CN114032208B - In-vitro cytokine storm model and construction method and application thereof - Google Patents
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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 in vitro cell factor storm model constructing system contains TNF-alpha factor, IL-1 beta factor, IL-6 factor and vascular endothelial cell. The in vitro cytokine storm model formed above. The in vitro cell factor storm model constructing process includes the incubation of vascular endothelial cell with TNF-alpha factor, IL-1 beta factor and IL-6 factor. The application of the in vitro cytokine storm model in screening the anti-cytokine release syndrome medicines. The application of the in vitro cytokine storm model in preparing and screening the anti-cytokine release syndrome medicine. The in vitro cell factor storm model is obtained by treating vascular endothelial cells by combining TNF-alpha, IL-1 beta and IL-6, can be prepared into the in vitro cell factor storm model with high apoptosis rate and obviously reduced cell activity, and provides a better basis for the research of inflammatory reaction related to multiple factors.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an in-vitro cytokine storm model and a preparation method and application thereof.
Background
Cytokine Release Syndrome (CRS), also known as Cytokine Storm (Cytokine Storm), is a serious life-threatening Syndrome that causes rapid and abundant production of various cytokines in body fluids after the body is infected with microorganisms, such as TNF-alpha, IL-1, IL-6, IL-12, IFN-alpha, IFN-beta, IFN-gamma, MCP-1 and IL-8, etc., and is involved in the sustained activation and expansion of lymphocytes and macrophages and the secretion of a large amount of cytokines, which may cause diseases such as systemic inflammatory response, multiple organ failure, methemoglobinemia and acute respiratory distress Syndrome. In addition to microbial infections such as bacteria and viruses, CAR-T cell therapy is also prone to cytokine storms, and cytokine storms may also occur when immune checkpoint inhibitor therapy is performed, but with very low probability.
In the prior art, microorganisms are commonly used to infect animals, or stimulate macrophage secretion after tumor killing (Liu Y, et al,. Gasdermin E-mediated target cell transfection by CAR T cells triggerers cytokine release syndrome. Sci Immunol.2020Jan 17, 43) to simulate endogenous storms of cytokine animals, so that the concentration of cytokines is not sufficiently determined, and the concentration of factor secretion after each tumor cell killing is unstable, so the animal body is also unstable.
While in vitro cytokine storm models have been reported:
(1) Blouzhen Kai et al (H) 2 O 2 Experimental study on LPS and TNF-alpha induced injury of human umbilical vein endothelial cells) using hydrogen peroxide (H) at different concentrations 2 O 2 ) Lipopolysaccharide (LPS) and tumor necrosis factor (TNF-alpha) stimulate human umbilical vein endothelial cells, and the incubation lasts for different times, and the research shows that H 2 O 2 LPS and TNF-alpha can damage human umbilical vein endothelial cells in vitro, successfully establish a human umbilical vein endothelial cell damage model, and each damage factor presents concentration and time dependence on the human umbilical vein endothelial cell damage degree in a certain concentration range.
(2) Speciale, A et al (Silibinin as a functional tool against SARS-Cov-2.
(3) Chen, tielong et al (Quercetin inhibitors TNF-. Alpha.induced HUVECs apoptosis and inflammation visual descending NF-kB and AP-1 signalling pathway in vitro, medicine.
Because only a single factor is used for preparing a cytokine storm model in vitro in the field at present, such as TNF-alpha is used, the obtained model is simpler, the model requirement of the research on inflammation induced by a specific cytokine in the medical field cannot be met, and no report of multi-factor combined modeling exists, the preparation of the multi-factor induced in vitro cytokine storm model is urgently needed.
Disclosure of Invention
Aiming at the problems, the invention provides an in vitro cytokine storm model, a construction method and application thereof, which solve the defects that in the prior art, the in vitro cytokine storm cell model is usually molded by single cytokine treatment, the obtained model is simpler and is difficult to meet the increasingly-growing requirements in the field of medicine, and the simulation effect is not very good.
In order to solve the problems, the invention adopts the following technical scheme:
the in vitro cell factor storm model constructing system contains TNF-alpha factor, IL-1 beta factor, IL-6 factor and vascular endothelial cell.
In some embodiments, there is also a culture medium; the vascular endothelial cells are human umbilical vein endothelial cells.
In some embodiments, the medium is endothelial cell medium.
The second technical scheme provided by the invention is as follows: an in vitro cytokine storm model formed from the above bodies.
