Application of ganoderenic acid B in promoting activation and proliferation of T lymphocytes

Abstract

The invention discloses application of ganoderenic acid B in promoting activation and proliferation of T lymphocytes. Ganodeenoic acid B is lanostane type triterpenes separated from ganoderma lucidum, and can promote proliferation and activation of T lymphocytes in a concentration range of 5-50 [mu] M. The invention discloses that GAB has a good application prospect in enhancing the immune response effect of T lymphocytes by promoting the activation and proliferation of the T lymphocytes.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of ganoderic acid B in promoting the activation and proliferation of T lymphocytes. Background Art

[0002] Thymus-dependent lymphocytes (T lymphocytes) are the main components of lymphocytes. They have multiple biological functions, such as directly killing target cells, assisting or inhibiting B cells to produce antibodies, responding to specific antigens and mitogens, and producing cytokines. They are an important cell type in the immune system.

[0003] With the increasing number of FDA-approved cancer immunotherapy drugs, immunotherapy has become the subject of intensive research, especially for cancer treatment. Among the breakthroughs in tumor immunotherapy, immune checkpoint inhibitors are the fastest and most mature. However, immune checkpoint inhibitors have only achieved clinical success in some cancer types, but limited remission rates, safety issues and complex pharmacokinetics remain the main limiting factors. Immunomodulatory small molecule drugs are expected to become an effective supplement to current immunotherapy due to their ability to enhance tissue permeability, improve bioavailability and reach intracellular targets. The development of low molecular weight immunopotentiators based on natural products is considered one of the most promising approaches, and Ganoderma lucidum has been shown to have a wide range of pharmacological properties, including anti-tumor, immunomodulatory and anti-inflammatory activities, which plays an important role in the development of future anti-tumor immunotherapy drugs.

[0004] Lingzhi (scientific name: Ganoderma lucidum (Curtis) P. Karst.) is a fungus of the Ganoderma family and the genus Ganoderma. Most of the fruiting bodies of Lingzhi are annual, and a few are perennial. It is a precious traditional Chinese medicinal material for strengthening the body and nourishing the body. It is reported that Lingzhi has unique effects in regulating immune function and inhibiting tumor growth.

[0005] Ganoderenic acid B (GAB) is a lanostane-type triterpenoid isolated from Ganoderma lucidum. Previous studies have reported that GAB can effectively reverse the multidrug resistance of the ABCB1-mediated hepatocellular carcinoma resistant cell line HepG2 / ADM to doxorubicin, vincristine, and paclitaxel. However, to date, there have been no reports on its application in promoting the proliferation and maturation of immune-related T lymphocytes. Summary of the invention

[0006] The purpose of the present invention is to provide the use of GAB in promoting the activation and proliferation of T lymphocytes. GAB can be used as an immune activator to improve immune activity by promoting the proliferation and activation of T lymphocytes.

[0007] To achieve the above object, the present invention adopts the following technical solution: A ganoderma lucidum extract having the activity of promoting the activation and proliferation of T lymphocytes; the ganoderma lucidum extract is ganoderic acid B, and its structural formula is: .

[0008] The concentration of the above-mentioned Ganoderma lucidum extract in promoting the activation and proliferation of T lymphocytes is 5 to 50 μM.

[0009] The above-mentioned Ganoderma lucidum extract is used to promote the activation and proliferation of T lymphocytes for non-disease diagnosis purposes.

[0010] The above-mentioned Ganoderma lucidum extract is used in the preparation of a product for promoting the activation and proliferation of T lymphocytes.

[0011] The application of the above-mentioned Ganoderma lucidum extract in the preparation of an immune activator.

[0012] The beneficial effects of the present invention are: The results of the present invention reveal that GAB can improve the body's immunity by promoting the proliferation and activation of T lymphocytes. GAB can act on multiple biological targets of T lymphocytes, including key molecules in cell signaling pathways, thereby regulating the proliferation, differentiation and activation of T lymphocytes at multiple levels, in order to promote the effect of anti-tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :The results of GAB's effect on T lymphocyte proliferation in vitro.

