Application, extraction method and composition of Ganoderma lucidum exosomes in the treatment of brain glioma

Abstract

The present application relates to the technical field of biological products, and provides an application of Ganoderma lucidum exosomes in the treatment of brain glioma, an extraction method and a composition, and the application of Ganoderma lucidum exosomes in any one or more of the following: a) preparing a product that promotes apoptosis of brain glioma cells; b) preparing a product that inhibits proliferation of brain glioma cells; c) preparing a product that inhibits autophagy of brain glioma cells; d) preparing a product for treating brain glioma. The present application verifies through cell and animal experiments that Ganoderma lucidum exosomes can effectively induce apoptosis of brain glioma cells and inhibit autophagy of brain glioma cells to induce glioma death, and act on brain glioma cells through the AKT / mTOR signaling pathway and inhibiting the production of autophagosomes in brain glioma cells.

Description

Technical Field

[0001] The present application relates to the technical field of biological products, and more specifically, to an application, extraction method and composition of Ganoderma lucidum exosomes in the treatment of brain glioma. Background Art

[0002] As a traditional Chinese medicinal material with a long history of medicinal use, the extraction and application of its active ingredients have always been a hot topic of research. Ganoderma lucidum and its polysaccharides (peptides) can directly act on mononuclear macrophages, dendritic cells and cytotoxic T cells in the body, promote the expression of TNFαmRNA and IFNγmRNA, increase the production of TNFα and IFNγ, enhance the activity of cytokines, and then inhibit the immune escape of tumor cells, thereby inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and ultimately killing tumor cells. Therefore, the anti-tumor effect of Ganoderma lucidum does not act directly on tumor cells, and the anti-tumor effect of Ganoderma lucidum is relatively limited. Summary of the invention

[0003] The present application is provided to solve the above-mentioned defects existing in the prior art. An application, extraction method and composition of Ganoderma lucidum exosomes in the treatment of glioma are needed. The present application verifies through cell and animal experiments that Ganoderma lucidum exosomes can effectively induce apoptosis of glioma cells and inhibit autophagy of glioma cells, induce death of glioma cells, and act on glioma cells through the AKT / mTOR signaling pathway and inhibiting the production of autophagosomes in glioma cells.

[0004] In a first aspect of the present application, a method for using Ganoderma lucidum exosomes in any one or more of the following is provided:

[0005] a) preparing a drug for promoting apoptosis of brain glioma cells;

[0006] b) preparing a drug for inhibiting the proliferation of brain glioma cells;

[0007] c) preparing a drug for inhibiting autophagy of brain glioma cells;

[0008] d) preparing drugs for treating brain glioma.

[0009] In a second aspect of the present application, a method for extracting exosomes of Ganoderma lucidum is provided, the method comprising: (1) grinding the soaked Ganoderma lucidum to obtain a grinding liquid; (2) coarsely filtering the grinding liquid to obtain a coarse filtrate; (3) mixing the supernatant after centrifugation of the coarse filtrate with an enzyme preparation for enzymolysis to obtain an enzymolysis liquid; (4) removing impurities, filtering and concentrating the enzymolysis liquid to obtain Ganoderma lucidum exosomes.

[0010] In a third aspect of the present application, a composition is provided, wherein the active ingredients of the composition at least include Ganoderma lucidum exosomes, and the composition has any one or more of the following applications:

[0011] a) Promote apoptosis of brain glioma cells;

[0012] b) inhibiting the proliferation of glioma cells;

[0013] c) Inhibit autophagy in glioma cells;

[0014] d) Treatment of brain glioma.

