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Application of atpif1 gene silenced T cells in preparation of antitumor drugs

An anti-tumor drug and gene silencing technology, applied in the field of biomedicine, to achieve the effect of enhancing the killing function

Active Publication Date: 2022-06-28
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 experiment

[0026] 1), 8 mice were taken from group A and group B, and B16 tumor cells were injected into the armpits. The 8 mice in group A were divided into groups A1 and A2, and the 8 mice in group B were Divided into B1 group and B2 group;

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

[0028] 3) After 14 days, the mice in groups A1, A2, B1, and B2 were sacrificed to remove the melanoma;

[0029] The size and weight of the melanoma of the mice in each group were compared, and the results are shown in the appendix. figure 1 , among which, Figure A is the size control picture of the melanomas taken out of the two mice, and Figure B is the weight comparison of the melanomas. It can be seen that ATPIF1 - / - Compared with wild-type mice, the tumor was smaller...

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, labeled as A1, A2, A3...A17, B1, B2, B3...B17, add pre-warmed PBS (0.1%BSA + 0.6%) to each cell culture dish Sodium citrate) 5 mL, a cell strainer was placed in each cell culture dish, 17 mice in each of groups A and B were taken and killed, the spleens were taken out and placed in the strainer respectively, and the tissue was ground with the back of a syringe. Rinse the filter 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 erythrocytes

[0038] 1) Each spleen was resuspended with 5 ml of erythrocyte ...

Embodiment 3

[0062] Fluorescein 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 resuspend the T cells in each tube in 5% Heat-inactivated serum (FBS) in 1 mL of 1640 medium at a T cell concentration of 5 x 10 6 / ml;

[0064] 2) Thoroughly resuspend each tube of T cells in 1 mL of 1640 medium containing 2% serum, and carefully place them into the bottom of a fresh (unwetted) 15 mL centrifuge tube;

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

[0066] 4) Carefully add 110 μL of PBS to the unwetted 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) Quickly ca...

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Abstract

The invention discloses a method for enhancing the activity of T cells. By silencing the ATPIF1 gene in T cells, the T cells can be rapidly multiplied and activated in the immune process, the effector function of T cells can be improved, and the killing effect of T cells on tumor cells can be enhanced. , the present invention also discloses the application of ATPIF1 gene silenced T cells in the preparation of anti-tumor drugs, which provides a new direction 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 T cells obtained by the method in adoptive immunotherapy of tumors. [0002] technical background [0003] The efficacy of PD-1 / PD-L1 antibody in the clinical use shows that tumor immunotherapy has become an effective strategy for the treatment of advanced or traditional treatment-resistant cancer patients; Although identification specificity is crucial, some other intrinsic properties of T cells, such as persistence, longevity, and functionality, also have an important impact on the effect of immunotherapy; in recent years, more and more studies have found that T cells Cellular energy metabolism pathways play an important role in regulating these properties, and metabolic activity is regulated by cell signaling pathways and epigenetics, which also profoundly affects the trajectory of T cell diff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10A61K35/17A61P35/00C12N15/10C12N15/90
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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