CN114032208A - In-vitro cytokine storm model and construction method and application thereof - Google Patents

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

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CN114032208A
CN114032208A CN202111196081.3A CN202111196081A CN114032208A CN 114032208 A CN114032208 A CN 114032208A CN 202111196081 A CN202111196081 A CN 202111196081A CN 114032208 A CN114032208 A CN 114032208A
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CN114032208B (en
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胡豫
梅恒
姚惟琦
石磊
董海波
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Tongji Medical College of Huazhong University of Science and Technology
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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 multifactorial related inflammatory reaction.

Description

In-vitro cytokine storm model and construction method and application thereof
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, cytokine storms are also likely to occur with CAR-T cell therapy, and cytokine storms are also likely to occur with immune checkpoint inhibitor therapy, but at 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,. Gasderm min E-mediated target cell transfection by CAR T cells triggerers cytokine release synthesis. Sci Immunol.2020Jan 17; 5(43): eaax7969.) to simulate endogenous storms of cytokine animals, so that the concentration of cytokines is not enough to be confirmed, and the concentration of factors secreted after each tumor cell killing is not stable, so the animals are also not stable.
While in vitro cytokine storm models have been reported:
(1) blouzhen Kai et al (H)2O2Experimental study on LPS and TNF-alpha induced injury of human umbilical vein endothelial cells) using hydrogen peroxide (H) at different concentrations2O2) 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 H2O2LPS 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 pair in a certain concentration rangeThe degree of human umbilical vein endothelial cell damage is concentration and time dependent.
(2) Speciale, A et al (Silibinin as a functional tool against SARS-Cov-2: In silico spike receptor-binding domain and main protease binding analysis, and In vitro endogenous protective effects. Phytoyopathy research. 2021; 35: 4616. sup. 4625.) examined the effect of Silybin In vitro on cytokine (TNF-. alpha.) induced inflammation and dysfunction of Human Umbilical Vein Endothelial Cells (HUVECs).
(3) Chen, Tieling et al (Quercetin inhibitors TNF- α induced HUVECs apoptosis and inflammation of visual descending NF-kB and AP-1signaling pathway in vitro, Medicine: September 18,2020-Volume 99-Issue 38-p e22241) reported that Quercetin inhibits TNF-a induced HUVECs apoptosis and inflammatory response by down-regulating NF-kB and AP-1signaling pathways.
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 the TNF- α factor, IL-1 β factor, and IL-6 factor is present at a concentration of 100 ng/ml; the incubation time was 24-168 h.
In some embodiments, more specifically, any one of the TNF- α factor, IL-1 β factor, and IL-6 factor is present at a concentration of 100 ng/ml; the incubation time was 120-168 h.
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 the 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 invention has the beneficial effects that:
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 72 h;
FIG. 2 is a photograph of a fluorograph taken 24h after treatment with the three-factor treatment group;
FIG. 3 is a photograph of a fluorograph taken 48h after the three-factor treatment group treatment;
FIG. 4 is a photograph of a fluorograph taken 48h after treatment with 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 72 h;
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 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 168 h.
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.
The in vitro cytokine storm model is 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 of the TNF- α factor, IL-1 β factor, and IL-6 factor is present at a concentration of 100 ng/ml; the incubation time was 24-168 h.
In some embodiments, more specifically, any one of the TNF- α factor, IL-1 β factor, and IL-6 factor is present at a concentration of 100 ng/ml; the incubation time was 120-168 h.
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 the 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-TL 513; the recombinant human IL-6 protein has the product number GMP-TL 512; and the recombinant human TNF-alpha protein has the product number GMP-TL303) are combined by using three factors, the cell factors are prepared at present, and the cell culture medium is used for diluting to 100 ng/ml. Adding a normal culture medium into the control group HUVEC cells, adding a three-factor culture medium into the three-factor treatment group HUVEC cells for culture, and culturing in a culture plate according to a conventional culture mode of the HUVEC cells (the culture condition is 37 ℃, and the culture flow is 95% CO)2、5％O2) 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:
firstly, preparing a stationary liquid: methanol: mixing glacial acetic acid (3:1), and shaking. It is used as it is.
② the Hoechst 33258 dye liquor (Standard Reagent, P4403, 1ml of Hoechst 33258 dye liquor is added into 99ml of PBS and kept away from light) is placed on a magnetic stirrer to be stirred for more than 15 min.
And thirdly, absorbing and discarding the culture medium in the hole of the 12-hole plate culture plate, and adding PBS for rinsing for 3 times.
Fourthly, adding the prepared stationary liquid and fixing for 15 min.
Fifthly, absorbing and removing the fixative, adding new fixative and continuing to fix for 15 min. After being discarded, the plates were naturally dried. (completely dry).
Sixthly, sucking 1 ml/hole of Hoechst 33258 dye solution. And dyeing for 30min in dark.
Seventhly, adding pure water to wash away redundant dye liquor after the dye liquor is sucked and discarded, and slightly shaking and repeating for 3 times.
Sucking the 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 carrying out apoptosis rate statistics.
(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. Denotes p <0.05 compared to the blank control group, and denotes 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) Experimental setup:
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
Inoculating cells into a 96-well plate at a concentration of 100 mu l/well, treating the cells with drugs with different concentrations the next day of culture, and continuously culturing for 72 hours;
② sucking out the culture solution, adding 110 mul of mixed cck-8 culture solution (100 mul of culture solution +10 mul of CCK-8;
thirdly, incubating for 2 hours in an incubator;
and fourthly, measuring the absorbance at 450nm by using a microplate reader.
(3) Results of the experiment
The specific results are shown in FIG. 6. Figure 6 results show that the cell viability was decreased in the three-factor treated HUVEC cells at different concentrations, and that the 50ng/ml, 100ng/ml, 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 was differentially significant compared to 50ng/ml, p <0.05, 200ng/ml was differentially significant 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 more the cell viability increased and then decreased rapidly, and the more the cell viability decreased with the increase in time. The synergistic effect of the first three factors on promoting the increase of the endothelial cell viability can be found, and the endothelial cells are in a growth stimulating state at first along with the time, but the death rate is increased by a certain time, which is similar to the clinical characteristics. 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 (10)

