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Application of ATPIF1 gene silenced T cells in preparation of antitumor drugs

An anti-tumor drug, gene silencing technology, applied in the field of biomedicine

Active Publication Date: 2019-12-24
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Mitochondria (Mitochondrion, MT) is the processing plant of cellular energy metabolism. It obtains the chemical energy of nutrients through the oxidation of substrates such as glucose and fatty acids. Through the phosphorylation of ADP, it is converted into energy that can be used by cells and stored in high-energy phosphoric acid. In the compound ATP, it provides energy for cell activities; as an organelle with a dynamic double-membrane structure in eukaryotic cells, mitochondria can be divided into four functional areas from outside to inside, namely, the outer mitochondrial membrane (OMM), the mitochondrial membrane space, Inner mitochondrial membrane (IMM) and mitochondrial matrix; Complex V on the inner mitochondrial membrane is ATP synthase, whose main function is to synthesize ATP; however, follow-up studies have shown that ATP synthase both synthesizes and hydrolyzes ATP, It has a bidirectional regulatory effect on cellular ATP levels; the activity of ATP synthase is regulated by the inhibitory factor (ATPIF1), which was discovered 60 years ago and encoded by the first nuclear gene that can interact with ATP synthase and is highly evolved A conserved mitochondrial protein, by interacting with the F1 region of ATP synthase, it can selectively inhibit ATP synthase from hydrolyzing ATP or synthesizing ATP under different physiological and pathological conditions, thus affecting intracellular oxidation Phosphorylation level, glycolysis level, cell membrane potential, generation of reactive oxygen species, etc.; current research on ATPIF1 in cell energy metabolism mainly focuses on tumor cells, neurons and stem cells, and its role in T cell metabolism and Its effect on T cell anti-tumor immune function has not been reported yet

Method used

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  • Application of ATPIF1 gene silenced T cells in preparation of antitumor drugs
  • Application of ATPIF1 gene silenced T cells in preparation of antitumor drugs
  • Application of ATPIF1 gene silenced T cells in preparation of antitumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Mouse B16 tumor inoculation experiments

[0026] 1), take 8 mice from group A and group B respectively, inject B16 tumor cells in the armpit, divide the 8 mice in group A into group A1 and group A2 equally, and the 8 mice in group B are all Divided into group B1 and group B2;

[0027] 2) Three days later, mice in groups A2 and B2 were intraperitoneally injected with CD8 antibody, and then injected intraperitoneally with CD8 antibody every three days to eliminate CD8 in mice. + The role of T cells;

[0028] 3), 14 days later, kill the mice in groups A1, A2, B1, and B2 to take out the melanoma;

[0029] Compare the size and weight of melanoma in each group of mice, see the attached figure 1 , among them, Figure A is the size comparison picture of the melanoma taken out of the two kinds of mice, and Figure B is the weight comparison of the melanoma, it can be seen that ATPIF1 - / - Compared with wild-type mice, the tumors in mice were smaller and lighter, and tumor growt...

Embodiment 2

[0031] Isolation and purification of mouse CD8 + T cells

[0032] 1. Preparation of spleen cells

[0033] 1) Take 34 5cm cell culture dishes, respectively labeled as A1, A2, A3...A17, B1, B2, B3...B17, add PBS (0.1% BSA + 0.6% Sodium citrate) 5 mL, put a cell strainer in each cell culture dish, take 17 mice from each group A and B and kill them, take out the spleen and put them in the strainer, grind the tissue with the back of the syringe, Rinse the filter screen regularly with PBS;

[0034] 2) Transfer the ground spleen cell suspension to a 15ml centrifuge tube with a pipette, fill it with cold PBS, and centrifuge at 1300rpm for 10 minutes;

[0035] 3) Discard the supernatant, resuspend the cell suspension in 15ml of cold PBS, and centrifuge for 10 minutes;

[0036] 4) Repeat the previous step and wash 3 times;

[0037] 2. Lysis of mouse red blood cells

[0038] 1) Each spleen was resuspended with 5ml erythrocyte lysis buffer;

[0039] 2) Incubate on ice for 5 minute...

Embodiment 3

[0062] Carboxyfluorescein diacetate succinimidyl ester (CFSE) monitoring T lymphocyte proliferation

[0063] 1) Take the A1, A2, A3, A4, A5, A6 and B1, B2, B3, B4, B5, B6 centrifuge tubes in Example 2, centrifuge and discard the supernatant, and the T cells in each tube are resuspended in 5% Heat-inactivated serum (FBS) in 1 mL of 1640 medium with T cell concentration at 5 x 10 6 / ml;

[0064] 2) Thoroughly resuspend each tube of T cells in 1 mL volume of 1640 medium containing 2% serum, carefully put into the bottom of a fresh (not wetted) 15 mL centrifuge tube;

[0065] 3) Place the tube horizontally (using an unwetted tube will prevent the 1 mL cell suspension from moving and mixing with the CFDA, SE solution prematurely);

[0066] 4) Carefully add 110 μL PBS to the non-wetted part of the plastic at the top of the tube, making sure it does not come into contact with the cell solution;

[0067] 5) Resuspend 1.1 μL of 5mM CFDA in 110 μL PBS;

[0068] 6) Cap the tube quick...

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Abstract

The invention discloses a method for enhancing the activity of T cells. By silencing an ATPIF1 gene in the T cells, the T cells can rapidly proliferate, activate, improve the effector function of theT cells and enhance the killing effect of the T cells on tumor cells in the immune process. The invention further discloses application of the ATPIF1 gene silenced T cells in the preparation of antitumor drugs. A new direction is provided for the research and development of tumor adoptive immunotherapy.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for enhancing the activity of T cells and the application of the T cells obtained by the method in adoptive immunotherapy of tumors. [0002] technical background [0003] The curative effect of PD-1 / PD-L1 antibody in a large number of clinical applications shows that tumor immunotherapy has become an effective strategy for the treatment of advanced or traditional treatment-resistant cancer patients; Recognition specificity is of course crucial, but some other intrinsic characteristics of T cells, such as persistence, longevity and functionality, also have an important impact on the efficacy of immunotherapy; more and more studies in recent years have found that T cells Cellular energy metabolism pathways play an important role in regulating these characteristics. Metabolic activity is regulated by cell signaling pathways and epigenetics, which also profou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61P35/00C12N15/10C12N15/90
CPCA61K35/17A61P35/00C12N5/0636C12N15/907C07K14/47C12N2510/00C12N2310/20
Inventor 钟根深王莹叶建平王辉梁银明熊熙文吴敏娜
Owner XINXIANG MEDICAL UNIV
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