[0014] Figure 2 : Effect of GAB on the expression of Ki67 in T lymphocytes. (A) Overlay of peaks of FITC-Ki67 expression detected by flow cytometry in dendritic cells of GAB, PMA, DMSO and control groups; (B) Quantitative statistics (A) Median of relative fluorescence of FITC-Ki67 in dendritic cells of each group. n = 3, *** p<0.001 vs. Control.

[0015] Figure 3 :The results of GAB's effect on T lymphocyte subsets differentiation.

[0016] Figure 4 :The results of GAB's effect on the expression of co-stimulatory and co-inhibitory molecules in T lymphocytes.

[0017] Figure 5: Effects of GAB on mouse body weight and spleen weight index. (A) Changes in mouse body weight during 14 days of treatment; (B) Photos of mouse spleens isolated after 14 days of treatment; (C) Mouse spleen weight index (spleen weight / mouse body weight). n = 5, * p<0.05, ** p<0.001 vs. Control.

[0018] Figure 6 :Flow cytometry was used to detect the differentiation of peripheral blood T lymphocyte subsets in each group.

[0019] Figure 7 : Quantitative Statistics Figure 6 The percentage of T lymphocyte subset differentiation in each group. n = 3, * p < 0.001 vs. Control.

[0020] Figure 8 :Flow cytometry was used to detect the differentiation of T lymphocyte subsets in spleen cells of each group.

[0021] Fig. 9 : Quantitative Statistics Figure 8 The percentage of T lymphocyte subsets in spleen cells of each group. n = 3.

[0022] Fig.10 :The percentage of Tregs differentiation in peripheral blood of each group detected by flow cytometry.

[0023] Fig.11 :The percentage of Tregs differentiation of spleen cells in each group detected by flow cytometry.

[0024] Fig.12 : Quantitative Statistics Fig.10 and Fig.11 The percentage of Tregs differentiation in each group. n = 3, * p < 0.05, ** p < 0.001 vs. Control. DETAILED DESCRIPTION

[0025] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0026] The experimental materials involved in the following embodiments are as follows: The ganoderic acid B in the following examples was purchased from Wuhan Tianzhi Biotechnology Co., Ltd., with a product number of CFN92991, a batch number of CFS202301, and a purity of ≥98%; ganoderic acid B was weighed and dissolved in DMSO to prepare a ganoderic acid B mother solution with a concentration of 100 mM, and diluted to the corresponding concentration in T lymphocyte complete medium for use in the following examples 1-4. Ganoderic acid B was weighed and dissolved in DMSO to prepare a ganoderic acid B solution with a concentration of 100 mg / mL, and the required volume of ganoderic acid B solution was taken according to the group, and then made up to a total volume of 200 μL with PBS for use in examples 5-8.

[0027] CD3 antibody was purchased from Takara, catalog number T210; Ficoll Paque PREMIUM was purchased from cytiva, catalog number 17544202; Lonza X-VIVO 15 culture medium was purchased from Lonza, catalog number 04-418Q; fetal bovine serum (FBS) was purchased from Cegrogen, catalog number A0500-3011; IL-2 was purchased from Sino Biological, catalog number GMP-11848-HNAE; IFN-γ was purchased from Sino Biological, catalog number GMP-11725-HNAE; STEMCELL CD3 / CD28 antibody was purchased from STEMCELL, catalog number 10971; hydroxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Beyotime, catalog number C0051; phorbol myristate (PMA) was purchased from MCE, catalog number 16561-29-8; permeabilization solution was purchased from BD, catalog number 554714; Ki67-FITC antibody was purchased from Biolegend, catalog number 350501; CD3-PC5, CD4-PC7 and CD8-ECD antibodies were all purchased from Beckman, catalog numbers A07749, 737660 and 6604728 respectively; 12.5% ​​PAGE gel rapid preparation kit was purchased from Yazyme Biotechnology, catalog number PG113; 4-1BB antibody was purchased from Cell Signaling Technology, catalog number 34594T; CD40L antibody was purchased from Cell Signaling Technology, Catalog No. 15094T; TIM-3 antibody was purchased from Cell Signaling Technology, Catalog No. 45208T; VISTA antibody was purchased from Cell Signaling Technology, Catalog No. 64953T; LAG-3 antibody was purchased from Cell Signaling Technology, Catalog No. 15372T; protease inhibitor (PMSF) was purchased from Bio-Time, Catalog No. ST507-10ml; lysis buffer (RIPA) was purchased from Yazyme Bio, Catalog No. PC101; Alexa Fluor 700-CD3 was purchased from BioLegend, Catalog No. 100216; PC7-CD4 was purchased from Invitrogen, Catalog No. 25-0041-82; APC-CD8a antibody was purchased from eBioscience, Catalog No. 0081-82; FITC- CD4 was purchased from Biolegend, catalog number 100405; PE-CD127 was purchased from eBioscience, catalog number 12-1273-82; PC61.5-CD25 was purchased from eBioscience, catalog number 35-0251-82.

