Application of TNFSF15 protein in preparation of products for promoting mast cell activation

Abstract

The invention discloses application of TNFSF15 protein in preparation of a product for promoting mast cell activation, and belongs to the technical field of medicines. The invention finds that the TNFSF15 protein promotes mast cell degranulation in vitro and increases release of inflammatory factors which play a role in regulating the occurrence and development of cancers. The released inflammatory factors are active in the tumor microenvironment, so that the macrophages with the tumor cell killing function are activated, and then the tumor immune microenvironment is activated. Meanwhile, in the process, apoptosis of mast cells is promoted, and the number of the mast cells playing roles is reduced. The deep research of the discovery has important significance in the anti-tumor basis and clinical medical research, and a new thought can be provided for clinical tumor treatment in the future.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of TNFSF15 protein in preparing products for promoting mast cell activation. Background Art

[0002] Tumors are a major disease faced by all human beings. With the increase of environmental pollution and life pressure, the incidence of tumors has increased year by year. The main methods of treating tumors are surgical resection and radiation therapy for local tumors, as well as the use of chemical drugs to kill tumor cells. These methods have the disadvantages of being unable to eradicate the root cause, easy to relapse, and having large side effects. With the development of modern immunology, people have realized that the stability of immune function plays an important role in the occurrence, development, metastasis, reversal, and regression of tumors. Tumors are a complex system. In addition to their own tumor cells, their microenvironment also contains many resident cell types, such as adipocytes and fibroblasts, macrophages, neutrophils, and mast cells. Among them, tumor-associated mast cells (TAMCs) are components of the microenvironment of almost all solid tumors and hematological tumors. They are a key type of tissue-resident immune cells that exist in almost all peripheral tissues of all vertebrates and are highly heterogeneous. There are a large number of lysosome-like secretory granules in the cytoplasm of mast cells, the main components of which are bioactive mediators proteases (including trypsin and chymase), histamine, cytokines, chemokines, and angiogenic factors. As the "sentinels" of the immune system, mast cells have a wide range of surface and cytoplasmic receptors that enable them to respond quickly to various stimuli when pathogens invade the body, releasing histamine and neurotransmitters to regulate peripheral vascular and nerve functions, while increasing in number through cell proliferation, recruitment, survival, and maturation of mast cell precursors, thereby regulating the body's immunity. Pathological studies have shown that mast cells can directly affect tumor cells and the surrounding extracellular matrix, and change the pathogenesis of tumors through a variety of mechanisms. For example, when stem cell factor (SCF) binds to the c-Kit receptor on the surface of mast cells, the c-Kit pathway is activated, resulting in the release of pre-synthesized particles of mast cells, and at the same time, a large number of newly synthesized particles are secreted outside the cell, and a large amount of TNF-α contained in them will be cytotoxic to tumor cells; histamine is another secretory mast cell factor that has the effect of inhibiting cancer cell proliferation or promoting tumor growth; after mast cell activation, it will also release a variety of proteases, such as tryptase, which can activate matrix metalloproteinases, thereby degrading the extracellular matrix and tissues around the tumor, promoting tumor growth, angiogenesis and metastasis. Cytokines such as VEGF, PDGF and IL-6 released by mast cells can attract more mast cells and make them migrate to the site of angiogenesis, thereby promoting the invasion and metastasis of cancer cells. In recent years, more and more evidence supports the great potential of mast cells in cancer treatment. At the same time, based on the complex role of mast cells in cancer, the treatment method and application of mast cells as a target is a scientific issue worthy of continued research.

[0003] Tumor necrosis factor superfamily-15 (TNFSF15, also known as TL1A) is an angiogenesis inhibitory factor mainly secreted by mature vascular endothelial cells. Studies have found that it is not only a negative vascular regulator that can inhibit angiogenesis and tumor growth in tumors, but also an immune activator that promotes T cell activation, dendritic cell maturation, and macrophage polarization. At present, there are no reports on the role of TNFSF15 protein as a mast cell activator. Summary of the invention

[0004] The purpose of the present invention is to provide an application of TNFSF15 protein in the preparation of a product for promoting mast cell activation, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the use of TNFSF15 protein in the preparation of products promoting mast cell activation.

[0007] The second technical solution of the present invention is a product for promoting mast cell activation, wherein the product contains TNFSF15 protein.

