Preparation method and application of a novel regulatory dendritic cell

A dendritic cell and regulatory technology, applied in the generation of new regulatory dendritic cells and in the field of immunotherapy, to improve the quality of life, remove tumor cells, and enhance the effect of killing

Active Publication Date: 2019-08-09
微能生命科技集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve some of the problems existing in the existing technology for treating malignant tumors, and to provide a novel regulatory dendritic cell and its production method

Method used

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  • Preparation method and application of a novel regulatory dendritic cell
  • Preparation method and application of a novel regulatory dendritic cell
  • Preparation method and application of a novel regulatory dendritic cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Main experimental reagents and consumables

[0073] (1) Main experimental reagents

[0074] Penicillin, streptomycin and trypsin-EDTA were purchased from GIBCO.

[0075] Human lymphocyte separation medium (Ficoll, Tianjin Haoyang Biological Products Co., Ltd.)

[0076] Cytokines rhIL-2, rhIL-12, IL-4, IL-7, GM-CSF, TNF-σ (Peprotech);

[0077] Human recombinant soluble CD40 ligand (rh sCD40-L; Bender Company);

[0078] Antibody: mouse anti-human CD11c, CD80, CD40, CD86, CD29, CD34, CD44, CD73, CD90, CD105, CD106, CD117, Flk-1, HLA-ABC, CD3 monoclonal antibody, CD28 monoclonal antibody and HLA-DR antibody , anti-mouse NK1.1mAb, anti-mouse CD8+T cell mAb, rabbit anti-mouse CD16 / CD56, CD8, CD3, CD19 flow antibody (Becton Dickinson Company, biolegend Company);

[0079] Magnetic bead sorting kit: CD34, CD4, CD8, NK cells (MiltenyiBiotec);

[0080] ELISA kit: IFN-γ (BD Pharmingen);

[0081] CFSE staining solution kit (Beiyuntian Company)

[0082] Cell culture ...

Embodiment 2

[0086] Example 2, In vitro induction method of irf4+irf8+regulatory dendritic cells (irf4+irf8+regDC)

[0087] Step 1. Isolation, cultivation and identification of third-generation mesenchymal stem cells derived from human bone marrow ( Figure 1-2 )

[0088] (1) Separation

[0089] 1. Aseptically collect 5-10ml of bone marrow from healthy volunteers in a sterile heparin tube.

[0090] 2. Take a sterile centrifuge tube and properly dilute the bone marrow with D-Hanks solution.

[0091] 3. Take a new centrifuge tube and add Ficoll which has been brought back to room temperature and the above-mentioned diluted bone marrow respectively. Careful operation should not damage the interface when adding. The ratio of the two is 1:1.

[0092] 4. After balancing the centrifuge tubes above, put them into a desktop centrifuge at room temperature, and centrifuge at 1800 rpm for 20 minutes at 20°C. After the centrifuge tube was taken out, the buffy coat layer was aseptically aspirated ca...

Embodiment 3

[0127] Example 3, Morphology and Identification of irf4+irf8+ Regulatory Dendritic Cells (irf4+irf8+regDC)

[0128] (1) Form ( Figure 7 )

[0129] Regulatory dendritic cells (regDC) generated from hematopoietic stem progenitor cells induced by mesenchymal stem cells were observed under a light microscope, and synapses could be seen on the cell membrane.

[0130] (2) Identification of surface marker factors ( Figure 8 )

[0131] Detection of the expression of relevant cell surface marker factors and co-stimulatory molecules indicated that the induced new regulatory dendritic cells (regDC) had low expression of CD11c and weak expression of CD80, CD86, and CD40.

[0132] (3) Induce the number of regulatory dendritic cells ( Figure 9 )

[0133] After 7 days of co-culture of mesenchymal stem cells and hematopoietic stem progenitor cells, the suspension cells were harvested. Flow cytometry showed that a single group of cells was obtained, accounting for 56.9% of all suspend...

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Abstract

The invention provides a preparation method of a novel regulatory dendritic cell and a DC-CIK combined tumor immunotherapy method. The preparation method comprises the following steps: separating mesenchymal stem cells and hematopoietic stem cells of a mammal; together culturing the mammal's mesenchymal stem cells and hematopoietic stem cells under conditions suitable for cell growth and proliferation; and after culturing for 1-7 days, recovering from a suspension culture medium and identifying so as to obtain the novel regulatory dendritic cells. The invention is characterized in that the novel regulatory dendritic cell is converted to mature regulatory dendritic cells when induced by CpGODN (CpG oligonucleotides) or an inducer with the same induction function, wherein expression level of irf4+ and irf8+ is increased and the cell has an immunoregulation function. The novel regulatory dendritic cell can suppress tumor growth and can be combined with DC-CIK for tumor immunotherapy.

Description

technical field [0001] The present invention relates to the field of generation of novel regulatory dendritic cells and their use in immunotherapy. Specifically, the present invention relates to a method for using mesenchymal stem cells to induce hematopoietic stem progenitor cells to differentiate into irf4+irf8+ regulatory dendritic cells regDC, and a new immunotherapy combined with DC-CIK. Background technique [0002] Despite the rapid development of modern medicine, malignant tumors are still a major problem that cannot be overcome. At present, the methods for treating malignant tumors are mainly surgery, chemotherapy, radiation therapy and biological therapy. The first three treatment modalities provide options for treating most early localized tumors as well as chemoradiotherapy-sensitive tumors. However, for relapsed and refractory tumors, residual tumors, and drug-resistant tumors, biological therapy has irreplaceable advantages of other treatments. Among them, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0784A61K35/50A61K35/28A61K35/12A61K39/00A61P35/00
Inventor 陈桦
Owner 微能生命科技集团有限公司
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