Method for simulating cytokine storm caused by septicopyemia and determining specific cytokines

Abstract

The invention belongs to the field of micro-fluidic chips, and discloses a method capable of simulating cytokine storm caused by septicopyemia and determining specific cytokines. The human primary umbilical vein endothelial cell experiment is adopted, the natural characteristics of endothelial cells are reserved, and the kit is sensitive to various inflammatory factors, drugs and stimulus reactions, is closer to the real in-vivo environment, is widely applied to drug screening, therapeutic effect research and biological agent testing, and provides data with more biological significance for septicopyemia diseases. According to the method capable of simulating the cytokine storm model caused by septicopyemia, key factor screening scientificity is high, concentration setting conforms to the pathological level, and the authenticity is enhanced through the LPS synergistic effect. According to the method for specifically removing the cytokines, a specific removal strategy which is difficult to realize clinically is simulated, and the technical problem that various inflammatory factors in the body of a sepsis patient exist at the same time and interact with one another, and respective functional contributions are difficult to distinguish is solved.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic chips and relates to a method for simulating a cytokine storm caused by sepsis and determining specific cytokines. Background Art

[0002] Sepsis, a life-threatening systemic disease triggered by an extreme host response to infection, remains a major challenge for modern medicine. Its clinical diagnosis is primarily based on the Sequential Organ Failure Assessment (SOFA) score, with a confirmed diagnosis when the patient has a SOFA score ≥2 and clear evidence of infection. In the early stages of infection, a moderate immune response helps eliminate pathogens and promote tissue repair. However, if damaging factors persist or become excessive, the local inflammatory response cannot maintain homeostasis, leading to overactivation of immune cells and the release of large amounts of inflammatory mediators, creating a positive feedback loop that ultimately triggers uncontrolled systemic inflammation, a phenomenon known as the cytokine storm. This storm, by increasing vascular permeability and damaging endothelial cells, further releases damage-associated molecular patterns (DAMPs), exacerbating the inflammatory response, thus creating a vicious cycle between sepsis and the cytokine storm.

[0003] Currently, because the key effectors of cytokine storm and their specific mechanisms of action in organ damage have not been fully elucidated, clinical treatment still mostly relies on nonspecific broad-spectrum blood purification techniques. However, such methods may also deplete beneficial molecules (such as albumin and coagulation factors) due to non-selective removal, which not only affects the treatment effect but may also have an adverse effect on patient prognosis. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention aims to provide an in vitro model and method for simulating the cytokine storm produced by sepsis, and to identify the key cytokines that play a major role in it through targeted removal. This approach is expected to not only break this pathological cycle and improve clinical outcomes, but also significantly reduce medical costs, representing an important development direction for the future treatment of sepsis.

[0005] The technical solution of the present invention:

[0006] 1. A method for simulating a cytokine storm induced by sepsis and determining specific cytokines, comprising the following steps:

[0007] The first step is to build a biomimetic microfluidic vascular chip;

[0008] The biomimetic microfluidic vascular chip is divided into three parts, including an upper chip layer, a middle chip layer, and a lower substrate layer. The upper chip layer, the middle chip layer, and the lower substrate layer are sealed using plasma technology.

[0009] The specific structure of the bionic microfluidic vascular chip is as follows:

[0010] The upper chip layer and the middle chip layer are made of PDMS material, and the lower substrate layer is made of a transparent carrier film;

[0011] The upper chip layer measures 25mm*25mm*4mm and has two elliptical through-holes. Each elliptical through-hole has a major axis of 15mm and a minor axis of 6mm. Culture medium is placed in the elliptical through-holes to provide the nutrients needed for cell culture.

[0012] The middle chip layer measures 25mm*25mm*5mm and contains three parallel curved channels. Each channel is 12mm long, 400um wide, and 200um high. Both ends of each channel are open. These channels are used to culture primary human umbilical vein endothelial cells and subsequently add inflammatory factors prepared in culture medium. The concentration settings are based on clinical test results.

[0013] The size of the lower substrate layer is 25mm*75mm*1mm.

[0014] The biomimetic microfluidic vascular chip uses a shaker to provide fluid shear force; fluid simulation shows that the fluid shear force in the chip channel is 2-5 dyne / cm 2 The shear force exerted by the blood flow in the veins on the endothelial cells is about 1-6 dyne / cm 2 Close, with high biomimetic properties.

