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Preparation method of human cytokine-induced killer cells

A cytokine and cell killing technology, applied in the fields of biotechnology and medicine, can solve the problem of low proportion of CD3/CD56 double-positive cells, and achieve the effect of enhancing tumoricidal activity and improving tumoricidal activity

Inactive Publication Date: 2012-10-17
SHENZHEN YOUNGCELL BIO TECH
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Problems solved by technology

[0007] Aiming at the problem that the in vitro expansion efficiency of peripheral blood mononuclear cells and the proportion of CD3 / CD56 double-positive cells in CIK cells are both low in the prior art, the present invention provides a method for improving the in vitro expansion efficiency of human peripheral blood mononuclear cells and Method for preparing human cytokine-induced killer cells according to the proportion of CD3 / CD56 double positive cells in CIK cells

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  • Preparation method of human cytokine-induced killer cells
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  • Preparation method of human cytokine-induced killer cells

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Embodiment 1

[0032] 1. Obtaining the target gene

[0033] According to the human ICAM-1 and FN gene sequences, two pairs of primers were designed: forward primer P1 and reverse primer P2 of human ICAM-1; forward primer P3 and reverse primer P4 of human FN. And the target gene sequence was obtained by two-step PCR. The primer sequences are as follows:

[0034] Pl: 5'-CATCATCATCATCATCAT CAGACATCTGTGTCCCC-3' (SEQ ID NO: 3)

[0035] P2: 5'- GTAGGGGGCCGAGGTGTTCT-3' (SEQ ID NO: 4)

[0036] P3: 5'- CCCACTGACCTGCGATTCAC-3' (SEQ ID NO: 5)

[0037] P4: 5'-AGACTGCAGGTCGAC TTATGTGGAAGGAACATCCAAGAT-3' (SEQ ID NO: 6)

[0038] The 5' end of the P1 primer introduces the NdeI restriction site (underlined and italic part), and the 5' end of the P2 primer introduces the complementary sequence (boxed part) of the flexible peptide GGGGSGGGGS (SEQ ID NO: 1) cDNA to contain the human ICAM-1 cDNA The sequenced plasmid (purchased from Origene) was used as a template to amplify the extracellular I and ...

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Abstract

The invention discloses a preparation method of human cytokine-induced killer cells, comprising the following steps: coating a cell culture flask with a coating buffer containing effective amount of fusion protein and human CD3 monoclonal antibody before culturing precursor cells of human CIK cells, and adding the human CD3 monoclonal antibody in the whole process of inducing and culturing the human CIK cells, wherein the fusion protein is human intercellular adhesion molecule-1 functional domain and human fibronectin functional domain fusion protein, and the concentration of the human CD3 monoclonal antibody in the cell culture solution is lower than the concentration of the human CD3 monoclonal antibody in the coating buffer. According to the invention, ex-vivo expansion efficiency of peripheral blood mononuclear cells and the proportion of CD3 / CD56 double positive cells in the CIK cells are significantly raised, the cytotoxicity activity of the CIK cells is enhanced, thus the effect of cellular immunity treatment is raised.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular, the invention relates to a preparation method of human cytokine-induced killer cells. Background technique [0002] The incidence of tumors has been on the rise since cancer was recorded, especially in the past two or three decades, the incidence of tumors has been increasing at a rate of 3%-5% per year. With the increasing incidence of tumors, the treatment options for tumors are also constantly developing. Immunotherapy is one of the important treatment methods. It is of great significance to improve the survival rate of patients and relapse. Among them, cell biological therapy has already shown its strength and has become an important development direction of tumor treatment. Following lymphokine-induced killer cells (LAK), tumor-infiltrating lymphocytes (TIL) and CD3 monoclonal antibody-activated killer cells (CD3AK), the tumor-killing effect of cytokine-induced kil...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0786C12N15/62C12N15/70C07K19/00
Inventor 李志惠
Owner SHENZHEN YOUNGCELL BIO TECH
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