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
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[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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