Novel preparation method of dendritic cell (DC) vaccine

A technology of dendritic cells and vaccines, applied in the field of cellular immunology, can solve the problem of reducing DC cells, achieve the effect of increasing cell survival time and cell survival rate, and improving tumor killing effect

Inactive Publication Date: 2018-04-20
BEIJING TRICISIONBIO THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the presence of PGE2 significantly reduced the level of IL-12 secreted by DC cells

Method used

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  • Novel preparation method of dendritic cell (DC) vaccine
  • Novel preparation method of dendritic cell (DC) vaccine
  • Novel preparation method of dendritic cell (DC) vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] In vitro transcription and synthesis of Bcl-2, IL-12p70mRNA

[0050] 1. Construct plasmids containing Bcl-2 and IL-12p70 respectively, the nucleotide sequences of both are obtained from GeneBank;

[0051] 2. Design primers according to the Bcl-2 and IL-12p70 gene sequences, clone the fragments of the two by PCR, and connect the cloned genes to the psp73 vector;

[0052] 3. Treat the psp73 vector connected with Bcl-2 and IL-12p70 genes with speI endonuclease to linearize it;

[0053] 4. Synthesis of Bcl-2 and IL-12p70 mRNA by in vitro transcription

Embodiment 2

[0055] Preparation and detection of human dendritic cell vaccine

[0056] 1. Collect 100ml of peripheral blood from the patient, dilute blood cells with PBS at a ratio of 2:1, add Ficoll-Paque to the diluted blood at a ratio of 1:2 into a centrifuge tube, and centrifuge at 700g for 20 minutes. After centrifugation, carefully aspirate the buffy coat cells, and wash the cells twice with calcium-magnesium-free PBS or HBSS at 500g for 10 minutes and 300g for 10 minutes, respectively.

[0057] 2. Resuspend the mononuclear cells isolated above in 10ml of AIM-V or X-VIVO-15 medium, transfer the cells into a T175cm culture flask, and supplement the medium to 30ml. The culture flask was placed in a 37°C, 5% carbon dioxide incubator for 2 hours to adhere to the wall.

[0058] 3. Gently tap the culture bottle to re-suspend the non-adhered cells, collect the non-adhered cells into a centrifuge tube; add PBS to the bottle, gently shake the culture bottle to wash away the non-adhered cells...

Embodiment 3

[0069] Tumor antigen-specific T cell responses induced by DC cells in vitro

[0070] 1. One day before DC cell electroporation, thaw the frozen non-adherent lymphocytes, resuspend the cells in 15ml of AIM-V or X-VIVO-15 medium, transfer them to a T75 culture flask, and place at 37°C, 5% Recover overnight in a carbon dioxide incubator.

[0071] 2. On the second day, DC cells and T cells 2 hours after electroporation were mixed and cultured at a ratio of 1:10. The remaining DCs were then frozen.

[0072] 3. On the third day of co-cultivation, IL-2 was added with a final concentration of 50U / ml, and half of the cells were replaced with fresh medium containing 50U / ml IL-2 every 2 days.

[0073] 4. After 7-10 days of co-culture, DC cells were revived and counted. At the same time, co-cultured T cells were collected and counted.

[0074] 5. Mix and co-culture the recovered DC cells with the collected T cells at a ratio of 1:10.

[0075] 6. After re-stimulating the T cells in ste...

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Abstract

The invention belongs to the technical field of cell immunology and provides a novel preparation method of a dendritic cell (DC) vaccine. According to the novel preparation method of the dendritic cell (DC) vaccine, while tumor antigen is loaded, exogenous Bcl-2 mRNA and IL-12P70 mRNA are transferred into DC cells in an electrotransfection manner. The method has the advantages that the prepared DCcells have higher vitality and longer survival time, and can secrete abundant IL-12, therefore, the antigen presenting capacity of the DC is remarkably increased, and the specific immune response ofthe antigen is effectively induced. The DC vaccine prepared by the method is mainly used for treatment for tumor or prevention of cancer high-risk people, and the preparation method has the features that the preparation process is simple, the cost is low, the specificity is strong, the curative effects are significant, etc.

Description

technical field [0001] The invention belongs to the technical field of cellular immunity, in particular to a preparation method of a dendritic cell tumor vaccine. Background technique [0002] Tumor has become the second biggest killer of human health after cardiovascular disease. Conventional tumor treatment methods include surgery, radiotherapy and chemotherapy. However, the existing tumor treatment methods cannot well solve the problems of tumor recurrence and migration. As the fourth emerging tumor treatment method, tumor immunotherapy has shown great application potential and prospects in tumor treatment and prevention due to its good efficacy and safety. [0003] Tumor immunotherapy can be divided into active immunotherapy and passive immunotherapy. Dendritic cell vaccines are active immunotherapy. It can carry tumor antigen information, activate specific T cells to generate a response, and produce an anti-tumor effect. Dendritic cells (DCs, Dendritic Cells) are t...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P35/00C12N5/0784
CPCA61K39/0011A61K2039/5154C12N5/0639C12N2501/02C12N2501/22C12N2501/2301C12N2501/2304C12N2501/2306C12N2501/25
Inventor 不公告发明人
Owner BEIJING TRICISIONBIO THERAPEUTICS INC
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