The in vitro cytokine storm model is formed by a construction system of any one of the in vitro cytokine storm models.
The third technical scheme provided by the invention is as follows: the preparation method of the in vitro cytokine storm model.
The in vitro cell factor storm model constructing process includes the incubation of vascular endothelial cell with TNF-alpha factor, IL-1 beta factor and IL-6 factor.
In some embodiments, any of TNF- α factor, IL-1 β factor, and IL-6 factor is present at a concentration of 100-400ng/ml; the incubation time is 24-168h.
In some embodiments, more specifically, any of TNF- α factor, IL-1 β factor, and IL-6 factor is present at a concentration of 100-200ng/ml; the incubation time is 120-168h.
In some embodiments, the concentrations of TNF-alpha factor, IL-1 beta factor, and IL-6 factor are the same; the incubation temperature was 37 ℃; the medium incubated was endothelial cell medium.
The fourth technical scheme provided by the invention is as follows: the application of the in vitro cytokine storm model according to the second technical scheme in screening anti-cytokine release syndrome drugs.
The application of the in vitro cytokine storm model in preparing and screening the anti-cytokine release syndrome medicine.
In a specific embodiment of the invention, the vascular Endothelial cells are Human Umbilical Vein Endothelial Cells (HUVEC).
In the present invention, the Medium for incubation may be selected as is conventional in the art, and in some cases is Endothelial Cell Medium (ECM).
In a preferred embodiment of the present invention, the method further comprises a step of separating the prepared in vitro cytokine storm model from the culture fluid after the incubation is finished, and the separation can be a separation method which is conventional in the art, such as fluid change.
In some cases, the cytokine release syndrome is a cytokine release syndrome triggered by TNF- α, IL-1 β, and IL-6.
The fifth aspect of the invention relates to a preparation method of the medicine for resisting the cytokine release syndrome, which comprises the following steps
Screening components with inhibiting effect on in-vitro cytokine storm through an in-vitro cytokine storm model; the screened effective components are used for preparing anti-cytokine release syndrome medicines.
The former paragraph refers to the existing pharmaceutical technology as one way to apply the effective components to the pharmaceutical process.
The beneficial effects of the invention are:
the in vitro cytokine storm model is obtained by treating vascular endothelial cells by combining TNF-alpha, IL-1 beta and IL-6, can be prepared into the in vitro cytokine storm model with high apoptosis rate and obviously reduced cell activity, and provides a better basis for the research of multifactorial related inflammatory reaction. The cell activity of the in vitro cytokine storm model is remarkably reduced, and the requirement of higher treatment effect of in vitro research medicines is met.
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FIG. 1 is a photograph of fluorescence photographs of the blank control treated for 24h, 48h and 72h;
FIG. 2 is a photograph of a 24h fluorescence photograph taken with the three factor treatment group;
FIG. 3 is a photograph of a 48h fluorescence photograph taken with the three factor treatment group;
FIG. 4 is a 48h fluorescence photograph of the three factor treatment group;
FIG. 5 is a graph of the rate of apoptosis in HUVEC cells at various time points using three factor (100 ng/ml each) drug treatments.
FIG. 6 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 72h;
FIG. 7 shows cell viability of HUVEC cells treated with three factors at different concentrations for 24 h;
FIG. 8 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 96 h;
FIG. 9 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 120 h;
FIG. 10 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 168h.
Detailed Description
The first aspect of this section explains the scheme of the present invention:
the in vitro cell factor storm model constructing system contains TNF-alpha factor, IL-1 beta factor, IL-6 factor and vascular endothelial cell.
The construction system is a composition preparation system of the storm model, substances in the construction system interact with each other, and the storm model can be formed through the construction system. The construction system is a prophase system in the process of preparing the storm model and is mainly used for representing the material composition for preparing the storm model. Of course, other embodiments having the same architecture of construction should also be considered as falling within the scope of the invention.
In some embodiments, the construction system further comprises a culture medium; the vascular endothelial cells are human umbilical vein endothelial cells.
In some embodiments, the medium is endothelial cell medium.
The second technical scheme provided by the invention is as follows: an in vitro cytokine storm model formed from the above bodies.
An in vitro cytokine storm model formed by a construction system of any one of the in vitro cytokine storm models. Compared with other single-factor or multi-factor models, the storm model has better simulation effect.
The third technical scheme provided by the invention is as follows: the preparation method of the in vitro cytokine storm model.
