Method for establishing a cell model of acute lung injury caused by spike protein in COVID-19 and its application

Abstract

The present invention discloses a method for establishing a cell model of acute lung injury induced by a spike protein, and an application thereof, comprising the following steps: S10, obtaining human lung macrophages, human alveolar epithelial cells and SARS-CoV-2 recombinant spike protein; S20, co-culturing human lung macrophages and human alveolar epithelial cells for a first preset time to obtain a co-culture system; S30, adding the spike protein to the co-culture system and co-culturing for a second preset time to obtain a cell model of acute lung injury of COVID-19. The co-culture system can simulate a more realistic lung structure and pathological characteristics of acute lung injury. In addition, the use of non-infectious spike protein to replace the infectious SARS-CoV-2 live virus does not require the use of a biosafety level 3 (BSL-3) laboratory, and can induce significant pathophysiological changes of viral pneumonia and acute lung injury while avoiding infection of experimental personnel. This modeling method meets the ethical desire to reduce the use of experimental animals, has low experimental conditions, short cycle, and low price, and is suitable for popularization and application in the screening of therapeutic drugs for acute lung injury of COVID-19.

Description

Technical Field

[0001] The present invention relates to the field of cell model technology, and in particular to a method for establishing a cell model of COVID-19 acute lung injury induced by spike protein and the technical field of its application. Background Art

[0002] In recent years, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute lung injury / acute respiratory distress syndrome (ARDS) is the main cause of death in severe COVID-19, and its pathophysiological basis and pathogenesis still have a lot of unsolved problems. Compared with the typical ARDS in the traditional sense, diffuse alveolar damage associated with "inflammatory cytokine storm" is the most distinctive feature of COVID-19 acute lung injury / ARDS, and there is currently a lack of corresponding effective treatment methods in clinical practice. Therefore, it is of practical urgency to establish a suitable disease model, explore its pathogenesis from a new perspective, and find new effective therapeutic targets.

[0003] Animal models of host-pathogen interactions are essential for understanding the pathogenesis of COVID-19 ALI / ARDS and evaluating the efficacy of treatments. Appropriate animal models produce the desired disease features with clinical symptoms that closely resemble those observed in humans. Experimental studies have shown that mice, hamsters, cats, ferrets, and non-human primates can be infected with SARS-CoV-2 and may develop ALI / ARDS. Human angiotensin-converting enzyme 2 (hACE2) transgenic mice and hamsters are small animal models that are easily available, but translating knowledge from rodent studies to clinical applications can be challenging. Ferrets are excellent models for virus transmission, pathogenesis, and testing of therapeutic countermeasures, but their availability in some countries can be an issue and require special housing and breeding facilities. Cats are the natural reservoir of SARS-CoV-2 and can transmit the virus well but do not show typical disease symptoms. Non-human primates constitute the model that is genetically closest to humans, however, such models are expensive and time-consuming to establish. In general, animal models of COVID-19 acute lung injury / ARDS induced by live virus infection have the following limitations: the experiments may be difficult and dangerous, must be conducted in a biosafety level 3 (BSL-3) laboratory, have complex experimental technical conditions, relatively long experimental cycles, are difficult to repeat, are expensive, and are difficult to apply on a large scale. Summary of the invention

[0004] The main purpose of the present invention is to propose a method for establishing a cell model of COVID-19 acute lung injury induced by spike protein and its application, aiming to solve the problems in the prior art that model establishment is difficult and dangerous, must be carried out in a biosafety level 3 (BSL-3) laboratory, has complex experimental technical conditions, long experimental cycle and high cost.

[0005] To achieve the above objectives, the present invention proposes a method for establishing a cell model of acute lung injury induced by spike protein in COVID-19, comprising the following steps:

[0006] S10, obtain human lung macrophages, human alveolar epithelial cells and SARS-CoV-2 recombinant spike protein;

[0007] S20, co-culturing the human lung macrophages and the human alveolar epithelial cells for a first preset time to obtain a co-culture system;

[0008] S30. Add the SARS-CoV-2 recombinant spike protein to the co-culture system and co-culture for a second preset time to obtain a COVID-19 acute lung injury cell model.