[0028] Example 1 Effect of GAB on T lymphocyte proliferation (1) Dilution of CD3 monoclonal antibody: Dilute CD3 monoclonal antibody with PBS solution to a final concentration of 10 μg / mL, and add 1 mL of the diluted CD3 antibody solution to a 6-well plate. Seal the plate with sealing film and place it in a 4°C refrigerator for overnight coating.

[0029] (2) Extraction of T lymphocytes: On the second day, extract 4 mL of normal peripheral blood, centrifuge at 2000 rpm for 5 min, discard the upper serum, collect the lower blood cells, and then add PBS equal to the volume of blood cells to mix and dilute. Add 4 mL of FicollPaque PREMIUM to a new 15 mL centrifuge tube, then use a Pasteur pipette to draw the diluted blood cells and add them slowly against the tube wall, centrifuge at 2000 rpm for 20 min, and adjust the centrifuge acceleration to 8 and the deceleration to 0. After centrifugation, the layers are clearly separated. Slowly extend the pipette to the surface of the white film layer, slowly draw the white film layer in a circle, and draw as much as possible and transfer it to another new 15 mL centrifuge tube containing 10 mL PBS. Centrifuge at 1000 rpm for 10 min, remove the supernatant, collect the cell pellet, and wash twice with 5 mL PBS.

[0030] (3) Take 2 mL of T lymphocyte complete medium (Lonza X-VIVO 15 medium + fetal bovine serum with a final concentration of 10% + IL-2 with a final concentration of 25 μg / mL + IFN-γ with a final concentration of 50 μg / mL) to resuspend T lymphocytes to obtain T lymphocyte suspension, and then add STEMCELL CD3 / CD28 antibody solution at a volume of 25 μL / mL. Take out the above 6-well plate coated with CD3 antibody, aspirate the CD3 antibody dilution, mix the T lymphocyte suspension and add it, and place it in a constant temperature incubator at 37°C and 5% CO2 for culture. In the first 4 days, 0.5-1 mL of T lymphocyte complete medium can be appropriately added according to the growth of cells. On the 4th to 5th day, the T lymphocytes are transferred to a T75 culture flask for expansion culture. After 7 days, the experimental detection of T lymphocytes can be carried out.

[0031] (4) CFSE is a fluorescent dye that can penetrate the cell membrane. Follow the steps in the kit instructions: 1) Prepare CFSE storage solution and cell labeling solution: dilute 20× cell labeling solution to 1× cell labeling solution with PBS, and then dilute 1000× CFSE storage solution to 2× CFSE storage solution with 1× cell labeling solution.

[0032] 2) Count the number of cells using a cell counter, and pipette the T lymphocyte suspension into a 15 mL centrifuge tube. Centrifuge at 2000 rpm for 5 min, discard the supernatant, collect the cell pellet, and then label 1×10 cells with 1 mL of 1× cell labeling solution. 6 Add 1× cell labeling solution to the cell pellet to resuspend it and mix it well. Immediately add the same volume of 2× CFSE storage solution, mix it well, and incubate it in a 37°C, 5% CO2 constant temperature incubator for 15 min.