The in vitro cell factor storm model constructing process includes the incubation of vascular endothelial cell with TNF-alpha factor, IL-1 beta factor and IL-6 factor.
In some embodiments, any one of TNF-alpha, IL-1 beta, and IL-6 is present at a concentration of 100-400ng/ml; the incubation time is 24-168h.
In some embodiments, more specifically, any of TNF- α factor, IL-1 β factor, and IL-6 factor is present at a concentration of 100-200ng/ml; the incubation time is 120-168h.
In some embodiments, the concentrations of TNF-alpha factor, IL-1 beta factor, and IL-6 factor are the same; the incubation temperature was 37 ℃; the medium incubated was endothelial cell medium.
The fourth technical scheme provided by the invention is as follows: the application of the in vitro cytokine storm model described in the second technical scheme in screening anti-cytokine release syndrome drugs.
The application of the in vitro cytokine storm model in the preparation and screening of anti-cytokine release syndrome medicines.
In a specific embodiment of the invention, the vascular Endothelial cells are Human Umbilical Vein Endothelial Cells (HUVEC).
In the present invention, the Medium for incubation may be selected as is conventional in the art, and in some cases is Endothelial Cell Medium (ECM).
In a preferred embodiment of the present invention, the method further comprises a step of separating the prepared in vitro cytokine storm model from the culture fluid after the incubation is finished, and the separation can be a separation method which is conventional in the art, such as fluid change.
In some cases, the cytokine release syndrome is a cytokine release syndrome triggered by TNF- α, IL-1 β, and IL-6.
The fifth aspect of the invention relates to a preparation method of the medicine for resisting the cytokine release syndrome, which comprises the following steps
Screening components with inhibiting effect on in-vitro cytokine storm through an in-vitro cytokine storm model; the screened effective components are used for preparing anti-cytokine release syndrome medicines.
The former paragraph refers to the existing pharmaceutical technology as one way to apply the effective components to the pharmaceutical process.
The second aspect of this section is explained with some specific examples:
experimental example 1 Effect of cytokine treatment time on Molding
HUVEC (human umbilical vein endothelial cells) cells are cultured by using a Sciencell ECM culture medium, human TNF-alpha, IL-1 beta and IL-6 (which are purchased from Beijing Hokkaiyuan, wherein the recombinant human IL-1 beta protein has the product number GMP-TL513; the recombinant human IL-6 protein has the product number GMP-TL512; and the recombinant human TNF-alpha protein has the product number GMP-TL 303) are combined by using three factors, the cell factors are prepared at present, and the cell culture medium is used for diluting to 100ng/ml. The control group HUVEC cells were cultured in a normal culture medium, the three-factor treated group HUVEC cells were cultured in a three-factor culture medium, and the culture was carried out in a plate according to a conventional culture method for HUVEC cells (culture conditions 37 ℃,95% CO) 2 、5％O 2 ) Culturing for 24h, 48h and 72h after corresponding time, and detecting the apoptotic bodies by using a fluorescent staining method.
HUVEC cells were treated for 24h, 48h, 72h (i.e., the length of co-incubation) at a cytokine concentration of 100ng/ml each, while a control was set and apoptotic bodies were detected using a fluorescent staining method.
(1) Experimental setup:
A. blank control group (HUVEC cells are not treated)
B. Three factor treatment group (HUVEC + TNF-. Alpha. + IL-1. Beta. + IL-6 (100 ng/ml each))
(2) The fluorescent staining detection method comprises the following steps:
(1) preparing a stationary liquid: methanol: glacial acetic acid (3:1) was mixed and shaken well. It is used as it is.
(2) The Hoechst 33258 dye solution (Standard Reagent, P4403,1ml Hoechst 33258 dye solution was added to 99ml PBS, and stored in the dark) was stirred on a magnetic stirrer for 15min or more.
(3) The medium in the wells of the 12-well plate was discarded by pipetting and rinsed 3 times with PBS.
(4) Adding the prepared stationary liquid, and fixing for 15min.
(5) The fixative was removed by suction and a new fixative was added and fixation continued for 15min. After aspiration, the plates were allowed to air dry naturally. (completely dry).
(6) Sucking up Hoechst 33258 dye solution, 1 ml/hole. And dyeing for 30min in dark.
(7) After the absorption, pure water is added to wash away the redundant dye liquor, and the process is repeated for 3 times with gentle shaking.
(8) And (4) sucking out pure water in the holes, naturally drying, transferring the culture plate to a fluorescence inverted microscope for observation under the condition of keeping out of the sun, and photographing for counting the apoptosis rate.