[0015] The application, extraction method and composition of Ganoderma lucidum exosomes in the treatment of glioma provided in various embodiments of the present application have been proved through experiments that Ganoderma lucidum exosomes can be taken up by glioma cells and enriched in the cells; can inhibit the proliferation of glioma cells, but have no effect on the proliferation of microglial cell lines, and have no obvious cytotoxicity to normal microglial cells; have anti-glioma effects and the effect of inducing apoptosis of glioma cells; can effectively inhibit the occurrence of autophagy in glioma cells and induce glioma death through the AKT / mTOR signaling pathway and inhibiting the production of autophagic bodies in glioma cells, and at the same time have no obvious cytotoxicity to normal microglial cells at corresponding concentrations; and have good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar parts. The accompanying drawings generally illustrate various embodiments by way of example and not limitation, and together with the specification and claims, are used to illustrate the embodiments applied for. When appropriate, the same reference numerals are used throughout the drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

[0017] Figure 1 The NTA test result diagram of the size and concentration of the exosomes of Ganoderma lucidum according to Example 1 of the present application is shown;

[0018] Figure 2 It is shown that the Ganoderma lucidum exosomes treated with brain glioma cells according to Example 3 of the present application can be taken up into the brain glioma cells;

[0019] Figure 3 A graph showing the cell proliferation results after treating brain glioma and microglia with Ganoderma lucidum exosomes and Ganoderma lucidum extract according to Example 4 of the present application;

[0020] Figure 4 A schematic diagram showing the development of a tumor after intratumoral injection in a glioma CDX model according to Example 5 of the present application is shown;

[0021] Figure 5A schematic diagram showing the apoptosis of brain glioma cells induced by Ganoderma lucidum exosomes according to Example 6 of the present application is shown;

[0022] Figure 6 A graph showing the results of Western Blotting showing that the Ganoderma lucidum exosomes according to Example 7 of the present application can inhibit the expression of TM9SF1 and inhibit the autophagy-related pathway;

[0023] Figure 7 An electron microscopic result showing that the Ganoderma lucidum exosomes according to Example 8 of the present application can inhibit the occurrence of autophagosomes in brain glioma cells;

[0024] Figure 8 The LC3-Cherry dual fluorescence result display diagram shows that the Ganoderma lucidum exosomes treated brain glioma cells according to Example 9 of the present application can inhibit the occurrence of autophagosomes.

[0025] Fig. 9 It is shown that the treatment of brain glioma cells with Ganoderma lucidum exosomes according to Example 10 of the present application did not cause significant pathological tissue abnormalities in the liver, kidney, heart, lung, brain, and spleen of mice. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0027] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements.

[0028] According to the embodiments of the present application, a use of Ganoderma lucidum exosomes in any one or more of the following is provided:

[0029] a) preparing a drug for promoting apoptosis of brain glioma cells;

[0030] b) preparing a drug for inhibiting the proliferation of brain glioma cells;

[0031] c) preparing a drug for inhibiting autophagy of brain glioma cells;

[0032] d) preparing drugs for treating brain glioma.

[0033] Ganoderma exosomes are important bioactive substances in Ganoderma lucidum. They are small vesicles secreted by cells, and their diameter is usually between 30-150nm. Studies have shown that exosomes can carry a variety of specific proteins, RNA, DNA and other substances. Exosomes play an important role in regulating the balance of the intracellular and extracellular environment, signal transduction, immune response and other physiological processes.

[0034] Experimental studies have shown that exosomes can be taken up by glioma cells and have anti-glioma cell effects, and have low toxicity to microglia.

[0035] In some embodiments, the application of the drug for preparing the inhibition of autophagy in brain glioma cells at least shows: inhibiting the autophagy pathway and inhibiting the production of autophagosomes in brain glioma cells through the AKT / mTOR signal axis. Inhibiting the autophagy pathway through the AKT / mTOR signal axis. Since the autophagy pathway is inhibited through the AKT / mTOR signal axis, the production of autophagosomes in brain glioma cells can be inhibited. In some embodiments, the Ganoderma exosomes can act on autophagy pathway proteins including: LC3B1 / 2, AKT, P-AKT, mTOR, P-mTOR, p62 and / or beclin1, etc. Ganoderma exosomes can induce apoptosis of brain glioma cells by inhibiting autophagy pathway proteins and autophagy pathway-related proteins TM9SF1.

