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Immunologic tolerance dendritic cell, preparation method thereof and special culture medium

A dendritic cell, immune tolerance technology, applied in blood/immune system cells, biochemical equipment and methods, tissue culture, etc., can solve problems such as limited functional characteristics research, low cell number, etc., to reduce inflammation. , reduce inflammatory factors, simple operation effect

Inactive Publication Date: 2009-08-26
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the early study of DC, due to the lack of understanding of its origin, differentiation, development, and maturation, DCs could only be isolated from different tissues. The number of cells obtained in this way was extremely small, and the study of its functional characteristics was greatly limited.

Method used

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  • Immunologic tolerance dendritic cell, preparation method thereof and special culture medium
  • Immunologic tolerance dendritic cell, preparation method thereof and special culture medium
  • Immunologic tolerance dendritic cell, preparation method thereof and special culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. The mesenchymal stem cells can either be purchased commercially (Nanjing KGI Biotech Co., Ltd.), or they can be isolated and cultured.

[0032] 1. Take 2ml of healthy adult bone marrow, dilute with the same amount of PBS, add to the lymphocyte separation solution (percoll), centrifuge at 900g for 20 minutes, suck up the mononuclear cell layer, wash twice with PBS and discard the supernatant, use 10% FBS, Resuspend 100U / ml penicillin and 100U / ml streptomycin in DMEM-LG (Gibco company) to 1-2×10 6 cells / cm 2 High density inoculation to 75cm 2 Culture flask (Costa company), 37℃, 5% CO 2 Culture in an incubator. After 48 hours, the medium was changed to remove the suspended cells. After that, the medium was changed in full every 3 to 4 days. After the cells grew close to fusion, they were digested with 0.25% pancreatin (Sigma) and frozen for experiment.

Embodiment 2

[0033] Example 2. Induction of immune tolerant dendritic cells (MSC-DC) and DC and IL-10-DC

[0034] 1. Resuscitate mesenchymal stem cells, inoculate a 24-well plate, 5×10 4 / hole.

[0035] 2. Get CD34 + Cells can be purchased from commercial sources (Nanjing KGI Biotech Co., Ltd.), or CD34 from cord blood can be isolated by the MACS method + cell. The discarded cord blood obtained from the hospital is added with an equal volume of PBS, then a quarter of 5% methylcellulose is added, and after mixing, it is centrifuged at 1000 rpm for 10 min. Discard the supernatant, add PBS (pH 7.4) to resuspend, and then slowly add it to a test tube containing 5ml Ficoll at 1500rpm for 20min. Take the middle mononuclear cell layer, wash twice with PBS, 1000rpm, 10min. Buffer A (containing 0.5% BSA; 2mM EDTA-2Na + PBS solution) and count after resuspension. Add 300μl buffer A / 1×10 after centrifugation 8 Cells, 100ul blocking antibody (FcRBlocking Reagent) / 1×10 8 Cells, 100μl CD34 magnetic bead ant...

Embodiment 3

[0039] Example 3. Identification of Immune Tolerant Dendritic Cells (MSC-DC)

[0040] 1. Observation of cell morphology

[0041] In the process of inducing culture in Example 1, such as figure 1 Shown ( figure 1 A, B, and C are the cell morphology photos of MSC-DC at 0, 7, and 14 days. Among them, B is taken on the first day after IL-4 is added, and C is on the second day after LPS is added. taking pictures; figure 1 The D, E, and F are the cell morphology photos of conventionally induced DC at 0, 7, and 14 days). It can be found that MSC-DC began to proliferate and a small amount of protrusions protruded around 7 days after induction, while conventionally induced DCs The cell body becomes larger, the number of protrusions increases significantly, and the cells are clustered together. At 14 days, the two groups of cells had different degrees of proliferation, but the MSC-DC group still maintained a round shape, while the control group had a large number of protrusions.

[0042]...

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Abstract

The invention discloses an immunologic tolerance dendritic cell, a preparation method thereof and a special culture medium. The culture medium is formed by the way that mesenchyma stem cells are added into a dendritic cell culture medium. The preparation method comprises: the precursors and the inducible factors of dendritic cells are added into the culture medium. The co-culture proportion of the mesenchyma stem cells to the dendritic cells is (1-2) to (1-10), preferably 1 to 1; the precursors of the dendritic cells are CD34 cells; and the inducible factors comprise GM-CSF, TNF- alpha, IL-4 and LPS. The invention provides the application of the immunologic tolerance dendritic cell during preparing immunologic tolerance medicine for organisms. The immunologic tolerance dendritic cell can lowly express co-stimulatory molecule, reduce inflammatory factors, increase the secretion of suppressor cytokines, have vasculogenesis activity, and inhibit the multiplication of variant T cells. The invention has the advantages of simple operation, convenience and practicality, and establishes a stable culture technical system for selective activation dendritic cells.

Description

Technical field [0001] The invention relates to a dendritic cell and a preparation method thereof and a special culture medium, in particular to an immune tolerance type dendritic cell, a preparation method thereof and a special culture medium. Background technique [0002] Dendritic cell (DC) is one of the most important antigen-presenting cells, named after many dendritic or pseudopod-like protrusions when they mature. It participates in the recognition, processing and presentation of antigens. It is the initiator of the body's immunity. It has a unique and important role in the induction of immune responses. It can activate T cells and antigen-specific killer T cells, and is used in anti-tumor and Play an important role in the microbial immune response. In recent years, DC-related research has gradually become a hot spot in immunology, and the basic research and clinical application of DC have received more and more attention. In the early study of DC, due to the lack of under...

Claims

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Application Information

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IPC IPC(8): C12N5/08A61K35/44A61K39/00A61P29/00A61P37/02A61K35/15C12N5/0784
Inventor 张毅苏永锋江小霞霍思维刘元林吴英毛宁
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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