[0008] The third technical solution of the present invention is a method for promoting mast cell activation, using the product to treat mast cells.

[0009] A fourth technical solution of the present invention is the use of TNFSF15 protein in the preparation of drugs for regulating the number of mast cells in the tumor microenvironment.

[0010] Based on the above technical solution, the present invention has the following technical effects:

[0011] (1) The TNFSF15 protein of the present invention promotes mast cell degranulation to release inflammatory factors, thereby enhancing the activity of macrophages, thereby achieving the purpose of tumor treatment, and can be widely used in tumor treatment.

[0012] (2) The TNFSF15 of the present invention can also inhibit the proliferation of mast cells and promote their apoptosis, that is, the protein can effectively reduce the number of mast cells that play a role in the tumor microenvironment.

[0013] (3) Compared with chemotherapy, TNFSF15, as a biological protein, has the characteristics of less toxicity and higher safety.

[0014] (4) The TNFSF15 purification method of the present invention is simple, large-scale, and has a relatively low economic cost. It can be combined with other treatment methods and has a broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The growth changes of mast cell line LAD2 after TNFSF15 protein treatment. Among them, A is the effect of TNFSF15 on LAD2 cell viability detected by CCK-8; B is the detection of LAD2 cell proliferation in TNFSF15-treated group and control group; C is the detection of LAD2 cell cycle in TNFSF15-treated group and control group; D is the Western blot detection of Bax and Bcl-2 protein expression levels in LAD2 in TNFSF15-treated group and control group. ; E is the flow cytometry detection of LAD2 cell apoptosis rate after TNFSF15 treatment for 48 hours; F is the flow cytometry detection of LAD2 cell apoptosis rate after TNFSF15 treatment for 72 hours.

[0017] Figure 2 The activation of mast cell line LAD2 after TNFSF15 protein treatment. A is a typical image of mast cell line LAD2 under optical microscope in TNFSF15 treatment group and control group; B is a typical image of mast cell line LAD2 stained with toluidine blue in TNFSF15 treatment group and control group; C is qPCR detection of CD63 expression at transcriptional level in LAD2 cells in TNFSF15 treatment group and control group; D is Western blot detection of CD63 protein content in LAD2 cells in TNFSF15 treatment group and control group; E is flow cytometry detection of CD63 + Mast cell ratio.

[0018] Figure 3 The release of inflammatory factors in the mast cell line LAD2 after TNFSF15 protein treatment. AJ respectively detected the expression of (A) TNF-α, (B) IL-6, (C) IL-8, (D) IL-10, (E) IL-13, (F) IFN-γ, (G) CCL2, (H) TGF-β, (I) VEGF, and (J) SCF mRNA levels in LAD2 cells treated with different concentrations of TNFSF15 by qPCR. KQ respectively detected the protein levels of (K) TNF-α, (L) IL-6, (M) IL-10, (N) IFN-γ, (O) TGF-β, (P) VEGF, and (Q) CCL2 in LAD2 cells treated with TNFSF15 by Western blot.

[0019] Figure 4 This is the activation of the signaling pathway after the mast cell line LAD2 was treated with TNFSF15 protein. Among them, AC represents the effect of TNFSF15 on the expression of mast cell surface receptor DR3; DF represents the activation of mast cells mediated by TNFSF15 through the MAPK / NF-κB / IκB signaling pathway; GI represents the activation of mast cells mediated by TNFSF15 through the PLCγ / PI3K / Akt signaling pathway; J represents the inhibition of TNFSF15-mediated mast cell activation by MAPK signaling pathway inhibitors, specifically, qPCR detection of the mRNA expression levels of CD63, TNF-α, IL-6, IL-8, IFN-γ, TGF-β, and VEGF in LAD2 cells in the treatment groups under different conditions and the control group; K represents the inhibition of TNFSF15-mediated mast cell activation by MAPK signaling pathway inhibitors, specifically, qPCR detection of the mRNA expression levels of CD63, TNF-α, IL-6, IL-8, IFN-γ, TGF-β, and VEGF in LAD2 cells in the treatment groups under different conditions and the control group.

