Method for detecting immunosuppression function of human regulatory T cells

A technology of immunosuppression and detection method, applied in the direction of measuring devices, instruments, individual particle analysis, etc., can solve the problems affecting the maturation and function of Treg cells, insufficient to define the function and phenotype of Treg cells, etc.

Inactive Publication Date: 2017-07-07
山东大学深圳研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the early days, it was thought that the transcription factor FoxP3 was the most specific marker of Treg cells, but the expression of FoxP3 alone was not enough to define Tre...

Method used

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  • Method for detecting immunosuppression function of human regulatory T cells
  • Method for detecting immunosuppression function of human regulatory T cells
  • Method for detecting immunosuppression function of human regulatory T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Treg cell detection

[0021] The materials used in this embodiment are mainly as follows:

[0022] (1) Specimen source: In this example, the peripheral blood approved and authorized by the family members of children with new-onset ALL and normal healthy children was used as the source of mononuclear cells. A total of 10 samples of ALL peripheral blood and 10 samples of normal control peripheral blood were collected. Add heparin for anticoagulation, 3-5ml per serving;

[0023] (2) Main reagents: mouse anti-human CD4-FITC and FoxP3-APC antibodies were purchased from ebiosicence Company in the United States, mouse anti-human CD25-PE-Cy5 and Helios-PE antibodies were purchased from Biolegend Company in the United States, and FoxP3 Fix / Perm buffer was purchased from ebioscience, red blood cell lysate was purchased from BD Biosciences, USA, the cell counter was Beck-mancoulter (U.S.), and the GuavaeasyCyte 6HT flow cytometer was produced by EMD Millipore, USA;

[0024] (3) ...

Embodiment 2

[0026] Isolation of mononuclear cells and sorting of Treg cells

[0027] (1) Specimen source: In this example, the peripheral blood approved and authorized by the family members of children with new-onset ALL and normal healthy children was used as the source of mononuclear cells. A total of 5 samples of ALL peripheral blood and 5 samples of normal control peripheral blood were collected. Add heparin for anticoagulation, 10ml each;

[0028] (2) Main reagents: lymphocyte separation fluid was purchased from Sigma, CD4 + CD25 + Treg cell separation kit, immunomagnetic bead sorter (autoMACS Pro Separator), MACS buffer, LD and MS MACS separation columns were purchased from Miltenyi Biotec, Germany;

[0029] (3) Separation of mononuclear cells: extract 10ml of heparin-anticoagulated peripheral blood, add an equal amount of PBS solution to the peripheral blood to dilute, take a 50ml centrifuge tube, carefully add 20ml of lymphocyte separation solution to each tube, and place 20ml o...

Embodiment 3

[0032] Detection of the immunosuppressive function of Treg cells

[0033] (1) Main reagents: Co-cultivation medium: Add 10% fetal bovine serum to 1640 medium, 100U / ml penicillin + chain

[0034] Mycin, 50μM / L 2-mercaptoethanol and 2mM / L L-glutamine, 200IU / mL rhIL-2. CD3 / CD28dynbeads were purchased from Invitrogen, USA, and CFSE was purchased from Dojin Chemical Research Institute, Japan;

[0035] (2) The CD4 sorted out in Example 2 + CD25 - Cells were washed and adjusted to 1×10 by adding PBS 7 Concentration, label 5μM CFSE according to CFSE instructions, incubate at 37°C for 15-30 minutes, wash twice with PBS, take 1×10 6 According to the ratio of cell number 2:1, Treg cells in experimental group 1, experimental group 2, and control group were added to 6-well plates for co-cultivation. The co-cultivation medium was used as the medium, and CD3 / CD28 dynbeads were cultured for 72 hours and cell proliferation was detected by flow cytometry, and the results were analyzed by ...

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Abstract

The invention discloses a method for detecting an immunosuppression function of human regulatory T cells. The method comprises the following steps: collecting peripheral blood for extracting a mononuclear cell; using fluorescein marked anti-CD4 and anti-CD25 antibodies for marking a cell surface antigen; using a fixing membrane-breaking agent for fixing the cell surface antigen; after breaking the membrane, using fluorescein marked anti-Foxp3 and anti-Helios antibodies for marking endonuclear transcription factor Foxp3 and Helios; and finally, using a flow cytometry for detecting CD4+CD25+FoxP3+Helios+Treg cell population. According to the method, the function of the regulatory T cells in the peripheral blood is directly analyzed in the manner of quickly detecting the expression condition of the children peripheral blood transcription factor Helios. According to the method, the anti-Helios, anti-CD4, anti-CD25 and anti-Foxp3 flow antibodies can be used for more quickly and accurately marking the peripheral blood Treg cells with higher immunosuppression function.

Description

technical field [0001] The invention relates to a detection method for rapidly evaluating the immunosuppressive function of regulatory T cells in peripheral blood. The method is used for monitoring the inhibitory activity of regulatory T cells and belongs to the field of biotechnology. Background technique [0002] The human immune system is subject to multiple precise adjustments, among which regulatory T cells (regulatory T cells, Treg) play an important role in negative immune regulation, and are the main immune cells that block immune surveillance and inhibit effective anti-tumor immunity. The proportion of Treg cells in the body is very small, and only CD4 cells in the peripheral blood of normal people + The proportion of Treg cells with immunosuppressive ability is even lower. Treg cells express a variety of cell surface molecules, such as CD4, CD25, FoxP3, CTLA4, GITR, and CD103. How to identify and detect specific Treg cells with immunosuppressive functions is the ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N15/14
CPCG01N33/56966G01N15/14G01N2015/149G01N2333/001G01N2333/4704
Inventor 鞠秀丽李雪
Owner 山东大学深圳研究院
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