[0009] Optionally, in step S20, the cell number ratio of human lung macrophages to human alveolar epithelial cells is 1:(2-8).

[0010] Optionally, in step S20, the first preset time is 12 to 48 hours.

[0011] Optionally, in step S30, the mass concentration of the spike protein in the co-culture system is 5-15 μg / mL.

[0012] Optionally, in step S30, the second preset time is 3 to 5 days.

[0013] The present invention also proposes the application of the cell model established by the method in the screening of therapeutic drugs for COVID-19 acute lung injury.

[0014] The present invention proposes a method for establishing a cell model of acute lung injury induced by spike protein in COVID-19, wherein human lung macrophages (AM) and human alveolar epithelial cells (HPAEpic) are co-cultured and then exposed to spike protein to induce inflammatory response and oxidative stress, wherein human lung macrophages (AM) and human alveolar epithelial cells (HPAEpic) are the two most important cells that directly contact SARS-CoV-2 in actual human COVID-19 pneumonia, and through virus-host and cell-cell interactions, while clearing the virus, excessive inflammatory response, i.e., "inflammatory factor storm", and oxidative stress may induce acute lung injury. Co-culture of human lung macrophages (AM) and human alveolar epithelial cells (HPAEpic) can maintain the immune homeostasis of the cell model, and the two can interact with each other, so as to simulate a more realistic lung structure and acute lung injury pathological characteristics. In addition, the SARS-CoV-2 recombinant spike protein, also known as the spike protein, is a non-infectious viral protein. It can induce obvious pathophysiological changes of viral pneumonia and acute lung injury while avoiding infection of experimental personnel. Therefore, it avoids the risk of transmission of live SARS-CoV-2 virus, allowing the cell model of COVID-19 acute lung injury to be established in an ordinary laboratory without the need for laboratory conditions with a higher level of safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. It is worth noting that the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the contents shown in these drawings without paying any creative work.

[0016] Figure 1 A schematic diagram of a method for establishing a cell model of acute lung injury induced by spike protein in COVID-19 provided in one embodiment of the present invention;

[0017] Figure 2 This is a diagram for modeling and verification of a cell model of acute lung injury induced by spike protein in COVID-19 provided in one embodiment of the present invention.

[0018] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0019] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0020] In recent years, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute lung injury / acute respiratory distress syndrome (ARDS) is the main cause of death in severe COVID-19, and its pathophysiological basis and pathogenesis still have a lot of unsolved problems. Compared with the typical ARDS in the traditional sense, diffuse alveolar damage associated with "inflammatory cytokine storm" is the most distinctive feature of COVID-19 acute lung injury / ARDS, and there is currently a lack of corresponding effective treatment methods in clinical practice. Therefore, it is of practical urgency to establish a suitable disease model, explore its pathogenesis from a new perspective, and find new effective therapeutic targets.

[0021] Animal models of host-pathogen interactions are essential for understanding the pathogenesis of COVID-19 acute lung injury / ARDS and evaluating the efficacy of treatments. Appropriate animal models produce the desired disease features with clinical symptoms that closely resemble those observed in humans. Experimental studies have shown that mice, hamsters, cats, ferrets, and non-human primates can be infected with SARS-CoV-2 and may develop acute lung injury / ARDS. Human angiotensin-converting enzyme 2 (hACE2) transgenic mice and hamsters are small animal models that are easily available, but translating knowledge from rodent studies to clinical applications can be challenging. Ferrets are excellent models for virus transmission, pathogenesis, and testing of therapeutic countermeasures, but their availability in some countries can be an issue and require special housing and breeding facilities. Cats are the natural reservoir of SARS-CoV-2 and can transmit the virus well but do not show typical disease symptoms. Non-human primates constitute the model that is genetically closest to humans, however, such models are expensive and time-consuming to establish. In general, animal models of COVID-19 acute lung injury / ARDS induced by live virus infection have the following limitations: the experiments may be difficult and dangerous and must be conducted in a biosafety level 3 (BSL-3) laboratory; the experimental technical conditions are complex; the experimental cycle is relatively long and difficult to repeat; and it is expensive and difficult to apply on a large scale.