[0015] Primary human umbilical vein endothelial cells were seeded onto the curved channels of the middle chip layer and cultured for seven days;

[0016] In the second step, an in vitro method was performed to simulate the cytokine storm produced by sepsis patients;

[0017] Normal culture medium ECM was set as the control group;

[0018] A serum group of sepsis patients was set up. Fresh serum samples from sepsis patients were collected and allowed to stand at room temperature until the blood clots shrank and coagulated completely, and the upper serum layer turned light yellow. The upper serum layer was transferred to a sterile centrifuge tube and centrifuged at 4°C for 10 minutes. The upper liquid was again aspirated into a new centrifuge tube and centrifuged a second time.

[0019] Cytokine combined detection was performed on serum samples after secondary centrifugation, and the detection results of seven cytokines were obtained, including IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A;

[0020] A combined stimulation group was set up. Recombinant human cytokines were added to the normal ECM culture medium according to the specific values of sepsis patients detected clinically, and lipopolysaccharide (LPS) was supplemented to simulate the effects of residual inflammatory factors produced by the signal cascade effect triggered by Gram-negative bacterial infection. The modeling was successful when the fluorescence intensity of the combined stimulation group was consistent with that of the sepsis patient serum group.

[0021] The concentration of lipopolysaccharide (LPS) was determined by analyzing the fluorescence intensity of ZO-1 on tight junctions of endothelial cells.

[0022] The third step is to further identify the cytokines with the strongest damaging effects;

[0023] Normal culture medium ECM was set as the control group;

[0024] A combined stimulation group was set up with 1 μg / mL LPS combined with a cytokine combination containing the cytokines determined in the second step;

[0025] Based on the established 1μg / mL LPS combined with cytokine stimulation protocol, an experimental verification system was established using a strategy of targeted elimination of target factors:

[0026] Experimental group design:

[0027] Experimental group I: cytokine A was targetedly deleted, while LPS and other factors except A were retained;

[0028] Experimental group II: targeted deletion of cytokine B; LPS and other factors except B were retained;

[0029] Experimental group III: targeted deletion of cytokine C; LPS and other factors except C were retained;

[0030] By analogy, experimental groups were set up in sequence according to the cytokines determined in the second step;

[0031] After 24 hours, the fluorescence intensity of ZO-1 in endothelial cell tight junctions was analyzed.

[0032] Beneficial effects of the present invention:

[0033] (1) The present invention uses primary human umbilical vein endothelial cells for experiments, which fully retains the natural characteristics of endothelial cells and is sensitive to a variety of inflammatory factors, drugs and stimuli. It is closer to the real environment in the body and can be widely used in drug screening, treatment effect research and testing of biological agents. It can provide more biologically meaningful data for sepsis and improve the reliability of medical research.

[0034] (2) Fluid shear force (2-5 dyne / cm 2 ) and the actual shear stress range of human veins (1-6dyne / cm 2 ), demonstrating that the chip accurately reproduces venous hemodynamic characteristics. It can essentially simulate fluid flow in venous vessels, creating a highly realistic physiological environment simulation that can be used to study the effects of shear stress on endothelial cells, overcoming the limitation of traditional models that cannot measure fluid flow.

[0035] (3) The present invention has developed a method for simulating a cytokine storm model caused by sepsis. The screening of key factors is scientific, the concentration setting is consistent with the pathological level, and the synergistic effect of LPS enhances authenticity. It is a precise modeling based on clinical data and a quality control method verified by fluorescence intensity. It achieves high-fidelity reproduction of the inflammatory environment of sepsis, has scientificity, controllability and translational potential, and provides a reliable tool for the study of the pathogenesis of sepsis and the development of treatment.

[0036] (4) The present invention provides a method for specific cytokine removal, simulating a specific removal strategy that is difficult to achieve clinically. This method addresses the technical difficulty of distinguishing the functional contributions of multiple inflammatory factors that coexist and interact with each other in sepsis patients. By removing specific factors one by one, the independent impact of each cytokine on endothelial cell damage, inflammatory response, etc. can be clarified, providing a basis for targeted therapy and promoting the development of sepsis treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the structure of a highly biomimetic microfluidic vascular chip; in the figure: 1 is the upper chip, 1a and 1b are reservoirs for culture medium, 2 is the middle chip, 2a is the human primary umbilical vein endothelial cell culture channel, 2b is the perforated area, and 3 is the lower substrate.