[0020] Figure 5 The effect of conditioned medium collected from mast cell line LAD2 treated with TNFSF15 protein on macrophage activation in macrophage cell line RAW264.7. AD is qPCR detection of mRNA expression levels of IL-6, iNOS, CD80, and CD86 in LAD2 cells of treatment group and control group under different conditions. E is flow cytometry detection of CD86 + The proportion of macrophages; F is the detection of MHCⅡ by flow cytometry + The proportion of macrophages.

[0021] Figure 6 It is the related signaling pathways and cytokines in the activated and resting states of mast cells. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0028] The embodiment of the present invention provides the use of TNFSF15 protein in preparing a product for promoting mast cell activation.

[0029] In some specific embodiments, the product includes a drug and a culture medium.

[0030] In some specific embodiments, the concentration of TNFSF15 protein in the product is 3-10 μg / mL.

[0031] In some specific embodiments, the components of the culture medium are: RPMI 1640, 10% FBS and 3-10 μg / mL TNFSF15 protein.

[0032] An embodiment of the present invention further provides a product for promoting mast cell activation, wherein the product contains TNFSF15 protein.

[0033] The embodiment of the present invention also provides a method for promoting mast cell activation, and the mast cells are treated using the product.

[0034] In some specific embodiments, the method comprises the step of culturing the mast cell line LAD2 in a culture medium comprising TNFSF15 protein.

[0035] In some specific embodiments, the mast cell line LAD2 is placed in an incubator (37° C., 5% CO 2 ), and fresh culture medium containing TNFSF15 (RPMI 1640+10% FBS+3 μg / mL TNFSF15 protein) is added for 0-24 h.

[0036] In some specific embodiments, mast cells were treated with different concentrations of TNFSF15 recombinant protein (3 μg / mL or 10 μg / mL), and toluidine blue staining observed that the blue-purple particles in the LAD2 cytoplasm of the TNFSF15-treated group were significantly reduced, the cell morphology changed, and the degranulation of mast cells was visually observed. At the same time, the expression of CD63, a surface marker of mast cell activation, increased. Further qPCR and Western blot experimental results showed that within a period of time after TNFSF15 treatment, intracellular TNF-α, IL-6, IL-8, IL-10, IL-13, IFN-γ, CCL2, TGF-β, VEGF, SCF and other cytokines were synthesized in large quantities and released outside the cells. This has a certain effect on the tumor immune microenvironment.

[0037] The embodiments of the present invention also provide the use of TNFSF15 protein in the preparation of a drug for regulating the number of mast cells in a tumor microenvironment.

[0038] In some specific embodiments, the TNFSF15 protein regulates the number of mast cells in the tumor microenvironment by inhibiting mast cell proliferation and promoting mast cell apoptosis.

[0039] The embodiments of the present invention also provide the use of TNFSF15 protein in preparing a drug for promoting mast cell degranulation.

[0040] In some specific embodiments, the TNFSF15 protein promotes mast cell degranulation, especially the release of inflammatory factors such as TNF-α, IL-6, IL-8, IL-10, IL-13, IFN-γ, CCL2, TGF-β, VEGF, and SCF.

[0041] The embodiments of the present invention also provide the use of TNFSF15 protein in promoting mast cell degranulation, activating anti-tumor macrophages through released inflammatory factors, and enhancing immune activity.

[0042] In some specific embodiments, TNFSF15 protein activates mast cells to produce factors that promote MHC II + ,CD86 + The increase of macrophages can enhance the anti-tumor effect.

[0043] In some specific embodiments, mast cell culture medium (RPMI 1640 + 10% FBS + 3 μg / mL TNFSF15 recombinant protein) was collected and temporarily stored at -20°C, and the macrophage concentration was adjusted to 5x10 5 / well, after the cells were cultured in complete medium for 12 hours, the old medium was discarded, and the cells were gently washed once with PBS. The corresponding stimuli were added: 1 mL of complete medium (containing 3 μL Buffer), 1 mL of complete medium (containing 3 μL TNFSF15), 1 mL of complete medium and LAD2-3 μg / mL mixed solution (ratio 1:1), and 1 mL of complete medium and LAD2-TNFSF15-3 μg / mL mixed solution (ratio 1:1). Return to the incubator and continue to culture for 24 hours. Total RNA was extracted from the cells, and the expression of IL-6, iNOS, CD80, and CD86 was detected by qPCR, and the expression of MHCⅡ and CD86 was detected by flow cytometry.

[0044] The mast cells used in the present embodiment are mast cell line LAD2, which were purchased from the American type culture collection (ATCC).

