution was added to the control group. After 24 hours of culture, the chamber was taken out, and the bottom of the chamber was repeatedly wiped with a cotton swab to remove the cells, and then the cells at the bottom were fixed with 4% paraformaldehyde solution for 15 minutes. After the cell fixation time was over, the membrane was washed twice with PBS solution and permeabilized with methanol for 10 minutes. After that, the membrane was washed twice with PBS solution, and the cells at the bottom of the chamber were stained with crystal violet stain. After the chamber was dried, the membrane of the chamber was carefully cut off with a sharp knife, and the membrane was sealed between the slide and the coverslip with a sealing agent to make a glass slide. It was then observed and photographed under a microscope. The stained cells represent the cells that have invaded. Five fields of view were randomly selected for counting and statistics. The results are as follows. Figure 5 As shown in A: It can be seen that the number of invading cells in the glioma tumor-derived exosomes-treated group was significantly reduced compared with the number of invading cells in the control group, indicating that glioma tumor-derived exosomes-treated group can significantly inhibit the invasion of glioma cells.

[0044] Example 4 Glioma intracystic fluid-derived exosomes effectively inhibit the proliferation of glioma cells

[0045] A cell cloning experiment was used to explore the effect of exosomes on the proliferation of glioma cells. Cell preparation: U251 cells in the logarithmic growth phase were obtained, digested with 0.25% trypsin to form a single cell suspension, resuspended in complete medium (basal medium + 10% FBS) and counted; cell seeding: 1000 cells were seeded per well and gently shaken to disperse evenly; culture and observation: Cultured in a 37°C, 5% CO2 incubator for 14 days, with the medium changed every 7 days. Avoid blowing cells when changing the medium, and terminate the culture when the number of cells in the clone is greater than 50; fixation and staining: discard the medium, wash with PBS, and fix with 4% paraformaldehyde for 20 minutes; add crystal violet stain for 10 minutes, wash the residual stain with PBS, and let it dry; colony counting: count clones with a diameter greater than 0.3 mm or containing greater than 50 cells under a microscope or by naked eye, and calculate the number of clones formed in the two groups. The results are shown in the figure below. Figure 5 As shown in B, it can be seen that the number of cell clones in the glioma tumor-derived exosomes-treated group was significantly reduced compared with the number of cell clones in the control group, indicating that glioma tumor-derived exosomes-treated group can significantly inhibit the proliferation of glioma cells.

[0046] At the same time, CCK-8 experiments were used to explore the effect of exosomes on glioma cell proliferation. We conducted in vitro experiments to verify the effect of glioma tumor-derived exosomes on the proliferation of glioma cell line U251, and then drew the corresponding growth curve. U251 cells were divided into two groups and seeded into 96-well cell culture plates, with 5 replicates in each group and a cell density of 1x10 3 cells / well, and 100μL of DMEM / F12 culture medium containing 10% fetal bovine serum was added to each well, and then placed in a 37℃ carbon dioxide incubator for overnight culture. The next day, 5 wells were selected from each group for detection, and 10uL of CCK-8 reaction solution was added to each well. After culturing for 1 hour, the absorbance value of each well at a wavelength of 450nm was detected using a microplate reader. Then, 1uL of the extracted exosomes was added to each well of the exosome treatment group, and 1uL of PBS solution was added to no well of the control group. CCK-8 (Cell Counting Kit-8) was added to each group at the 2nd, 3rd, 5th, and 7th days, and the absorbance value of the cells at 450nm was detected, thereby detecting the cell proliferation ability. The results are as follows Figure 5 As shown in C, it can be seen that on the 5th and 7th days, the OD450 value of the cells in the glioma tumor-derived exosomes-treated group was significantly lower than that in the control group, indicating that the glioma tumor-derived exosomes treatment can significantly inhibit the proliferation of glioma cells.

[0047] Example 5 Glioma Cyst Fluid-Derived Exosomes Inhibit Glioma Growth

[0048] NOD / SCID mice were randomly divided into two groups. The mice were injected intracranially with glioma cell line U87 cells (after the mice were anesthetized by intraperitoneal injection and fixed on a stereotaxic instrument, the bregma was used as the positioning point, and the skull of the corresponding point was moved 2mm to the right and 1.5mm forward. A small opening was drilled through the skull, and the needle tip of a micro-syringe was used to align the positioning point. The injection was made at a depth of 4mm, and then after retreating 2mm, 200,000 cells were injected into each mouse. The total volume was 5uL). Three days later, the tumor-bearing mice were treated with tail vein injection. Each mouse was injected with 5uL of the above exosomes. The control group was given an equal volume of solvent PBS solution. The drugs were administered twice a week for 4 consecutive weeks. During this period, the living conditions of the mice in each group were observed daily, and the time of death of the mice was recorded. At the same time, small animal magnetic resonance imaging was used to detect the growth of gliomas inoculated in situ in the brain of the mice. The results are shown in Figure 2. Figure 5 As shown in Figure D, the orthotopic glioma growth in mice treated with exosomes derived from glioma intracystic fluid was significantly lower than that in the control group, indicating that exosomes derived from glioma intracystic fluid can also inhibit glioma growth in glioma animal models.

