阅读说明：本技术 Tnfsf15蛋白作为巨噬细胞免疫增强剂的用途及其活化方法 (Application of TNFSF15 protein as macrophage immunopotentiator and activation method thereof ) 是由 张强哲 李鲁远 赵灿灿 于 2021-07-29 设计创作，主要内容包括：本发明公开了一种TNFSF15蛋白作为巨噬细胞免疫增强剂的用途及其活化方法,属于医药技术领域,在该用途中,TNFSF15蛋白能在体外活化小鼠M-CSF或GM-CSF诱导的骨髓来源巨噬细胞、小鼠腹腔巨噬细胞和巨噬细胞系Raw264.7为M1型,增强其杀伤肿瘤细胞功能；且在体内TNFSF15蛋白能增强巨噬细胞免疫活性抑制肿瘤生长,可为日后临床肿瘤治疗提供新的思路。(The invention discloses an application of TNFSF15 protein as a macrophage immunopotentiator and an activation method thereof, belonging to the technical field of medicines, wherein in the application, the TNFSF15 protein can activate mouse M-CSF or GM-CSF-induced bone marrow-derived macrophages, mouse abdominal cavity macrophages and a macrophage system Raw264.7 in vitro to be M1 type, so that the function of killing tumor cells is enhanced; and the TNFSF15 protein can enhance the immune activity of macrophages in vivo and inhibit the growth of tumors, thereby providing a new idea for clinical tumor treatment in the future.)

Use of TNFSF15 protein as macrophage immunopotentiator in preparing medicine for treating tumor.

2. The use of claim 1, wherein the therapeutically effective amount of TNFSF15 protein is mixed with a pharmaceutically acceptable carrier and/or excipient to form a composition.

3. The use according to claim 1, wherein the macrophages are activated to M1 type.

4. A method of in vitro activation of macrophages to M1-type macrophages, comprising the step of culturing the macrophages in a polarized medium comprising TNFSF15 protein.

5. The method of claim 4, wherein said TNFSF15 protein is present in the polarizing medium at a concentration of no more than 10 μ g/mL.

6. The method of claim 4, wherein the macrophage is a mouse macrophage including a mouse M-CSF or GM-CSF-induced myeloid-derived macrophage, a mouse peritoneal macrophage, and/or a macrophage line Raw264.7.

7. The method according to any one of claims 4 to 5, characterized in that it comprises in particular the steps of: culturing macrophage in incubator at 37 deg.C and 5% CO for 12 hr₂The medium was aspirated, and a fresh polarizing medium containing TNFSF15 was added to the medium and cultured for 24h to activate macrophages to M1.

8. A polarized culture medium for in vitro macrophage activation, wherein the polarized culture medium comprises TNFSF15 protein.

9. The polarization medium of claim 8, wherein the concentration of TNFSF15 protein in the polarization medium is no greater than 10 μ g/mL.

10. A polarised medium according to claim 8, characterised in that the composition of the polarised medium is: RPMI 1640+ 10% FBS + 3. mu.g/mL TNFSF15 protein.

Technical Field

The invention relates to the technical field of medicines, in particular to application of TNFSF15 protein as a macrophage immunopotentiator and an activation method thereof.