[0022] In vitro models include cell models, explants, and organoids based on primary or immortalized cell cultures. They meet the ethical desire to reduce the use of animal models and allow the study of specific cell targets, which is not possible in in vivo macroscopic systems. Explants derived from patient organs (ex vivo models) or organoids generated from induced stem cells have the advantage of retaining tissue structure and complexity, and the results obtained from tissues are conducive to describing their possible response to SARS-CoV-2 infection and finding new therapeutic targets. However, the disadvantages are technical difficulties and ethical restrictions. Human airway epithelial primary cells are isolated from human lung donors or individuals who have received lung transplants, with differentiated ciliated cells, goblet cells, and basal cells, and maintain the highest similarity to the physiological conditions of human airway epithelium in vivo. The advantages of using primary cell models are mainly more realistic virus-host interactions, but models based on monolayer cultures of immortalized cell lines are easier to use, less time-consuming, inexpensive, easily reproducible, and bypass the ethical issues associated with the use of animals and human tissues. However, the cell models constructed with single immortalized cell lines (Vero E6, Huh-7, A549-ACE2, etc.) have relatively simple functions and cannot fully simulate the complex process of acute lung injury in COVID-19.

[0023] in, Figure 1The flowchart of the preparation method of one embodiment of the present invention is as follows. To achieve the above-mentioned purpose, the present invention proposes a method for establishing a cell model of acute lung injury induced by spike protein in COVID-19, comprising the following steps:

[0024] S10, obtain human lung macrophages, human alveolar epithelial cells and SARS-CoV-2 recombinant spike protein;

[0025] S20, co-culturing the human lung macrophages and the human alveolar epithelial cells for a first preset time to obtain a co-culture system;

[0026] S30. Add the SARS-CoV-2 recombinant spike protein to the co-culture system and co-culture for a second preset time to obtain a cell model of COVID-19 acute lung injury induced by the spike protein.

[0027] In the technical scheme of the present invention, human lung macrophages (AM) and human alveolar epithelial cells (HPAEpic) are co-cultured and then exposed to spike protein to induce inflammatory response and oxidative stress, wherein human lung macrophages (AM) and human alveolar epithelial cells (HPAEpic) are the two most important cells that are in direct contact with SARS-CoV-2 in actual human COVID-19 pneumonia. Through virus-host and cell-cell interactions, while clearing the virus, excessive inflammatory response, i.e., "inflammatory factor storm", and oxidative stress may induce acute lung injury. The co-culture of human lung macrophages (AM) and human alveolar epithelial cells (HPAEpic) can maintain the immune homeostasis of the cell model, and the two can interact with each other to simulate more realistic lung structures and pathological characteristics of acute lung injury. In addition, the SARS-CoV-2 recombinant spike protein is a non-infectious viral protein. It can induce obvious pathophysiological changes of viral pneumonia and acute lung injury while avoiding infection of experimental personnel. Therefore, it avoids the risk of infection by live SARS-CoV-2 virus, so that the cell model of COVID-19 acute lung injury can be established in an ordinary laboratory without the need for laboratory conditions with a higher level of safety. The experimental cycle is short, convenient and fast, and inexpensive, making it suitable for popularization and application.

[0028] Furthermore, in step S20, the ratio of the number of co-cultured cells of human lung macrophages and human alveolar epithelial cells is 1:(2-8). Under this ratio, the immune homeostasis of the cell model can be maintained and a more realistic lung structure and acute lung injury pathological characteristics can be simulated.

[0029] Furthermore, in step S20, the first preset time is 12 to 48 hours. Within this time range, the activated human lung macrophages and human lung epithelial cells can fully interact with each other, which is beneficial for the co-culture system to induce inflammatory response and oxidative stress after exposure to spike protein, thereby causing acute lung injury.

[0030] Furthermore, in step S30, the mass concentration of the spike protein in the co-culture system is 5-15 μg / mL. Within this concentration range, significant inflammatory response and oxidative stress can be induced, which is closer to the actual pathogenesis of COVID-19 acute lung injury in humans. The mass concentration of the spike protein in the co-culture system can be 5 μg / mL, 10 μg / mL or 15 μg / mL.