[0038] Figure 2These are bright field images of the human primary umbilical vein endothelial cells after 7 days of flow culture in Example 1; wherein, (a) is a bright field image of the cells just after flow culture until confluence, (b) is a bright field image of the cells after flow culture until confluence on the first day, (c) is a bright field image of the cells after flow culture until confluence on the second day, (d) is a bright field image of the cells after flow culture until confluence on the third day, (e) is a bright field image of the cells after flow culture until confluence on the fourth day, (f) is a bright field image of the cells after flow culture until confluence on the fifth day, (g) is a bright field image of the cells after flow culture until confluence on the sixth day, and (h) is a bright field image of the cells after flow culture until confluence on the seventh day.

[0039] Figure 3 Characterization of tight junctions in primary human umbilical vein endothelial cells in Example 1; wherein (a) shows the expression of tight junction protein ZO-1 in vascular endothelial cells, (b) shows the expression of DAPI in the nuclei of vascular endothelial cells, and (c) shows the Merge diagram of vascular endothelial cells;

[0040] Figure 4 The results of the LPS concentration screening in Example 2 are shown in FIG. (a) is a fluorescence image showing the damaging effects of different concentrations of LPS combined with inflammatory factors on ZO-1 of endothelial cells, and (b) is the fluorescence intensity value showing the damaging effects of different concentrations of LPS combined with inflammatory factors on ZO-1 of endothelial cells.

[0041] Figure 5 Evaluation of the effect of removing inflammatory factors in Example 3; wherein (a) is a fluorescence image showing the damage effect of inflammatory factors on ZO-1 of endothelial cells, and (b) is the fluorescence intensity value showing the damage effect of inflammatory factors on ZO-1 of endothelial cells. DETAILED DESCRIPTION

[0042] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0043] Example 1: Construction of a highly biomimetic microfluidic vascular chip

[0044] S1. Prepare a microfluidic chip template, then pour PDMS prepolymer and curing agent on the template at a mass ratio of 9:1. After curing, cut the chip according to the shape to obtain the following: Figure 1 Chip 1 and Chip 2 are shown. For Chip 1, 1a and 1b were cut using a custom blade. For Chip 2, a 1.5mm punch was used to punch holes in the circular area 2b. Layers 1, 2, and 3 were then plasma-treated and sealed together to create the vascular chip model.

[0045] S2. Pre-treat the assembled chip by sterilizing it under high temperature and high pressure, drying it in an 80°C oven, and then transferring it to a clean bench. Add 100 μg / ml of poly-lysine to each channel to enhance the attachment of primary cells to the glass slide through the interaction of positive charges with negative cell membrane charges. Place the chip in an 80°C oven to dry, then rinse the channels three times with sterile water. Then, add Matrigel coating solution to the channels and incubate at 37°C for at least 2 hours before seeding the cells.

[0046] S2, in the channel of the chip with 4×10 6 The core was inoculated at a density of 1000 cells / ml, and ECM culture medium was added to the elliptical reservoir. After 6 hours of inoculation, the chip was placed on a shaker for liquid flow.

[0047] S3. The chip was cultured on a shaker, the medium was changed every day, and dynamic morphology was monitored by microscopy. Images were collected at the time points of the cells just forming a continuous monolayer and from 1 to 7 days of continuous fluid stimulation. Three repeated shots were taken at a fixed observation point on the chip, and image stitching and morphological parameter analysis were performed. The results are shown in Figure 3. Figure 2 As shown in Figure 2, after the application of fluid shear force, cell morphology undergoes significant dynamic changes. From days 1 to 3 of confluence, cells gradually elongate along the flow field direction. After day 7 of confluence, more than 80% of cells exhibit significant orientation.