Background

Tumors are a common serious disease faced by human beings, and the incidence of the tumors is increased year by year along with the increase of environmental pollution and life pressure. The methods for treating the tumor mainly comprise surgical excision and radioactive ray killing aiming at local tumor and methods for killing tumor cells by using chemical drugs, and the methods have the defects of incapability of eradicating the disease, easy relapse and great side effect. With the development of modern immunology, it is recognized that stabilization of immune function plays an important role in tumorigenesis, development, metastasis, reversal, and regression. Tumors are a complex system whose microenvironment, in addition to their own tumor cells, contains many resident cell types, such as adipocytes, fibroblasts, macrophages, neutrophils and mast cells. Among them, macrophages often play a crucial role in tumorigenesis, development, and metastasis. Macrophages are important cellular components of the body's innate immune system and are highly heterogeneous. Macrophages are essentially classified into classical activated macrophages (type M1) having pro-inflammatory and killing effects and Alternative activated macrophages (type M2) having anti-inflammatory and repairing functions, depending on the activation mode. Pathological studies show that many diseases occur in association with macrophage polarity, such as arthritis, atherosclerosis, obesity, etc. due to the large amount of M1 macrophages in the focus, proinflammatory factors secreted by the macrophages aggravate body injury; in addition, the macrophage M2 has too strong polarity, which can also cause diseases such as asthma, fibrosis, parasitic infection and tumor, so that the maintenance of the polarity balance of the macrophages M1 and M2 is important for the health of the organism. Most intratumoral macrophages exhibit the M2 type, promoting tumor growth, and thus, knocking out intratumoral macrophages is the method tried by many researchers. Research shows that the growth of tumor can be inhibited by blocking CSF1R signal transduction through a macrophage specific receptor CSF1R monoclonal antibody or a small tyrosine kinase inhibitor and then eliminating TAM of the tumor. In addition, CCR2 receptor is blocked, macrophage recruitment by tumors is prevented, so that the amount of TAM in the tumors is reduced, and the aim of inhibiting tumor growth can be fulfilled. However, this method cannot accurately distinguish all macrophages in the tumor from macrophages in other regions, and simple and rough elimination can cause side effects and long-term toxicity; in addition, after macrophage elimination, the immune system in the tumor lacks an important link and can not activate other immune cells in the body. Therefore, the conversion of TAM to M1 macrophages with anti-tumor function is considered a better potential therapeutic approach.

Tumor necrosis factor superfamily member 15(Tumor necrosis factor super family-15, TNFSF15, also known as TL1A) is a blood vessel growth inhibitory factor secreted mainly by mature vascular endothelial cells. Researches find that the compound is not only a blood vessel negative regulation factor, and can inhibit angiogenesis and tumor growth in tumors; can also be used as an immune activator to promote T cell activation and dendritic cell maturation. At present, no relation between TNFSF15 and macrophages has been reported.

Disclosure of Invention

The invention aims to provide application of TNFSF15 protein as a macrophage immunopotentiator and an activation method thereof, which are used for solving the problems in the prior art, and the protein can enhance the polarity of macrophage M1, so that the macrophage M1 has a killing effect, and provides a treatment scheme for tumor treatment.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an application of TNFSF15 protein as a macrophage immunopotentiator in preparing a medicament for treating tumors.

Further, in the use, the TNFSF15 protein with effective treatment quantity is mixed with a pharmaceutically acceptable carrier and/or excipient to prepare a composition.

Further, the macrophages are activated to M1 type.

In this use, the TNFSF15 protein increases the proportion of macrophages in the tumor, particularly M1 macrophages positive for antigen presenting proteins (MHC2 and CD86), and inhibits tumor growth. When macrophages, especially M1 macrophages, in tumors are eliminated by macrophage scavengers, the tumor growth inhibitory effect of TNFSF15 protein is impaired.

Specifically, TNFSF15 overexpression plasmid was constructed, LLC cells were infected with lentivirus, cell lines overexpressing TNFSF15 were selected, and control cells were added at 5X 10 cells per mouse⁵Two weeks after inoculation of LLC cells in the C57BL/6 shoulder, TNFSF15 protein was observed to inhibit tumor growth and to increase infiltration of macrophages, especially M1 macrophages, in the tumor when these macrophages are infiltratedAfter macrophage scavenger (CL) clearance, TNFSF15 decreased its tumor growth-inhibiting effect.

The present invention also provides a method for in vitro activation of macrophages to M1-type macrophages, the method comprising the step of culturing the macrophages in a polarized medium comprising TNFSF15 protein.

Further, the concentration of the TNFSF15 protein in the polarized medium is not higher than 10 μ g/mL.

Further, the macrophage is mouse macrophage, including mouse M-CSF or GM-CSF induced bone marrow derived macrophage, mouse peritoneal macrophage and/or macrophage line Raw264.7.

Further, the method specifically comprises the following steps: culturing macrophage in incubator at 37 deg.C and 5% CO for 12 hr₂The medium was aspirated, and a fresh polarizing medium containing TNFSF15 was added to the medium and cultured for 24h to activate macrophages to M1.

Specifically, the method comprises the following steps:

taking out mouse bone marrow cells, resuspending the bone marrow cells in (RPMI 1640+ 15% FBS +50ng/mL M-CSF/GM-CSF) culture medium, and adjusting the cell concentration to 2X 10⁶2mL per well, into a well plate (e.g., 6 well plate), and in an incubator (37 ℃, 5% CO)₂) The culture was carried out for 7 days. When the proportion of the bone marrow cells induced into macrophages reaches more than 85%, the culture medium is removed by aspiration, and fresh polarized culture medium (RPMI 1640+ 10% FBS + 3. mu.g/mL TNFSF15 protein) containing TNFSF15 is added for 24h of culture.