[0045] Example 1

[0046] Effect of TNFSF15 protein on the growth of mast cells

[0047] Effect of TNFSF15 on mast cell proliferation

[0048] (1) Cell treatment: Collect mast cells into a 15 mL centrifuge tube, wash with PBS, dilute and count. Calculate the required cell volume, aspirate the cell suspension into a new centrifuge tube, centrifuge at 200 g for 3 min, and discard the supernatant. After centrifugation, resuspend the remaining cells in complete culture medium, inoculate them into a cell culture dish, and culture them for later use.

[0049] (2) Staining: After centrifugation and discarding the supernatant, the cells were resuspended in 1 mL of PBS, and 1 mL of CFSE dye (prepared in RPMI1640 basal medium, final concentration of 2.5 μM) was added to the cells and mixed by pipetting. Incubate at 37°C in the dark for 15 min, and mix by inverting every 5 min. Add 800 μL of FBS and continue incubating at 37°C in the dark for 10 min to terminate the staining. After the staining is terminated, centrifuge at 200 g for 3 min and discard the supernatant. The cells were resuspended and washed twice in the medium before plating.

[0050] (3) Preparation of TNFSF15: Calculate the volume of TNFSF15 required for each concentration and prepare complete culture medium of the corresponding concentration. Add 500 μL of culture medium containing the corresponding amount of TNFSF15 or buffer to each well of a clean 12-well plate. The concentration of TNFSF15 in the culture medium is 3 μg / mL or 10 μg / mL, respectively.

[0051] (4) After staining, 2×10 cells were collected per well. 5 Evenly add 100 cells to the 12-well plate with the drug added, shake gently, and carefully transfer to a 37°C cell incubator with 5% CO2 for culture.

[0052] (5) After 24 h of culture, the cell suspension in the corresponding wells was collected and centrifuged at 200 g for 3 min. The supernatant was discarded. After resuspending and washing with PBS, the cells were resuspended in 200 μL PBS, passed through a 200-mesh nylon sieve, and transferred to a 5 mL open-top round-bottom flow tube. The cell fluorescence intensity was detected by flow cytometry.

[0053] (6) After 48 h of culture, the cells were treated in the same manner as at 24 h, and the cell fluorescence intensity was detected by flow cytometry.

[0054] Effect of TNFSF15 on mast cell apoptosis

[0055] (1) Preparation of TNFSF15: Calculate the volume of TNFSF15 required for each concentration and prepare complete culture medium of corresponding concentration. Unseal a sterile 12-well plate and add 500 μL of culture medium containing the corresponding amount of TNFSF15 or buffer to each well. The concentration of TNFSF15 in the culture medium is 3 μg / mL or 10 μg / mL, respectively.

[0056] (2) Cell plating: After mast cells have been passaged 2-3 times, the number of cells is sufficient and the growth is healthy. The cells in the logarithmic growth phase are resuspended with PBS, washed, and counted. 2×10 cells are plated per well. 5 Take out the required cell suspension, centrifuge, resuspend with the required volume of complete medium, and evenly drop into the 12-well plate, with a volume of 1mL per well. After inoculating the cells, shake them to make them uniform. Carefully transfer them to the cell culture incubator for culture.

[0057] (3) After the cells were cultured for 48 h, the cell suspension was collected and centrifuged at 1000 g for 5 min to precipitate the cells. The supernatant was discarded and the cells were resuspended and washed with pre-cooled PBS.

[0058] (4) According to the instructions of the apoptosis kit, resuspend each tube of cells with 100 μL 1× binding buffer, add 5 μL Annexin V / FITC, mix with a gun, and incubate at room temperature in the dark for 5 minutes. Then add 5 μL propidium iodide solution (PI), immediately pass through a 200-mesh nylon sieve and transfer to a 5 mL round-bottom flow tube. Add an appropriate volume of PBS according to the number of cells and mix. Immediately test on the machine. Pay attention to setting blank wells and single-stained wells.

[0059] (5) After the cells were cultured for 72 h, they were treated in the same way as at 48 h, and the apoptotic rate of cells in each group was detected by flow cytometry.

