Mushroom-derived immunostimulant and derivative thereof, DC vaccine and preparation method thereof

Abstract

The invention provides a shiitake mushroom source immunologic stimulant, a shiitake mushroom source immunologic stimulant derivative, a DC vaccine and a preparation method, and provides a shiitake mushroom source immunologic stimulant preparation method, the method comprises purification, carboxylation and other links, and the extracted carboxymethyl-beta-glucan can be used as an immunologic stimulant through experimental verification. Therefore, the shiitake mushroom source immunologic stimulant extracted based on the method can still have strong immunocompetence after being improved and processed, and can cope with various environments. Meanwhile, the invention also develops a lentinus edodes-derived immunostimulant derivative which comprises tumor antigen while stimulating APC cells, and is more beneficial to the tumor antigen presentation function of APC.

Description

Technical Field

[0001] The present invention relates to the field of immunostimulants, and particularly to an immunostimulant and derivatives derived from Lentinula edodes, a DC vaccine, and a preparation method thereof. Background Art

[0002] Since the traditional Chinese medicine era, fungi have been an important source of drug precursors. With the progress of extraction techniques for various natural drugs and the clarification of their active ingredients, fungal derivatives have been proven to play important anti-inflammatory and anti-tumor roles through multiple pathways such as immunity and metabolism.

[0003] To date, fungi and their derivatives have become an important part of the food and pharmaceutical industries. Among them, a particularly concerned component is β-glucan. β-glucan (β-Glucan, β-Glu) is the main component of the basic skeleton of the fungal cell wall. It is quite conserved during evolution. Whether it is a simple unicellular fungus (such as yeast) or a large fungus (Lentinula edodes), β-glucan is widely present in its cell wall structure. These conserved molecules can act as pathogen-associated molecular patterns (PAMPs). The mammalian innate immune system can effectively recognize them as non-self molecules and help eliminate microbial pathogens. The specific receptors for β-glucan are Toll-like receptors (TLRs) and C-type lectin-like receptors: among the latter, dectin-1 is the best-characterized receptor, mainly expressed on the surface of monocytes / macrophages, neutrophils, dendritic cells, and natural killer (NK) cells. Intracellular signal transduction follows the receptor recognition step, leading to the activation of nuclear factor k-light-chain-enhancer (NF-κB), the transcription of inflammatory immune genes, the production of cytokines, nitric oxide (NO), and reactive oxygen species (ROS).

[0004] β-glucan can also regulate inflammatory IL-2 and IL-11, and upregulate IFN-1 and IFN-γ after viral attack. Therefore, β-glucan has also been applied to the preparation of vaccines against viruses such as SARS and COVID-19. After being ingested as food or supplements, β-glucans reach the small intestine without any gastric digestion; they are internalized by intestinal epithelial cells and / or macrophages and presented to immune cells in Peyer's patches and distal lymphoid organs. In lymph nodes, β-glucans stimulate monocytes / macrophages, neutrophils, dendritic cells, and natural killer cells, thus achieving the effect of stimulating the body's immunity.

[0005] Moreover, due to its good safety and the property of stimulating APCs, β-glucan has been applied in the research and development field of small molecule drugs. A self-adjuvant nano-vaccine (CPBG-Al@OVA) was constructed by functionalizing aluminum hydroxide with β-1,3-glucan. β-1,3-glucan can recognize pattern recognition receptors through Dectin-1, thus achieving the ability to accurately and efficiently deliver antigens. Carboxymethyl phosphorylated β-1,3-glucan (CPBG) has been synthesized and optimized to achieve excellent adjuvanticity while maintaining water solubility. Subsequently, this experiment demonstrated that CPBG-Al@OVA could effectively drain into lymph nodes in vivo and play an antigen-presenting role.

[0006] However, most β-glucans are connected through carbon atoms at positions 1 and 3 of glucose. However, there can also be arrangements of glucose connected at 1,4 or 1,6 on the backbone of β-glucan. These two arrangements different from the 1,3 connection cause a bias in the running of the glucan chain: that is, branches deviating from the 1,3 backbone are formed. Among them, β-1,3-glucan can bind to the Dectin-1 receptor on the surface of APC cells to exert immune efficacy. Therefore, the content of β-1,3-glucan in various fungi determines the level of immune stimulation ability. The content of β-glucan in common yeast is generally 5%-15%, and there are few 1,3 bonds and a large number of 1,6 bond branches in its structure, so its immune efficacy is greatly reduced.

[0007] All in all, β-glucan has very wide applications in immune regulation and diseases. However, how to extract β-glucan from suitable fungi will directly affect the subsequent research and application of β-glucan.

[0008] Glioma, as the most common "brain cancer", has an unsatisfactory prognosis under the current mainstream treatment plan (surgical resection + postoperative radiotherapy and chemotherapy + comprehensive treatment methods). Its median survival time is only 14.6 months, and the 5-year survival rate is only 4.8% or even lower. Therefore, new treatment methods have always been a research hotspot in the field of glioma internationally.

[0009] Dendritic cells (DCs) are the only professional antigen-presenting cells (APCs) that can induce the maturation of naive T cells. The effective activation of DCs is a key link for various different forms of tumor vaccine products to achieve effective memory immune induction. Different from microbial vaccines, tumor vaccines have the characteristics of weak antigen heterogenicity and low immune activation efficacy. Therefore, in the process of preparing tumor vaccines, the activation of DCs depends on the application of exogenous adjuvants. Adjuvants mainly have two functions: one is to promote DCs to uptake antigen components in vaccine preparations; the other is to directly induce DCs to transform into a mature phenotype, so as to efficiently present antigens to CD4 + 、CD8+ T lymphocytes present antigens, and this function of adjuvants depends on the recognition of foreign substances by the innate immune system of the human body. Therefore, microbial components with strong foreign signals are often important sources for the development of adjuvants.

[0010] Currently, there are already cases of developing DC vaccines loaded with A2B5-positive cell antigens. For example, Chinese patent application CN116496986A discloses a dendritic cell targeting A2B5-positive tumors. In this scheme, after screening out A2B5-positive cells, the tumor is first inactivated by X-ray irradiation (6 Gy), and then the tumor cells are disrupted by a grinder. This process will lose some water-insoluble antigens. Secondly, this scheme uses IFN-γ and LPS to stimulate the maturation of DC vaccines. LPS cannot form a tight link with the surface of DC cells, and LPS may spread to other cells after intravenous injection of the vaccine, resulting in "collateral damage" to T cells and potentially leading to a series of autoimmune diseases in severe cases.