The mouse macrophage cell line Raw264.7 cells were resuspended in (RPMI 1640+ 10% FBS) medium and the cell concentration was adjusted to 2X 10⁵1mL per well, into a well plate (e.g., 12 well plate), and in an incubator (37 ℃, 5% CO)₂) After 12 hours of incubation, the medium was aspirated and fresh polarized medium containing TNFSF15 (RPMI 1640+ 10% FBS + 3. mu.g/mL TNFSF15 protein) was added and incubated for 24 h.

Peritoneal cells were removed from the mouse peritoneal cavity and resuspended in (RPMI 1640+ 10% FBS) medium to adjust the cell concentration to 2X 10⁶2mL per well, into a well plate (e.g., 6 well plate) inIncubator (37 ℃, 5% CO)₂) After 6 hours of culture, the medium was aspirated, washed twice with PSB to wash away nonadherent cells, and fresh polarized medium containing TNFSF15 (RPMI 1640+ 10% FBS + 3. mu.g/mL TNFSF15 protein) was added and cultured for 24 hours.

The invention also provides a polarized culture medium for in vitro macrophage activation, wherein the polarized culture medium comprises TNFSF15 protein.

Further, the concentration of TNFSF15 protein in the polarization medium is not higher than 10. mu.g/mL.

Further, the composition of the polarization medium is: RPMI 1640+ 10% FBS + 3. mu.g/mL TNFSF15 protein.

The invention discloses the following technical effects:

(1) the TNFSF15 protein can enhance the polarity of macrophage M1 in a tumor microenvironment to achieve the purpose of tumor treatment, and can be widely applied to tumor treatment.

(2) Compared with chemotherapy, TNFSF15 has the advantages of low toxicity and high safety as biological protein.

(3) The TNFSF15 can effectively enhance the polarity of M1 of macrophages from different sources, and can be applied to related diseases with over-strong polarity of M2, such as asthma, fibrosis, parasitic infection and bacterial infection.

(4) The TNFSF15 purification method is simple and mass, has low economic cost, can be used for combined treatment with other treatment modes, and has wide application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the change of polarity of the TNFSF15 protein after treating bone marrow-derived macrophages.

FIG. 2 shows the change of polarity of the TNFSF15 protein after it treated peritoneal macrophages.

FIG. 3 shows the change of polarity of the macrophage cell line Raw264.7 after the TNFSF15 protein is treated.

FIG. 4 shows the change of TNFSF15 protein in phagocytosis of E.coli by macrophage Raw264.7 cells, wherein A is the fluorescence photograph of macrophage Raw264.7 treated group and untreated group, and B is the statistical analysis of the mean fluorescence intensity of macrophage phagocytosis of E.coli.

FIG. 5 shows the tumor cell killing effect of TNFSF15 protein on Raw264.7 macrophage cell line, where A is the change of LLC tumor cell swallowing effect of TNFSF15 protein on Raw264.7 macrophage cell line, and B is the change of LLC tumor cell apoptosis induced by TNFSF15 protein on Raw264.7 macrophage cell line.

FIG. 6 shows that TNFSF15 protein up-regulates the ratio of M1 macrophages to inhibit tumor growth, wherein A is the effect of TNFSF15 protein on LLC transplantable tumors; b is the influence of TNFSF15 protein on M1 macrophage in tumor; c is the influence of TNFSF15 protein on M2 macrophage in tumor; d is the influence of TNFSF15 protein on the ratio of M1/M2 macrophages in tumors.