[0060] 3 Experimental results

[0061] Depend on Figure 1 It can be observed in A to C that after 48 hours of treatment with 10 μg / mL TNFSF15, the peak shifted to the right, indicating that the fluorescence intensity was higher than that of the control group, and the number of cells was significantly less than that of the control group. At the same time, compared with the control group, the proportion of cells in the S phase in the TNFSF15-treated group was significantly reduced, while the proportion of cells in the G2 / M phase was significantly increased, which means that a large number of cells were blocked in the G2 / M phase, that is, TNFSF15 can inhibit the proliferation of mast cells; Figure 1 As shown in Figures D to F, compared with the control group, the apoptosis rate of LAD2 cells in the group treated with 10 μg / mL TNFSF15 for 48 h was significantly increased, and the anti-apoptotic protein Bcl-2 was significantly decreased under the stimulation of TNFSF15, which suggests that TNFSF15 has good potential in inducing mast cell apoptosis.

[0062] Example 2

[0063] 1 A culture medium for promoting mast cell activation, the composition of the culture medium is: RPMI 1640+10% FBS+3μg / mL TNFSF15 protein.

[0064] 2. A method for activating mast cells, comprising the following steps: adding mast cells to the above-mentioned culture medium for promoting mast cell activation and culturing for 24 hours.

[0065] Effect of TNFSF15 protein on mast cell activation

[0066] 3.1 Morphological observation of mast cell activation

[0067] (1) TNFSF15 preparation and cell plating: same as Example 1.

[0068] (2) After the cells were cultured for 24 hours, the cell suspension was collected and centrifuged at 800g for 5 minutes to precipitate the cells. The supernatant was discarded and the cells were gently washed 2-3 times with pre-cooled PBS and the supernatant was discarded. The cells were fixed with 200μL 4% paraformaldehyde and incubated at 4℃ overnight. Centrifuged at 800g for 5 minutes in a 4℃ centrifuge and the supernatant was discarded. After washing once with PBS, the cells were resuspended with 200μL PBS.

[0069] (3) Use a 200 μL pipette to draw up the cell suspension and drop it onto a clean glass slide, allowing the liquid to spread freely but not to flow off the slide. Let it dry at room temperature, or place it in a fume hood to dry.

[0070] (4) Add toluidine blue dye solution and let stand at room temperature for 2 min. Rinse with running water for 30 s to remove excess dye solution.

[0071] (5) Add the color separation solution and let it stand at room temperature for 4 minutes. Rinse with running water for 30 seconds.

[0072] (6) Observe the cell staining. If the staining is too dark, repeat step (5).

[0073] (7) Shake off the residual water on the slide and seal the slide.

[0074] (8) Observe under a microscope and take photos.

[0075] 3.2 Detection of mast cell surface marker proteins

[0076] (1) TNFSF15 preparation and cell plating: same as Example 1.

[0077] (2) After the cells were cultured for 12 h, the cell suspension was collected and centrifuged at 1000 g for 5 min at 4 °C to precipitate the cells. The supernatant was discarded and the cells were resuspended and washed with PBS.

[0078] (3) According to the instructions, add 100 μL PBS and CD63 flow cytometry antibody (5 μL / 10 6 cells), incubated at 4°C in the dark for 30 min, and mixed by inversion every 10 min. After incubation, the cells were centrifuged at 1000 g for 5 min to precipitate the cells, the supernatant was discarded, and the cells were resuspended and washed with PBS. The cells were resuspended with 200 μL PBS, passed through a 200-mesh sieve, and transferred to a 5 mL round-bottom flow tube. The proportion of CD63-positive cells was detected by flow cytometry.

[0079] 3.3 Results Analysis

[0080] Depend on Figure 2 In A and B, it can be observed that there are a large number of blue-purple metachromatic granules in the cytoplasm of LAD2 cells in the control group. After the treatment of TNFSF15 recombinant protein, the blue-purple granules in the cytoplasm of LAD2 cells are significantly reduced, the cell morphology is changed, and the degranulation of mast cells is visually observed; Figure 2 As shown in Figures C to E, the CD63 content in the TNFSF15-treated group was significantly higher than that in the control group, both at the transcriptional level and the protein level. That is, TNFSF15 increased the expression of mast cell activation surface marker proteins, which confirmed that mast cells were activated under the stimulation of TNFSF15.

[0081] Example 3

[0082] Regulatory effect of TNFSF15 protein on inflammatory factors associated with mast cell degranulation

[0083] 1 A culture medium for promoting mast cell activation, the composition of the culture medium is: RPMI 1640+10% FBS+3μg / mL TNFSF15 protein.