[0031] Furthermore, in step S30, the second preset time is 3 to 5 days. Within this time range, significant inflammatory response and oxidative stress can be induced, which more realistically simulates the natural course of COVID-19 acute lung injury.

[0032] The present invention also proposes the application of the cell model established by the method for establishing a cell model of COVID-19 acute lung injury induced by the spike protein in the screening of therapeutic drugs for COVID-19 acute lung injury. The cell model of COVID-19 acute lung injury induced by the spike protein established by the present invention can be used to screen therapeutic drugs for COVID-19 acute lung injury, simplifying the experimental procedures, without the need for strict ethical approval related to the use of human tissues, and the storage conditions are also relatively simple. Recombinant spike protein is used to replace the infectious SARS-CoV-2 live virus, and the modeling laboratory conditions are not demanding, and there is no need to use a biosafety level 3 (BSL-3) laboratory and expensive instruments and equipment. The experimental cycle is short, easy to repeat, and low in price, which is suitable for promotion and application in ordinary biological laboratories. The cell model established by this method can fully simulate the pathogenesis of diffuse alveolar damage, excessive inflammatory response and oxidative stress of COVID-19 acute lung injury.

[0033] The cell model of COVID-19 acute lung injury induced by spike protein provided by the present invention can be verified by the following methods: Figure 1 and Figure 2 As shown:

[0034] Cell recovery and culture

[0035] Take human lung AM and HPAEpic cells frozen in a -80℃ refrigerator, thaw in a 39℃ water bath, transfer the cells in the cryopreserved tube together with the cryopreserved solution into a new sterile EP tube, centrifuge at room temperature at 1000rpm for 5 minutes, and discard the supernatant. Take a small amount of cell culture solution (preheated at 37℃ for 30 minutes in advance) to gently resuspend the cells, transfer to a T25 cell flask (a small amount of cell culture solution has been added), add culture solution to a total volume of 5mL in the cell flask, place in a 5% CO2 incubator, and culture at 37℃.

[0036] Experimental Grouping

[0037] Different concentrations of SARS-CoV-2 recombinant S1 protein (0.1 μg / mL, 1 μg / mL, 10 μg / mL) were added to co-cultured (co-culture ratio 1:5) human lung macrophages (AM) + human alveolar epithelial cells (HPAEpic), and samples were collected 1d, 3d, and 5d after treatment. LPS (1 μg / mL) was used as a positive control to establish the dose-effect and time-effect curves of cell damage rate, inflammation and oxidative stress indicators.

[0038] BCA protein quantification

[0039] Preparation of protein standards

[0040] 1) Take 0.6mL of protein standard solution and add it to a tube of protein standard (15mg BSA bovine serum albumin). After fully dissolving, prepare a 25mg / mL protein standard solution. It can be used immediately after preparation or stored at -20℃ for a long time.

[0041] 2) Protein samples and standards can be diluted with PBS, and the diluted standards can be stored at -20℃ for a long time.

[0042] 3) Use PBS (phosphate buffered saline) as the diluent to dilute and package the standard into concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and 1 mg / mL.

[0043] Preparation of BCA working solution

[0044] Using a conventional BCA kit, 200 μL of BCA working solution is required to measure one sample. According to the number of samples, prepare an appropriate amount of BCA working solution at a volume ratio of 50:1 between BCA reagent A and BCA reagent B (for example, mix 5 mL of BCA reagent A and 100 μL of BCA reagent B to make 5.1 mL of BCA working solution) and mix thoroughly.

[0045] Protein concentration detection

[0046] 1) Preparation of standard curve: Set up 3 parallel samples for each standard, take the average value of the 3 parallel samples to prepare the standard curve, take 1 tube of each standard with different concentrations, and add 20 μL / well to the standard wells of a 96-well plate.

[0047] 2) Each sample was prepared with 3 parallel samples and the average value of the 3 parallel samples was used to calculate the concentration.

[0048] 3) Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 minutes.

[0049] 4) Measure the absorbance at a wavelength of 595 nm using an enzyme-labeled instrument.

