Sorting and amplification method of human peripheral blood CD4+CD25+Foxp3+ regulatory T cells

A human peripheral blood, foxp3 technology, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problem of small number and so on

Inactive Publication Date: 2015-05-27
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, CD4+CD25+Foxp3+regulatory T cells only account for 5%-10% of normal human peripheral blood CD4+T lymphocytes, and their number is extremely small. They must be able to be expanded in large quantities in vitro to meet the needs of scientific research and clinical applications.

Method used

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  • Sorting and amplification method of human peripheral blood CD4+CD25+Foxp3+ regulatory T cells
  • Sorting and amplification method of human peripheral blood CD4+CD25+Foxp3+ regulatory T cells
  • Sorting and amplification method of human peripheral blood CD4+CD25+Foxp3+ regulatory T cells

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Embodiment 1

[0027] 1. The materials used in this embodiment are mainly as follows:

[0028] (1) Source of specimens: Peripheral blood specimens were collected from healthy volunteers, a total of 3 cases. Aged 25-32 years old, median age 26 years old, 1 male and 2 females.

[0029] (2) Main reagents: Lymphocyte separation medium was purchased from Sigma, CD3 / CD28 monoclonal antibody-coated Dynalbeads were purchased from Dynal Inc. (Oslo, Norway), CD4+CD25+Treg cell sorting kit, immunomagnetic bead sorting kit AutoMACS Pro Separator, MACS buffer, LD and MS MACS separation columns were purchased from Miltenyi Biotec, Germany. Cell culture reagents: RPMI 1640 cell culture liquid, penicillin and streptomycin double antibodies, fetal bovine serum were purchased from Gibco, flow cytometry antibodies: CD4-FITC, CD25-APC, Foxp3-PE and PE isotype control monoclonal antibodies, fixed Agents and membrane breakers were purchased from BD Pharmigin. The cell counter was Beck-man coulter (USA), and th...

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Abstract

The invention discloses a sorting and amplification method of human peripheral blood CD4+CD25+Foxp3+ regulatory T cells, and belongs to the field of cell sorting amplification. The sorting and amplification method of the human peripheral blood CD4+CD25+Foxp3+ regulatory T cells specifically comprises the following steps: (1) separating peripheral blood mononuclear cells; (2) performing MACS (magnetic activated cell sorting) on CD4+CD25+Foxp3+Treg cells; and (3) performing in-vitro culture and amplification on the CD4+CD25+Foxp3+Treg cells, and finally performing amplification and culture to obtain the human peripheral blood CD4+CD25+Foxp3+ regulatory T cells. According to the sorting and amplification method disclosed by the invention, human peripheral blood CD4+CD25+Treg cells are successfully separated by using an immuno-magnetic bead two-step process, and the purity of the human peripheral blood CD4+CD25+Treg cells is subjected to flow identification to ensure that the purity of the finally-obtained CD4+CD25+Treg cells can reach 97%, and the activity of the cells can reach more than 95%.

Description

technical field [0001] The invention belongs to the field of cell sorting and expansion, and in particular relates to a method for sorting and expanding human peripheral blood CD4+CD25+Foxp3+ regulatory T cells. Background technique [0002] CD4+CD25+Foxp3+ regulatory T cell (Regulatory T cell, Treg) is a cell population with immune regulation function, which can inhibit the immune response of autoreactive T cells, inhibit the activation of traditional T cells and promote some inhibitory functions. The secretion of cytokines, etc., play an important role in maintaining the stability of the internal environment of the body and inducing the tolerance of the graft. It is an important way for the body to actively acquire and maintain self-tolerance. In 1995, Sakaguchi et al first reported the existence of a T cell subset expressing IL2 receptor α chain (IL2Ra, CD25) in autoimmune tolerance, and named this type of CD4+CD25+ T cells as regulatory T cells (Regulatory T cell, Treg)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 张建平陈慧祝丽琼刘梅兰刘玉昆谭剑平陈立斌刘颖琳王蕴慧张睿
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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