[0011] CN101481677A discloses a method for stimulating the maturation of dendritic cells in vitro. After isolating tumor stem cells from the human glioma U87 cell line, they are irradiated with a dose of 6 Gy, and then repeatedly frozen and thawed to obtain tumor antigens for stimulating the maturation of DC cells. However, this method will lose some water-soluble antigens during repeated freezing and thawing and cannot provide complete antigens like live cells.

[0012] Different from these schemes, β-glucan can be used as an immune stimulant for the preparation of DC vaccines. Currently, there are also application cases of developing β-glucan as a vaccine adjuvant. For example, Chinese patent application CN102600467A discloses a method for inducing the maturation of DC pulsed vaccines by soluble β-glucan in vitro, but the production process of β-glucan is not disclosed in this scheme, nor is the configuration of "β-glucan" analyzed and verified.

[0013] In summary, there are still gaps in the current research on the preparation of β-glucan and its application in the field of DC vaccines. Summary of the Invention

[0014] The purpose of the present invention is to provide an immunostimulant and derivative derived from Lentinula edodes, a DC vaccine, and a preparation method. An immunostimulant that can stimulate APC cells is obtained by extracting β-glucan from Lentinula edodes. Based on this immunostimulant, an immunostimulant derivative derived from Lentinula edodes that can kill tumor cells and stimulate APC cells can be prepared, and an adjuvant for a DC vaccine that preserves A2B5-positive antigens can be prepared based on the immunostimulant derivative derived from Lentinula edodes to stimulate T cells to achieve the killing of glioma tumor cells.

[0015] To achieve the above objectives, in a first aspect, the present technical solution provides a method for preparing an immune stimulant derived from Lentinula edodes, comprising the following steps:

[0016] S1: Extract Lentinan: Add Lentinula edodes medicinal materials to purified water and extract in a high-temperature environment to obtain an extract. After cooling the extract to room temperature, add ethanol for alcohol precipitation to obtain a precipitate. After centrifuging and collecting the precipitate, perform ultrafiltration treatment to obtain lentinan;

[0017] S2: Prepare an immune stimulant derived from Lentinula edodes: Suspend lentinan in ethanol and add chloroacetic acid to obtain a mixed solution. After magnetically stirring the mixed solution, collect the precipitate. Wash the precipitate with ethanol and then perform dialysis to obtain a dialysate. Concentrate and lyophilize the dialysate to prepare an immune stimulant derived from Lentinula edodes.

[0018] The immune stimulant derived from Lentinula edodes prepared by this solution is carboxymethyl-β-glucan. The content of β-glucan in Lentinula edodes exceeds 50% and most of it is β-1,3-glucan. Therefore, this solution selects Lentinula edodes as the fungal source to extract carboxymethyl-β-glucan as an immune stimulant. The carboxymethyl-β-glucan extracted by this solution can be used as an immune stimulant through experimental verification. The reaction formula for carboxymethylating lentinan to obtain an immune stimulant derived from Lentinula edodes in this solution is as follows:

[0019]

[0020] In the step of "S1. Extract Lentinan", weigh Lentinula edodes medicinal materials and extract twice. Each time, add purified water and extract at 100 °C for 2 h. Combine and concentrate the two extracts. After cooling to room temperature, add ethanol for alcohol precipitation. After the alcohol precipitation ends, centrifuge and collect the precipitate. Add water to fully dissolve the precipitate and transfer it to a ceramic membrane storage tank. After ultrafiltration through a 50 nm ceramic membrane, collect the permeate and transfer it to a spiral wound membrane storage tank. After ultrafiltration through a PW spiral wound membrane, collect the retentate, concentrate and lyophilize to obtain lentinan.

[0021] In the step of "Prepare an immune stimulant derived from Lentinula edodes", weigh lentinan, suspend it in ethanol, add chloroacetic acid with the same mass, place it on a magnetic stirrer and react for 2 h. Then collect the precipitate, wash the precipitate twice with ethanol to obtain the precipitate. Take the precipitate, dissolve it in water and centrifuge. Transfer the supernatant to a dialysis bag for dialysis. Collect the dialysate, concentrate and lyophilize to obtain carboxymethyl β-glucan.

[0022] In a second aspect, the present solution provides an immune stimulant derived from Lentinula edodes, which is prepared according to the above method for preparing an immune stimulant derived from Lentinula edodes and is carboxymethyl-β-glucan extracted from Lentinula edodes.

[0023] The applicant would like to emphasize that although there are research reports on the biological properties of β-glucan, especially its anti-inflammatory, antioxidant and anti-tumor effects, the research conclusions related to β-glucan in the prior art are often inconsistent or even contradictory. Therefore, although the immune stimulation of β-glucan on the human body is clear, the specific and complete mechanism is still not clear. The research team of this application extracted β-glucan from Lentinula edodes to obtain an immune stimulant of Lentinula edodes origin, and after completely exploring its immune stimulation effect on the human body, it was found that the immune stimulant of Lentinula edodes origin has a stimulating effect on APCs.

[0024] In some embodiments, the immune stimulant of Lentinula edodes origin synthesized by the above method is used to stimulate APCs. Specifically, the immune stimulant of Lentinula edodes origin alone can cause antigen-presenting cells to transform into a mature phenotype, and has a stronger ability to present antigens and activate naive T cells.

[0025] In some embodiments, the concentration of the immune stimulant of Lentinula edodes origin for stimulating APCs is 0 - 2000 μg / ml. Preferably, the concentration of the immune stimulant of Lentinula edodes origin is 1000 μg / ml.

[0026] In addition, due to the poor solubility, permeability and stability of drugs, a large number of orally administered anti-cancer / cytosine drugs are difficult to enter the systemic circulation to exert their efficacy. However, the immune stimulant of Lentinula edodes origin provided in this solution has good water solubility, and this carboxymethyl-β-glucan also exhibits high viscosity at a very low concentration (1%), and is stable within a wide pH range, which perfectly adapts to the pH environment of the gastrointestinal tract. Therefore, the carboxymethyl-β-glucan extracted in this solution is also expected to be used as a mainstream oral drug carrier.

[0027] In the third aspect, the present solution provides a preparation method of a derivative of the immune stimulant of Lentinula edodes origin, including the following steps: The immune stimulant of Lentinula edodes origin prepared in the first aspect, BOP and triethylamine are added to an organic solvent containing dissolved poly-D-lysine and reacted for a period of time, and then dialyzed, and the dialysate is freeze-dried to obtain a derivative of the immune stimulant of Lentinula edodes origin, wherein the derivative of the immune stimulant of Lentinula edodes origin is β-glucan-poly-D-lysine.