[0050] The protein concentration of the sample is calculated based on the standard curve and the sample volume used. BCA protein quantification can standardize experimental data, reduce the result deviation caused by differences in cell numbers, and make the results of different experiments under the same conditions comparable and reproducible.

[0051] Cell damage rate detection-lactate dehydrogenase (LDH) release experiment

[0052] Lactate dehydrogenase (LDH) is abundant in the cytoplasm and can be released outside the cell when the cell is damaged or dead. The released LDH is in the culture medium supernatant and can be detected by enzyme reaction. Use the Lactate Dehydrogenase Cytotoxicity Assay Kit to detect LDH levels.

[0053] 1) Take AM and HPAEpic cells from different groups that are in the logarithmic growth phase and in good growth condition, digest them routinely, and resuspend the cells in DMEM complete medium containing 10% FBS. Adjust the concentration of the cell suspension to 2×10 4 cells / mL.

[0054] 2) Add 100 μL of the above cell suspension to each well of a 96-well plate, i.e., 2000 cells / well, and place the 96-well plate in an incubator for culture.

[0055] 3) Aspirate the culture medium and wash once with PBS. Replace with fresh culture medium and divide each culture well into the following groups: culture medium blank group, NC group (control group, i.e., untreated group), S protein treatment group (0.1μg / mL, 1μg / mL, 10μg / mL), LPS group (1μg / mL), and maximum lysis solution group. Give appropriate drug treatment according to experimental needs and add 10μL of specific drug stimulation, and continue to incubate in the cell culture incubator.

[0056] 4) Take out the cell culture plate from the cell culture incubator, add cell lysis buffer to the maximum lysis buffer group, the amount added is 10% of the original culture buffer volume, repeatedly pipette and beat several times to mix, and continue to incubate in the cell culture incubator.

[0057] 5) Centrifuge the cell culture plate at 400 g for 5 min using a multi-well plate centrifuge, take 120 μL of the supernatant from each well and add it to the corresponding well of a new 96-well plate.

[0058] 6) Add 60 μL of LDH detection working solution to each well, mix well, and incubate at room temperature (about 25°C) in the dark for 30 minutes. Use a multifunctional microplate reader to measure the absorbance at 490 nm. Use 600 nm as the reference wavelength for dual wavelength measurement.

[0059] Standardized cell damage rate calculation method:

[0060] ① The absorbance of each group measured should be subtracted from the average absorbance of the background blank control well group.

[0061] ②A Cell damage rate (%) = absorbance of each well of the treated sample group / average absorbance of the cell maximum enzyme activity group × 100.

[0062] ③B Calculate the total protein ratio = total protein in each well / average total protein in all wells.

[0063] ④Standardized cell damage rate = A / B.

[0064] Inflammation indicators: Detection of inflammatory factors (IL-6, TNF-α)

[0065] 1) The cells of each treatment group were centrifuged at 1000 g for 20 minutes, and the supernatant was taken for detection.

[0066] 2) Take out the ELISA plate, discard the liquid, add the biotinylated antibody working solution directly to each well, cover with the sealing film and incubate at 37°C for 1 hour.

[0067] 3) Discard the liquid, add 300 μL 1x washing solution to each well, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat 3 times.

[0068] 4) Then add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37℃ for 30 minutes. Discard the liquid and wash again 5 times, then add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 minutes.

[0069] 5) Take out the ELISA plate, add 50 μL of stop solution directly to each well, and measure the absorbance of each well at 450 nm using a multifunctional ELISA reader.

[0070] 6) Calculate the concentration of IL-6 and TNF-α in each well according to the standard curve and normalize it by the average value of total protein in each well / total protein in all wells.

[0071] Oxidative stress indicator: detection of intracellular glutathione peroxidase 4 (GPX4)

[0072] The level of intracellular glutathione peroxidase 4 (GPX4) was detected using human glutathione peroxidase 4 (GPX4) enzyme-linked immunosorbent assay kit.

[0073] 1) The cells of each treatment group were centrifuged at 2000-3000 rpm / min for 20 minutes and the supernatant was collected.