[0049] Example 6 Glioma intracystic fluid-derived exosomes can alter the ribosome function of glioma cells

[0050] Electron microscopy was used to detect the changes in the number of ribosomes in cells. U251 cells in the logarithmic growth phase were digested and collected, and 1x10 6 Cells were inoculated per well and treated with 10uL of exosomes from the glioma cyst fluid and 10uL of control PBS liquid for 24 hours. The two groups of cells were fixed with 3% glutaraldehyde at 4°C for 2 hours to maintain cell morphology, rinsed with PBS 3 times for 10 minutes each time to remove the residual fixative, and fixed again with 1% osmium acid for 1 hour to enhance conductivity. Gradient dehydration was then performed: 30% → 100% ethanol gradient dehydration for 15 minutes each time. Isoamyl acetate was used to replace ethanol, and a critical point dryer was used to remove moisture to avoid surface collapse. Spray-coating a conductive layer: A 5-10nm gold or platinum layer was sprayed on a vacuum coating instrument to eliminate charge accumulation and enhance the secondary electron signal. The sample was placed on the scanning electron microscope sample stage, the acceleration voltage was adjusted, and the number of ribosomes in the cell was observed. The results are shown in Figure 6 A, The results showed that the number of ribosomes in U251 cells treated with glioma intracystic fluid-derived exosomes was significantly higher than that in the control group, indicating that glioma intracystic fluid-derived exosomes can significantly increase the number of ribosomes in glioma cells.

[0051] Protein synthesis efficiency is used to detect changes in ribosome synthesis function in cells. HPG (L-homopropargylglycine) is a methionine analog that can replace methionine and be incorporated into newly synthesized proteins during protein synthesis. It is combined with a fluorescent probe (such as azide-labeled AF594) through a click reaction to achieve labeling and quantitative detection of newly synthesized proteins (Thermo Fisher Click-iT TM HPG kit). U251 cells were counted and seeded onto confocal microplates at a density of 60%-80%. After 24 hours of treatment, 10 μL of glioma intracystic fluid-derived exosomes and 10 μL of a control PBS solution were administered. HPG (100× stock solution) was diluted to a working concentration (typically a final concentration of 50 μM) and incubated with the cells for 0.5 hour. The culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 15-30 minutes at room temperature. The cells were then washed three times with PBS. Treatment with 0.5% Triton X-100 was performed for 10 minutes to enhance probe permeability. To prepare the reaction solution, click reaction buffer, CuSO4, Azide 594 (e.g., AF594 azide), and Click Additive (a small molecule that aids the reaction) were mixed according to the kit's proportions. 500 μL of the reaction solution was added to each well of a 6-well plate and incubated at room temperature for 60 minutes in the dark. Nuclear staining: Add DAPI and incubate for 10 minutes, wash with PBS, seal and store in the dark. Microscope observation: Red fluorescence (AF594) under fluorescence microscope represents new protein, and blue fluorescence DAPI marks the cell nucleus. The results are shown in Figure 6 B. The results showed that the protein synthesis efficiency of U251 cells in the group treated with glioma intracystic fluid-derived exosomes was significantly higher than that in the control group, indicating that glioma intracystic fluid-derived exosomes can significantly increase the ribosomal protein synthesis function of glioma cells.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. Exosomes derived from cystic fluid in gliomas, characterized by: The exosomes are derived from the cystic fluid inside the glioma, the particle size of the exosomes is 60-130 nm, and the exosomes specifically express CD63, CD81, and TSG101.

2. The exosomes derived from glioma cyst fluid according to claim 1, characterized in that: The exosome preparation method comprises the following steps: (1) Centrifuge the glioma intracystic fluid at 300 g and 4°C for 10 minutes, remove the precipitate, and retain the supernatant; (2) centrifuging the supernatant obtained in step (1) at 3000 g and 4° C. for 10 minutes, removing the precipitate and retaining the supernatant; (3) The supernatant of step (2) was centrifuged at 10,000 g and 4° C. for 30 minutes, and the precipitate was removed and the supernatant was retained; (4) The supernatant of step (3) was centrifuged at a centrifugal force of 100,000 g and 4° C. for 70 minutes. After completion, the supernatant was removed and the precipitate was retained. The process was repeated once and the precipitate was collected again. The precipitate was the exosomes derived from the cystic fluid inside the glioma.

3. Use of the exosomes derived from the cystic fluid of a glioma according to claim 1 or 2 in inhibiting the proliferation of glioma cells.

4. Use of the exosomes derived from the cystic fluid of a glioma according to claim 1 or 2 in the preparation of an anti-glioma drug.

5. The use according to claim 3, characterized in that: The anti-glioma drug comprises the exosomes and pharmaceutically acceptable excipients.

6. Use of the exosomes derived from the cystic fluid of a glioma according to claim 1 or 2 in the preparation of an anti-glioma drug delivery vector.

7. A drug for treating glioma, characterized in that: The drug comprises the exosomes derived from the cystic fluid inside the glioma according to claim 1 or 2.

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