[0028] In some embodiments, poly-D-lysine is dissolved in DMSO as the organic solvent containing dissolved poly-D-lysine, and then the immune stimulant of Lentinula edodes origin, BOP and triethylamine are added to the organic solvent containing dissolved poly-D-lysine and reacted for a period of time. The reaction solution is filled into a dialysis bag and dialyzed with pure water to obtain a dialysate, and the dialysate is concentrated and freeze-dried to obtain β-glucan-poly-D-lysine.

[0029] In a specific embodiment, poly-D-lysine was weighed and dissolved in DMSO, and carboxymethylated dextran, BOP and 60 mg of triethylamine were added. The reaction was carried out at 40 °C for 2 h, and then it was loaded into a dialysis bag for dialysis (cut-off molecular weight 30 kDa), dialyzed in pure water for 24 h, and the dialysis solution was collected and freeze-dried to obtain β-dextran-poly-D-lysine.

[0030] More specifically, 150 mg of ε-poly-D-lysine was weighed and dissolved in 1.5 mL of DMSO, 250 mg of carboxymethylated β-dextran, 100 mg of BOP and 60 mg of triethylamine were added. The reaction was carried out at 40 °C for 2 h, and then it was loaded into a dialysis bag for dialysis (cut-off molecular weight 10 kDa), dialyzed in pure water for 24 h, and the dialysis solution was collected and freeze-dried to obtain 103 mg of the product, which was β-dextran-poly-D-lysine.

[0031] Fourthly, the present solution provides a lentinula edodes-derived immunostimulant derivative, which is prepared according to the preparation method of the lentinula edodes-derived immunostimulant derivative mentioned in the third aspect. The lentinula edodes-derived immunostimulant derivative is β-dextran-poly-D-lysine, which is formed by coupling carboxymethyl-β-dextran derived from lentinula edodes and poly-D-lysine.

[0032] In some embodiments, the lentinula edodes-derived immunostimulant derivative is used to kill tumor cells. Specifically, the lentinula edodes-derived immunostimulant derivative of the present solution can adhere to the surface of tumor cells, and can also enhance the antigen-presenting ability of antigen-presenting cells and the ability to activate naive T cells. In some embodiments, for 10 6 the concentration of the lentinula edodes-derived immunostimulant derivative selected for tumor cells is less than 1000 μg / ml. Preferably, for 10 6 the concentration of the lentinula edodes-derived immunostimulant derivative selected for tumor cells is 500 μg / ml.

[0033] Fifthly, the present solution provides a preparation method of a DC vaccine based on a lentinula edodes-derived immunostimulant derivative, including the following steps:

[0034] S1: Isolate PBMC from human peripheral blood;

[0035] S2: Prepare A2B5-positive glioma cells;

[0036] S3: Culture PBMC in a medium to obtain immature DC cells, and co-culture the A2B5-positive glioma cells and the lentinula edodes-derived immunostimulant derivative in the fourth aspect in the medium to obtain mature DC cells as the DC vaccine.

[0037] The carboxymethyl-β-glucan in the immunostimulatory agent derivative derived from Lentinus edodes can bind to the Dectin-1 receptor on the surface of dendritic cells and trigger a series of signal transduction pathways within dendritic cells, leading to the activation of immune cells and the release of cytokines. For DC cells, after the carboxymethyl-β-glucan activates DC cells, it can enhance the antigen presentation ability of DC cells. In the process of vaccine preparation, the antigen presentation function of DC cells is a key link. A better antigen presentation ability means that tumor antigens (such as A2B5-positive glioma cell antigens) can be presented to T lymphocytes more effectively, thereby triggering a stronger anti-tumor immune response. At the same time, compared with β-glucans from other sources, the carboxymethyl-β-glucan derived from Lentinus edodes can be ingested by the human body in the long-term diet, so that the carboxymethyl-β-glucan from Lentinus edodes has better safety performance in the human body.

[0038] Regarding the acquisition of immature DC cells: Specifically, in step S3, after culturing PBMC in a medium for a period of time, the supernatant is obtained, the adherent cells in the supernatant are collected, and the adherent cells are added to a 1640 medium for culturing to obtain immature DCs.

[0039] In some embodiments, the 1640 medium for culturing adherent cells contains 15% serum, 100 ng / ml rhGM-CSF, 100 ng / ml rhIL-4, and immature DCs are obtained by culturing in the 1640 medium for 5 days.

[0040] Regarding the culture of PBMC cells, the collected PBMC is cultured in a 1640 medium at 37 °C and 5% CO 2 for 4 h in the 1640 medium under the conditions.

[0041] In addition, in step S3, corresponding to 1*10 6 A2B5-positive glioma cells, the concentration of β-glucan-poly-D-lysine is less than 1000 μg / ml. The research team of this application verified that when the concentration of β-glucan-poly-D-lysine is greater than 1000 μg / ml, the number of dead tumor cells increases significantly. It can be speculated that β-glucan-poly-D-lysine has a killing effect on normal cells when exceeding this concentration. Therefore, this scheme specifically sets the concentration of β-glucan-poly-D-lysine to be less than 1000 μg / ml.

[0042] Corresponding to 1*10 6For A2B5 positive glioma cells, the concentration of β-glucan-poly-D-lysine is 25, 50, 100, 200, 500, 1000 ug / ml. Preferably, the concentration of β-glucan-poly-D-lysine is 500 ug / ml, at which the β-glucan-poly-D-lysine will not lose normal cells, while also ensuring that the maturation of immature DC cells is greatly promoted.

[0043] In addition, in this protocol, A2B5-positive glioma cells and β-glucan-poly-D-lysine were added to the culture medium and placed at 37°C and 5% CO 2 Under the same conditions, co-culture for 1 to 48 hours to obtain mature DC cells as DC vaccine.

[0044] Regarding the acquisition of PBMC:

[0045] In step S1, human peripheral blood is obtained in a heparin anticoagulant tube and PBS is added to dilute the blood at a ratio of 1:1. The diluted blood is slowly added along the wall of the test tube above the lymphocyte separation solution so that the diluted blood overlaps the lymphocyte separation solution and forms a clear interface with the lymphocyte separation solution. After centrifugation at room temperature, the middle white film-like mononuclear cell layer is carefully aspirated to aspirate all the mononuclear cells as much as possible, and PBS is added with a volume of 5 times or more to wash twice, each centrifugation is performed at 1500rpm for 10 minutes to obtain PBMC.