FIG. 7 shows the effect of eliminating TNFSF15 protein on tumor macrophages and tumor growth by macrophage depletion, where A is TNFSF15 protein and macrophage scavenger clodronate on MHC II in LLC transplanted tumors⁺The effect of M1 macrophages, B is TNFSF15 protein and the macrophage scavenger clodronate on LLC transplantable intratumoral CD86⁺The effect of M1 macrophages, wherein C is LLC transplanted tumor images of different treatment groups of TNFSF15 protein and macrophage scavenger clodronate, and D is the change condition of LLC transplanted tumor volume of different treatment groups of TNFSF15 protein and macrophage scavenger clodronate.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 Effect of TNFSF15 protein on macrophage activation

1) Obtaining bone marrow-derived macrophages

(1) And (3) preparation: placing clean surgical instruments (at least two small scissors and small forceps), gauze (8 layers, 20 pieces), and nylon membrane (70 μm pore diameter, 10 pieces) in a lunch box, autoclaving at 120 deg.C for 20 min;

(2) and taking out bone marrow cells: the male mice were sacrificed for 5 weeks, the hind leg bones were removed, and three connected bones were removed from one leg, the surrounding tissues were removed with gauze, the joints at both ends of the bones were cut off, and the bone marrow cells were flushed out with PBS aspirated by a 1mL syringe. Collecting the flushed bone marrow cells together to form red sheets, and blowing and beating the red sheets by using a 1mL syringe and a pipette gun to disperse the red sheets into single cell suspension;

(3) filtering the collected bone marrow cells with a 70-micron nylon net, centrifuging the filtered bone marrow cells for 5min at 400g and 4 ℃, and removing supernatant;

(4) and lysing erythrocytes: adding 2mL of erythrocyte lysate, resuspending, placing on ice, adding 10mL of PBS solution containing 2% FBS after 3min, and uniformly mixing;

(5) centrifuging at 4 ℃ for 5min at 400g, removing the supernatant, adding 10mL of 2% FBS PBS solution, and mixing uniformly;

(6) counting: 10. mu.L of the cell suspension was aspirated, diluted by a certain factor, and counted under a microscope using a cell counting plate. 10mL of cells, total of four large cells/4X 10⁴X multiple × 10 Ml;

(7) centrifuging at 400g for 5min at 4 deg.C, discarding the supernatant, resuspending in (RPMI 1640+ 15% FBS +50ng/mL M-CSF or GM-CSF factor) culture medium, adjusting cell concentration to 2 × 10⁶2mL per well, into a well plate (e.g., 6 well plate), and in an incubator (37 ℃, 5% CO)₂) Culturing for 7 days;

(8) half-volume liquid change is carried out on culture media (RPMI 1640+ 15% FBS +50ng/mL M-CSF or GM-CSF factor) at 3d and 5d, when cells are harvested at 7d, flow detection is carried out by taking F4/80 as a Marker, F4/80⁺The proportion reaches more than 80 percent, and the macrophage derived from the bone marrow is considered to be obtained.

2) Peritoneal macrophage acquisition

(1) 5 days before macrophage separation, 1mL of sterile 4% thioglycollate medium is injected into the abdominal cavity of each mouse to aggregate macrophages;

(2) on the day of macrophage separation, placing a cervical-broken pretest mouse on a dissection plate, injecting 5mL of precooled serum-free RPMI-1640 culture medium into the abdominal cavity, massaging the abdomen of the mouse for 5min gently, and then standing for 5 min;

(3) the abdomen of the mouse is dissected under the aseptic condition, and the abdominal cavity fluid (more macrophages in yellow) is extracted by a 5mL syringe;

(4) sieving the peritoneal fluid with a 200-mesh sieve, collecting filtrate by using a precooled culture dish, and then collecting cell suspension in the culture dish into a 15mL precooled centrifuge tube;

(5) centrifuging at 1200rpm for 5min at 4 ℃;

(6) removing the supernatant, pouring out the supernatant as clean as possible, adding 2mL of erythrocyte lysate, resuspending, placing on ice, adding 10mL of PBS solution containing 2% FBS after 3min, and uniformly mixing;

(7) centrifuging at 4 ℃ and 1200rpm for 5min, and performing next dyeing flow analysis; if the macrophage is continuously cultured, the following steps are continued;

(8) the supernatant was discarded, and the pellet was resuspended in 10% FBS-containing RPMI-1640 medium to adjust the cell concentration to 2X 10⁶Per mL, at 4X 10 per well⁶Cells were plated in six well plates at 37 ℃ with 5% CO₂Culturing;

(9) after 6 hours, the supernatant is discarded and washed for 2 times by RPMI-1640 without FBS;

(10) using 10% FBS-containing RPMI-1640 medium, 5% CO at 37 ℃₂And continuing culturing in the incubator.