[0036] In some embodiments, the drug further comprises at least one inactive pharmaceutical ingredient. The inactive pharmaceutical ingredient may be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to common methods, the drug may be prepared into a dosage form in the form of an oral preparation, an external preparation, a suppository, or a sterile injection solution such as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0037] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0038] In some embodiments, the carriers, excipients and diluents include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0039] In certain embodiments, the medicine of the present invention can be administered to the body in a known manner. For example, it can be delivered systemically by vein. It can be optionally administered via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, routes of administration, modes of administration, etc.

[0040] In some embodiments, the drug administration subjects are humans and non-human mammals, such as rats, mice, rabbits, monkeys, etc.

[0041] According to an embodiment of the present application, a method for extracting Ganoderma lucidum exosomes is also provided, the extraction method comprising: (1) grinding the soaked Ganoderma lucidum to obtain a grinding liquid; (2) coarsely filtering the grinding liquid to obtain a coarse filtrate; (3) mixing the supernatant after centrifugation of the coarse filtrate with an enzyme preparation for enzymolysis to obtain an enzymolysis liquid; (4) removing impurities, filtering and concentrating the enzymolysis liquid to obtain Ganoderma lucidum exosomes.

[0042] In some embodiments, the soaking in step (1) is to soak the Ganoderma lucidum in PBS buffer solution. In some embodiments, the Ganoderma lucidum can be cut into pieces before soaking, and preferably the Ganoderma lucidum pieces are less than or equal to 1 cm 3 .

[0043] In some embodiments, the soaking time is 2-24 hours; preferably 6-8 hours.

[0044] In some embodiments, the enzyme preparation includes one or more of pectinase, cellulase, β-1,3-glucanase, and ligninase.

[0045] In some embodiments, the grinding process is performed at a speed of 1000-3000 rpm / min. In some embodiments, the grinding liquid is coarsely filtered using three or two layers of gauze. The coarse filtrate after coarse filtration is centrifuged at a speed of 800-2000 rpm for 5-30 min.

[0046] In some embodiments, the mass of the enzyme preparation used for enzymolysis is 0.1-3% of the mass of Ganoderma lucidum; the mass fractions of the components in the enzyme preparation are pectinase 10-30%, cellulase 30-50%, β-1,3-glucanase 5-15%, and ligninase 20-40%.

[0047] In some embodiments, the enzymatic hydrolysate is sequentially frozen, centrifuged and filtered to remove impurities. In some embodiments, the filter screen is 80-120 mesh. In some embodiments, the filtrate after impurity removal is concentrated by tangential flow filtration to obtain Ganoderma lucidum exosomes. In some embodiments, the filter diameter of the tangential flow filtration is 30-1000nm; preferably, the filter diameter of the tangential flow filtration is 30-600nm.

[0048] According to an embodiment of the present application, a composition is further provided, wherein the active ingredients of the composition at least include Ganoderma lucidum exosomes, and the composition has any one or more of the following applications:

[0049] a) Promote apoptosis of brain glioma cells;

[0050] b) inhibiting the proliferation of glioma cells;

[0051] c) Inhibit autophagy in glioma cells;

[0052] d) Treatment of brain glioma.

[0053] In some embodiments, the concentration of Ganoderma lucidum exosomes in the composition is greater than or equal to 4×10 9 Particles / mL. 4×10 9 Particles / mL means 4E+9 Particles / mL, and the concentration of Ganoderma lucidum exosomes in the composition is 4×10 9 Particles / mL can significantly kill brain glioma cells. 9 Particles / mL of Ganoderma lucidum exosomes had no obvious cytotoxicity to microglia.

[0054] Example 1

[0055] A1. Wash fresh Ganoderma lucidum and sterilize it by irradiation.

[0056] A2. Chop the cleaned fresh Ganoderma lucidum into small pieces less than 1 cm 3 of Lingzhi blocks;

[0057] A3, soak the Ganoderma lucidum block obtained in A2 in PBS for 6 hours;

[0058] A4. Transfer the Ganoderma lucidum exosomes soaked in A3 to a food processor for shearing, grinding and juicing at a speed of 3000 rpm / min.