1. The in vitro cytokine storm model construction system is characterized by comprising TNF-alpha factor, IL-1 beta factor, IL-6 factor and vascular endothelial cells.
2. The system for constructing an in vitro cytokine storm model according to claim 1, further comprising a culture medium; the vascular endothelial cells are human umbilical vein endothelial cells.
3. The system for constructing an in vitro cytokine storm model according to claim 2, wherein the culture medium is an endothelial cell culture medium.
4. The in vitro cytokine storm model construction method is characterized in that vascular endothelial cells are incubated with TNF-alpha factor, IL-1 beta factor and IL-6 factor together.
5. The in vitro cytokine storm model construction method of claim 4, wherein any one of the concentrations of TNF- α factor, IL-1 β factor and IL-6 factor is 400 ng/ml; the incubation time was 24-168 h.
6. The in vitro cytokine storm model construction method of claim 5, wherein any one of the concentrations of TNF- α factor, IL-1 β factor and IL-6 factor is 100-200 ng/ml; the incubation time was 120-168 h.
7. The method for constructing an in vitro cytokine storm model according to any one of claims 4-6, wherein the concentrations of TNF- α factor, IL-1 β factor and IL-6 factor are the same; the incubation temperature was 37 ℃; the medium incubated was endothelial cell medium.
8. An in vitro cytokine storm model, characterized by being formed by the construction system of the in vitro cytokine storm model according to any one of claims 1 to 3 or prepared by the construction method of the in vitro cytokine storm model according to any one of claims 4 to 6.
9. Use of the in vitro cytokine storm model of claim 8 for the preparation of a medicament for screening for anti-cytokine release syndrome.
10. Use of the in vitro cytokine storm model of claim 8 for the screening of anti-cytokine release syndrome drugs, characterized by the fact that it is used for screening anti-cytokine storm agents.
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