Method for promoting proliferation of NK cell by using CD3+CD8+CD56+T cell subtype

A kind of NK cell and cell technology, applied in the biological field, can solve the problems of unsatisfactory NK cell therapy and scarcity of NK cells

Active Publication Date: 2015-10-28
普华赛尔生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the rare number of NK cells, which only account for about 5% of peripheral blood lymphocytes, even after in vitro expansion, it is still impossible to obtain a satisfactory number of NK cells for later treatment

Method used

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  • Method for promoting proliferation of NK cell by using CD3+CD8+CD56+T cell subtype
  • Method for promoting proliferation of NK cell by using CD3+CD8+CD56+T cell subtype
  • Method for promoting proliferation of NK cell by using CD3+CD8+CD56+T cell subtype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1, CD3 + CD56 + Preliminary Study on the Effect of T Cells on the Proliferation of NK Cells

[0032] This example will preliminarily study CD3 + CD56 + The effect of T cells on the proliferation of NK cells. According to the CD4 / CD8 classification, CD3 + CD56 + T cells are divided into CD3 + CD4 + CD56 + T cells, CD3 + CD8 + CD56 + T cells and CD3 + CD4 - CD8 - CD56 + Three groups of T cells. As CD3 + CD4 - CD8 - CD56 + The proportion of T cells in peripheral blood is very low, so it is not considered in this study.

[0033] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0034] Obtain peripheral blood mononuclear cells (PBMC) from the same healthy volunteer (a healthy person who has been clinically identified as having no major disease) as follows:

[0035] Using human lymphocyte separation solution, the use of density gradient centrifugation to separate peripheral blood mononuclear cells (PBMC). This is a centrifugal method that separates by density in a...

Embodiment 2

[0060] Example 2: CD3 during proliferation + CD8 + CD56 + Optimization of the initial ratio of T cells and NK cells

[0061] In this example, on the basis of example 1, we will further study CD3 in the process of promoting proliferation. + CD8 + CD56 + The initial ratio of T cells and NK cells affects the proliferation of NK cells.

[0062] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0063] Same as Step 1 of Example 1, except that the peripheral blood mononuclear cells (PBMC) are from another healthy volunteer different from Example 1.

[0064] Two, flow cytometric sorting

[0065] Use flow cytometry to separate NK cells (CD3) from the peripheral blood mononuclear cells (PBMC) obtained in step 1. - CD56 + Lymphocytes) and CD3 + CD8 + CD56 + T cells. The specific operation is as follows, refer to step 2 of embodiment 1.

[0066] Three, cell culture

[0067] The two cell populations obtained in step 2 are in accordance with CD3 + CD8 + CD56 + T cells and NK cells are divide...

Embodiment 3

[0072] Example 3, CD3 + CD56 + CD8 + Study on the mechanism of T cell promoting NK cell proliferation

[0073] The increase in the number of cells is mainly caused by two factors, one is the acceleration of cell mitosis; the other is the slowdown of cell apoptosis. To clarify CD3 + CD56 + CD8 + The mechanism by which T promotes the proliferation of NK cells in vitro, the inventors of the present invention have studied NK cells under different culture conditions from two aspects.

[0074] 1. CD3 + CD56 + CD8 + The influence of T cells on NK cell mitosis

[0075] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0076] Refer to step one in Example 1.

[0077] 2. Flow cytometric sorting

[0078] Use flow cytometry to isolate NK cells (CD3) from the peripheral blood mononuclear cells (PBMC) obtained in step 1. - CD56 + Lymphocytes) and CD3 + CD8 + CD56 + T cells. The specific operation is as follows, refer to step 2 of embodiment 1.

[0079] 3. Cell culture

[0080] The inventors o...

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Abstract

The invention discloses a method for promoting proliferation of an NK cell. The invention provides a new use of a CD3+CD8+CD56+T cell in preparation of NK cell proliferation promotion products, or preparation of NK cell mitosis promotion products, or preparation of NK cell apoptosis resistance enhancement products. An NK cell mixed culturing system is established through co-culturing the following two immune cells with strong killing functions: the CD3+CD8+CD56+T cell and the NK cell, by using the NK cell proliferation promotion characteristic of the CD3+CD8+CD56+T cell. The method provides a new idea for in vitro massive proliferation culturing of the NK cell.

Description

Technical field [0001] The invention belongs to the field of biotechnology and relates to a method for promoting the proliferation of NK cells. Background technique [0002] At the end of 2013, Science magazine selected ten breakthrough scientific achievements, and tumor immunotherapy ranked first. Compared with traditional anti-tumor treatments, cell therapy has a wide range of applications, especially for micrometastasis, and its side effects are significantly lower than those of radiotherapy and chemotherapy. With the development of tumor biology, molecular biology and immunology, immune cell therapy is currently the most promising treatment method for breakthroughs, and has become the fourth tumor treatment mode after traditional tumor treatment methods, surgery, radiotherapy and chemotherapy. At present, the most widely used tool cell is T lymphocytes. However, because lymphocytes from tumor patients have received immune editing from the tumor microenvironment for a long ti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 马洁袁伟张兴华韩志楷王铮
Owner 普华赛尔生物医疗科技有限公司
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