[0046] In a specific embodiment, 5 mL of fresh peripheral blood is added to PBS to dilute the blood at a ratio of 1:1, and 5 mL of lymphocyte separation solution is taken into a 15 mL sterile centrifuge tube for use. After aspirating the diluted blood, it is slowly added along the wall of the test tube 1 cm above the lymphocyte separation solution, so that the diluted blood overlaps on the lymphocyte separation solution and forms a clear interface with the lymphocyte separation solution. Centrifuge at room temperature at 2000 rpm for 20 minutes. At this time, 5 layers are formed in the centrifuge tube: the top is plasma, and between the plasma layer and the lymphocyte separation solution is a lymphocyte layer in the form of a white film. Carefully aspirate the mononuclear cell layer in the middle in the form of a white film, and try to aspirate all the mononuclear cells. Add PBS with a volume of 5 times or more and wash twice, each time centrifuging at 1500 rpm for 10 minutes.

[0047] In step S2, the glioma tissue is inactivated and lysed to obtain a single cell suspension, the single cell suspension is magnetically sorted, and the A2B5-positive cells are irradiated with X-rays (25 Gy 1 min), thereby obtaining attenuated A2B5-positive cells as A2B5-positive glioma cells.

[0048] In a sixth aspect, the DC vaccine based on shiitake mushroom-derived immunostimulant derivatives prepared according to the above-mentioned preparation method has a killing effect on glioma cells.

[0049] Compared with the prior art, the present technical solution has the following characteristics and beneficial effects:

[0050] The present solution proposes a preparation method of an immune stimulant derived from Lentinula edodes, which includes purification, carboxylation and other steps. The extracted carboxymethyl-β-glucan has been experimentally verified to be used as an immune stimulant. Moreover, the immune stimulant derived from Lentinula edodes extracted based on this method can still have strong immune activity after being modified and processed, and can cope with various environments. At the same time, the present invention also develops a derivative of the immune stimulant derived from Lentinula edodes, which contains tumor antigens while stimulating APC cells, and is more conducive to the tumor antigen presentation function of APCs.

[0051] The present solution selects the derivative of the immune stimulant derived from Lentinula edodes to prepare a DC vaccine against glioma tumor cells. Among them, the carboxymethyl-β-glucan derived from Lentinula edodes can bind to the Dectin-1 receptor on the surface of DC cells, activate DC cells and enhance their antigen presentation ability, effectively present glioma antigens to T lymphocytes, stimulate a strong anti-tumor immune response, directly act on glioma cells, inhibit their growth and spread, thereby playing a killing role in glioma, and providing an innovative and potential immunotherapy approach for the treatment of glioma.

[0052] In addition, in the cell culture step, the culture of PBMCs needs to be carried out in RPMI 1640 medium at 37 °C and 5% CO 2 for 4 hours. This specific environment provides a suitable bed for the initial growth and stability of PBMCs, ensures cell viability and the potential for subsequent differentiation, and can accurately induce the differentiation of PBMCs into immature DC cells, ensuring that the immature DC cells have good basic immunomodulatory functions; in addition, A2B5-positive glioma cells and carboxymethyl-β-glucan-poly-D-lysine are added to the medium and co-cultured at 37 °C and 5% CO 2 for 1-48 hours to obtain mature DC cells as the DC vaccine. It is preferably co-cultured for 48 hours. During this process, the temperature of 37 °C and the concentration of 5% CO 2 simulate the in vivo physiological environment, enabling the cells to stably interact and transform functions. The determination of the co-culture time has been rigorously verified. 48 hours can not only ensure that A2B5-positive glioma cells fully release antigens and are effectively taken up by DC cells, but also allow carboxymethyl-β-glucan-poly-D-lysine to fully play the role of an adjuvant, activate DC cells and promote their maturation, so that the mature DC vaccine can accurately identify and efficiently kill glioma cells. This strict control of the culture conditions and co-culture conditions lays a solid foundation for the immunotherapy of glioma, and is expected to improve the treatment effect while minimizing the risk of cell function abnormalities or treatment failure caused by inappropriate conditions, bringing new hope and possibilities for the recovery of glioma patients. Description of the Drawings

[0053] Figure 1 It is the one-dimensional nuclear magnetic resonance diagram of carboxymethyl-β-glucan.

[0054] Figure 2 It is the two-dimensional nuclear magnetic resonance diagram of carboxyl-β-glucan.

[0055] Figure 3 It is the molecular weight and surface potential diagram of β-glucan-polylysine.

[0056] Figure 4 It is the hydrogen spectrum of β-glucan-polylysine.

[0057] Figure 5 It is the carbon spectrum of β-glucan-polylysine.

[0058] Figure 6 It is the COSY spectrum of β-glucan-polylysine.

[0059] Figure 7 It is the TOCSY spectrum of β-glucan-polylysine.

[0060] Figure 8 It is the HSQC spectrum of β-glucan-polylysine.

[0061] Figure 9 It is the ROESY spectrum of β-glucan-polylysine.

[0062] Figure 10 It is the HMBC spectrum of β-glucan-polylysine.

[0063] Figure 11 It is the figure of isolated PBMC observed under the microscope.

[0064] Figure 12 It is the figure of A2B5-positive glioma cells under the microscope after irradiation with 25 Gy.

[0065] Figure 13 It is the figure of immature DC observed under the microscope after 5 days of culture.

[0066] Figure 14 It is the figure of the mixed system of mature DC+G-PL observed under the microscope after adding G-PL to immature DC cells and culturing for 48 h.

[0067] Figure 15 It is the schematic diagram of confocal monitoring of the attachment of FITC-labeled G-PL to the membrane of A2B5-positive glioma cells.

[0068] Figure 16It is a schematic diagram showing the attachment of G-PL to the cell membrane of A2B5-positive glioma observed by scanning and transmission electron microscopy.

[0069] Figure 17 It is a schematic diagram of the fluorescence intensity after co-incubation of FITC-labeled G-PL with the tumor for different times.

[0070] Figure 18 It is a result graph showing the effects of different concentrations of β-glucan on CD83 and CD86.

[0071] Figure 19 It is a result graph showing the expression of CD83 and CD86 analyzed by flow cytometry in the control group, tumor group, and glucan group when using 1000 μg / ml β-glucan.

[0072] Figure 20 It is based on Figure 19 The result graph of the statistical sample analysis.

[0073] Figure 21 It is a schematic diagram of the difference in the ability of DC cells in different experimental groups to secrete TNF-α and IFN-β when using 1000 μg / ml β-glucan.

[0074] Figure 22 It is a flow cytometry result analysis graph of the stimulation of T cell secretion by DC vaccines in different experimental groups when using 1000 μg / ml β-glucan.

