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DC cells and antigen-specific T cells with high antigen presentation and their preparation methods and applications

A specific, cellular technology, applied in cell culture active agents, biochemical equipment and methods, medical preparations containing active ingredients, etc., can solve the problem of low antigen presentation efficiency and unsatisfactory T cell specific immune response question

Active Publication Date: 2020-05-15
深圳华云生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, at present, there are defects such as low antigen presentation efficiency and unsatisfactory effect of T cell-specific immune response between DC cells and T cells cultured in vitro.

Method used

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  • DC cells and antigen-specific T cells with high antigen presentation and their preparation methods and applications
  • DC cells and antigen-specific T cells with high antigen presentation and their preparation methods and applications
  • DC cells and antigen-specific T cells with high antigen presentation and their preparation methods and applications

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preparation example Construction

[0032] see figure 2 , a method for preparing DC cells with high antigen presentation according to one embodiment, comprising the following steps S110-S130.

[0033] S110. Providing DC cells.

[0034] Specifically, DC cells are normal DC cells in organisms, which can be obtained by extracting cells from peripheral blood, or by subcultured DC cell lines.

[0035] In this embodiment, the DC cells are human DC cells.

[0036] Specifically, DC cells are obtained by the following preparation methods: extract peripheral blood from healthy volunteers, anticoagulate with heparin, and separate mononuclear cells from peripheral blood with lymphocyte separation medium. Then resuspend the mononuclear cells, and after culturing for 1 hour to 4 hours, wash and collect the adherent cells to obtain DC cells.

[0037] Specifically, the collected DC cells are cultured in a medium containing GM-CSF (granulocyte-macrophage colony stimulating factor, granulocyte-macrophage colony stimulating fa...

Embodiment 1

[0084] Preparation of Gene Expression Silencers

[0085] (1) Design shRNA of Tao-1 protein phosphokinase

[0086] Using the shRNA sequence online design program on the Thermo website (http: / / rnaidesigner.thermofisher.com / rnaiexpress / setOption.do?designOption=shrna&pid=-2900031043253144145), the Tao-1 shRNA oligos sequence was designed as follows:

[0087] Chain of Justice:

[0088] 5'-CCGGGGAAGTCAAGTTTCTACAAAGCTCGAGCTTTGTAGAAACTTGACTTCCTTTTT-3' (SEQ ID No. 1).

[0089] Antisense strand:

[0090] 5'-AATTGGAAGTCAAGTTTCTACAAAGCTCGAGCTTTGTAGAAACTTGACTTCC-3' (SEQ ID No. 2).

[0091] (2) Preparation of pLKO-Tet-On / Tao-1-shRNA lentivirus

[0092] The shRNA of the Tao-1 protein phosphokinase designed above was sent to the gene company for synthesis, and the lentiviral vector pLKO-Tet-On was cut with AgeI and EcoR1, and the annealed Tao-1-shRNA oligonucleotide chain was connected to In the pLKO-Tet-On vector, the DNA ligation reaction system is as follows:

[0093]

[0094] Af...

Embodiment 2

[0096] Co-culture of DC cells and T cells to prepare DC cells with high antigen presentation and antigen-specific T cells

[0097] 1. Culture of DC cells and T cells

[0098] (1) 50 mL of peripheral blood was extracted from healthy volunteers, anticoagulated with heparin, and peripheral blood mononuclear cells (PBMC) were separated with lymphocyte separation medium.

[0099] (2) Resuspend PBMC cells with Alys-505 culture medium containing 10% autologous plasma, and divide into six-well plates, 5×10 6 pieces / mL, 2mL / well, placed in saturated humidity, 37°C, 5.0% CO 2 Incubate for 2 h in an incubator.

[0100] (3) Washing and collecting the unattached cells in (2), the unattached cells are T cells. Unattached cells were stimulated with Alys-505 culture solution containing 50ng / mL CD3 monoclonal antibody, 1000U / mL IL-2 and 0.5% autologous plasma, and the cell density was adjusted to 1×10 6 / mL, transferred to a six-well plate, 2mL / well, while adding 1000U / mL IFN-γ to each wel...

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Abstract

The invention discloses a DC (dendritic) cell with high antigen presentation and a T (thymus) cell with antigen specificity and a preparation method and application thereof. The preparation method is characterized in that the DC cell is loaded with an antigen, and a gene expression silencing agent is transfected into the DC cell, so as to obtain the DC cell with high antigen presentation; in a coculture process of the DC cell and the T cell, tetracycline is added to regulate and control the use and unuse of the gene expression silencing agent in the DC cell, the gene expression of Tao-1 protein phosphokinase with negative regulating and control function in the DC cell is inhibited in the process of mutual contact of the DC cell and the T cell and forming of an immunological synapse structure, the forming time of the immunological synapse is prolonged, the strength of the immunological synapse is improved, and the stability of the formed immunological synapse is improved. Results show that the preparation method has the advantages that the antigen presentation efficiency between the DC cell with the high antigen presentation and the T cell is higher, and the more matured and activated T cell with antigen specificity is obtained through stimulation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DC cell with high antigen presentation, an antigen-specific T cell and a preparation method and application thereof. Background technique [0002] In recent years, autologous immune cell therapy has become the most promising tumor treatment method after surgery, radiotherapy and chemotherapy. DC cells (Dendritic cells, dendritic cells) are the most important antigen-presenting cells known in the human body, and their role in mediating tumor antigen-specific cellular immunity and tumor immune response has become a hot research topic. Tumor-specific antigen presentation and T cell immune response mediated by DC cells are not only an effective means for the body to fight against tumors in the early stage of tumor pathogenesis, but also an important mechanism for tumor immune escape. [0003] There is a key step in the process of antigen transmission between DC cells and T cells (Thym...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N5/0783A61K39/00A61P35/00
CPCA61K39/00C12N5/0636C12N5/0639C12N9/12C12N2501/999C12N2502/1121
Inventor 刘韬陈雪梅周美玲
Owner 深圳华云生物技术有限公司
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