[0048] S4. Expression of tight junction ZO-1 in vascular endothelial cells after seven days of growth was performed. Figure 3 As shown, ZO-1 protein exhibits a continuous, clear linear distribution pattern at the junctions between endothelial cells, with clearly visible cell boundaries. This indicates that by day 7 of culture, endothelial cells have established a complete tight junction network, forming a vascular endothelial barrier structure with typical morphological and functional characteristics, providing a reliable in vitro model for subsequent inflammatory factor injury experiments.

[0049] Example 2: A method for simulating cytokine storm in patients with sepsis in vitro

[0050] S1. Blood samples come from the hospital. Informed consent was obtained before collection, and the samples were anonymized. The hospital ethics committee approved the plan. Samples of the acute phase of sepsis (within 24 hours after diagnosis) were obtained from the hospital using disposable vacuum blood collection tubes. The samples were transported to the laboratory within 1 hour using an ice box, then sprayed with alcohol on the surface, transferred to a clean bench, and left to stand at room temperature for 30 minutes. After the blood clots shrank and coagulated completely, the upper liquid became light yellow. The upper serum was transferred to a sterile centrifuge tube and centrifuged at 1000g at 4°C for 10 minutes. The upper liquid was aspirated again into a new centrifuge tube and centrifuged a second time. After centrifugation, it was stored at -20°C for a short period of time and at -80°C for a long period of time. The serum was also tested for cytokines (7 joint tests) to obtain specific values of the 7 cytokines.

[0051] S2. Quantitatively analyze the peripheral blood cytokine profile of patients in the acute phase of sepsis using a combined cytokine assay to screen for key inflammatory mediators. The results confirm that IL-6, TNF-α, and IL-10 contribute 99.5% of the total inflammatory mediators.

[0052] S3. Set up a control group, a patient serum group, and a combined stimulation group. The combined stimulation group added recombinant human cytokines (950 pg / ml IL-6, 2 pg / mL TNF-α, and 15 pg / mL IL-10) based on clinically tested values to ECM basal culture medium, and supplemented with LPS to simulate the effects of residual inflammatory factors produced by the signal cascade triggered by Gram-negative bacterial infection. The concentration of LPS requires further research. Modeling was successful when the ZO-1 fluorescence intensity in the combined stimulation group was essentially consistent with that in the patient serum group.

[0053] S4. The study used a combination stimulation strategy of LPS (100 ng / mL, 1 μg / mL, 10 μg / mL) combined with the above three cytokines, and verified the model by analyzing the ZO-1 fluorescence intensity of endothelial cells and observing cell morphology. Figure 4 The ZO-1 fluorescence intensity in the 10 μg / mL LPS co-stimulation group was lower than that in the patient serum group, accompanied by increased cell shedding, indicating an excessive injury effect. However, the ZO-1 fluorescence intensity in the 100 ng / mL LPS co-stimulation group was higher than that in the patient serum group, failing to effectively simulate the degree of injury in clinical samples. The ZO-1 fluorescence intensity in the 1 μg / mL LPS co-stimulation group was essentially consistent with that in the patient serum group. Based on these analyses, the 1 μg / mL LPS combined with the three-factor stimulation regimen was ultimately determined to be an effective in vitro surrogate model for the effects of sepsis serum.

[0054] Example 3: Method for further determining the most damaging cytokine

[0055] S1. For the established 1 μg / mL LPS combined with three factors in vitro sepsis pathological model in Example 2, a IL-6 / TNF-α / IL-10 targeted depletion model was further constructed.

[0056] S2. Set up control groups, the established 1 μg / mL LPS combined with three-factor in vitro sepsis pathological model in Example 2 (1 μg / mL LPS + 950 pg / ml IL-6 + 2 pg / mL TNF-α + 15 pg / mL IL-10), the targeted IL-6 removal group (1 μg / mL LPS + 2 pg / mL TNF-α + 15 pg / mL IL-10), the targeted TNF-α removal group (1 μg / mL LPS + 950 pg / ml IL-6 + 15 pg / mL IL-10), and the targeted IL-10 removal group (1 μg / mL LPS + 950 pg / ml IL-6 + 2 pg / mL TNF-α)

[0057] S3. In the above step S2, the final result is characterized by the fluorescence intensity of ZO-1. The result is as follows Figure 5 As shown, the fluorescence intensity of the IL-6 depletion group was significantly higher than that of the in vitro sepsis model, increasing by 10.8%. However, there was no significant difference between the TNF and IL-10 depletion groups and the in vitro sepsis model. This preliminarily suggests that IL-6 plays a core regulatory role in the pathological process of sepsis and that its targeted depletion can significantly alleviate endothelial damage, while the individual depletion of TNF-α and IL-10 did not produce a significant protective effect.