[0075] Figure 23 It is a result analysis graph of the degree of T cell division in different experimental groups when using 1000 μg / ml β-glucan.

[0076] Figure 24 It is a schematic diagram of the killing results of different concentrations of β-glucan-polylysine on tumor cells.

[0077] Figure 25 It is a flow cytometry sample analysis result graph of the stimulating effects on CD83 and CD86 in different experimental groups when using 500 μg / ml β-glucan-polylysine.

[0078] Figure 26 It is a schematic diagram of the difference in the ability of DC cells in different experimental groups to secrete TNF-α and IFN-β when using 500 μg / ml β-glucan-polylysine.

[0079] Figure 27 It is a result analysis graph of the degree of T cell division in different experimental groups when using 500 μg / ml β-glucan-polylysine.

[0080] Figure 28It is a diagram showing the test results of the ability of whole-cell tumor adjuvants prepared with different concentrations of G-PL to activate DC cells in vitro.

[0081] Figure 29 It is a schematic diagram of the results of the differences in the ability of different experimental groups to stimulate DC cells to secrete TNF-a and IFN-b.

[0082] Figure 30 It is a schematic diagram of the results of DC cells in different experimental groups activating T cells.

[0083] Figure 31 It is a schematic diagram of the in vivo homing phenomenon. Detailed implementation manners

[0084] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present invention.

[0085] I. Preparation of an immune stimulant derived from Lentinus edodes:

[0086] (1) Extraction of lentinan:

[0087] Weigh 2 kg of Lentinus edodes medicinal materials, extract twice, add 20 kg of purified water each time, and extract at 100 °C for 2 h. Combine the two extraction solutions and concentrate them to 300 ml. After cooling to room temperature, add 750 ml of ethanol for alcohol precipitation. After the alcohol precipitation is completed, centrifuge at 4000 r / min for 5 minutes, collect the precipitate, dissolve it fully with water, transfer it to a ceramic membrane storage tank, ultrafilter it through a 50-nm ceramic membrane, collect the permeate and transfer it to a spiral wound membrane storage tank, ultrafilter it through a PW spiral wound membrane, and collect the retentate, concentrate and freeze-dry it to obtain 20 g of lentinan.

[0088] (2) Carboxymethylation of lentinan

[0089] Weigh 20 g of lentinan, suspend it in 40 ml of ethanol, add 40 ml of chloroacetic acid with the same mass, place it on a magnetic stirrer and react at 60 °C and 200 r / min for 2 h. Subsequently, collect the precipitate and wash it twice with 20 ml of ethanol. Dissolve the precipitate in water and centrifuge at 5000 r / min for 5 min. Transfer the supernatant into a dialysis bag and dialyze it for 24 h (cut-off molecular weight 500 Da). Collect the dialysate, concentrate and freeze-dry it to obtain 7.9 g of carboxymethyl β-glucan.

[0090] Perform physical and chemical property detection on the synthesized product of this specific embodiment, and the detection results are as Figure 1 and Figure 2 shown. Figure 1 It is the one-dimensional nuclear magnetic resonance diagram of carboxymethyl β-glucan.Figure 2 It is the two-dimensional nuclear magnetic resonance diagram of carboxymethyl β-glucan. For carbohydrate compounds, their nuclear magnetic resonance spectra will show obvious characteristic signals. For example, the protons at the anomeric position generally appear in the range of 4.2 - 5.8 ppm in the hydrogen spectrum. Among them, those with α-configuration are generally greater than 4.8 ppm, while those with β-configuration are generally in the range of 4.2 - 4.8 ppm. In the carbon spectrum, the anomeric carbon of carbohydrate substances with α-configuration will be in the range of 98 - 103 ppm, and that with β-configuration will be in the range of 103 - 106 ppm. From Figure 2 it can be seen that after the glucan of the lentinan polysaccharide extracted by this scheme is carboxymethylated, its solubility in heavy water is significantly improved. In the nuclear magnetic resonance hydrogen spectrum, the characteristic proton signals of the polysaccharide in the range of 3.5 - 5.0 ppm can be clearly observed. Among them, the anomeric proton signal of the sugar ring exactly overlaps with the solvent (HDO) peak at 4.79 ppm and can be identified with the help of the subsequent HSQC spectrum. The remaining proton signals in the spectrum should come from the corresponding groups of the sugar ring and carboxymethyl. Combining the carbon spectrum and HSQC spectrum, first, the signals at 4.82 / 106.6 ppm corresponding to the hydrogen / carbon at the anomeric position (i.e., position 1) of the sugar ring can be observed. According to the aforementioned chemical shift rules, it shows that the anomeric configuration of this sugar ring is β-type, that is, the lentinan polysaccharide extracted by this scheme is β-glucan; with the help of the COSY spectrum, the proton signals at positions 2, 3, and 4 in the sugar ring can be attributed in sequence. For the attribution of the hydrogen signals at positions 5 and 6, it can be completed through the correlation signals between the hydrogens at different positions in the TOCSY spectrum. With the help of the HSQC spectrum, the chemical shifts of the corresponding hydrogen / carbon at position 3 of the sugar ring are 3.78 / 88.1 ppm in sequence, indicating that an obvious glycosylation shift has occurred at this position. Combining the correlation signals at 4.82 / 3.78 ppm in the ROESY spectrum, it shows that the sugar ring connection mode in the structure of this glucan is β-1→3 connection, that is, the lentinan polysaccharide extracted by this scheme is β-1,3-glucan; for the attribution of carboxymethyl, first, the methylene signal at 3.99 / 74.5 ppm in the HSQC spectrum can be clearly identified, and the carbonyl of carboxymethyl has a chemical shift of 182.3 ppm in the carbon spectrum. After the sugar ring is modified by carboxymethylation, due to the smaller steric hindrance at position 6, carboxymethylation preferentially occurs at this position, which can be clearly verified in the HSQC spectrum. After carboxymethylation at position 6 of the sugar ring, obvious glycosylation shifts also occur in the hydrogen spectrum and carbon spectrum, and the corresponding shifts are 4.35, 4.11 / 75.3 ppm respectively. With the help of the correlation between 4.35 / 3.99 in the ROESY spectrum, it can be confirmed that carboxymethylation has occurred at position 6 of the sugar ring. The attribution of this carboxymethyl-β-glucan is shown in Table 1.

