Culture method of rapamycin induced regulatory gamma delta T cells

A technology of rapamycin and culture method, applied in the field of immunology research, to achieve the effects of strong feasibility, high induction efficiency and low cost

Active Publication Date: 2013-12-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently there are few in vitro induction and expansion methods for γδTreg. Therefore, finding a new in vitro induction and culture method for γδTreg and improving its in vitro induction and expansion efficiency are of great significance to the further research and clinical application of γδTreg in the future.

Method used

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  • Culture method of rapamycin induced regulatory gamma delta T cells
  • Culture method of rapamycin induced regulatory gamma delta T cells
  • Culture method of rapamycin induced regulatory gamma delta T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation of human peripheral blood-derived mononuclear cells

[0021] 1. First dilute the collected heparin anticoagulated peripheral blood (Zhejiang Blood Center) with the same amount of PBS (phosphate buffer saline) 1:1;

[0022] 2. Take a number of 15ml centrifuge tubes, add 5ml of human lymphocyte separation medium to each, then slowly superimpose the diluted peripheral blood on the lymphocyte separation medium along the tube wall, add 10ml of peripheral blood to each centrifuge tube, that is, 5ml of lymphocyte Cell separation solution + 10ml diluted peripheral blood (1:2); horizontal centrifugation, 500g×20 minutes, room temperature;

[0023]3. After centrifugation, the inside of the tube is divided into three layers, the upper layer is plasma and PBS buffer, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separation fluid, and there is a layer of white mononuclear cells at the interface between the upper an...

Embodiment 2

[0026] Example 2 Induction and culture of regulatory γδT cells

[0027] 1. Divide the cultured cells into three groups, including IL-2 culture group alone (γδT group), TGF-β1 / IL-2 / IL-15 induction group (TGF-β1 group), and combined TGF- β1 / IL-2 / IL-15 and rapamycin induction group (combined with rapamycin group); all cytokines were added on the 0th day of cell culture, and the final concentrations after addition are shown in Table 1 below; Rapamycin was also added at day 0 of cell culture.

[0028]

[0029]

[0030] 2. On the 3rd, 6th, and 9th day, the medium was changed in half, and the corresponding cytokines were added to keep the final concentration of each cytokine consistent.

[0031] 3. The storage concentrations of each cytokine are: zoledronic acid (56 mmol / ml), IL-2 (2×10 4 U / ml), IL-15 (10 μg / ml), TGF-β1 (1 μg / ml), rapamycin (10 μM); store at -20°C.

Embodiment 3

[0032] Example 3 Analysis and comparison of the induction of regulatory γδT cells under different culture conditions

[0033] At present, the in vitro induction of regulatory γδT cells is generally adopted by the method of IL-2 / IL-15 / TGF-β1 induction. In this experiment, on the basis of IL-2 / IL-15 / TGF-β1, further combined application Synergistic induction with rapamycin, detection and comparison of changes in induction efficiency.

[0034] 1. Collect the cells cultured for 15 days under different induction culture conditions, that is, the IL-2 only culture group (γδT group, as a control), and the TGF-β1 / IL-2 / IL-15 culture group (TGF-β1 group) ) and the combined rapamycin and TGF-β1 / IL-2 / IL-15 culture group (combined rapamycin group), the content of regulatory γδT cells was analyzed by flow cytometry.

[0035] 2. The method for detecting the content of regulatory γδT cells by flow cytometry is as follows:

[0036] (1) Harvest cells and place them in flow tubes, 10 cells per...

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Abstract

The invention provides a culture method of rapamycin induced regulatory gamma delta T cells. The culture method is characterized in that mononuclear cells derived from human peripheral blood are amplified by uniting multiple cell factors with macrolides immunosuppressor-rapamycin through induction in vitro, so that the lots of regulatory gamma delta T cells are cultured. The culture method is simple in the whole process of induction, amplification and enrichment, short in cell culture period and low in cost, has strong feasibility, high induction efficiency and good repeatability and can be applied to the culture of the regulatory gamma delta T cells. The regulatory gamma delta T cells induced by the method have good activity, a relatively strong immunosuppression function and very high popularization value and can be used for ensuring the further research.

Description

technical field [0001] The invention belongs to the field of immunology research, and relates to a method for culturing regulatory γδT cells induced by rapamycin, which is to apply various cytokines (TGF-β1 / IL-15 / IL-2) combined with macrolides to immunize The inhibitory drug, rapamycin, synergistically induces the generation and expansion of regulatory γδ T cells. Background technique [0002] γδT cells are a special subpopulation of T lymphocytes, which have unique structures and biological functions. Different from general αβT cells, γδT cell surface receptors are composed of two glycoprotein chains, γ chain and δ chain, hence the name; this group of cells does not need to be presented by histocompatibility antigen molecules on the cell surface to recognize antigens. Direct recognition of protein, peptide and non-peptide antigens, that is, the process is non-HLA (human leukocyte antigen) restricted, is an important biological characteristic of γδT cells. Although the pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 黄河顾嫣珺胡永仙
Owner ZHEJIANG UNIV
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