Method for preparing dendritic cell vaccine

A technology of dendritic cells and vaccines, applied in the field of preparation of dendritic cell vaccines, can solve the problems of poor specificity, low loading efficiency, and low targeting, and achieve high loading efficiency, no immune tolerance, Sensitive and specific effects

Inactive Publication Date: 2014-07-30
江苏和泽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the existing DC vaccine preparation methods use tumor tissue blocks or cancer-related proteins as targeting substances, which have low loading efficiency, low targeting, and poor specificity

Method used

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  • Method for preparing dendritic cell vaccine
  • Method for preparing dendritic cell vaccine
  • Method for preparing dendritic cell vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Preparation of DCs derived from umbilical cord blood

[0019] (1) Separation of umbilical cord blood mononuclear cells:

[0020] Cut the umbilical cord blood bag, measure the umbilical cord blood with a 50ml syringe, dilute the blood with an equal amount of compound electrolyte injection; slowly add the diluted cell suspension to the Ficoll at a ratio of 2:3 between the Ficoll separation medium and the cell suspension The upper layer of the separation solution; centrifuge, 900g, 25min; absorb the cells near the buffy coat at the boundary, and resuspend in the compound electrolyte injection; centrifuge 400g, 8min; absorb the supernatant, and resuspend the cell pellet in the compound electrolyte injection; centrifuge 400g, 8min; Aspirate the supernatant, resuspend the cell pellet and add 10ml of culture medium, and then count; culture DC according to the counting results.

[0021] (2) DC adherent culture

[0022] Use RPMI-1640 medium containing 1% FBS to adjus...

Embodiment 2

[0025] Example 2 Detection of Immunophenotype of DC Cells After Improving Maturation

[0026] Take DC cells after maturation (on the 6th day, mix the cells and take samples), wash them twice with PBS without calcium and magnesium, take 1×105 / mL each, and add them to the corresponding FCM tubes. Add 5 μl of monoclonal antibodies to be tested, including CD11C, HLA-DR, CD83, and CD86 antibodies, incubate at 4°C in the dark for 30 minutes, and shake once every 10 minutes to fully contact the cells with the antibodies. Washed twice with PBS, resuspended in 400 μl of PBS, and detected by flow cytometer FASCSCalibur (BD Biosciences), the results are shown in Table 1, Figure 2-4 .

[0027] Table 1

[0028]

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Abstract

The invention belongs to the cellular immunity field, and concretely relates to a preparation method of dendritic cell vaccine. The method comprises the following steps: separating cord blood to obtain cord blood monocyte; screening DC cell from the cord blood monocyte, culturing and performing amplification, then performing single epitope multiple antigen peptide (SEA-MVP) for loading, and then incubating overnight to obtain the DC vaccine. The method has the beneficial effect that the single epitope multiple antigen peptide (SEA-MVP) is used for loading DC cells, the single epitope multiple antigen peptide (SEA-MVP) is obtained by in vitro synthesis, the composition is clear and single, the purity is high; alkaline amino acid is taken as a frame for connecting the single epitope, so that the orientation of the connected epitope peptide has consistency and the epitope peptide is easily recognized and presented by DC cells, the sensitivity and singularity are strong, the loading efficiency is high, and immune tolerance is not generated; compared with the current method, the antineoplastic cell capability of the dendritic cell vaccine for activating the CD4+ and CD8+ cells is increased by more than ten thousand times.

Description

technical field [0001] The invention belongs to the field of cellular immunity, in particular to a preparation method of a dendritic cell vaccine. Background technique [0002] Dendritic cells (DCs) are the most powerful antigen-presenting cells (Antigenpresenting cells, APCs) in the body, which can stimulate the proliferation of initial T cells and initiate immune responses; they can also present antigens to MHC-class I-restricted CD8+ and MHC-class II restricted CD4+ T lymphocytes, which induce specific immune responses, are called "natural immune adjuvants", so they have a unique position in inducing immune responses. In recent years, DC-based immunotherapy has received more and more attention in clinical applications, and is a research hotspot at home and abroad. [0003] Most of the existing DC vaccine preparation methods use tumor tissue blocks or cancer-related proteins as targeting substances, which have low loading efficiency, low targeting, and poor specificity. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00C12N5/0784A61P35/00
Inventor 刘乐锋
Owner 江苏和泽生物科技有限公司
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