[0033] 3) After 15 min, remove the cells and add 10 mL of T lymphocyte complete medium to terminate the labeling. Mix by inverting, centrifuge at 2000 rpm for 5 min, discard the supernatant, and collect the cell pellet. Add 5 mL of T lymphocyte complete medium, blow the cells evenly, centrifuge at 2000 rpm for 5 min, discard the supernatant, and collect the cell pellet. Add another 5 mL of T lymphocyte complete medium, blow the cells evenly, and incubate in a cell culture incubator at 37°C for 5 min. Centrifuge at 2000 rpm for 5 min, discard the supernatant, and collect the cell pellet. Finally, add the corresponding volume of T lymphocyte complete medium to resuspend the cells and dilute the cell concentration to 5×10 5 / mL, inoculated in 12-well plates, 2 mL per well. T lymphocytes were divided into control group, DMSO group, PMA group and GAB group. The control group was not treated with other treatments; the DMSO group was treated by adding DMSO with a final concentration of 1 μL / mL to the T lymphocytes; the PMA group was treated by adding phorbol ester (PMA) with a final concentration of 10 ng / mL to the T lymphocytes; the GAB group was treated by adding GAB with a final concentration of 10 μM to the T lymphocytes. The above four groups of cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 96 h, the cells were collected, centrifuged at 2000 rpm for 5 min, the supernatant was discarded, the cell pellet was collected, and the cell pellet was washed with PBS (containing 3% FBS) for 2-3 times. Then 300 μL PBS (containing 3% FBS) was added to the washing solution to resuspend the cells and transfer them to the flow tube, and then the machine was tested.

[0034] After CFSE enters the cell, when the cell divides and proliferates, the fluorescent cytoplasmic protein is evenly distributed to the second generation of cells, and its fluorescence intensity will be reduced by half compared with the first generation of cells; the fluorescence intensity of the third generation of cells obtained by division will be weakened again. By detecting the continuous decrease in cell fluorescence intensity, further analysis can be used to obtain the situation of cell division and proliferation. Phorbol ester is currently recognized as an immune cell activator, and PMA is used as the positive control group here. The results showed that the T lymphocytes in the control group and the DMSO-treated group only divided for 2 generations, while the T lymphocytes in the PMA group and after GAB treatment divided for 4 generations, indicating that GAB can promote the division and proliferation of T lymphocytes (such as Figure 1 ).

[0035] Example 2 Effect of GAB on the expression of Ki67 in T lymphocytes After obtaining T lymphocytes by the same extraction method as in Example 1, 1×10 6 / well were seeded in a 12-well plate, and the T lymphocytes were divided into control group, DMSO group, PMA group, GAB group and Isotype group; the control and Isotype groups were induced for 48 h without other treatments, the DMSO group was induced for 48 h by adding DMSO with a final concentration of 1 μL / mL to the T lymphocytes, the PMA group was induced for 48 h by adding PMA with a final concentration of 10 ng / mL to the T lymphocytes, and the GAB group was induced for 48 h by adding GAB with a final concentration of 10 µM to the T lymphocytes.

[0036] After culturing in a 37°C, 5% CO2 incubator for 48 hours, the cells of each group were collected, centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, the cell pellets of each group of samples were collected in a centrifuge tube, and 1 mL of 4% paraformaldehyde solution precooled at 4°C for 3 hours was added to each centrifuge tube, mixed, and fixed at 4°C for 24 hours. The next day, centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, the cell pellets were collected, and the cell pellets were washed once with PBS (containing 3% FBS). 200 µL of membrane permeabilization solution was added to each of the four groups, and the mixture was mixed by pipetting, and incubated in a 4°C refrigerator, wrapped with tin foil and protected from light for 20 minutes. Centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, the cell pellets were collected, and washed twice with PBS (containing 3% FBS).

[0037] Prepare Ki67-FITC antibody diluent: Add 50 μL PBS (containing 3% FBS) and 2 μL Ki67-FITC antibody to each sample group to prepare antibody diluent. Add 50 μL of the above antibody diluent to each sample tube of each group, of which only 50 μL PBS (containing 3% FBS) was added to the Isotype group, and flicked to mix the cells for labeling, and incubated at room temperature in the dark for 30 min. Centrifuge at 2000 rpm for 5 min, discard the supernatant, collect the cell pellet, and wash twice with 0.5 mL PBS (containing 3% FBS). Resuspend in 300 μL PBS (containing 3% FBS) and transfer to the flow tube, and then detect the expression of Ki67 in T lymphocytes on the machine.