3) Preparation of Raw264.7 cells

The mouse macrophage cell line Raw264.7 cells were resuspended in (RPMI 1640+ 10% FBS) medium and the cell concentration was adjusted to 2X 10⁵1mL per well, into a well plate (e.g., 12 well plate), and in an incubator (37 ℃, 5% CO)₂) And (5) culturing.

4) Induction of M1 macrophages

Macrophage medium was replaced with an induction medium containing TNFSF15 protein (RPMI 1640+ 10% FBS + 3. mu.g/mL TNFSF15 protein) and cultured for 24 h.

5) And (4) analyzing results:

as can be seen from fig. 1, TNFSF15 protein activates bone marrow-derived macrophages to M1 type compared to the control group; as shown in FIG. 2, TNFSF15 protein activated peritoneal macrophages to M1 type; as shown in FIG. 3, the TNFSF15 protein activated macrophage cell line Raw264.7 was M1 type. From this, it is known that TNFSF15 protein can activate macrophages to M1 type.

Example 4 Effect of TNFSF15 protein on macrophage phagocytosis of bacteria

1) Treatment of Raw264.7 cells

Raw264.7 is paved in a twenty-four pore plate with a reptile round plate at the bottom, and after 12 hours, Buffer and TNFSF15 are added for processing for 24 hours;

2) phagocytosis operation

(1) Dissolving a certain amount of fluorescent particles in a sterile serum-free culture medium to make the final concentration of the particles be 10⁷Performing ultrasonic treatment for 30min, and paying attention to light shielding and sterility;

(2) removing cell supernatant by aspiration, washing with PBS once, and adding 300 mu L of serum-free culture medium dissolved with fluorescent particles into each hole;

(3) incubating at 37 ℃ for 10min, 30min and 60 min;

(4) after the incubation is finished, washing twice by using precooled PBS;

(5) adding 300 mu L of 4% paraformaldehyde into each hole, and fixing for 20min at room temperature;

(6) removing 4% paraformaldehyde by suction, and washing the PBST twice;

(7) adding 300 μ L PBS containing 0.1% Triton-100 and 5% BSA, standing at room temperature for 15 min;

(8) absorbing and discarding, and PBST cleaning twice;

(9) 200. mu.L of Biotin-F4/80 antibody dilution (1:100) was added to each well; incubating at 37 ℃ for 60 min;

(10) adding PBST, standing for 5min, removing by suction, and repeating for 3 times;

(11) adding 200 mu L of PE-Streptavidin (1:150)) antibody diluent into each well, and incubating for 60min at room temperature in a dark place;

(12) absorbing and discarding the secondary antibody, adding PBST, standing for 5min, absorbing and discarding, and repeating for 3 times;

(13) adding 200 mu L of DAPI diluent (1:1000) into each hole, and keeping out of the sun for 10min at room temperature;

(14) absorbing and discarding, and washing the PBST once;

(15) taking out the climbing slice wafer by using a pair of tweezers, and sealing the slice;

(16) shooting by using a laser scanning confocal microscope, and storing pictures;

(17) phagocytosis Index (PI) is the total number of phagocytosed fluorescent particles/total number of macrophages, and the phagocytosed fluorescent particles cannot be counted clearly, so that the fluorescence intensity is often used instead.

3) And (4) analyzing results:

as can be seen from fig. 4, TNFSF15 protein significantly increased the phagocytosis of e.coli by raw264.7 cells, and thus TNFSF15 protein was able to enhance the phagocytic ability of macrophages.

Example 5 Effect of TNFSF15 protein on killing tumor cells by macrophages

1) Effect of TNFSF15 protein on phagocytosis of tumor cells by macrophages

(1) Spreading Raw264.7 cells in a six-hole plate for 12h, and then adding Buffer and TNFSF15 for processing for 24 h;

(2) and when the LLC, 4T1 density reaches more than 90%, discarding the cell culture medium, and performing cell culture according to the following steps of 1: 4000 Calcein was dissolved in serum-free medium (final concentration: 1mM), added to LLC and 4T1, and incubated at 37 ℃ for 30 min;

(3) separately digesting Raw264.7, LLC and 4T1, washing, counting, and according to macrophage: mixing Raw264.7 in Control group and Raw264.7 in TNFSF15 group with LLC and 4T1 respectively at a ratio of 1:2, suspending in serum-free medium, spreading in 24-well ultra-low adhesion plate, and incubating at 37 deg.C for 2 h;

