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Application of gamma interferon-induced lysosomal sulfydryl reductase GILT in enhancing anti-tumor immune response

A gamma interferon and anti-tumor immune technology, applied in the field of immunomedicine, can solve the problems of undisclosed GILT breast cancer cell association

Pending Publication Date: 2022-04-12
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, prior art does not disclose the association of GILT in immune cell killing of breast cancer cells

Method used

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  • Application of gamma interferon-induced lysosomal sulfydryl reductase GILT in enhancing anti-tumor immune response
  • Application of gamma interferon-induced lysosomal sulfydryl reductase GILT in enhancing anti-tumor immune response
  • Application of gamma interferon-induced lysosomal sulfydryl reductase GILT in enhancing anti-tumor immune response

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Construction of GILT overexpression breast cancer cell model

[0021] 1. Experimental materials:

[0022] Cell lines: human breast cancer cell lines MDA-MB-231, BT-549

[0023] Reagent consumables: RPMI-1640 culture medium, DMEM culture medium, trypsin cell digestion solution, fetal bovine serum, lentiviral vector, Polybrene, protein lysate, rabbit-derived anti-human GILT antibody

[0024] Instruments: cell incubator, ultra-clean bench, fluorescence microscope, electrophoresis apparatus

[0025] 2. Experimental method:

[0026] The full-length sequence of the GILT gene was cloned into the lentiviral vector GV287 to form an overexpression lentiviral vector of GILT (GV287-GILT), and the breast cancer cell lines (MDA-MB-231, BT-549 cells ) were planted in a 6-well cell culture plate, with 100,000 cells per well. After the cells adhered to the wall, breast cancer cells were cultivated in a conventional medium containing GV287-GILT virus. In this experiment,...

Embodiment 2

[0029] Example 2: T cell sorting and activation

[0030] 1. Experimental materials:

[0031] Cells: Human peripheral blood lymphocytes (PBMC), T cells

[0032] Reagent consumables: Ficoll Paque density gradient separation medium, T cell culture medium, IL-2 (50-150IU / mL), CD3 / CD28 T cell activator, EasySepTM Buffer, Human CD3 Positive Selection CocktailII, EasySepTM Dextran RapidSpheresTM 50100, EasySepTM Magnet , BD Pharmingen Stain Buffer.

[0033] Instruments: cell incubator, ultra-clean bench, flow cytometer, centrifuge

[0034] 2. Experimental method:

[0035] PBMC isolation:

[0036] (1) Centrifuge the collected blood for 5min at 300g, and discard the plasma;

[0037] (2) Put the centrifuged blood cell pellet and an equal volume of PBS into a 10mL centrifuge tube, invert the centrifuge tube several times to fully mix the blood cells and PBS;

[0038] (3) Invert the bottle of Ficoll Paque density gradient separation solution several times, mix thoroughly, use a syri...

Embodiment 3

[0060] Example 3: GILT enhances the ability of T cells to kill tumor cells

[0061] 1. Experimental materials:

[0062] Cell lines: activated T cells, human breast cancer cell lines MDA-MB-231, BT-549

[0063] Reagent consumables: cytotoxic lactate dehydrogenase (LDH) assay kit, T cell culture medium, cell fixative, crystal violet solution, cell culture plate

[0064] Instruments: cell incubator, ultra-clean bench, microplate reader

[0065] 2. Experimental method:

[0066] LDH experiment:

[0067] (1) Draw 100 μL of breast cancer cells (cell number: 5000) and inoculate them into 96-well cell culture plates, at 37°C, 5% CO 2 Overnight pre-cultivation in the incubator;

[0068] (2) Add 100 μL of medium containing 200,000 T cells to the breast cancer cells, and continue to store at 37°C, CO 2 Cultured in an incubator.

[0069] (3) Add 10 μL Lysis Buffer to the breast cancer cells after co-cultivation, at 37°C, 5% CO 2 Incubate in the incubator for 30 minutes;

[0070] (...

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Abstract

The invention discloses an application of gamma interferon induced lysosomal sulfydryl reductase GILT in enhancement of anti-tumor immune response. It is found that high expression of GILT can promote infiltration of T cells in breast cancer tissue. GILT is overexpressed in breast cancer cells, and the killing ability of T cells to the breast cancer cells can be enhanced. The GILT provided by the invention can effectively promote T cell mediated anti-tumor immune response, and the breast cancer immunotherapy effect can be improved by activating GILT expression.

Description

technical field [0001] The invention relates to the technical field of immunomedicine, in particular to the application of gamma interferon-induced lysosomal sulfhydryl reductase GILT in improving anti-tumor immune response for tumor immunotherapy. Background technique [0002] Breast cancer is considered to be an immune "cold" tumor. Only a small number of patients are effective to the existing immune checkpoint inhibitors, and the treatment response rate is poor. Therefore, it is urgent to explore new and effective immunotherapy options. Interferon-gamma-induced lysosomal sulfhydryl reductase, abbreviated as GILT, is a protein induced by interferon-gamma, which can process antigens in professional antigen-presenting cells. In recent years, it has been used in tumor research It was found that the expression of GILT in tumor cells is related to the prognosis of patients. Our previous studies found that the lower the expression of GILT in breast cancer cells, the worse the pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/44A61K45/00A61P35/00
Inventor 余之刚黄淑亚郭明明相玉娟刘丽媛
Owner SHANDONG UNIV
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