[0038] like Figure 2As shown in the figure, compared with the control group and DMSO group, the Ki67 expression level of T lymphocytes treated with 10 µM GAB increased significantly, indicating that the proliferation ability of T lymphocytes was significantly improved. Combined with the experimental results of CFSE and Ki67, it further shows that GAB has the effect of promoting the proliferation of T lymphocytes.

[0039] Example 3 Effect of GAB on T lymphocyte subset differentiation After obtaining T lymphocytes by the same extraction method as in Example 1, 1×10 6 / well were inoculated in a 12-well plate, and T lymphocytes were divided into control group, DMSO group, PMA group, 5 µM group, 10 µM group and 50 µM group. The control group was induced for 48 hours without other treatments, the DMSO group was induced for 48 hours by adding DMSO with a final concentration of 1 μL / mL to the T lymphocytes; the 5µM GAB group was induced for 48 hours by adding GAB with a final concentration of 5µM to the T lymphocytes; the 10µM GAB group was induced for 48 hours by adding GAB with a final concentration of 10µM to the T lymphocytes; and the 50µM GAB group was induced for 48 hours by adding GAB with a final concentration of 50µM to the T lymphocytes. After the induction was completed in a 37°C, 5% CO2 constant temperature incubator, T lymphocytes of each group were collected, centrifuged at 2000 rpm for 5 min, the supernatant was discarded, the cell pellet was collected, and washed twice with PBS (containing 3% FBS). Centrifuge at 2000 rpm for 5 min, discard the supernatant, and temporarily store the cell pellet in a 4°C refrigerator. Add 1.5 μL CD3-PC5, 1.5 μL CD4-PC7, 1.5 μL CD8-ECD antibodies and 50 μL PBS (containing 3% FBS) to each sample tube to prepare a CD3 / CD4 / CD8 antibody mixture. Add 50 μL of the above CD3 / CD4 / CD8 antibody mixture to each sample tube, mix well, incubate at room temperature in the dark for 30 min, centrifuge at 2000 rpm for 5 min, discard the antibody mixture, and wash twice with PBS (containing 3% FBS). Finally, add 300 μL PBS (containing 3% FBS) to resuspend the cells and transfer them to the flow tube, and then analyze them on the machine.

[0040] After 48 h of GAB induction, CD3 + CD4 + Helper T lymphocytes and CD3 + CD8 + The percentage of cytotoxic T lymphocytes. Figure 3 It can be seen that after the action of 5 μM GAB, CD3 + CD4 +The proportion of helper T lymphocytes reached 47.5%, compared with 43.4% in the control group and 35.9% in the DMSO group. After 5 μM GAB treatment, CD3 + CD4 + The proportion of helper T lymphocytes increased; 10 μM and 50 μM GAB concentrations CD3 + CD4 + The proportion of T lymphocytes was 45.2% and 43.2% respectively, which was an increase but not significant. In addition, we also found that when GAB at 5 μM, 10 μM and 50 μM concentrations acted on T lymphocytes, CD3 + CD8 + The proportions of cytotoxic T lymphocytes were 53.0%, 52.9% and 50.0%, respectively, which were all higher than 49.5% in the control group.