(3) The experimental results are as follows:
the fluorescence photographs of the apoptotic bodies are shown in figures 1-4 (only one photograph is shown at a time point, the apoptotic bodies are circled, it is to be noted that the photograph is only shown for the appearance of the culture, the number of the cells photographed in each visual field and the number of the stained apoptotic bodies are different, and the specific apoptosis rate statistics are shown in figure 5), wherein the normal control group A has no apoptotic cells, and the three-factor treatment group B and the three-factor treatment group have apoptotic cells. The specific statistical analysis is shown in FIG. 5, and the apoptosis rate is highest 24h after the treatment. * Indicates p <0.05 compared to the blank control group and indicates p <0.001 compared to the blank control group. The apoptosis rate at different time points was decreasing, with the highest at 24h and the lowest at 72 h. Both were of differential significance compared to the blank control group, with p <0.001 for 24h and 72h, p <0.05 for 48 h; compared with 48h and 24h and 72h and 48h, the apoptosis rate has difference significance, and p is less than 0.05; p is <0.001 for 72h versus 24 h.
Since the inflammatory reaction is usually characterized in that the metabolic pathways which are affected mutually after the combined action of a plurality of cytokines cause cell death, after the three factors are treated, the cell death rate of HUVEC is increased along with the time, and the in-vitro cytokine storm model induced by the TNF-alpha + IL-1 beta + IL-6 three factors is successfully prepared in the embodiment.
Experimental example 2 Effect of cytokine concentration on Molding
HUVEC cells were treated with a combination of human TNF-alpha, IL-1 beta, and IL-6 at set concentrations of 50ng/ml, 100ng/ml, and 200ng/ml for 72h, while a control group was set and cell viability was observed.
(1) Experiment setting:
A. blank control group (HUVEC cells are not treated)
B.50ng/ml treatment group (HUVEC + TNF-. Alpha. + IL-1. Beta. + IL-6 (50 ng/ml each))
C.100ng/ml treatment group (HUVEC + TNF-. Alpha. + IL-1. Beta. + IL-6 (100 ng/ml each))
D.200ng/ml treatment group (HUVEC + TNF-. Alpha. + IL-1. Beta. + IL-6 (200 ng/ml each))
(2) CCK-8 detection of cell viability
(1) Inoculating the cells into a 96-well plate at a concentration of 100 mu l/well, culturing for the next day, treating with different concentrations of drugs, and continuously culturing for 72h;
(2) aspirate the broth and add 110. Mu.l of mixed cck-8 broth (100. Mu.l broth + 10. Mu.l CCK-8;
(3) incubating in an incubator for 2 hours;
(4) absorbance at 450nm was measured with a microplate reader.
(3) Results of the experiment
The specific results are shown in FIG. 6. The results in fig. 6 show that the three factor treated HUVEC cells at different concentrations showed decreased cell viability, and that the 50ng/ml, 100ng/ml, and 200ng/ml treated groups were significantly different from the blank control group, indicating that p <0.001 was observed in each treated group compared to the control group, and that the difference was not significant in the 50ng/ml and 100ng/ml treated groups; 200ng/ml had differential significance compared to 50ng/ml, p <0.05, 200ng/ml had differential significance compared to 100ng/ml treatment group, p <0.05.
Experimental example 3 Effect of cytokine combination treatment concentration and treatment time on Molding
HUVEC were treated with a combination of human TNF-alpha, IL-1 beta, and IL-6 at the same concentration, with different experimental groups set at 100ng/ml, 200ng/ml, and 400ng/ml, respectively, at 24h, 96h, 120h, and 168h, and with the same treatment and detection methods as in example 2. The results are shown in FIGS. 7-10. Wherein cell viability reflects cell mortality, i.e. the lower the cell viability, the higher the cell mortality.
FIG. 7 shows the cell viability at 24h of combined treatment. Compared with a blank control group, the p is less than 0.001, and the difference is very obvious; compared with a blank control group, the p of 200ng/ml is less than 0.001, and the difference is very obvious; the difference between 400ng/ml and the blank control group is not significant, the difference between 100ng/ml and the 400ng/ml treatment group is p <0.05, and the difference is significant, and the difference between 100ng/ml and 200ng/ml is not significant compared with 200ng/ml and 400 ng/ml.