[0059] A5. Filter the grinding liquid obtained in A4 through three layers of sterile gauze.

[0060] A6. Transfer the crude filtrate obtained in A5 to a 50 ml sterile centrifuge tube and centrifuge to remove impurities. The centrifugal speed is 1000 rpm and the centrifugal time is 20 min.

[0061] A7. Perform enzymolysis treatment on the supernatant obtained in A6 with a composite enzyme preparation of 20% pectinase, 40% cellulase, 10% β-1,3-glucanase and 30% ligninase at a ratio of 0.1-0.3% to obtain an enzymolysis solution.

[0062] A8. The enzymatic hydrolysis product obtained in A7 is transferred to a freezing and impurity removal device, and the impurities are removed by freezing centrifugal filtration, and the filter screen is 100 mesh.

[0063] A9. The filtrate obtained in A8 is concentrated by tangential flow filtration to obtain plant exosome-like vesicles with a filter diameter of 30-600 nm.

[0064] A10. Sterilize the Ganoderma lucidum exosomes obtained in A9 by irradiation.

[0065] Example 2 Particle size and electron microscopy analysis of Ganoderma lucidum exosomes

[0066] (1) Particle size analysis

[0067] 1 mL of Ganoderma lucidum exosome suspension (the suspension consists of Ganoderma lucidum exosomes and PBS suspension) was added to the sample pool, and the sample pool was placed in NanoSight (Nano-ZS) for testing and analysis, and a particle size map was drawn to obtain the following: Figure 1 As shown in the particle size distribution diagram in part B, the average diameter of Ganoderma lucidum exosomes is 155.4 nm.

[0068] (2) Transmission electron microscopy (TEM) analysis

[0069] For TEM imaging, 30 μL of the Ganoderma lucidum exosome suspension was added to the discharge treated sample grid, left horizontally to allow the exosomes to adsorb, and then the liquid was carefully blotted with filter paper. The sample was stained with 2% phosphotungstic acid dye for 2 min, and the liquid was carefully blotted with filter paper. After drying for 30 min, the sample was sent for analysis and TEM observation and photography were obtained. The transmission electron microscope micrographs are shown in the figure. Figure 1 Part A of both pictures is shown. Figure 1 Two transmission electron microscope micrographs can be obtained that are consistent with the morphology of Ganoderma lucidum exosomes.

[0070] Example 3: Detection experiment of exosome uptake in brain glioma cells

[0071] U87 cells, a glioma cell line in the logarithmic growth phase, were inoculated with a cell suspension (containing MEM medium) at 2000 / well in a Cell Carrier 96-well cell culture plate, and the cultured cell suspension was obtained by overnight attachment for 12 hours. According to the instructions of the PKH26 dye kit, the Ganoderma lucidum exosomes were incubated at 37°C for 37 minutes using the PKH26 dye kit (Dolemi, DL22065) for staining, and the concentration of the stained Ganoderma lucidum exosomes was adjusted to 4×10 9 Particles / mL.

[0072] According to the instructions of DiO live cell dye (Biyuntian, C1993s) and Hoechst nuclear dye, the cell suspension after culture was incubated at 37°C for 15 minutes to stain the membrane structure and nucleus, respectively. U87 cells were stained with the cell suspension obtained from culture, and then washed twice with PBS. The culture medium containing Ganoderma lucidum exosomes after the above staining was added, and the Ganoderma lucidum exosome channel, membrane channel and nuclear channel were photographed using the water microscope of the live cell workstation, and the corresponding Figure 2 Figure A in Figure 2 Figure B in the figure, Figure 2 Figure C in the figure is merged as Figure 2 As shown in Figure D of the present application, the only experimental variable in the experimental group is the addition of Taishan red Ganoderma lucidum exosomes. Figure 2 It can be found that Taishan red Ganoderma lucidum exosomes can be taken up by glioma cells and enriched inside the cells.