[0091] Table 1 Attribution of Carboxymethyl-β-Glucan

[0092] 。

[0094] II. Preparation of Immunostimulatory Agent Derivatives from Lentinus edodes:

[0095] Weigh 150 mg of poly-D-lysine and dissolve it in 1.5 mL of DMSO. Add 250 mg of carboxymethylated dextran, 100 mg of BOP, and 60 mg of triethylamine. React at 40 °C for 2 h, then load it into a dialysis bag (cut-off molecular weight 10 kDa) and dialyze in pure water for 24 h. Collect the dialysate and freeze-dry to obtain 103 mg of the product.

[0096] The synthesis process and molecular structural formula of this β-glucan-polylysine are shown as follows:

[0097]

[0098] Use a Malvern particle size analyzer to detect the molecular weight and surface potential of β-glucan-polylysine as Figure 3 shown. It can be seen that the surface potential of G-PL is 20 mV and the particle size is 449 nm, providing a theoretical basis for the adhesion of G-PL to the cell surface and providing an electronic driving force.

[0099] The nuclear magnetic resonance spectra of the β-glucan-polylysine synthesized in this example are as Figures 4 to 10 shown, Figure 4 which is the hydrogen spectrum of β-glucan-polylysine, Figure 5 which is the carbon spectrum of β-glucan-polylysine, Figure 6 which is the COSY spectrum of β-glucan-polylysine, Figure 7 which is the TOCYS spectrum of carboxymethyl-β-glucan-polylysine, Figure 8 which is the HSQC spectrum of carboxymethyl-β-glucan-polylysine, Figure 9 which is the ROESY spectrum of carboxymethyl-β-glucan-polylysine, Figure 10This is the HMBC spectrum of carboxymethyl-β-glucan--polylysine. It can be seen that in the hydrogen spectrum and carbon spectrum, the hydrogen / carbon signals at the anomeric position of glucan can be first identified as 4.81 / 105.3 ppm, indicating that it is in the β configuration. Similarly, the signal assignments of other positions of glucan can be completed successively with the help of other two-dimensional spectra such as COSY, TOCSY, and HSQC spectra. Among them, there is an obvious glycosylation shift in the carbon signal at the 3-position of the sugar ring, and the corresponding hydrogen / carbon shifts are 3.80 / 86.0 ppm. At the same time, the relevant signals of 4.81 / 3.80 can be clearly observed in the ROESY spectrum, and the connection mode of glucan in this complex is β-1→3. The information provided by the HSQC spectrum can be used to confirm that the methylene signal in the carboxymethyl group is 3.99 / 73.2 ppm, and the -O-6 of the sugar ring with signals of 4.36, 4.07 / 73.9 ppm undergoes carboxymethylation. The corresponding characteristic signals of polylysine can also be assigned in this complex. The three groups of characteristic methylene groups at 1.43~1.48 ppm, 1.71~1.81 ppm, and 3.01 ppm are consistent with the structure of the aforementioned α-polylysine that has been analyzed. The remaining methine signal can be identified with the help of 4.30 / 56.6 ppm in the HSQC spectrum, and the correlation peaks between the methylene signal in the high-field region and this methine signal can be observed in both the COSY and TOCSY spectra. Thus, the characteristic signals in lysine are 4.30 / 56.6 ppm, 3.02 / 42.0 ppm, 1.78 / 33.3 ppm, 1.71 / 29.1 ppm, and 1.44 / 25.0 ppm respectively.

[0100] After the side-chain amino group of the α-polylysine reacts with carboxymethyl glucan to form an amide, due to the excessive molecular weight and the increased degree of freedom of the groups, the key correlation signals between the side-chain methylene of lysine and that from carboxymethyl glucan cannot be observed in the ROESY and HMBC spectra. However, compared with the hydrogen spectrum of polylysine before the reaction, it can be found that after reacting with carboxymethyl glucan to form a polymer with a larger molecular weight, the peak shapes of each characteristic signal of polylysine have changed significantly, indicating that the α-polylysine is modified on the carboxylic acid of carboxymethyl β-glucan.

[0101] III. Preparation of DC vaccine made from immunostimulant derivatives derived from Lentinula edodes:

[0102] S1: Isolate PBMC from human peripheral blood:

[0103] 1. Draw human peripheral blood into a heparin anticoagulant tube, take 5 mL of fresh blood from it, and add PBS to dilute the blood at a ratio of 1:1. 2. Take 5 mL of lymphocyte separation solution and place it in a 15 mL sterilized centrifuge tube for later use. 3. Aspirate the diluted blood and slowly add it along the tube wall 1 cm above the lymphocyte separation solution, so that the diluted blood overlaps on the lymphocyte separation solution and forms a distinct interface with the separation solution. 4. At room temperature, centrifuge at 2000 rpm for 20 min. At this time, 5 layers are formed in the centrifuge tube: the plasma is on the top, and between the plasma layer and the lymphocyte separation solution is the lymphocyte layer in a white film shape. 5. Carefully aspirate the mononuclear cell layer in the middle in a white film shape and try to aspirate all the mononuclear cells. Add PBS with a volume more than 5 times to wash 2 times, and centrifuge at 1500 rpm for 10 min each time. The PBMC isolated here is as Figure 11 shown.

[0104] S2: Prepare A2B5-positive glioma cells:

[0105] 1. Digest and lyse the glioma tissue to obtain a single-cell suspension. 2. Sort the single-cell suspension in step 1 with magnetic beads to obtain A2B5-positive glioma cells. 3. Irradiate the A2B5-positive glioma cells in step 2 with X-rays (25 Gy, 1 min) to obtain attenuated A2B5-positive cells as A2B5-positive glioma cells for subsequent G-PL synthesis. The obtained A2B5-positive glioma cells are as Figure 12 shown.

[0106] S3: Culture PBMC in a medium to obtain immature DC cells, and co-culture the A2B5-positive glioma cells and the lentinula edodes-derived immunostimulant derivative prepared above in the medium to obtain mature DC cells as DC vaccines. The lentinula edodes-derived immunostimulant derivative is carboxymethyl-β-glucan-poly-D-lysine formed by coupling carboxymethyl-β-glucan derived from lentinula edodes and poly-D-lysine:

[0107] Culture the collected PBMC in RPMI 1640 medium in a culture plate at 37 °C and 5% CO 2 for 4 h; 2. Gently aspirate the liquid supernatant in step 1, collect the adherent cells, add RPMI 1640 medium containing 15% serum, 100 ng / ml rhGM-CSF, and 100 ng / ml rhIL-4, and culture for 5 d to obtain immature DC cells, as Figure 13 shown; 3. Co-culture the A2B5-positive glioma cells with FITC-labeled β-glucan-poly-D-lysine (G-PL) for 48 h to obtain mature DC cells loaded with whole tumor stem cell antigens, as Figure 14 shown.

