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Preparation method for efficiently amplifying NK cells by utilizing trophoblasts

A technology for NK cells and trophoblasts, applied in the fields of genetic engineering and cell biology, can solve the problems of low NK cell expansion multiples, poor repeatability, and low purity

Active Publication Date: 2020-01-14
山东德升生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims at problems such as low amplification factor, low purity, and poor repeatability of NK cells proposed in the background technology, and simultaneously uses K562 cells expressing IL-21, 4-1BBL, and MICA molecules as trophoblast cells in the present invention Cultivate NK cells to improve the amplification factor and purity of NK cells is also one of the problems discussed in the present invention

Method used

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  • Preparation method for efficiently amplifying NK cells by utilizing trophoblasts
  • Preparation method for efficiently amplifying NK cells by utilizing trophoblasts
  • Preparation method for efficiently amplifying NK cells by utilizing trophoblasts

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Construction of pHR-mbIL-21, pHR-4-1BBL and pHR-MICA plasmid vectors

[0079] 1. Obtain the whole gene coding region sequence of mbIL-21, 4-1BBL and MICA

[0080] 1) Query the human mbIL-21 gene sequence, human 4-1BBL gene sequence, and human MICA gene sequence on the NCBI website;

[0081] 2) The signal peptide region sequence in the mbIL-21 gene sequence, the human IL-21 sequence, the hinge region sequence, the human Fc fragment sequence, and the human CD8 transmembrane region sequence in the mbIL-21 gene sequence were connected by PCR amplification method, and then in the mbIL-21 gene sequence The restriction endonuclease BamHI was introduced into the first part of the sequence, and the restriction endonuclease NotI was introduced into the tail part to prepare the sequence of the coding region of the whole gene of mbIL21 for future use;

[0082] 3) The signal peptide region sequence in the 4-1BBL gene sequence, the human IL-21 sequence, the hinge region sequence, th...

Embodiment 2

[0089] Preparation of pHR-mbIL-21 recombinant lentivirus, pHR-4-1BBL recombinant lentivirus and pHR-MICA recombinant lentivirus

[0090] 1. Preparation of 293FT cells:

[0091] 1) 293FT cells in the logarithmic growth phase were introduced into a 6-well plate, and each well contained 0.6x10 6 1 cells and 2ml of DMEM complete culture medium, mixed evenly, placed in a 37°C incubator for overnight culture, meanwhile prepare 20ml of DMEM complete culture medium and cultured overnight under the same conditions for later use.

[0092] 2) When the confluence of 293FT cells reaches 50%-60% under the microscope, discard the culture solution in the 6-well plate, and then add preheated DMEM complete culture solution on the 6-well plate, the addition amount is 2ml / Wells, used to remove unattached cells, spare;

[0093] 2. Preparation of recombinant lentivirus

[0094] Preparation of pHR-mbIL-21 recombinant lentivirus:

[0095] 1) According to the mass ratio, take 3ug of pHR-mbIL-21 p...

Embodiment 3

[0113] Preparation of K562-mbIL-21-4-1BBL-MICA trophoblast cells

[0114] 1. The preparation method of K562-mbIL-21-4-1BBL-MICA trophoblast cells includes:

[0115] 1) Add pHR-mbIL-21 recombinant lentivirus, pHR-4-1BBL recombinant lentivirus, pHR-MICA recombinant lentivirus sequentially according to the final MOI value of the composite recombinant lentivirus at 5, and mix evenly to form a composite recombinant lentivirus;

[0116] 2) Infect K562 cells with a composite recombinant lentivirus for 72 hours, and clone by flow cytometry and limiting dilution to obtain K562-mbIL-21-4-1BBL-MICA engineered cell line;

[0117] 3) The K562-mbIL-21-4-1BBL-MICA engineered cell strain was sterilized by cobalt-60 irradiation, and the K562-mbIL-21-4-1BBL-MICA engineered cell strain was sterilized by cobalt-60 irradiation, and cobalt- The dose of cobalt in 60 irradiation sterilization was 100Gy, and the K562-mbIL-21-4-1BBL-MICA trophoblasts were prepared, and the cell density of the K562-mbI...

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Abstract

The invention relates to the field of gene engineering and cytobiology, in particular to a preparation method for efficiently amplifying NK cells by utilizing trophoblasts. The construction method comprises the following steps: (1) constructing a pHR-mbIL-21 plasmid vector, a pHR-4-1BBL plasmid vector and a pHR-MICA plasmid vector; (2) preparing recombinant lentiviruses by using pHR-mbIL-21, pHR-4-1BBL and pHR-MICA plasmid vectors respectively; (3) preparing K562-mbIL-21-4-1BBL-MICA trophoblasts; (4) extracting PBMC cells; and (5) carrying out in-vitro amplification on NK cells. The inventionprovides the preparation method of NK cells. The method employs the step of independently constructing plasmid vectors for expressing mbIL-21, 4-1BBL and MICA molecules, K562 is infected with recombinant lentivirus, the NK cells can be prepared, the preparation method overcomes the defects in the prior art, the K562 cells simultaneously expressed by IL-21, 4-1BBL and MICA molecules are used as trophoblasts for amplification culture of the NK cells, the amplification multiple of the NK cells reaches up to 890 times, the purity of the prepared NK cells reaches 92.2%, and the repeatability of theamplification multiple between different PBMC cells is good.

Description

technical field [0001] The invention relates to the fields of genetic engineering and cell biology, in particular to a preparation method for efficiently expanding NK cells by using trophoblasts. Background technique [0002] At present, the incidence of tumors in the world is developing rapidly. However, traditional treatment methods, such as surgery, radiotherapy and chemotherapy, cannot achieve good results. Tumor immunotherapy is currently the fourth cancer treatment method recognized by the medical community. Natural killer cells (NK) are the main effector cells of human innate immunity, which can kill tumor cells without MHC restriction by using perforin, granzyme and other mechanisms without pre-stimulation. In recent years, NK immunotherapy technology is becoming a broad-spectrum treatment method for malignant tumors due to its majorhistocompatibility complex (MHC) restriction, small side effects and strong anti-tumor activity. [0003] NK cells are mainly distribu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/10C12N15/867C12N7/01
CPCC12N5/0646C12N5/0694C12N7/00C12N15/86C12N2501/2302C12N2501/2321C12N2501/50C12N2501/599C12N2502/30C12N2510/00C12N2740/15021C12N2740/15043
Inventor 郭红陈佃雷汤文玲刘蕾
Owner 山东德升生物工程有限公司
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