[0073] Example 4 Detection of the Anti-Glioma Cell Proliferation Effect of Ganoderma Lucidum Exosomes

[0074] The brain glioma cell lines U87 and U251 cells and the microglial cell line HMC3 in the logarithmic growth phase were inoculated with 6000 cell suspensions in 96-well cell culture plates and allowed to adhere overnight for 12 h. A blank control group (the culture medium was inoculated into the 96-well cell culture plates inoculated with glioma cells) and a Ganoderma lucidum exosome test group (the mixture of Ganoderma lucidum exosome suspension and culture medium was inoculated into the 96-well cell culture plates inoculated with glioma cells, the volume ratio of Ganoderma lucidum exosome suspension in the mixture was 10%, and the concentration of Ganoderma lucidum exosome suspension was 4×10 9Particles / mL) and a Ganoderma lucidum extract treatment group (Ganoderma lucidum extract treatment group, a mixture of Ganoderma lucidum extract and culture medium was added to a 96-well plate inoculated with glioma cells, and the volume ratio of Ganoderma lucidum extract in the mixture was 10%). The preparation method of Ganoderma lucidum extract includes: 100g of Ganoderma lucidum is first crushed, then added with 100mL of ultrapure water and soaked for 30min, and then heated to a water temperature of 100 degrees Celsius after soaking, and boiled in a water bath for 2h to obtain a mixed solution, and the mixed solution is filtered with 0.45 μm and 0.22 μm filters successively to obtain Ganoderma lucidum extract. The culture medium is MEM culture medium supplemented with 10% FBS. Nine replicates were set up, with a parallel control group of 5 wells each. The volume of Ganoderma lucidum exosome suspension and Ganoderma lucidum extract added to each well of the Ganoderma lucidum exosome test group and Ganoderma lucidum extract treatment group was the same, and the culture was continued at 37°C. Cell viability was detected at 0h, 24h, and 48h respectively; the detection method was CCK8 (10μl / well), 4h, incubated at 37°C, placed in the dark for 30 min, and the OD450 absorbance was measured by an enzyme marker to calculate the cell viability. The results were as follows Figure 3 The results shown, Figure 3 The relative cell survival rate on the vertical axis indicates the survival ratio relative to the initial cell survival number. At 0 h, the cell survival number is set to 1. After 24 h and 48 h, the cell survival number / initial cell survival number is the corresponding vertical axis value. NC-U87 means only glioma cells U87 and culture medium; PLENs-U87 means that a mixture of Ganoderma lucidum exosome suspension and culture medium is added to the culture wells containing glioma cells U87 and culture medium; Extract-U87 means that a mixture of Ganoderma lucidum extract and culture medium is added to the culture wells containing glioma cells U87 and culture medium; NC-U251 means only glioma cells U251 and culture medium medium; PLENs-U251 means that a mixture of Ganoderma lucidum exosome suspension and culture medium was inoculated into the culture wells containing glioma cells U251 and culture medium; Extract-U251 means that a mixture of Ganoderma lucidum extract and culture medium was added into the culture wells containing glioma cells U251 and culture medium; NC-HMC3 means that only microglia HMC3 and culture medium were present; PLENs-HMC3 means that a mixture of Ganoderma lucidum exosome suspension and culture medium was inoculated into the culture wells containing microglia HMC3 and culture medium; Extract-HMC3 means that a mixture of Ganoderma lucidum extract and culture medium was added into the culture wells containing microglia HMC3 and culture medium.

[0075] pass Figure 3 As can be seen from the photo on the left, the apoptosis rate of glioma cells (U87 and U251 cells) in the Ganoderma lucidum exosome test group was significantly increased compared with the blank control group and the Ganoderma lucidum extract treatment group. Figure 3From the relative cell activity curve, it can be obtained that the relative cell activity of U87 and U251 cells decreased, while the relative cell activity of the microglial cell line HMC3 did not decrease. Moreover, the Ganoderma lucidum extract added in equal proportion did not show an inhibitory effect on glioma proliferation, indicating that the Ganoderma lucidum extract has no effect on glioma cells (U87 and U251 cells).