(4) resuspending the cells in each well, transferring to a 1.5mL EP tube, and centrifuging for 5min at 200 g;

(5) adding 100 mu L of Biotin-F4/80 antibody diluent (1:100) into each tube, and incubating for 30min at 4 ℃;

(6) centrifuging at 200g for 5min, discarding the primary antibody, and washing with PBS once;

(7) adding 100 mu L of APC-Streptavidin (1:150)) antibody diluent into each tube, and incubating for 30min at 4 ℃;

(8) centrifuging at 200g for 5min, adding cell fixing solution for resuspension, sieving with 70 μm sieve, and detecting by flow-type machine.

(9) Percent phagocytosis ═ macrophage phagocytosed tumor cells/total macrophages × 100%.

2) Effect of TNFSF15 protein on promotion of tumor cell apoptosis by macrophage

Obtaining Raw264.7 supernatant

Raw264.7 was adjusted to a density of 5X 10⁵Spreading the cells in a six-hole plate, adding corresponding Buffer and TNFSF15 into the holes after 12h for stimulation, sucking and collecting cell supernatant in a centrifuge tube after 24h, centrifuging for 10min at 500g, removing precipitate, transferring the supernatant into a new centrifuge tube, and storing at-20 ℃ for later use;

treatment of LLC tumor cells

(1) Firstly, uniformly drawing three horizontal lines and three vertical lines at the bottom of an unused 6-hole plate by using a Marker pen, wherein the intervals are kept consistent;

(2) at 5X 10⁵(ii)/well Density LLC was plated in six well plates;

(3) when the cells are overgrown, marking marks on the cells horizontally and vertically by using a yellow gun head along the marks;

(4) the culture medium is sucked and discarded, the scratched cells are washed by PBS, the cells are shot under a microscope, and the scratch width and the cell growth state at 0 are recorded;

(5) and adding a normal culture medium or a Raw264.7 condition culture medium, culturing for 24 hours, shooting again, and recording the scratch width and the cell growth state for 24 hours.

(6) And collecting protein samples of LLC cells after different treatments, and detecting the expression condition of apoptosis-related protein Caspase 3.

3) And (4) analyzing results:

from FIG. 5, TNFSF15 protein increased LLC tumor cell phagocytosis by Raw264.7 cells, and LLC cells were activated by Caspase3 cleavage in the supernatant of Raw264.7 after TNFSF15 treatment, thus TNFSF15 protein was found to enhance macrophage killing ability.

Example 6 Effect of TNFSF15 protein on macrophages in tumors

1) Obtaining a cell line of high-expression TNFSF15 or preparing TNFSF15 recombinant protein

(1) Constructing plvx-puro-TNFSF15 plasmid; or TNFSF15 recombinant protein has been prepared and applied for patent CN107541536A (here is its new medicinal effect).

(2) After plvx-puro-TNFSF15, pspAX2 and pMD.2G were transfected into 293T cells, the supernatant virus solution was collected and LLC cells were transfected. Further screening an over-expressed TNFSF15 cell strain by a limiting dilution method;

2) establishment of TNFSF15 overexpression tumor model

(1) Recovering and culturing LLC of the over-expressed hTNFSF15 and the control LLC cell line to certain amount, digesting, washing twice with PBS, and further washing with 5 × 10⁵The density per mL was resuspended in serum-free medium.

(2) 5X 10 per mouse⁵At a rate of 100. mu.L, cells were seeded subcutaneously in C57BL/6J mice. Tumor size was measured every two days and mouse body weight was recorded until tumors were harvested. Tumor volume (mm)³) Length × width/2.

3) Analysis of macrophage polarity in tumors

(1) The tumor tissue was removed from the mice, minced with small scissors, added with pancreatin, digested in a 37 ℃ water bath for 30min, shaken every 10 min.

(2) After digestion, neutralizing the mixture by using a culture medium containing 10% of serum, and centrifuging;

(3) washed once with PBS, then gently ground on a stainless steel 70 μm cell screen with an EP tube lid, and washed with PBS to disperse into individual cells.