[0084] 2. A method for activating mast cells, comprising the following steps: adding mast cells to the above-mentioned culture medium for promoting mast cell activation and culturing for 24 hours.

[0085] Effect of TNFSF15 on the mRNA expression of inflammatory factors related to mast cell degranulation

[0086] (1) TNFSF15 preparation and cell plating: same as Example 1.

[0087] (2) Remove the cells from which RNA extraction is required from the incubator, collect the cell suspension into labeled 1.5 mL or 2 mL centrifuge tubes, centrifuge at 300 g for 5 min, discard the supernatant, add 1 mL of PBS to wash once, centrifuge and discard the supernatant.

[0088] (3) Add 1 mL of Trizol reagent to each tube, blow evenly with a gun, and let stand at room temperature for 10 min.

[0089] (4) Add 200 μL of chloroform to each tube, shake vigorously to mix, and let stand at room temperature for 10 min.

[0090] (5) Centrifuge at 4°C, 12,000 rpm for 10 min. Clear stratification of the liquid can be observed. Transfer the upper aqueous phase as completely as possible to a new sterile, enzyme-free 1.5 mL centrifuge tube and record the volume of the supernatant.

[0091] (6) Add an equal volume of isopropanol to the supernatant, mix immediately, and incubate at 37°C for 10 min.

[0092] (7) Centrifuge at 12,000 rpm at 4°C for 30 min and discard the supernatant.

[0093] (8) Wash each tube with 1 mL of 75% ethanol, centrifuge at 4°C, 12,000 rpm for 5 min, and discard the supernatant. Repeat twice.

[0094] (9) Open the lid and place in a fume hood to air dry.

[0095] (10) Add 10-20 μL DEPC water to each tube to dissolve RNA, measure the concentration and record it.

[0096] (11) Reverse transcription.

[0097] (12) qPCR was used to detect the expression of inflammatory factors TNF-α, IL-6, IL-8, IL-10, IL-13, IFN-γ, CCL2, TGF-β, VEGF, and SCF at the mRNA level.

[0098] 4. Effect of TNFSF15 on the fate of inflammatory factors associated with mast cell degranulation

[0099] (1) TNFSF15 preparation and cell plating: same as Example 1.

[0100] (2) Prepare a box of ice, take out the cell plate that needs to be protein extracted from the cell culture incubator, collect the cell suspension into a new 1.5 mL centrifuge tube after observation under a microscope, centrifuge at 800 g for 5 min, discard the supernatant, add 1 mL PBS to wash once, centrifuge and discard the supernatant. Place on ice.

[0101] (3) Add 200 μL of cell lysis buffer to each tube, vortex and place on ice for 30 min. Vortex every 6 min. Centrifuge at 12,000 rpm for 15 min at 4°C, collect the supernatant into a new 1.5 mL centrifuge tube, and record the volume. Take out 20 μL of supernatant from each tube for BCA protein concentration determination.

[0102] (4) Open the metal bath and heat it to 100°C in advance. Dilute the 4× protein loading buffer to 2× with ddH2O, add an equal amount of 2× protein loading buffer to the supernatant, and boil in the metal bath for 8 min.

[0103] (5) Remove from the metal bath, centrifuge briefly at high speed, cool to room temperature, and store in a -20°C refrigerator.

[0104] (6) Determine the sample protein concentration according to the instructions of the BCA protein concentration assay kit.

[0105] (7) Western blot was used to detect the expression of TNF-α, IL-6, IL-10, IFN-γ, TGF-β, VEGF, and CCL2 at the protein level.