[0076] Example 5 Detection of the anti-glioma effect of Ganoderma lucidum exosomes in vivo

[0077] Four-week-old nude mice were divided into two groups of 4 mice each, namely the blank control group and the Ganoderma lucidum exosome test group (200w (100μl) cells / mouse in the armpit) to establish the U87 cell glioma CDX in vivo xenogeneic model. After tumor formation was confirmed, 1μg / g / 48h (1μg of Ganoderma lucidum exosomes was given for every 48h of 1g of body weight) of Ganoderma lucidum exosomes were injected into the tumor within 14 days after tumor formation. The tumor size was measured and recorded every 3 days. Figure 4 As shown in part C, the tumor was dissected and taken 3-4 weeks later. Figure 4 As shown in Figure A and Figure B.

[0078] pass Figure 4 It can be seen that in the Ganoderma exosome injection group, the tumor volume was significantly smaller than that of the blank control group during the Ganoderma exosome administration.

[0079] Example 6 Detection of the effect of Ganoderma lucidum exosomes in inducing apoptosis of brain glioma cells

[0080] U87 cells, a brain glioma cell line in the logarithmic growth phase, were inoculated with 200 μl of cell suspension at 6000 / well in a 96-well cell culture plate and allowed to adhere overnight for 12 h. A blank control group and a Ganoderma lucidum exosome test group (inoculated with Ganoderma lucidum exosome suspension at a concentration of 4×10 9 Particles / mL), continue to culture for 24h, 5 μl Tunel staining solution per well, incubate at 37℃ for 30min, and take photos of cell apoptosis by living cell workstation. Figure 5 The results shown by Figure 5 It can be seen that the number of apoptotic cells in the Ganoderma lucidum exosome test group was more than that in the blank control group.

[0081] Example 7 Ganoderma lucidum exosomes inhibit the expression of TM9SF1 and inhibit the expression of autophagy-related pathway proteins

[0082] The brain glioma cell lines U87 and U251 in the logarithmic growth phase were inoculated with 2 ml of cell suspension at 200,000 cells / well in a 6-well cell culture plate and allowed to adhere overnight for 12 h. A blank control group and a Ganoderma lucidum exosome test group (inoculated with Ganoderma lucidum exosome suspension at a concentration of 4 × 109 Particles / mL), continue culturing for 24 h, add protein lysis buffer to lyse cells, 12000 rpm for 10 min, and take the supernatant;

[0083] Protein concentration was determined by BCA assay kit;

[0084] The total protein loading amount in each well was 30 μg, and an appropriate amount of 6× protein loading buffer was added and the mixture was boiled in water for 10 min;

[0085] PAGE Gel Express Kit to prepare separation gel and stacking gel:

[0086] Add an appropriate volume of protein sample and 6 μL / well of protein marker;

[0087] First adjust the voltage to 90V constant voltage, and when the bromophenol blue indicator of the electrophoresis sample passes through the separation gel, change to 110V constant voltage electrophoresis until the bromophenol blue indicator reaches the bottom of the separation gel;

[0088] According to the number of samples and the size of the target protein, cut out the PVDF membrane of the appropriate size and soak it in methanol for about 5 seconds. Soak the various materials in the transfer apparatus in the electrotransfer solution, sponge pad-filter paper-PAGE gel-PVDF membrane-filter paper-sponge pad, remove the bubbles, and transfer the membrane at 400mA constant current for 45 minutes.

[0089] After the transfer is completed, take out the PVDF membrane, put it in 53% skimmed milk powder and incubate it in a shaker at room temperature for 1 hour for blocking.