[0041] Example 4 Effect of GAB on the Expression of Co-stimulatory and Co-inhibitory Molecules in T Lymphocytes After obtaining T lymphocytes by the same extraction method as in Example 1, 1×10 6 / well were inoculated in a 12-well plate, and the T lymphocytes were divided into control group, DMSO group, 5 µM GAB group, 10 µM GAB group, and 50 µM GAB group. The control group was induced for 48 hours without other treatments, the DMSO group was induced for 48 hours by adding DMSO with a final concentration of 1 μL / mL to the T lymphocytes; the 5µM GAB group was induced for 48 hours by adding GAB with a final concentration of 5µM to the T lymphocytes; the 10µM GAB group was induced for 48 hours by adding GAB with a final concentration of 10µM to the T lymphocytes; and the 50µM GAB group was induced for 48 hours by adding GAB with a final concentration of 50µM to the T lymphocytes. Cell lysis buffer was prepared at a ratio of protease inhibitor (PMSF): lysis buffer (RIPA) = 1:100, and the cells were collected 48 hours later in the corresponding labeled centrifuge tubes. After thorough mixing, the cells were counted and centrifuged at 2000 rpm for 5 min to discard the supernatant. Then, 1×10 6Add 80 µL of cell lysis buffer to each cell, place the lysed sample on an ice box for 30-40 min to obtain a cell protein sample. Add 5×Loading Buffer to the cell protein sample at a ratio of 5×Loading Buffer: cell protein sample = 1:4, mix well to obtain a mixed solution, and boil the mixed solution in boiling water for at least 5 min to fully denature the protein. Prepare the electrophoresis separation gel according to the instructions of the 12.5% ​​PAGE gel rapid preparation kit, and transfer the upper and lower separation gels to the middle of two cleaned and clamped glass plates. After the separation gel solidifies, hold a comb with both hands and insert it horizontally into the upper gel. After the upper gel solidifies, remove the glass plate and place it in a vertical electrophoresis tank. Add 2 μL of prestained protein marker (purchased from Yazyme Bio, catalog number: WJ101) to the first loading well, and then add 10 μL of protein sample to the loading wells one by one. The initial electrophoresis voltage was 100 V, and when the experimental sample ran into a uniform straight line, the electrophoresis voltage was increased to 120 V. After the electrophoresis, the gel was transferred to the transfer tank for transfer (the transfer condition was 45 min at 280 mA). After the electrophoresis, 2.5 g of skim milk powder was dissolved in 50 mL of TBST buffer (PH7-7.4) to obtain 5% skim milk. The strips were immersed in the prepared 5% skim milk and incubated on a shaker for 1 h at room temperature. Use 5% skim milk to dilute the antibodies (4-1BB, CD40L, TIM-3, VISTA and LAG-3) according to the dilution ratio specified in the antibody manual. Soak the above strips in the diluted antibody solution at 4°C overnight. After the primary antibody incubation, the strips are transferred to TBST buffer (PH 7-7.4) and washed on a shaker for 3 times, 15 minutes each time. After washing, the strips are transferred to the diluted corresponding secondary antibodies (anti-4-1BB monoclonal antibody, anti-CD40L monoclonal antibody, anti-TIM-3 monoclonal antibody, anti-VISTA monoclonal antibody and anti-LAG-3 monoclonal antibody) and incubated on a shaker for 1 hour at room temperature. Continue to wash with TBST buffer (PH 7-7.4) on a shaker for 3 times, 15 minutes each time. After washing, take out the strips one by one, and add the prepared ECL developer to the strips under light-proof conditions, and make it fully cover the strips, stay for about 2-3 minutes, place the strips in the developer, and expose the strips at an appropriate exposure time.

[0042] The immunoregulatory ability of GAB on T lymphocytes was further explored by detecting the expression of co-stimulatory or co-inhibitory molecule proteins on the surface of T lymphocytes after different concentrations of GAB acted on them by western blot. 4-1BB and CD40L are co-stimulatory molecules on the surface of T lymphocytes, which can promote the survival and activation of effector T lymphocytes; TIM3, VISTA and LAG-3 are co-inhibitory molecules on the surface of T lymphocytes, which have the function of negatively regulating T lymphocytes, and can reduce the killing ability of T lymphocytes on tumors by downregulating the immune response of T lymphocytes and inhibiting the inflammatory response mediated by T lymphocytes, thereby producing exhaustion or tolerance.

[0043] After inducing T lymphocytes with GAB at three concentrations of 5 μM, 10 μM and 50 μM for 48 h, the results were as follows: Figure 4 As shown, cofilin was selected as the internal reference protein. Compared with the control and DMSO groups, the expression levels of VISTA and LAG-3 were downregulated after treatment with different concentrations of GAB, while the expression level of 4-1BB was increased after treatment with 10 μM and 50 μM GAB, indicating that GAB can effectively promote the immune activation of T lymphocytes.