[0106] 5. Results Analysis

[0107] Depend on Figure 3As shown in Figures A to J, the mRNA levels of TNF-α, IL-6, IL-8, IL-10, IL-13, IFN-γ, CCL2, TGF-β, VEGF, and SCF in the TNFSF15 treatment group increased significantly, especially when the treatment concentration was 10 μg / mL. It can be concluded that within a period of time after TNFSF15 treatment, a large amount of cytokines such as TNF-α, IL-6, IL-8, IL-10, IL-13, IFN-γ, CCL2, TGF-β, VEGF, and SCF were synthesized in cells, and this regulatory effect was concentration-dependent. Figure 3 From the KQ, we can see that LAD2 cells in the growth phase were divided into two groups, and treated with 10μg / mL TNFSF15 and an equal amount of Buffer, respectively. After 24 hours, the cells were collected, and the total protein was extracted to detect the intracellular protein content. The electrophoresis bands showed that the bands of TNF-α, IL-6, IL-10, IFN-γ, TGF-β, and CCL2 in the LAD2 cells of the TNFSF15 treatment group were significantly lighter, that is, the expression level was lower. Combining the results of qPCR and Western blot, we can see that mast cells synthesize a large number of cytokines after being stimulated by the outside world. These mediators are not accumulated in the cells after synthesis, but are transported to the outside of the cells or degraded through some or some pathways, which is most likely caused by degranulation after mast cells are activated.

[0108] Example 4

[0109] Study on the mechanism of TNFSF15 promoting mast cell degranulation 1 TNFSF15 preparation and cell plating

[0110] (1) Preparation of TNFSF15: Calculate the volume of TNFSF15 required for each concentration and prepare complete culture medium of the corresponding concentration. Add 500 μL of culture medium containing the corresponding amount of TNFSF15 or buffer to each well of a clean 6-well plate. The concentration of TNFSF15 in the culture medium is 3 μg / mL or 10 μg / mL, respectively.

[0111] (2) Dissolution of inhibitor: This step should be carried out strictly according to the reagent instructions.

[0112] (3) Cell plating: After resuspending in PBS and counting, 5×10 cells were plated per well. 5 Calculate the required number of cells, take out the corresponding amount of cell suspension, centrifuge, resuspend in complete medium, and inoculate evenly into 6-well plates at a volume of 500 μL per well.

[0113] (4) Cell addition: The cells were divided into three groups and stimulated, namely, Buffer, TNFSF15, and TNFSF15 + signaling pathway inhibitor. After adding the drugs, the cells were shaken to mix and cultured in a 37°C cell culture incubator for 24 h.

[0114] 2 RNA extraction and protein detection: same as Example 4.

[0115] 3 Results Analysis

[0116] Depend on Figure 4 As shown in A to C, when the TNFSF15 protein treatment concentration was 10 μg / mL, the DR3 expression in the treatment group was significantly higher than that in the control group, and there was a statistical difference. This indicates that at the transcriptional level, DR3 in mast cells is upregulated by TNFSF15, and the results at the protein level also confirm this. Figure 4 The results of D to F showed that the expression of MAPK, NF-κB and IκB showed a significant upregulation in the TNFSF15 protein treatment group, both at the transcriptional level and the translational level, and Western blot bands showed that the protein phosphorylation levels of these key signaling molecules were significantly higher than those in the control group, indicating that the binding of TNFSF15 to DR3 on the surface of mast cells activates the intracellular MAPK / NF-κB / IκB signaling pathway, thereby regulating the growth, proliferation and activation of cells. Figure 4 The protein level detection of G~I showed that although the treatment with TNFSF15 did not cause significant changes in the total protein content of PLCγ, PI3K, and Akt, their phosphorylation levels increased significantly, indicating that TNFSF15 promotes the phosphorylation of PLCγ, PI3K, and Akt, maintains the activation signal in the cell, and promotes the degranulation of mast cells. Figure 4 The reverse validation results of J and K signaling pathway inhibitors showed that 3 μg / mL TNFSF15 treatment can significantly promote the expression of CD63, TNF-α, IL-6, IL-10, TGF-β, and VEGF, and the expression levels of IL-8 and CCL2 also tend to increase. After co-treatment with SB203580 or LY294002, this upregulation of gene expression will be neutralized, and the expression levels of CD63, TNF-α, IL-6, CCL2, and TGF-β are even lower than those in the control group. The above results all indicate that the MAPK / NF-κB / IκB signaling pathway and the PLCγ / PI3K / Akt signaling pathway play an important role in the activation of mast cells.

[0117] Example 5

[0118] Effect of TNFSF15 protein on macrophage activation after mast cell activation

[0119] 1. Collection of Conditioned Medium

[0120] (1) LAD2 cells with good growth status were subcultured, and 10 mL of complete medium was added to each cell culture dish. Two concentrations of 3 μg / mL and 10 μg / mL were set, and TNFSF15 or an equal volume of buffer was calculated and added respectively.