[0090] The primary antibody was prepared with 3% skim milk powder and added to the antibody incubation box, with the front side of the membrane facing up, and incubated overnight at 4°C. After the primary antibody incubation, the membrane was washed with TBST 3 times × 5 min;

[0091] The membrane was incubated in the secondary antibody at 37°C for 30 min and washed with TBST three times × 105 min;

[0092] Prepare ECL luminescent liquid, add it dropwise on the front of the PVDF membrane, and develop and fix it.

[0093] The results of Example 7 are as follows Figure 6 As shown, Figure 6 P-MTOR (phosphorylated rapamycin target protein), P-AKT (phosphorylated protein kinase B), AKT (protein kinase B), LC3B1 / 2 (human microtubule-associated protein light chain 3II), TM9SF1 (transmembrane 9 superfamily protein 1), and ACTIN (internal reference protein) in the expression of autophagy pathway proteins or autophagy pathway-related proteins, respectively. Figure 6It can be obtained that the Ganoderma lucidum exosome test group can inhibit LC3B1 / 2, P-AKT, AKT, P-mTOR autophagy pathway proteins and autophagy pathway related protein TM9SF1 to induce apoptosis of brain glioma cells. It shows that the Ganoderma lucidum exosomes of the present application can act on brain glioma cells through the AKT / mTOR signaling pathway.

[0094] Example 8 Electron microscopic observation of Ganoderma lucidum exosomes inhibiting the occurrence of autophagosomes

[0095] U87 cells, a brain glioma cell line in the logarithmic growth phase, were inoculated with 2 ml of cell suspension at 20,000 cells / well in a 6-well cell culture plate and allowed to adhere overnight for 12 h. A blank control group and a Ganoderma lucidum exosome test group (4 × 10 9 Particles / mL), and continue to culture for 24h. Scrape the iron wall cells and add electron microscope fixative. After the sample is fixed, add it to the discharge-treated sample grid, let it stand horizontally to allow the exosomes to adsorb, and then carefully absorb the liquid with filter paper. Stain with 2% phosphotungstic acid dye for 2 min, and carefully absorb the liquid with filter paper. After drying for 30 min, send the sample for analysis, and observe and take pictures with TEM, as shown below. Figure 7 Transmission electron microscopy micrographs shown.

[0096] Example 9: Dual fluorescence detection of Ganoderma lucidum exosomes by mRFP-cherry-LC3 (autophagy double-labeled adenovirus) to inhibit the autophagy process

[0097] mRFP-cherry-LC3 is LC3 protein (microtubule-related protein light chain 3) labeled with mRFP fluorescent protein (monomer red fluorescent protein) and cherry fluorescent protein (cherry fluorescent protein). U87 cells were infected with lentivirus for autophagy detection of mRFP-cherry-LC3, and a stable cell line was constructed. CellCarrier 96-well plates were inoculated with 2000 cells / well and adhered to the wall overnight for 12 hours; a blank control group and a Ganoderma lucidum exosome test group (inoculated with Ganoderma lucidum exosome suspension, the concentration of Ganoderma lucidum exosome suspension was 4×10 9 Particles / mL), and continue to culture for 24 hours. Add 5 μl of DAPI working solution to each well, and use the water microscope of the living cell workstation to take pictures of the cell nuclear channel to obtain the following Figure 8 As shown in Group A, the mRFP fluorescent protein-autophagosome channel was photographed and obtained as follows Figure 8 As shown in Group B, the cherry fluorescent protein channel was photographed to obtain Figure 8 As shown in group C, the cell nucleus channel, mRFP fluorescent protein-autophagosome channel and cherry fluorescent protein channel, the merged photographic results are as follows Figure 8 As shown in Group D.

[0098] pass Figure 7 and Figure 8 It can be found that the treatment with Taishan red Ganoderma lucidum exosomes significantly reduced the number of autophagosomes in U87 cells, which suggests that Taishan red Ganoderma lucidum exosomes can significantly inhibit the occurrence of autophagy and induce cell death.

