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NK cell as well as preparation method and application thereof

A NK cell and cell technology, applied in the field of biomedicine, can solve the problems of high cost, large amount of trophoblast cells, and difficulty in meeting clinical needs in terms of NK amplification quantity, purity and NK cell viability, and achieves low usage and high expansion. The effect of increasing quantity and reducing cost

Pending Publication Date: 2021-04-30
HENAN HUALONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number, purity and viability of NK cells are difficult to meet the clinical needs
[0005] CN104894072A provides a preparation method for the proliferation and culture of autologous natural killer cells. The method includes using 3-phosphoinositide-dependent protein kinase 1 and CD122 to transfect and stably express K562 cells and interleukin 2 to amplify and activate cells from cancer patients The natural killer cells are obtained from the mononuclear cells themselves, and the multiplier of natural killer cells reaches more than 2500 times, which has strong killing toxicity, but the amount of trophoblast cells is large and the cost is high

Method used

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  • NK cell as well as preparation method and application thereof
  • NK cell as well as preparation method and application thereof
  • NK cell as well as preparation method and application thereof

Examples

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Embodiment 1

[0061]In this embodiment, an engineered K562 cell is prepared, and the preparation method comprises:

[0062](1) Artificial Synthesis SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 Gene Sequence (Saturchero Biological Engineering (Shanghai) Co., Ltd.), and will SEQ ID NO: 1 and SEQ ID NO: 2 Synthesis to a gene sequence, SEQ ID NO: 1 and SEQ ID NO: 2 gene sequence named KCD19CD86, SEQ ID NO: 3 gene sequence named KIL21CD8α, SEQ ID NO: 4 The gene sequence is named KCD64, and the SEQ ID NO: 5 is named CD137L, which connects the synthetic gene sequence to the HLU vector, and the enzyme dug is BamHi / EcoRI or BamHi / NOTI, and the enzyme-cutting reaction system is shown in Table 1, The enzyme digestion was 30 ° C for 1 h, and 1.5 h was 1.5 h at 37 ° C for 1.5 h to give HLU-KCD19CD86, HLU-KCD64 and HLU-CD137L expression vectors;

[0063]Table 1

[0064]

[0065]

[0066](2) A strain containing HLU-KCD19CD86, HLU-KIL21CD8α, HLU-KCD64, HLU-CD137L or LV5-EGFP carrier is performed,...

Embodiment 2

[0072]In this example, a NK cell is prepared, and the method of preparing the NK cell comprises the steps of:

[0073](1) Pulling the anticoagulant cap slit with iodophor, after drying, the anti-condensate cover is opened, and the umbilical cord blood in the anti-concentration pipe is pulled out of the pipette, and the 50 ml of centrifuge tube is added, no more than 35ml per tube. The centrifuge is flattened, 2900r / min is centrifuged for 10 min;

[0074](2) The upper plasma obtained from the center was transferred to 50 ml of centrifuge tube, and the water bath was 30 min, then 1500 r / min was centrifuged to remove precipitation, transfer the upper plasma to a new 50 ml of centrifuge tube, and placed in a 4 ° C refrigerator preservation spare (If there is a precipitation after the refrigerator is placed, then centrifuge again);

[0075](3) Use physiological saline by volume of 2: 1 dilution of cord blood, mix mix;

[0076](4) After the dilution, the blood is carefully added to the separator,...

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Abstract

The invention relates to an NK cell as well as a preparation method and an application thereof. The preparation method comprises the following steps: inducing and culturing umbilical cord blood mononuclear cells by utilizing engineered K562 cells to obtain the NK cells. The engineered K562 cells express cytokines CD19, CD86, IL21, CD64 and CD137L, umbilical cord blood mononuclear cells can be efficiently amplified and activated, the prepared NK cells have high amplification quantity and cell viability, the use amount of the engineered K562 cells is low, and the cost can be effectively reduced.

Description

Technical field[0001]The invention is within the field of biomedical technology, involving a NK cell and a preparation method thereof.Background technique[0002]Natural Killer Cell (NK) is an important immune cell, not only related to anti-tumor, anti-viral infection, and immunomodulation, but also the first defense line of human resistance cancer cells and viral infections, non-specific direct killing tumors Cells, also have immunomodulatory functions, which can interact with other plurality of immune cells of the body, and regulate the body's immunity and immune function. Clinical studies have found that NK cellular adverse sex immunotherapy has good application prospects for a variety of solid tumors and blood systems. Effective effects.[0003]The existing NK cell amplification culture technology mainly includes the factor method: extracting peripheral blood or using a single cell collector and combined with a lymphocyte separation to obtain peripheral blood mononuclear cells (PBMC...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/10A61K35/17A61P37/02A61P35/00A61P31/12
CPCC12N5/0646A61K35/17A61P37/02A61P35/00A61P31/12C12N2502/99C12N2501/599C12N2501/51C12N2501/2321C12N2501/505
Inventor 赵礼军熊建民
Owner HENAN HUALONG BIOLOGICAL TECH
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