[0058] In summary, the present invention, based on a highly biomimetic microfluidic vascular chip, has developed an in vitro method for simulating the cytokine storm produced by sepsis patients and a method for specifically clearing cytokines. This preliminary demonstration of the key role of IL-6 in the cytokine storm induced by sepsis provides a basis for achieving specific clearance in clinical practice.

[0059] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A method for simulating a cytokine storm caused by sepsis and determining specific cytokines, characterized in that: Here are the steps: The first step is to build a biomimetic microfluidic vascular chip; The biomimetic microfluidic vascular chip is divided into three parts, including an upper chip layer, a middle chip layer, and a lower substrate layer. The upper chip layer, the middle chip layer, and the lower substrate layer are sealed using plasma technology. The biomimetic microfluidic vascular chip uses a shaker to provide fluid shear force; Primary human umbilical vein endothelial cells were seeded onto the curved channels of the middle chip layer and cultured for seven days; In the second step, an in vitro method was performed to simulate the cytokine storm produced by sepsis patients; Normal culture medium ECM was set as the control group; A serum group of sepsis patients was set up. Fresh serum samples from sepsis patients were collected and allowed to stand at room temperature until the blood clots shrank and coagulated completely, and the upper serum layer turned light yellow. The upper serum layer was transferred to a sterile centrifuge tube and centrifuged at 4°C for 10 minutes. The upper liquid was again aspirated into a new centrifuge tube and centrifuged a second time. Cytokine combined detection was performed on serum samples after secondary centrifugation, and the detection results of seven cytokines were obtained, including IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A; A combined stimulation group was set up. Recombinant human cytokines were added to the normal culture medium (ECM) according to the specific values of sepsis patients detected clinically, and lipopolysaccharide (LPS) was supplemented to simulate the effects of the remaining inflammatory factors produced by the signal cascade effect triggered by Gram-negative bacterial infection. The modeling was successful when the fluorescence intensity of the combined stimulation group was consistent with that of the sepsis patient serum group. The concentration of lipopolysaccharide (LPS) was determined by analyzing the fluorescence intensity of ZO-1 on tight junctions of endothelial cells. The third step is to further identify the cytokines with the strongest damaging effects; Normal culture medium ECM was set as the control group; A combined stimulation group was set up with 1 μg / mL LPS combined with a cytokine combination containing the cytokines determined in the second step; Based on the established 1μg / mL LPS combined with cytokine stimulation protocol, an experimental verification system was established using a strategy of targeted elimination of target factors: Experimental group design: Experimental group I: cytokine A was targetedly deleted, while LPS and other factors except A were retained; Experimental group II: targeted deletion of cytokine B; LPS and other factors except B were retained; Experimental group III: targeted deletion of cytokine C; LPS and other factors except C were retained; By analogy, experimental groups were set up in sequence according to the cytokines determined in the second step; After 24 hours, the fluorescence intensity of ZO-1 in endothelial cell tight junctions was analyzed.

2. The method for simulating cytokine storm caused by sepsis and determining specific cytokines according to claim 1, characterized in that: The specific structure of the bionic microfluidic vascular chip is as follows: The upper chip layer and the middle chip layer are made of PDMS material, and the lower substrate layer is made of a transparent carrier film; The upper chip layer measures 25mm*25mm*4mm and has two elliptical through-holes. Each elliptical through-hole has a major axis of 15mm and a minor axis of 6mm. Culture medium is placed in the elliptical through-holes to provide the nutrients needed for cell culture. The middle chip layer measures 25mm*25mm*5mm and contains three parallel curved channels. Each channel is 12mm long, 400um wide, and 200um high. Both ends of each channel are open. These channels are used to culture primary human umbilical vein endothelial cells and subsequently add inflammatory factors prepared in culture medium. The concentration settings are based on clinical test results. The size of the lower substrate layer is 25mm*75mm*1mm.

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