[0044] Example 5 Effect of GAB on Mouse Body Weight and Spleen Weight Index In vitro experiments have shown that GAB has a good immunomodulatory effect. Therefore, the immunomodulatory effect of GAB in vivo was further evaluated by using normal and healthy 4-6 week old female BALB / c mice (purchased from Wushi Experimental Animal Trading Co., Ltd.). Levamisole hydrochloride can promote the function of T lymphocytes, macrophages and neutrophils, and has potential anti-tumor activity. Therefore, in this experiment, we used levamisole hydrochloride as a positive control drug. The BALB / c mice were divided into control group, positive group, 20 mg / kg GAB group and 40 mg / kg GAB group. The total volume of administration for all groups was 200 μL, control was injected with 200 μL PBS; positive group was administered with 80 mg / kg levamisole hydrochloride (solvent was PBS); 20 mg / kg GAB group was administered with 20 mg / kg GAB; 40 mg / kg GAB group was administered with 40 mg / kg GAB. The body weight of mice in each group was weighed and recorded before administration. The drug was administered by intraperitoneal injection once every 3 days, and the body weight was weighed and recorded. The drug treatment lasted for 14 days in total.

[0045] After the administration, the mice were weighed and then killed by dislocating the neck. After disinfection, the abdominal cavity was opened aseptically to remove the spleen of the mice and weigh it. Then, each mouse was placed in a small dish with 2 mL of normal saline for cleaning according to the group.

[0046] The results are as follows Figure 5 As shown in the figure, the weight of mice in the experimental group increased during the administration period, the weight gain rate of mice in the control group was significantly slower, and the weight gain rate of mice in the positive group and 40 mg / kg GAB group was the most significant ( Figure 5 A). The spleen of each group of mice was weighed and its weight index was calculated. Compared with the control group, the spleen index of the mice in the GAB 40 mg / kg group and the positive group was significantly increased. The GAB 20 mg / kg group had a slight upward trend in the spleen index, but it was not as significant as the positive group and the 40 mg / kg GAB group, indicating that GAB has a certain growth-promoting effect on the growth and development of the mouse spleen in vitro (see Figure 5 BC).

[0047] Example 6 Effect of GAB on T lymphocyte subsets in mice Peripheral blood mononuclear cells (PBMCs) are cells with a single nucleus in peripheral blood, mainly including lymphocytes and monocytes. PBMCs play a key role in the immune system, and they can protect the body from infection by viruses, bacteria and parasites. After 14 days of administration as in Example 5, the weight of the mice was measured, anticoagulation tubes were prepared, blood was collected from the eyeballs, and the blood was collected in anticoagulation tubes. Prepare 10 mL centrifuge tubes and mark them, transfer the blood to a new centrifuge tube, centrifuge at 2000 rpm for 5 min, discard the upper serum, collect the blood cells in the lower layer, and then add PBS equal to the volume of the blood cells to mix. Add 4 mL Ficoll Paque PREMIUM to a new 10 mL centrifuge tube, then use a Pasteur pipette to absorb the diluted blood cells and add them slowly against the tube wall, centrifuge at 2000 rpm for 20 min, and adjust the acceleration of the centrifuge to 8 and the deceleration to 0. After centrifugation, the cells were clearly separated into different layers. Slowly extend the pipette to the surface of the buffy coat layer, and slowly aspirate the buffy coat layer in a circle to remove as much as possible and transfer it to another new 15 mL centrifuge tube containing 10 mL PBS. Centrifuge at 1000 rpm for 10 min, remove the supernatant, collect the cell pellet, and wash twice with 5 mL PBS to obtain mouse PBMCs.