[0121] (2) After 24 h, the cell suspension was collected into a 15 mL centrifuge tube and centrifuged at 200 g for 3 min. The supernatant was collected into a new centrifuge tube and labeled as LAD2-3 μg / mL, LAD2-10 μg / mL, LAD2-TNFSF15-3 μg / mL, and LAD2-TNFSF15-10 μg / mL, respectively. The tubes were stored in a -20 °C refrigerator for short-term use.

[0122] Effect of TNFSF15 protein on macrophage activation after mast cell activation

[0123] (1) Cell plating: After resuspending in PBS and counting, 5×10 cells were plated per well. 5 Calculate the required number of cells, take out the corresponding amount of cell suspension, centrifuge, resuspend in complete medium, inoculate 500 μL per well evenly into 6-well plates, shake to mix, and culture in a 37°C cell culture incubator.

[0124] (2) Adding drugs to cells: After the cells are cultured for 12 hours, discard the old culture medium, wash gently with PBS once, and add the corresponding stimulus (taking the 3μg / mL treatment group as an example): 1mL of complete culture medium (containing 3μL Buffer), 1mL of complete culture medium (containing 3μL TNFSF15), 1mL of complete culture medium and LAD2-3μg / mL mixed solution (ratio 1:1), and 1mL of complete culture medium and LAD2-TNFSF15-3μg / mL mixed solution (ratio 1:1). The 10μg / mL treatment group was added with the corresponding stimulus in the same way. Return to the incubator and continue culturing.

[0125] (3) Total cell RNA was extracted and reverse transcribed according to the instructions provided by the reagent manufacturer, and then qPCR was used to detect the expression of IL-6, iNOS, CD80, and CD86.

[0126] (4) Flow cytometry detection of MHCⅡ and CD86 expression:

[0127] a. Collect RAW264.7 cells, transfer to 1.5 mL EP tube, centrifuge at 400 g for 5 min, and discard the supernatant;

[0128] b. According to the antibody instructions, dilute MHCⅡ, CD86 antibodies in PBS at a ratio of 1:100. After centrifugation, add 100 μL of antibody dilution solution to the cells and mix gently with a pipette;

[0129] c. Incubate at 4°C in dark for 30 min, centrifuge at 400 g for 5 min, discard the supernatant, and add 1 mL of PBS for washing;

[0130] d. Centrifuge at 400g for 5 min, discard the supernatant, add 200μL PBS to resuspend, pass through a 70μm sieve, transfer to a flow cytometer, and analyze on a flow cytometer.

[0131] 3 Results Analysis

[0132] Depend on Figure 5 It can be seen that compared with the single TNFSF15 group and the single LAD2 group, the expressions of IL-6, iNOS, CD80 and CD86 in the TNFSF15+LAD2 group were significantly increased, and the macrophage CD86 + and MHCII + The expression of TNFSF15 was also significantly increased. All of the above indicate that TNFSF15 achieves the effect of resisting tumor by promoting mast cell degranulation and then activating anti-tumor macrophages.

[0133] In summary, the present invention found that TNFSF15 protein promotes mast cell degranulation in vitro and increases the release of inflammatory factors that regulate the occurrence and development of cancer. The released inflammatory factors are active in the tumor microenvironment, activating macrophages with tumor cell-killing function, thereby activating the tumor immune microenvironment. At the same time, in this process, mast cell apoptosis is promoted, reducing the number of mast cells that play a role. In-depth research on this discovery is of great significance in basic and clinical medical research on anti-tumor, and can provide new ideas for future clinical tumor treatment.

[0134] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of TNFSF15 protein in the preparation of products promoting mast cell activation.

2. The use according to claim 1, characterized in that: The products include drugs and culture media.

3. The use according to claim 1, characterized in that: The concentration of TNFSF15 protein in the product is 3 to 10 μg / mL.

4. A product for promoting mast cell activation, characterized in that: The product contains TNFSF15 protein.

5. A method for promoting mast cell activation, characterized in that: The product according to claim 4 is used to treat mast cells.

6. Application of TNFSF15 protein in the preparation of drugs that regulate the number of mast cells in the tumor microenvironment.

7. The use according to claim 6, characterized in that: TNFSF15 protein regulates the number of mast cells in the tumor microenvironment by inhibiting mast cell proliferation and promoting mast cell apoptosis.

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