In-vitro cytokine storm model and construction method and application thereof

A technology of cytokines and construction methods, which is applied in the field of in vitro cytokine storm model and its preparation, can solve the problems of multiplication, poor simulation effect, and difficulty in meeting the needs of the medical field, and achieve reduced cell viability, high therapeutic effect, and cell The effect of decreased vitality

Active Publication Date: 2022-02-11
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] In view of the above problems, the present invention provides an in vitro cytokine storm model and its construction method and application, which solves the problem that the cell model of in vitro cytokine storm in the prior art is usually modeled by single cytokine treatment, and the obtained model is relatively simple and difficult to Deficiencies in meeting the ever-increasing demands of the medical field, and the simulation is not very good

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  • In-vitro cytokine storm model and construction method and application thereof
  • In-vitro cytokine storm model and construction method and application thereof
  • In-vitro cytokine storm model and construction method and application thereof

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preparation example Construction

[0060] The fifth aspect of the present invention relates to a method for preparing an anti-cytokine release syndrome drug, specifically comprising the following steps

[0061] The components with inhibitory effect on the cytokine storm in vitro are screened through the cytokine storm model in vitro; the effective components obtained from the screening are used to prepare anti-cytokine release syndrome medicines.

[0062] In the preceding paragraph, the active ingredient is used in the pharmaceutical process, referring to the existing pharmaceutical process.

[0063] The second aspect of this section is illustrated with some specific examples:

experiment example 1

[0064] Experimental example 1 Effect of cytokine treatment time on modeling

[0065] In this study, HUVEC (human umbilical vein endothelial cells) cells were cultured in Sciencell ECM medium, human TNF-α, IL-1β and IL-6 (all purchased from Beijing Tongli Haiyuan, and recombinant human IL-1β Protein product number GMP-TL513; recombinant human IL-6 protein product number GMP-TL512; recombinant human TNF-α protein product number GMP-TL303) three-factor combination, cytokines are ready-to-use, and diluted to 100ng / ml with cell culture medium. The HUVEC cells in the control group were added with the normal culture medium, and the HUVEC cells in the three-factor treatment group were cultured with the three-factor medium, and cultured in the culture plate. 95%CO 2 , 5% O 2 ), and 24h, 48h, and 72h after culturing for the corresponding time, the apoptotic bodies were detected by fluorescent staining.

[0066] The concentration of each cytokine was 100ng / ml to treat HUVEC cells for ...

experiment example 2

[0082] Experimental example 2 The influence of the concentration of cytokines on modeling

[0083] Human TNF-α, IL-1β, and IL-6 were combined to treat HUVEC cells at a concentration of 50ng / ml, 100ng / ml, and 200ng / ml for 72 hours, and a control group was set up to observe cell viability.

[0084] (1) Experimental settings:

[0085] A. Blank control group (HUVEC cells are not treated)

[0086] B.50ng / ml treatment group (HUVEC+TNF-α+IL-1β+IL-6 (each 50ng / ml))

[0087] C.100ng / ml treatment group (HUVEC+TNF-α+IL-1β+IL-6 (each 100ng / ml))

[0088] D.200ng / ml treatment group (HUVEC+TNF-α+IL-1β+IL-6 (each 200ng / ml))

[0089] (2) CCK-8 detection of cell viability

[0090] ① Cells were seeded in 96-well plates, 100 μl / well, treated with different concentrations of drugs on the second day of culture, and continued to culture for 72 hours;

[0091] ② Aspirate the culture solution, add 110 μl mixed cck-8 culture solution (100 μl culture solution + 10 μl CCK-8;

[0092] ③Incubate in t...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an in-vitro cytokine storm model and a construction method and application thereof. The construction system of the in-vitro cytokine storm model contains a TNF-alpha factor, an IL-1[beta] factor, an IL-6 factor and vascular endothelial cells. The in-vitro cytokine storm model is formed by the body. According to the in-vitro cytokine storm model construction method, vascular endothelial cells, TNF-alpha factors, IL-1[beta] factors and IL-6 factors are incubated together. The invention discloses application of an in-vitro cytokine storm model in screening of anti-cytokine release syndrome drugs. The invention relates to an application of an in-vitro cytokine storm model in preparing and screening anti-cytokine release syndrome drugs. The in-vitro cytokine storm model disclosed by the invention is obtained by jointly treating vascular endothelial cells through three factors, namely TNF-alpha, IL-1[beta] and IL-6, the in-vitro cytokine storm model with high cell apoptosis rate and obviously reduced cell activity can be prepared, and a better basis is provided for research on inflammatory response related to multiple factors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro cytokine storm model and its preparation method and application. Background technique [0002] Cytokine Release Syndrome (CRS), also vividly known as Cytokine Storm, refers to the rapid and massive production of various cytokines in body fluids after the body is infected with microorganisms, such as TNF-α, IL -1, IL-6, IL-12, IFN-α, IFN-β, IFN-γ, MCP-1 and IL-8, etc., is a serious life-threatening syndrome, which marks an uncontrolled and A dysfunctional immune response, involving persistent activation and expansion of lymphocytes and macrophages, secreting large amounts of cytokines, may lead to systemic inflammatory responses, multiorgan failure, methemoglobinemia, acute respiratory distress syndrome, and other diseases. In addition to microbial infections such as bacteria and viruses, CAR-T cell therapy is also prone to cytokine storms. In addition, cytoki...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/069G01N33/5064C12N2501/2301C12N2501/2306C12N2501/25C12N2503/02G01N2500/10
Inventor 胡豫梅恒姚惟琦石磊董海波
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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