[0108] The attachment of FITC-labeled G-PL to the membranes of A2B5-positive glioma cells was monitored by confocal microscopy as Figure 15 shown. Due to the electrokinetic force generated by the positive and negative charges between A2B5-positive glioma cells and FITC-labeled β-glucan-poly-D-lysine (G-PL) after co-culture, G-PL was adsorbed on the surface of tumor cells, while β-glucan could not be adsorbed. To further confirm the surface distribution of G-PL after adhering to tumor cells, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed on the tumor cells after G-PL adhesion. The attachment of G-PL to the membranes of A2B5-positive glioma cells was observed by scanning and transmission electron microscopy as Figure 16 shown. The time required for G-PL to coat the tumor and the duration were further tested, and the results are shown as Figure 17 shown. It was found that G-PL could complete coating immediately after mixing with tumor cells and could last for at least 48 hours.

[0109] IV. Stimulation of APC cells by carboxymethyl-β-glucan at different concentrations:

[0110] Considering that carboxymethyl-β-glucan at different concentrations has different stimulatory effects on APC cells, CD83 and CD86 are surface markers of mature DCs and can reflect the immune efficacy of carboxymethyl-β-glucan.

[0111] Figure 18 The effects of carboxymethyl-β-glucan at different concentrations on CD83 and CD86 are shown. It can be seen from the figure that there are significant differences in the stimulatory effects of carboxymethyl-β-glucan on DCs between 0 and 2000 μg / ml. Moreover, the results shown in the figure indicate that when carboxymethyl-β-glucan is at 1000 μg / ml, the transformation of DCs into mature forms is the most, suggesting that this concentration has the best immune effect.

[0112] In addition, to compare the stimulatory effects of different immune stimulants on DCs, the experiment was divided into three groups: a control group (only PBS), a tumor group (only U87), and a glucan group (added with 1000 μg / ml of carboxymethyl-β-glucan), as Figure 19 shown. The flow cytometry results of the three groups of experiments analyzed by flow cytometry are shown, Figure 19 where Control represents the control group, Tumor represents the tumor group, and β-Glucan represents the glucan group. The expression of CD83 and CD86 in the glucan group is the highest, as Figure 20As can be seen, there are statistical differences between the dextran group and the control group (CD83: control group vs. dextran group, p: 0.001; CD86: control group vs. dextran group, p < 0.0001).

[0113] DC vaccines do not directly kill tumor cells, but produce effects after stimulating the differentiation and proliferation of tumor-specific T cells in lymph nodes. Therefore, in addition to some exocrine factors of DC itself, the function of T cells is also an important indicator for evaluating DC stimulated by carboxymethyl-β-dextran. The experiment was still divided into three groups as above, the control group (only PBS), the tumor group (only U87), and the dextran group (added with 1000 μg / ml of carboxymethyl-β-dextran), as Figure 21 shown by the flow cytometry results, the TNF-a and IFN-b in the dextran group were significantly increased, and the differences were statistically significant (TNF-α: control group vs. dextran group, p < 0.001; IFN-β: control group vs. dextran group, p < 0.001). Figure 21 In the figure, PBS corresponds to the control group, Tumor corresponds to the tumor group, and β-Glucan corresponds to the dextran group.

[0114] In addition, after adding T cells to different experimental groups and culturing for 72H, as Figure 22 and Figure 23 shown by the flow cytometry monitoring, the T cell division in the dextran group was significantly increased (control group vs. dextran group, p < 0.001), which proved that carboxymethyl-β-dextran could stimulate APC cells to the greatest extent at 1000 μg / ml.

[0115] V. Killing of tumor cells by different concentrations of β-dextran-polylysine (G-PL):

[0116] Use 10 6 A2B5 + tumor cells and co-culture with 1 ml of G-PL at different concentrations. Use 7-Aminoactinomycin D (7-AAD) as a marker for tumor cell death. As Figure 24 shown, when the concentration of G-PL is greater than 1000 μg / ml, the number of dead tumor cells increases significantly. It can also be speculated that G-PL has cytotoxicity to normal cells when exceeding this concentration. Therefore, when co-incubating 10 6 tumor cells with G-PL, the concentration of G-PL should be less than 1000 μg / ml. In the subsequent experiments of this patent, a concentration of 500 μg / ml was used corresponding to 10 6 tumor cells.

[0117] Take 500 μg / ml of β-dextran-polylysine and 10 6Complexes composed of tumor cells stimulate APC cells. Using different stimuli to stimulate APC cells, the experiments were still divided into a control group (PBS), a tumor group (U87), and a β-glucan derivative group (500 μg / ml β-glucan-polylysine), as Figure 25 The experimental results shown Figure 25 In [Figure], Control corresponds to the control group, Tumor corresponds to the tumor group, and β-GPDL corresponds to the β-glucan derivative group. It can be seen that DC cells significantly transform into a mature phenotype after being stimulated by β-GPDL (derivative group vs. control group; CD83: p < 0.001; CD86: p < 0.001). As Figure 26 The results shown indicate that after co-culturing for 24 h, the contents of TNF-α and IFN-β in the supernatant of the β-GPDL group are significantly increased (derivative group vs. control group; CD83: p < 0.001; CD86: p < 0.001), and as Figure 27 The results shown indicate that when using 500 μg / ml β-GPDL, the degree of T cell division in the glucan derivative group is significantly higher than that in the control group, p < 0.0001. The experimental results prove that the derivative of β-GPDL combined with tumors at an appropriate concentration still has strong immunogenicity.

[0118] VI. Activation effect of complexes of different concentrations of β-glucan-polylysine (G-PL) and A2B5 cells on DC cells:

[0119] To detect the activation effect of different concentrations of G-PL on DC cells, glioma cells were incubated with different concentrations of G-PL (0, 25, 50, 100, 200, 500, 1000 μg / ml) for 20 min, and then a new type of whole-cell tumor adjuvant (hereinafter referred to as the Vaccine group) was prepared and mixed with an equal amount of DC cells. They were co-cultured for 48 h at 37 °C and 5% carbon dioxide, and the expression levels of CD80, CD86, and CD80 of CD11c+ DC cells were detected. As 6 shown, with the increase in the concentration of G-PL, CD80, CD86, and CD83 show an upward trend, indicating that the concentration of G-PL is positively correlated with the degree of DC maturation. However, since it has been previously proven that G-PL at a concentration exceeding 1000 μg / ml can damage normal cells, we selected a safe concentration of 500 μg / ml G-PL. At the same time, this concentration of G-PL also has a stronger activation effect on DC cells compared to soluble β-glucan. Figure 28