[0048] The experimental groups were set up in the same way as in Example 5. After blood was collected, the mice were killed by dislocating the neck. After disinfection, the abdominal cavity was aseptically cut open to remove the spleen of the mice. The spleen was placed in a small dish with 2 mL of saline according to the group, and the spleen was blown with a 1 mL syringe. After that, the spleen cells blown out were collected in a centrifuge tube, centrifuged at 2000 rpm for 5 min, the supernatant was removed, the cell pellet was collected, 2 mL of red blood cell lysis solution was added for 2 min, and then 2-3 times the volume of red blood cell lysis solution was added to terminate the reaction, centrifuged at 2000 rpm for 5 min, the supernatant was discarded, the pellet was collected, and washed with PBS twice to obtain mouse spleen cells. After that, the mouse spleen cells were divided into tubes for flow antibody labeling, and 1.5 μL Alexa Fluor 700-CD3, 1.5 μL PC7-CD4, 1.5 μL APC-CD8a antibodies and 50 μL PBS (containing 3% FBS) were added to each sample tube to prepare an antibody mixture. Add 50 μL of the above antibody mixture to each sample tube of PBMCs and spleen cells in turn and mix well, incubate in the dark for 30 min, centrifuge at 2000 rpm for 5 min, remove the antibody mixture, collect the cell pellet, wash twice with the prepared PBS (containing 3% FBS), add 300 μL PBS (containing 3% FBS) to resuspend the cells and transfer them to the flow tube, and then analyze on the machine.

[0049] PBMCs were isolated from peripheral blood of mice treated with drugs for 14 days, and splenocytes were isolated from spleen for flow cytometry. Figure 6-Figure 9 As shown, compared with the control group, the peripheral blood CD3 + CD4 + The proportion of helper T lymphocytes was 57.96%. The CD3 + CD4 + The proportions of helper T lymphocytes were 64.78%, 64.65% and 68.55% respectively, all showing an upward trend ( Figure 7 ). Compared with the control group, CD3 + CD4 + The proportion of helper T lymphocytes was 16.15%, and the CD3 + CD4 + The proportions of helper T lymphocytes were 17.32%, 18.30% and 18.55% respectively, all showing an upward trend. + CD8 +For cytotoxic T lymphocytes, only the GAB 20 mg / kg group accounted for 9.53% and 18.87% in spleen cells and peripheral blood, respectively, which showed an upward trend compared with 7.24% and 10.69% in the control group, while there were no obvious changes in other groups compared with the control group.

[0050] Example 7 Effect of GAB on Tregs in Mice Mouse PBMCs and spleen cells were obtained by the same operating steps as in Example 6. The experimental groups were set up as in Example 5. 1.5 μL of FITC-CD4, PE-CD127, PC61.5-CD25 antibodies and 50 μL of PBS (containing 3% FBS) were added to each sample tube to prepare an antibody mixture. 50 μL of the above antibody mixture was added to PBMCs and spleen cells in turn, and the mixture was mixed by flicking the bottom of the tube. After incubation at room temperature in the dark for 30 min, the antibody mixture was removed by centrifugation at 2000 rpm for 5 min, and washed twice with the prepared PBS (containing 3% FBS). 300 μL of PBS (containing 3% FBS) was added to resuspend the cells and transferred to the flow tube, and then analyzed on the machine.

[0051] Tregs (Regulatory T cells) are a type of cell with significant immunosuppressive effects, which can inhibit the immune response of other cells. Tregs were labeled with flow cytometry antibodies, including CD4 + CD25 + CD127 - The cell population is Tregs. Figure 10-12 As shown in the figure, compared with the control, the proportion of Tregs in the peripheral blood and spleen of mice treated with GAB was significantly reduced, and both were statistically significant. The above experimental results show that GAB has the effect of inhibiting Tregs differentiation and thus enhancing the immune function of mice. The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A ganoderma lucidum extract, characterized in that: The ganoderma lucidum extract has the activity of promoting the activation and proliferation of T lymphocytes; the ganoderma lucidum extract is ganoderic acid B, and its structural formula is: 。 2. The use according to claim 1, characterized in that: The concentration of the ganoderma lucidum extract in promoting T lymphocyte proliferation and activation is 5 to 50 μM.

3. Use of the Ganoderma lucidum extract as claimed in claim 1 in promoting activation and proliferation of T lymphocytes for non-disease diagnosis purposes.

4. Use of the Ganoderma lucidum extract as claimed in claim 1 in the preparation of a product for promoting the activation and proliferation of T lymphocytes.

5. Use of the Ganoderma lucidum extract as claimed in claim 1 in the preparation of an immune activator.

Images