FIG. 8 shows the cell viability at 96h of combined treatment. Compared with a blank control group, the difference of 100ng/ml is extremely obvious, p is less than 0.001, the difference of 200ng/ml is obvious compared with the blank control group, p is less than 0.05, and the difference of 400ng/ml is obvious compared with the blank control group, p is less than 0.05; in addition, 100ng/ml and 200ng/ml,100ng/ml and 400ng/ml, and 200ng/ml and 400ng/ml are not different in significance.
FIG. 9 shows the cell viability for 120h of the combined treatment. Compared with a blank control group, the 100ng/ml, 200ng/ml and 400ng/ml have obvious average difference, and p is less than 0.001; in addition, 100ng/ml and 200ng/ml,100ng/ml and 400ng/ml, and 200ng/ml and 400ng/ml are not different in significance.
FIG. 10 shows 168h cell viability of the combined treatment. Compared with a blank control group, the differences of 100ng/ml, 200ng/ml and 400ng/ml are very obvious, and p is less than 0.001; in addition, compared with 100ng/ml and 200ng/ml and 100ng/ml and 400ng/ml, the difference is significant, and p is less than 0.05; the difference was not significant compared to 200ng/ml and 400 ng/ml.
As can be seen from the above, the longer the three-factor incubation treatment, the cell viability increased first and then decreased rapidly, and decreased more with the increase in time. The synergistic effect of the first three factors on the increase of the endothelial cell viability can be found, and the endothelial cells are in a growth stimulation state at first along with the time, but the death rate rises after a certain time, which is similar to the clinical representation. Overall, the molding effect is best at 168h of treatment.
It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.
Claims (6)
1. The in vitro cytokine storm model construction method is characterized in that human umbilical vein endothelial cells are incubated with TNF-alpha factor, IL-1 beta factor and IL-6 factor; wherein the content of the first and second substances,
the concentration of the TNF-alpha factor, the IL-1 beta factor or the IL-6 factor is 100-400ng/ml, the concentrations of the TNF-alpha factor, the IL-1 beta factor and the IL-6 are the same, and the incubation time is 72-168h.
2. The in vitro cytokine storm model building method of claim 1, wherein the incubation time is 100-168h, the concentration of tnf-alpha factor, IL-1 beta factor or IL-6 factor is 100-200ng/ml; the medium incubated was endothelial cell medium.
3. The method for constructing an in vitro cytokine storm model according to any one of claims 1-2, wherein the incubation temperature is 37 ℃; the incubation time is 120-168h.
4. The application of the in vitro cytokine storm model in the preparation of products for screening anti-cytokine release syndrome drugs; wherein the in vitro cytokine storm model is prepared by the in vitro cytokine storm model construction method of any one of claims 1-2.
5. The use of the in vitro cytokine storm model of claim 4 in the preparation of a product for screening anti-cytokine release syndrome drugs, wherein the in vitro cytokine storm model is constructed by a system further comprising an endothelial cell culture medium.
6. The use of the in vitro cytokine storm model of claim 4 in the preparation of a product for screening anti-cytokine release syndrome drugs, characterized in that the method of use is for screening anti-cytokine storm substances.
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| CN105452861A (en) * | 2013-03-22 | 2016-03-30 | 皇家创新有限公司 | In vitro method for assessing cytokine storm responses |
| CN112656798A (en) * | 2020-12-28 | 2021-04-16 | 复旦大学附属肿瘤医院 | Application of CDK7 targeted inhibitor in preparation of drug for treating cytokine release syndrome |
| WO2021150824A1 (en) * | 2020-01-22 | 2021-07-29 | Amgen Research (Munich) Gmbh | Combinations of antibody constructs and inhibitors of cytokine release syndrome and uses thereof |
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| WO2021167324A1 (en) * | 2020-02-18 | 2021-08-26 | Cellivery Therapeutics, Inc. | Improved cell-permeable nuclear import inhibitor synthetic peptide for inhibition of cytokine storm or inflammatory disease and use thereof |
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| CN105452861A (en) * | 2013-03-22 | 2016-03-30 | 皇家创新有限公司 | In vitro method for assessing cytokine storm responses |
| WO2021150824A1 (en) * | 2020-01-22 | 2021-07-29 | Amgen Research (Munich) Gmbh | Combinations of antibody constructs and inhibitors of cytokine release syndrome and uses thereof |
| CN112656798A (en) * | 2020-12-28 | 2021-04-16 | 复旦大学附属肿瘤医院 | Application of CDK7 targeted inhibitor in preparation of drug for treating cytokine release syndrome |
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