A kind of cytokine particle and its application

A technology of cytokines and granules, applied in the field of bioengineering, can solve problems affecting cell state and vitality, slow cell proliferation, and increased proportion of aging cells

Active Publication Date: 2017-11-24
北京安图生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally, conventional cytokine proteins are used to stimulate mononuclear cells, because cytokine proteins are usually small molecular proteins, and the stimulation effect on cells is relatively slow, and the cell proliferation is slow, which will lead to aging cells in the total obtained cells. Ratio increases, thereby affecting the state and viability of the overall cell

Method used

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  • A kind of cytokine particle and its application
  • A kind of cytokine particle and its application
  • A kind of cytokine particle and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of expression vector system

[0037] 1. Preparation of recombinant protein gag plasmid

[0038] (1) Nucleic acid extraction to obtain RNA of gag gene: the nucleic acid of murine leukemia virus MLV was extracted using the QIAamp Ultrasens virus kit from QIAGEN.

[0039] Specific method: Take 1ml virus sample to 2ml EP tube and equilibrate the temperature to 15℃-25℃, add 800μl BufferAC, add 5.6μl carrier RNA to the lid of EP tube, then vortex for 10s to mix well; incubate at room temperature for 10 minutes, centrifuge at 3200rpm For 3 minutes, discard all the supernatant; add 300μl Buffer AR (preheated at 60°C) and 20μl proteinase K, and vortex to dissolve the precipitate; vortex once for 5 seconds during 10 minutes in a water bath at 40°C, and transfer it all into the QIAamp Spin Column6000rpm centrifugation for 1 minute

[0040] Add 500μl Buffer AW1 and centrifuge at 7500rpm for 1 minute to discard the waste solution, add 500μl BufferAW2 and cen...

Embodiment 2

[0084] Example 2 Preparation of cytokine granules

[0085] Mix 48 μl gag plasmid (0.1ug / μl) and 20 μl cytokine plasmid (0.1ug / μl); add 480 μl DMEM (dulbecco's modified eagle medium) medium and mix well, add 120 μl calcium phosphate transfection reagent (PolyFect Transfection Kit) Mix well and let stand at room temperature for 5-10 minutes. 293 cells were washed twice with 0.01M PBS, digested with 0.25% Trypsin EDTA, added complete medium (DMEM medium + 10% fetal calf serum) to disperse by blowing, counted, filled 8 million cells in a 75ml culture bottle and supplemented the culture medium for the final The volume is 9ml, the plasmid to be transfected is diluted with 3.6ml of complete medium, then added to the culture bottle, tapped gently, so that the plasmid complex is fully mixed in the cell suspension. Cultivate in a 37°C, 5% CO2 incubator for 6 hours, suck off the transfection mixture, gently wash the cells 3 times with PBS, and add DMEM medium to continue culturing for 7...

Embodiment 3

[0090] Example 3 Cytokine granule IL15-CD137L stimulates differentiation and expansion of mononuclear cells into NK cells

[0091] Mononuclear cells (PBMC) collection: Use (heparin / EDTA) anticoagulation to collect 50ml of peripheral blood, use lymphocyte separation medium (ficol) to separate PBMC (mononuclear cells), and use (DMEM / 1640 / normal saline / PBS) to wash 2 times; plasma was collected at the same time and inactivated at 56°C for 30 minutes.

[0092] Culture bottle treatment: Use PBS to prepare mAb-CD16 monoclonal antibody at a concentration of 1-5ug / ml, and coat to 75cm 2 Culture flasks overnight, then wash twice with saline. Store at 4°C until use.

[0093]The T551 medium was divided into two groups, the cytokine granule group containing cytokine IL15 and CD137 receptor ligand protein IL15-CD137L (800IU / ml) and the cytokine granule group containing conventional cytokine IL15 and CD137L (each 800IU / ml) conventional cytokine group. Culture conditions: temperature: 3...

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Abstract

The invention provides cytokine particles and an application thereof. The invention also provides a preparation method of the cytokine particles. The particles comprise particles formed by one or more cytokines and recombinant protein gag. The invention also provides a method for stimulating differentiation of single nuclear cells in-vitro by the cytokine particles. The method comprises the following steps of: stimulating single nuclear cells to differentiate into NK cells or T cells in-vitro by the cytokine particles, and amplifying the quantity of the NK cells or the T cells. Compared with the cytokine, the cytokine particles are stronger in stimulating action and obvious in effect.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to cytokine granules prepared by bioengineering technology and their application in stimulating monocyte differentiation and expansion in vitro. Background technique [0002] The development of immune cell therapy technology has brought about major changes in the application of immune science, and its efficacy against tumors is changing the survival period and quality of life of cancer patients. For example, in autologous immune cell therapy, mononuclear cells are isolated from the peripheral blood of tumor patients, and culture medium containing targeted stimulating factors is used to stimulate the proliferation and differentiation of lymphocytes to increase their number and have anti-tumor specificity. Then infuse it back into the tumor patient's body, and have obvious therapeutic effect on the tumor. These applied immune cells include: LAK cells, TIL cells, CIK cells, T cells, NK c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/52C07K14/475C12N15/85C12N5/0783
Inventor 张永辉林小靖
Owner 北京安图生物工程有限公司
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