[0099] Example 10 In vivo safety test of Ganoderma lucidum exosomes against brain glioma

[0100] Four-week-old nude mice were divided into two groups, 6 in each group, named blank control group and Ganoderma lucidum exosome test group (200w (100μl) cells / mouse in the armpit), to establish an in vivo xenogeneic model of U87 cytoplasmic tumor CDX. After tumor formation was confirmed, 1μg / g / 48h (1μg of Ganoderma lucidum exosomes was given for every 48h of 1g body weight) of Ganoderma lucidum exosomes were injected into the tumor within 14 days of tumor formation. After 3-4 weeks, the liver, kidney, heart, lung, brain and spleen were dissected and HE staining was performed to evaluate the histopathological changes.

[0101] Get as Fig. 9 The results shown, Fig. 9 It can be seen that the Taishan red Ganoderma lucidum exosomes used in this treatment of brain glioma did not cause significant pathological changes in the liver (LIVER), kidney (Kidney), heart (Heart), lung (Lung), brain (Brain), and spleen (Spleen), and the treatment is safe.

[0102] In addition, although exemplary embodiments have been described herein, the scope includes any and all embodiments based on the present application with equivalent elements, modifications, omissions, combinations (e.g., various embodiments intersecting schemes), adaptations or changes. The elements in the claims are to be interpreted broadly based on the language adopted in the claims, and are not limited to the examples described in this specification or during the practice of this application, and the examples are to be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered as examples only, and the true scope and spirit are indicated by the following claims and the full scope of their equivalents.

[0103] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more schemes thereof) may be used in combination with each other. For example, a person of ordinary skill in the art may use other embodiments when reading the above description. In addition, in the above-mentioned specific embodiments, various features may be grouped together to simplify the present application. This should not be interpreted as an intention that a feature of an application that does not require protection is necessary for any claim. On the contrary, the subject matter of the present application may be less than all the features of an embodiment of a particular application. Thus, the following claims are incorporated herein in the specific embodiments as examples or embodiments, wherein each claim is independently a separate embodiment, and it is considered that these embodiments may be combined with each other in various combinations or arrangements. The scope of the present invention should be determined with reference to the appended claims and the full scope of equivalent forms granted by these claims.

[0104] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. Application of Ganoderma lucidum exosomes in any one or more of the following: a) preparing a drug for promoting apoptosis of brain glioma cells; b) preparing a drug for inhibiting the proliferation of brain glioma cells; c) preparing a drug for inhibiting autophagy of brain glioma cells; d) preparing drugs for treating brain glioma; The method for extracting Ganoderma lucidum exosomes comprises: (1) grinding the soaked Ganoderma lucidum to obtain a grinding liquid; (2) coarsely filtering the grinding liquid to obtain a coarse filtrate; (3) The supernatant after centrifugation of the crude filtrate is mixed with an enzyme preparation for enzymolysis to obtain an enzymolysis solution; (4) After the enzymatic hydrolysate is removed from the enzymatic hydrolysate, filtered, and concentrated, Ganoderma lucidum exosomes are obtained.

2. The use according to claim 1, characterized in that: The application of the drug for preparing the drug for inhibiting the autophagy of brain glioma cells in the treatment of brain glioma at least shows: inhibiting the autophagy pathway and inhibiting the generation of autophagosomes of glioma cells through the AKT / mTOR signal axis.

3. The use according to claim 1, characterized in that: The medicament further comprises at least one pharmaceutically inactive ingredient.

4. The use according to claim 1, characterized in that: The drug administration subjects are humans and non-human mammals.

5. The use according to claim 1, characterized in that: The soaking in step (1) is to soak the Ganoderma lucidum in a PBS buffer solution; the soaking time is 2-24 hours; the enzyme preparation in step (3) includes one or more of pectinase, cellulase, β-1,3-glucanase and ligninase.

6. The use according to claim 1, characterized in that: The mass of the enzyme preparation used for the enzymolysis in step (3) is 0.1-3% of the mass of Ganoderma lucidum; the mass fractions of the components in the enzyme preparation are pectinase 10-30%, cellulase 30-50%, β-1,3-glucanase 5-15%, and ligninase 20-40%.

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