[0074] 2) Add 50 μL of the prepared sample and standard, then add 50 μL of biotin antigen working solution, shake gently, cover with sealing film, and incubate in a 37°C incubator for 30 minutes.

[0075] 3) Discard the liquid, add washing solution, wash 5 times, add 50 μL of avidin HRP to each well, incubate in a 37°C incubator for 30 minutes, and wash 5 times.

[0076] 4) Add 50 μL of color development solution A and 50 μL of color development solution B, and develop the color at 37°C in the dark for 10 minutes. Then add 50 μL of stop solution to each well to stop the reaction, and measure the absorbance of each well at 450 nm using a multifunctional microplate reader.

[0077] 5) Calculate the GPX4 concentration in each well based on the standard curve and normalize it with the average of the total protein in each well / total protein in all wells.

[0078] Oxidative stress indicator: detection of intracellular malondialdehyde (MDA) content

[0079] Malondialdehyde (MDA) is a polyunsaturated fatty acid decomposition product produced by the peroxidation of compounds such as fatty acids and fatty acid esters. It is formed in the human body due to oxidative stress. Oxidative stress can lead to excessive production of free radicals and increased cell and tissue damage. MDA levels were detected using an MDA kit.

[0080] 1) After centrifuging the cell supernatant of each treatment group at 2-8°C, 2500rpm for 5 minutes, add 50μL of the sample to be tested to the corresponding well, and then immediately add 50μL of the biotin-labeled detection antibody working solution to each well, gently shake the ELISA plate to mix for 1 minute, apply the film, and incubate at 37°C for 45 minutes.

[0081] 2) Remove the film, discard the liquid in the ELISA plate, tap it on clean absorbent paper 2-3 times, then add 350 μL of washing buffer to each well, soak for 1 minute, discard the liquid in the well, tap it on absorbent paper 3 times. Repeat 3 times.

[0082] 3) Add 100 μL of SABC working solution to each well, apply the film, incubate at 37°C for 30 minutes, and then wash 5 times.

[0083] 4) Then add 90 μL TMB color substrate to each well, apply the film, and incubate at 37°C in the dark for 20 minutes. After color development, add 50 μL of reaction stop solution to each well. Measure the OD value of each well at 450 nm.

[0084] The MDA concentration in each well was calculated based on the standard curve and normalized by the total protein in each well / the average of the total protein in all wells.

[0085] The detection data of cell damage rate, inflammatory factors (IL-6, TNF-α), intracellular glutathione peroxidase 4 (GPX4), and intracellular malondialdehyde (MDA) are plotted into a graph, such as Figure 2 shown.

[0086] Depend on Figure 2 It can be seen that human lung AM and HPAEpic cells were co-cultured at a ratio of 1:5 for 24 hours and then treated with 10 μg / mL of S1 protein. After 3 to 5 days, obvious cell damage, inflammatory response and oxidative stress can be produced, and the effect is better than the corresponding changes induced by 1 μg / mL LPS (positive control group). When the treatment time is 1 day, the induced acute lung injury cell model does not meet the requirements. The COVID-19 acute lung injury cell model established by the present invention has very good application prospects. When the S1 protein is below 1 μg / mL, the induced cell damage is not obvious enough.

[0087] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for establishing a cell model of COVID-19 acute lung injury induced by spike protein, characterized in that: The following steps are involved: S10, obtain human lung macrophages, human alveolar epithelial cells and SARS-CoV-2 recombinant spike protein; S20, co-culturing the human lung macrophages and the human alveolar epithelial cells for 12 to 48 hours to obtain a co-culture system; S30. Add SARS-CoV-2 recombinant spike protein to the co-culture system and co-culture for 3 to 5 days to obtain a spike protein-induced COVID-19 acute lung injury cell model; Wherein, in step S20, the cell number ratio of human lung macrophages to human alveolar epithelial cells is 1:(2-8); In step S30, the mass concentration of the spike protein in the co-culture system is 5 to 15 μg / mL.

2. Application of the cell model established by the method for establishing a cell model of COVID-19 acute lung injury induced by spike protein as described in claim 1 in screening therapeutic drugs for COVID-19 acute lung injury.

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