A method for inducing and expanding NK cells in combination with traditional Chinese medicines in vitro and its application

A technology of NK cells and traditional Chinese medicine, applied in the field of biomedicine, can solve the problems of cumbersome operation process of immunomagnetic bead sorting method, unsuitability for large-scale clinical application, and unguaranteed safety, so as to improve proliferation and killing activity, enhance Human immune activity and the effect of stimulating T cell proliferation

Active Publication Date: 2017-10-13
HANGZHOU S EVANS BIOSCI LTD
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  • Abstract
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  • Claims
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Problems solved by technology

At present, in vitro expansion of NK cells mainly adopts the method of co-culture of feeder cells and immunomagnetic bead sorting, but the co-culture of feeder cells is to add NK cells to the modified tumor cell line, so that the modification is clinically applicable. The safety is not guaranteed, and the immunomagnetic bead sorting method is cumbersome and costly, and is not suitable for large-scale clinical application

Method used

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  • A method for inducing and expanding NK cells in combination with traditional Chinese medicines in vitro and its application
  • A method for inducing and expanding NK cells in combination with traditional Chinese medicines in vitro and its application
  • A method for inducing and expanding NK cells in combination with traditional Chinese medicines in vitro and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] (1) Peripheral blood mononuclear cell separation: After the patient signed the informed consent, 50ml of peripheral blood and 10ml of heparin sodium anticoagulant were collected under aseptic conditions and mixed evenly, and evenly divided into two 50ml centrifuge tubes, each tube Centrifuge 30ml of blood sample at 800g for 10min. After centrifugation, discard the upper layer of plasma. The volume in the centrifuge tube is about 15ml±3ml. Add an equal volume of normal saline to the blood sample separated from plasma to 30ml, and mix well. Then add 15ml of lymphatic separation solution to each of the two new 50ml centrifuge tubes. The volume ratio of lymphatic separation solution and diluted blood sample is 1:2. Pipette the diluted blood sample and slowly add it to Ficoll along the tube wall at a distance of 1cm from the liquid surface. and then centrifuged at 700g for 20min. After centrifugation, pipette 2 tubes of buffy coat into a new 50ml centrifuge tube, dilute it t...

Embodiment 2

[0037] Embodiment 2 (control group)

[0038] Except in (3) cell expansion, no addition of traditional Chinese medicine extracts of astragalus polysaccharides and Lycium barbarum polysaccharides other specific operation steps as in Example 1.

Embodiment 3

[0039] Example 3: NK cell amplification multiple detection on the amplification product

[0040] Collect 500 μl of cells on the 1st, 7th, 14th, and 21st days respectively, and blow them into single cells, or centrifuge at 400g×5min, discard the supernatant, add 500μl of PBS, and use a Counterstar automatic blood cell counter to count the cells on the day according to the principle of trypan blue staining. The total number of cells divided by the number of cells before culture on the first day was the cell expansion factor. This method can be used to calculate the cell expansion fold. According to the observation of the cell state during the culture process, it was found that the cell state of the experimental group with the concentration of astragalus polysaccharide 200 μg / ml and the concentration of wolfberry polysaccharide 125 μg / ml was the best. test group.

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Abstract

The invention provides a method for inducing and amplifying NK cells in combination with traditional Chinese medicine extracts and cytokines in vitro, comprising adding cytokine IL-2, cytokine IL-15, astragalus polysaccharide and wolfberry polysaccharide to NK cell culture fluid , the concentration of astragalus polysaccharide is 150-250 μg / ml, and the concentration of wolfberry polysaccharide is 100-200 μg / ml. The NK cells obtained by this culture method have significantly improved expansion multiples, cell viability and killing effect, and all the stimulating factors used have been approved for safe clinical use. At the same time, the method is easy to operate, low in cost, and suitable for large-scale applications .

Description

technical field [0001] The invention relates to the field of biomedicine, and specifically refers to a method for inducing and amplifying NK cells by combining traditional Chinese medicine extracts of astragalus polysaccharides and wolfberry polysaccharides with cytokines in vitro and its application. Background technique [0002] According to the 2013 China Cancer Annual Report, the incidence and mortality of cancer in my country are increasing year by year, and cancer has become the number one killer of human beings. At present, the combination of surgery, radiotherapy, and chemotherapy is mainly adopted in clinical treatment of tumors in my country. Traditional treatment methods often seriously damage the patient's body function, sharply reduce the patient's immunity, and generally lead to poor quality of life. In recent years, with the development of immunology, cell biology, and tumor biology, and in-depth research on the mechanism of tumor generation and development, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
Inventor 吴晓星陈文珍焦明超
Owner HANGZHOU S EVANS BIOSCI LTD
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