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Application of TNFSF15 protein as macrophage immunopotentiator and activation method of TNFSF15 protein

An immune enhancer, macrophage technology, applied in the field of TNFSF15 protein as a macrophage immune enhancer, to achieve the effect of low toxicity, wide application prospects, and high safety

Active Publication Date: 2021-10-22
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no report on the relationship between TNFSF15 and macrophages

Method used

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  • Application of TNFSF15 protein as macrophage immunopotentiator and activation method of TNFSF15 protein
  • Application of TNFSF15 protein as macrophage immunopotentiator and activation method of TNFSF15 protein
  • Application of TNFSF15 protein as macrophage immunopotentiator and activation method of TNFSF15 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Effect of TNFSF15 protein on macrophage activation

[0041] 1) Acquisition of bone marrow-derived macrophages

[0042] (1) Preparation: Put clean surgical instruments (at least two small scissors and tweezers), gauze (8 layers, about 20 pieces in total), nylon membrane (70 μm pore size, 10 small pieces), in a lunch box, Autoclave, 120°C, 20min;

[0043] (2) Take out the bone marrow cells: kill the 5-week-old male mice, take out the hind leg bones, and take out three connected bones from one leg, remove the surrounding tissues with gauze, cut off the joints at both ends of the bones, and use a 1mL syringe to absorb PBS flush bone marrow cells. The washed-out bone marrow cells are aggregated together, presenting a red flake, and blown with a 1mL syringe and pipette gun to disperse them into a single-cell suspension;

[0044] (3) Filter the collected bone marrow cells with a 70 μm nylon mesh, centrifuge at 400 g for 5 min at 4° C., and discard the supernatant;...

Embodiment 4

[0067] Example 4 Effect of TNFSF15 protein on macrophage phagocytosis of bacteria

[0068] 1) Treatment of Raw264.7 cells

[0069] Raw264.7 was placed in a twenty-four-well plate with a slide disk at the bottom, and after 12 hours, Buffer and TNFSF15 were added for 24 hours;

[0070] 2) Phagocytosis operation

[0071] (1), take a certain amount of fluorescent particles and dissolve them in sterile serum-free medium, so that the final concentration of the particles is 10 7 / mL, sonicate for 30 minutes, avoid light and keep sterile;

[0072] (2) Discard the cell supernatant, wash it once with PBS, and add 300 μL of serum-free medium with fluorescent particles dissolved in each well;

[0073] (3) Incubate at 37°C for 10 min, 30 min, 60 min;

[0074] (4), after the incubation is completed, wash twice with pre-cooled PBS;

[0075] (5) Add 300 μL of 4% paraformaldehyde to each well and fix at room temperature for 20 minutes;

[0076] (6), absorb and discard 4% paraformaldehyde...

Embodiment 5

[0090] Example 5 Effect of TNFSF15 protein on killing tumor cells by macrophages

[0091] 1) The effect of TNFSF15 protein on the phagocytosis of tumor cells by macrophages

[0092] (1) Raw264.7 cells were plated in a six-well plate for 12 hours, and treated with Buffer and TNFSF15 for 24 hours;

[0093] (2) When the density of LLC and 4T1 reaches more than 90%, discard the cell culture medium, dissolve Calcein in serum-free medium at a ratio of 1:4000 (final concentration: 1mM), add it to LLC and 4T1, and keep at 37°C Incubate for 30 minutes;

[0094] (3), respectively digest Raw264.7, LLC, 4T1, wash, count, and control group Raw264.7 and TNFSF15 group Raw264.7 respectively with LLC, 4T1 according to the ratio of macrophage: tumor cell=1:2 Mix, resuspend in serum-free medium, spread in 24-well ultra-low adhesion plate, and incubate at 37°C for 2h;

[0095] (4) Resuspend the cells in each well, transfer to a 1.5mL EP tube, and centrifuge at 200g for 5min;

[0096] (5) Add ...

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Abstract

The invention discloses application of a TNFSF15 protein as a macrophage immunopotentiator and an activation method of the TNFSF15 protein, and belongs to the technical field of medicines, in the application, the TNFSF15 protein can activate mouse M-CSF or GM-CSF induced bone marrow-derived macrophages, mouse peritoneal macrophages and a macrophage line Raw264.7 into M1 type in vitro, so that the tumor cell killing function of the macrophages, the mouse peritoneal macrophages and the macrophage line Raw264.7 is enhanced; in addition, the TNFSF15 protein can enhance the immunocompetence of macrophages and inhibit tumor growth in vivo, and a new thought can be provided for clinical tumor treatment in the future.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to the use of TNFSF15 protein as a macrophage immune enhancer and its activation method. Background technique [0002] Tumor is a major disease faced by human beings. With the increase of environmental pollution and living pressure, the incidence of tumor is increasing year by year. The methods of tumor treatment mainly include surgical resection and radiation killing for local tumors, and the method of killing tumor cells with chemical drugs. These methods have the disadvantages of inability to eradicate the disease, easy recurrence, and large side effects. With the development of modern immunology, it is recognized that the stability of immune function plays an important role in the occurrence, development, metastasis, reversal and regression of tumors. A tumor is a complex system whose microenvironment contains many resident cell types in addition to its own tumor cells, such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/19A61P37/04A61P35/00C12N5/0786
CPCA61K38/191A61P37/04A61P35/00C12N5/0645C12N2501/50
Inventor 张强哲李鲁远赵灿灿
Owner NANKAI UNIV
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