(4) Centrifuging, washing once with PBS, adding erythrocyte lysate, and performing lysis on ice for 5min, wherein the quantity of erythrocytes can be shown by the shade of the color of the erythrocyte lysate because the erythrocyte lysate is colorless;

(5) neutralizing with PBS containing 2% serum, washing with PBS once, and passing through 70 μm nylon membrane to obtain tumor single cell suspension;

(6) then transferring the cells into a 1.5mL EP tube, centrifuging at 400g for 5min, and discarding the supernatant;

(7) diluting CD45, F4/80, CD86 and MHCII antibodies in PBS according to the antibody specification in a ratio of 1:100, centrifuging the cells, adding 100 mu L of antibody diluent, gently blowing and uniformly mixing the antibody diluent by using a pipette gun, and incubating the mixture for 30min at 4 ℃ in a dark place;

(8) centrifuging at 400g for 5min, discarding the supernatant, and adding 1mL PBS for washing;

(9) centrifuging at 400g for 5min, discarding the supernatant, adding 200 μ L PBS for resuspension, sieving with 70 μm sieve, transferring to flow tube, and performing detection and analysis on the machine.

As shown in fig. 6, the over-expression of TNFSF15 in tumor tissues inhibits tumor growth, increases the proportion of M1 macrophages, and inhibits the proportion of M2 macrophages, so that TNFSF15 protein increases the proportion of M1/M2 macrophages in tumors, thereby improving the tumor immune microenvironment.

Example 7 relationship of TNFSF15 protein to macrophage and tumor inhibition in tumors

1) Establishment of tumor model of macrophage-cleared TNFSF15 overexpression LLC

(1) Recovering and culturing LLC of the over-expressed hTNFSF15 and the control LLC cell line to certain amount, digesting, washing twice with PBS, and further washing with 5 × 10⁵The density per mL was resuspended in serum-free medium.

(2) 5X 10 per mouse⁵The amount of 100 mu L, the cells are planted into C57BL/6J mice subcutaneously, and LLC-TNFSF15 and LLC-Mock mice are randomly divided into two groups after tumors grow to 7 days;

(3) and macrophage scavenger (CL) and sterile PBS were taken out of the refrigerator and naturally returned to room temperature. Turning upside down and mixing evenly, injecting 200 mu L into the abdominal cavity of each mouse according to groups;

(4) and once every three days, measuring the size of the tumor every two days, and recording the weight of the mice until the tumor is collected. Tumor volume (mm)³) Length × width/2).

2) Analysis of macrophage polarity in tumors

(1) The tumor tissue was removed from the mice, minced with small scissors, added with pancreatin, digested in a 37 ℃ water bath for 30min, shaken every 10 min.

(2) After digestion, neutralizing the mixture by using a culture medium containing 10% of serum, and centrifuging;

(3) washed once with PBS, then gently ground on a stainless steel 70 μm cell screen with an EP tube lid, and washed with PBS to disperse into individual cells.

(4) Centrifuging, washing once with PBS, adding erythrocyte lysate, and performing lysis on ice for 5min, wherein the quantity of erythrocytes can be shown by the shade of the color of the erythrocyte lysate because the erythrocyte lysate is colorless;

(5) neutralizing with PBS containing 2% serum, washing with PBS once, and passing through 70 μm nylon membrane to obtain tumor single cell suspension;

(6) then transferring the cells into a 1.5mL EP tube, centrifuging at 400g for 5min, and discarding the supernatant;

(7) diluting CD45, F4/80, CD86 and MHCII antibodies in PBS according to the antibody specification in a ratio of 1:100, centrifuging the cells, adding 100 mu L of antibody diluent, gently blowing and uniformly mixing the antibody diluent by using a pipette gun, and incubating the mixture for 30min at 4 ℃ in a dark place;

(8) centrifuging at 400g for 5min, discarding the supernatant, and adding 1mL PBS for washing;

(9) centrifuging at 400g for 5min, discarding the supernatant, adding 200 μ L PBS for resuspension, sieving with 70 μm sieve, transferring to flow tube, and performing detection and analysis on the machine.

As can be seen from fig. 7, overexpression of TNFSF15 in tumor tissues increased the proportion of M1 macrophages and inhibited tumor growth, whereas the tumor-inhibiting effect of TNFSF15 was impaired after the proportion of M1 macrophages was decreased in the presence of macrophage scavenger, and thus, TNFSF15 inhibited tumor growth by enhancing macrophage immune activity in tumors.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.