[0120] VII. Activation effect of DC vaccine prepared from immunostimulant derivatives derived from Lentinula edodes on T cells:

[0121] Similarly, after preparing the new vaccine by the same method, it was combined with 1×10 6 6T cell co-culture. A total of 4 groups were set up, namely DC + PBS, DC + A2B5 positive cells, DC + A2B5 positive cells + soluble β-glucan, and DC + A2B5 positive cells coated with β-glucan-polylysine. At 48 hours of co-culture, the expressions of TNF-α and INF-β in the supernatant were detected simultaneously. The results are as Figure 29 The results shown in indicated that the secretion levels of tumor necrosis factor α (TNF-α) and interferon β (INF-β) in the co-culture system with G-PL added were the highest, significantly higher than those in the soluble β-glucan group. Moreover, the secretion level in the soluble β-glucan group was higher than that in the A2B5 positive cell group and the PBS group. Figure 29 In , PBS corresponds to DC + PBS (PBS group), A2B6 + Tumor corresponds to DC + A2B5 positive cells (A2B5 positive cell group), β-Glucan corresponds to DC + A2B5 positive cells + soluble β-glucan (soluble β-glucan group), and G-PL corresponds to DC + β-glucan-polylysine.

[0122] After co-incubating for 0 h, 12 h, 24 h, and 48 h respectively under the conditions of 37 °C and 5% carbon dioxide, the supernatant of the co-culture system was collected, and the secretion level of interleukin 2 (IL-2) was detected by flow cytometry. The results are as Figure 30 shown. It was found that the G-PL group showed the strongest IL-2 secretion function at the above different time points, and it increased continuously with time, reaching the highest level at 48 h. It can be seen that the DC vaccine prepared with the immunostimulant derivative from Lentinus edodes and whole tumor cell antigen in this protocol can activate the tumor-killing function of T cells in vitro.

[0123] VIII. In vivo homing phenomenon of the DC vaccine prepared with the immunostimulant derivative from Lentinus edodes:

[0124] After preparing the DC vaccine according to the above method, CD11c magnetic beads were used for sorting to obtain pure DC cells. After staining the DC cell membrane with Dialkylcarbocyanines (DID), PBMC-reconstituted mice were injected into the footpads at an order of magnitude of 2x10 5 . As Figure 31 shown, after 48 h, it can be seen that the red-labeled DC cells migrated towards the popliteal lymph nodes of the mice.

[0125] The present invention is not limited to the above best implementation mode. Anyone can obtain various other forms of products under the inspiration of the present invention. However, no matter what changes are made in its shape or structure, as long as it has a technical solution identical or similar to the present application, it falls within the protection scope of the present invention.

Claims

1. A method for preparing an immunostimulant derived from shiitake mushrooms, comprising the following steps: S1: Extracting Lentinan: adding Lentinus edodes medicinal materials into purified water and extracting at high temperature to obtain an extract, cooling the extract to room temperature and then adding ethanol to obtain a precipitate, collecting the precipitate by centrifugation and then performing ultrafiltration to obtain Lentinan; S2: Preparation of shiitake mushroom-derived immunostimulant: suspend shiitake mushroom polysaccharide in ethanol and add chloroacetic acid to obtain a mixed solution, magnetically stir the mixed solution and collect the precipitate, wash the precipitate with ethanol and dialyze to obtain a dialysate, concentrate and freeze-dry the dialysate to prepare the shiitake mushroom-derived immunostimulant, wherein the shiitake mushroom-derived immunostimulant is carboxymethyl-β-glucan.

2. A Lentinus edodes-derived immunostimulant, characterized in that: The method for preparing the shiitake mushroom-derived immunostimulant according to claim 1 is used to stimulate APC, and the concentration of the shiitake mushroom-derived immunostimulant that stimulates APC is 0 to 2000 ug / ml.

3. A method for preparing a derivative of an immunostimulant derived from Lentinus edodes, characterized in that: The method comprises the following steps: adding the shiitake mushroom-derived immunostimulant according to claim 1, BOP and triethylamine to an organic solvent in which poly-D-lysine is dissolved, reacting for a period of time, dialyzing, and freeze-drying the dialysate to obtain a shiitake mushroom-derived immunostimulant derivative, wherein the shiitake mushroom-derived immunostimulant derivative is β-glucan-poly-D-lysine.

4. A derivative of a Lentinus edodes-derived immunostimulant, characterized in that: The method for preparing the shiitake mushroom derived immunostimulant derivative according to claim 3 is used to kill tumor cells, corresponding to 10 6 The concentration of the shiitake mushroom-derived immunostimulant derivative selected for tumor cells was less than 1000 μg / ml.

5. A method for preparing a DC vaccine based on a derivative of an immunostimulant derived from Lentinus edodes, characterized in that: The following steps are involved: S1: PBMCs isolated from human peripheral blood; S2: Preparation of A2B5-positive glioma cells; S3: PBMCs are cultured in a culture medium to obtain immature DC cells, and A2B5-positive glioma cells and the shiitake mushroom-derived immunostimulant derivative according to claim 3 are added to the culture medium for co-culturing to obtain mature DC cells as a DC vaccine.

6. The method for preparing a DC vaccine based on a derivative of an immunostimulant derived from Lentinus edodes according to claim 5, characterized in that: After culturing PBMCs in the culture medium for a period of time, a supernatant is obtained, adherent cells in the supernatant are collected, and the adherent cells are added to 1640 culture medium for culture to obtain immature DCs.

7. The method for preparing a DC vaccine based on a derivative of an immunostimulant derived from Lentinus edodes according to claim 5, characterized in that: The collected PBMCs were cultured in 1640 medium at 37°C and 5% CO2 for 4 h.

8. The method for preparing a DC vaccine based on a derivative of an immunostimulant derived from Lentinus edodes according to claim 5, characterized in that: A2B5-positive glioma cells and β-glucan-poly-D-lysine were added to the culture medium and co-cultured at 37° C. and 5% CO 2 for 1 to 48 hours to obtain mature DC cells as DC vaccines.

9. A DC vaccine based on a derivative of an immunostimulant derived from Lentinus edodes, characterized in that: The DC vaccine is prepared according to the method for preparing a shiitake mushroom-derived immunostimulant derivative according to any one of claims 5 to 8 and is used to kill tumor cells.

10. The DC vaccine based on the derivative of Lentinus edodes-derived immunostimulant according to claim 9, characterized in that: Used to kill glioma tumor cells.

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