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A method for directional induced expansion of thymus-derived inkt cells

A technology of directional induction and cell proliferation, applied in the field of immunology, can solve the problems of small amount of iNKT cells, difficulty in directional acquisition, complex process, etc., to reduce cell morphology and function variation and pollution, reduce complicated and cumbersome steps, and low cost Effect

Active Publication Date: 2020-06-23
HEBEI UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to provide a method for the directional induction and expansion of thymus-derived iNKT cells to solve the problems of the existing methods such as complex process, long cycle, small amount of iNKT cells obtained and difficulty in directional acquisition of iNKT cells with specific functions

Method used

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  • A method for directional induced expansion of thymus-derived inkt cells

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Embodiment 1

[0034] Example 1: Targeted induction and expansion of iNKT cells

[0035] (1) Subcutaneously inject the stimulator α-Galcer (2 μg / mouse) into the base of the tail of 6-week-old DBA / 1 male mice;

[0036] (2) After 8 days, the mice were sacrificed by taking blood from the eyeballs and taking off their necks;

[0037] (3) Immerse the mouse completely in 75% alcohol for 1 min, then fix the limbs of the mouse on the dissection tray to take the thymus;

[0038] (4) Place the thymus in a petri dish containing PBS, and wash it 2-3 times with PBS to remove blood stains;

[0039] (5) Afterwards, place the thymus on a 200-mesh sieve (the sieve is placed in a petri dish containing PBS), cut the thymus into 2-3mm pieces with ophthalmic scissors, and then grind it. Transfer the tissue fluid into a 10ml centrifuge tube, centrifuge at 1000r / min for 5min, discard the liquid, and wash twice with PBS (1000r / min, 5min), (the whole process is performed on ice);

[0040] The frequency of iNKT de...

Embodiment 2

[0044] Example 2: Detection of killing activity of iNKT cells

[0045] iNKT cytotoxicity assay adopts 51 Cr release experiment. K562 is the target cell and interacts with Na 51 CrO 4 Incubate at 37°C for 1 h, wash with PBS, and adjust the target cell density to 5×10 5 For each cell, effector cells and target cells were added to a 96-well U-shaped culture plate at a ratio of 20:1, and several wells were set up. Placed at 37°C, 5vol% CO 2 Incubate in the incubator for 4 h, take it out, suck out 0.1ml of the supernatant of each well with a micropipette, add it to a small plastic test tube (do not suck out the cells), and use a γ-counter (Beckman LS 6500) to measure the primer in the supernatant. 51 Cr radioactivity. And calculate the cytotoxic activity: cell killing rate=(experimental well release amount-natural release amount) / (maximum release amount-natural release amount)×100%. After testing, the average cell killing rate was 125%.

[0046] Through the optimized combin...

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Abstract

The invention provides a method for orient induction amplification of thymus gland-derived iNKT (Invariant Natural Killer) cells. The method comprises two stages, namely a first stage, including stepsof performing in-vivo stimulation of iNKT cell proliferation, namely stimulating in-vivo iNKT cell proliferation of mice by using a specific stimulant alpha-Galcer; a second stage, including steps ofperforming in-vitro orient induction of iNKT cell proliferation, namely stimulating thymus glands of the mice by using alpha-Galcer, preparing a single-cell suspension, adding alpha-Galcer and an induction agent, assisting iNKT cell amplification, and inducing cell differentiation. With the combination of in-vivo proliferation stimulation and in-vitro induction amplification, orient iNKT cell induction is achieved, and the purpose that a great amount of orient differentiation iNKT cells with specific functions are obtained within a short time is achieved. Complex and tedious steps of in-vitrocell amplification culture are effectively reduced, the cell culture time is shortened, good repeatability is achieved, and the cost is relatively low.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a method for directional induction and expansion of thymus-derived iNKT cells. Background technique [0002] iNKT cells are a group of special immune cells with both NK cell function and T cell characteristics, and are a bridge between adaptive immunity and innate immunity. iNKT cells are different from traditional T cells in that their surface TCRs lack diversity, and their antigen recognition spectrum is narrow. They can recognize common lipid and glycolipid antigens (a-Galcer) presented by CD1 molecules on the surface of different target cells, and are not affected by MHC. limit. iNKT cells are innate immune cells, and their main biological functions are cytotoxicity and immune regulation. iNKT cells can act as the center of immune regulation to interact with other immune cells and play a vital role in the development of infection, tumor and autoimmune diseases. [0003] iNKT cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078
CPCC12N5/0646C12N2500/34C12N2500/38C12N2500/44C12N2501/2302C12N2501/2304
Inventor 孟明王园园刘慧芳陈冬志张金库
Owner HEBEI UNIVERSITY
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