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

A technology of dendritic cells and immune tolerance, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc. The effect of reducing inflammation and simple operation

Inactive Publication Date: 2011-11-02
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. Obtaining mesenchymal stem cells can be purchased commercially (Nanjing Kaiji Biological Co., Ltd.), or isolated and cultured.

[0032] 1. Take 2ml of healthy adult bone marrow, dilute it with an equal amount of PBS, add it to the lymphocyte separation medium (percoll), centrifuge at 900g for 20 minutes, absorb the mononuclear cell layer, wash twice with PBS, discard the supernatant, and wash with 10% FBS, 100U / ml penicillin, 100U / ml streptomycin in DMEM-LG (Gibco company) resuspended, with 1-2 × 10 6 cells / cm 2 High density inoculation up to 75cm 2 culture flask (Costa company), 37 ° C, 5% CO 2 cultured in an incubator. Change the medium after 48 hours, remove the suspended cells, and then change the medium every 3-4 days in full, and when the cells grow close to confluence, they are digested with 0.25% trypsin (Sigma Company) and frozen for use in experiments.

Embodiment 2

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

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

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

Embodiment 3

[0039] Example 3, Identification of immune tolerant dendritic cells (MSC-DC)

[0040] 1. Observation of cell morphology

[0041] In the process of embodiment 1 induction culture, as figure 1 as shown ( figure 1 A, B, and C are photos of cell morphology of MSC-DC at 0, 7, and 14 days, where B was taken on the first day after adding IL-4, and C was taken on the second day after adding LPS taking pictures; figure 1 D, E, and F are photos of the cell morphology of conventionally induced DCs at 0, 7, and 14 days), and it can be found that MSC-DCs begin to proliferate and protrude a small amount at about 7 days after induction, while conventionally induced DCs The cell body becomes larger, the number of processes increases significantly, and the cells gather together. At 14 days, the cells in the two groups proliferated to varying degrees, but the cells in the MSC-DC group still maintained a round shape, while the cells in the control group had protruded a large number of protru...

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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, a preparation method thereof and a special culture medium, in particular to an immune tolerance dendritic cell, a preparation method thereof and a special culture medium. Background technique [0002] Dendritic cell (DC) is the most important antigen-presenting cell, named for its many dendritic or pseudopodia-like processes when mature. It participates in the recognition, processing and presentation of antigens, and is the initiator of the body's immunity. It plays a unique and important role in the induction of immune responses, can activate T cells and antigen-specific killer T cells, and plays an important role in anti-tumor and important role in the immune response to microorganisms. In recent years, DC-related research has gradually become a hot spot in immunology, and more and more attention has been paid to the basic research and clinical application of DC. In the early study of DC, due to the lack o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071A61K35/15C12N5/0784
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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