Method for inducing immunosuppressive b-lymphocytes from human body peripheral blood

A technology of B lymphocytes and human peripheral blood, applied in the biological field, can solve problems such as method difficulties

Inactive Publication Date: 2013-03-20
JIANGSU UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, there are still difficulties in obtaining a large number of B cells with immunosuppressive function

Method used

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  • Method for inducing immunosuppressive b-lymphocytes from human body peripheral blood
  • Method for inducing immunosuppressive b-lymphocytes from human body peripheral blood

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Experimental program
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Embodiment Construction

[0014] 1. Purification of human peripheral blood mononuclear cells: Fresh human peripheral blood was provided by healthy volunteers. Lymphoprep for Human Peripheral Blood Mononuclear Cells TM (Axis-Shield) separation. Fresh human peripheral blood samples were diluted 1:1 with normal saline, and 3ml of diluted samples were slowly added to pre-added 5ml of Lymphoprep TM 15ml centrifuge tube (BD). After the centrifuge tube is balanced, put it into a centrifuge (Beckman Coulter), room temperature, centrifugal force 600g, and centrifuge for 20min. Carefully take out the centrifuge tube, and use a sterile pipette to take out the cell layer at a density of 1.077g / ml, which is the mononuclear cell layer. The cells were washed 2-3 times with normal saline, and counted with a cell counting plate. Freshly prepared PBMCs can be used directly to induce suppressor B lymphocytes or can be frozen in liquid nitrogen. The formulation of the cryopreservation solution is as follows: 15% DMSO...

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Abstract

The present invention relates to the technical field of biology, specifically to induced culture and purification of human suppressive b-lymphocytes and applications of a potential cell therapy method in autoimmune diseases. According to the method, human peripheral blood is sterilely sampled, LymphoprepTM (Axis-Shield) is adopted to separate human mononuclearcell, the separated cell is placed in a 10% FBS-containing RPMI1640 culture medium to culture, and concurrently 50 ng / ml of human recombinant BAFF cytokine is added at a first day, a third day and a fifth day to induce suppressive b-lymphocyte production. According to the present invention, the BAFF is adopted for differentiation of the human peripheral blood B cells into the suppressive b-lymphocytes through in vitro induction, wherein the suppressive b-lymphocytes express IL-10, and have T lymphocyte proliferation inhibition capability.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to the induction culture and purification of human suppressive B lymphocytes and the application of potential cell therapy in autoimmune diseases. Background technique [0002] In recent years, certain mouse B cell subsets can secrete regulatory cytokines and cells with immunosuppressive effects are called regulatory B cells. In various disease models in mice, especially in mouse models of autoimmune diseases, researchers have found that the existence of regulatory B cells and its relationship with the occurrence and development of diseases. So far, B cells with regulatory functions have been found to include B1a, T2-MZ precursor cells, B10 cells (CD19 hi CD1d hi CD5 + )Wait. Although their phenotypes are different, these B cells can all secrete IL-10 and / or TGF-β. Our recent studies have found that BAFF can induce the production of regulatory B10 cells in vitro, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781
Inventor 王胜军芮棵杨敏吕力为
Owner JIANGSU UNIV
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