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Use of gsk-3β inhibitor to induce human breg cells in vitro and methods for isolating and inducing breg cells

A technology of β inhibitors and B cells, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of unsatisfactory scientific research and clinical research, few sources of Breg cells, low in vitro induction efficiency, etc., to achieve The effect of large base, maintaining cell viability, and less possibility of contamination

Active Publication Date: 2019-07-30
夏永祥
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The main feature of Breg is the production of cytokines such as interleukin IL-10 and (or) transforming growth factor TGF-β, and then play a negative regulatory role, but it lacks specific cell phenotypes, among which CD19 + CD24 ++ CD27 + It is the most widely studied Breg at present, and this group of cells has significant changes in acute and chronic rejection of kidney transplant patients, and can even predict the occurrence of postoperative rejection
At present, the way to induce Breg in the world is mainly to induce ordinary B cells through BAFF (B-cell activating factor), but the induction purity of this technology is only about 20-30%, which cannot meet the needs of further scientific research and clinical research.
[0004] Cell therapy is a new type of solution that is expected to treat many difficult diseases. Recently, the adoptive infusion of Breg has become a new branch of cell therapy. However, the problem that has always plagued Breg adoptive therapy is that the source of Breg cells is relatively small, and the current in vitro induction efficiency is low. And its activity is also low

Method used

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  • Use of gsk-3β inhibitor to induce human breg cells in vitro and methods for isolating and inducing breg cells
  • Use of gsk-3β inhibitor to induce human breg cells in vitro and methods for isolating and inducing breg cells
  • Use of gsk-3β inhibitor to induce human breg cells in vitro and methods for isolating and inducing breg cells

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Effect test

Embodiment 1

[0041] Use of GSK-3β inhibitors to induce human Breg cells in vitro.

[0042] Further, the use of GSK-3β inhibitors to induce transformation of ordinary B cells into Breg cells.

[0043] Further, induction of common B cells into CD19 + CD24 ++ CD27 + Breg cell subsets.

[0044] GSK-3β inhibitors induce human Breg cells in vitro, and GSK-3β inhibitors induce the transformation of ordinary B cells into Breg cells, the use of GSK-3β inhibitors to induce Breg cells induces high purity image 3 , the secretion of Breg inflammatory cytokines decreased after induction Figure 4 , Breg suppressive function enhanced after induction see Figure 5 .

[0045] Such as Figure 6 Shown, a method for isolating and inducing Breg cells, comprising the following steps:

[0046] Step 1: Obtain mononuclear lymphocytes, use blood cell apheresis to obtain concentrated human peripheral blood mononuclear lymphocytes 50~80ml, mix with normal saline at a ratio of 1:1, slowly add 15ml mononuclear...

Embodiment 2

[0059] 1 Experimental materials

[0060] 1.1 Sample acquisition

[0061] Blood samples were obtained from Nanjing Medical University volunteers.

[0062] 1.2 Required reagents and experimental equipment

[0063] CD19 FITC, CD24 PE, CD27 APC flow cytometry antibody (Miltenyi Biotec);

[0064] Medium (RPMI 1640, 10% FBS, 100mg / ml streptomycin, 10000U / mlpenicilin, Gibco);

[0065] AUTOMACS (MACS);

[0066] Flow cytometer (BD);

[0067] Cell culture incubator (Thermo);

[0068] Microscope (Zeiss Axiovert).

[0069] 2 methods

[0070] 2.1 Obtaining peripheral blood mononuclear lymphocytes

[0071] Such as figure 1 As shown, first, volunteers (6 persons) were recruited, and after signing the informed consent, the peripheral blood mononuclear cell samples of the volunteers were collected by hematology professional physicians using a blood cell apheresis. After the specimen is obtained, the lymphocyte separation medium is used for centrifugation to extract the lymphocytes an...

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Abstract

The invention discloses application of a GSK-3beta inhibitor to in-vitro induction of human Breg cells and a method for inducing the human Breg cells in vitro by the GSK-3beta inhibitor. In the effects of inducing the human Breg cells in vitro by the GSK-3beta inhibitor and inducing transformation of common B cells into the Breg cells by the GSK-3beta inhibitor, the induction purity of inducing the Breg cells by the GSK-3beta inhibitor is high, secretion of Breg inflammatory cytokines is decreased after induction, and the Breg inhibiting function is enhanced after induction. By the method forinducing the human Breg cells in vitro by the GSK-3beta inhibitor, the operation is simple, a short time is consumed, the purity is high, the cell activity damage is high, the transformation efficiency is high, the cell activity is high, a source is wide, the pollution possibility is low, an insufficient amount of Breg in basic and clinical researches can be made up, and a novel experimental method is provided for further research on the Breg cells.

Description

technical field [0001] The invention relates to the field of separating and inducing cells, in particular to the use of GSK-3β inhibitors in inducing human Breg cells in vitro and the method for isolating and inducing Breg cells. Background technique [0002] At present, both animal models and clinical patients have confirmed that Breg is closely related to acute and chronic rejection of kidney transplantation. Breg can not only predict the risk of acute and chronic rejection in postoperative patients, but also found that adoptive infusion of Breg can treat rejection in animal experiments. reaction. At the same time, Breg can also regulate other immune cells by secreting related cytokines, realize the interaction of immune responses in the body, and maintain the immune balance of the body. However, because Breg is extremely small in the human body and cannot be effectively induced by ordinary B cells, current experiments are mostly limited to animal models, and research on ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0781
CPCC12N5/0635C12N2501/405
Inventor 夏永祥陆云杰吕凌高骥周浩明饶建华
Owner 夏永祥
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