Method for establishing multi-cell co-culture inflammation model of respiratory system

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a method for establishing a multi-cell co-culture inflammation model of a respiratory system. The establishment method of the inflammation model comprises the following steps: S1, carrying out cell culture; s2, inoculating the human umbilical vein endothelial cells to the outer side surface of the bottom of the Transswel cell; inoculating human alveolar epithelial cells on the inner side surface of the bottom of the Transwell chamber; s3, inducing human mononuclear / macrophages with phorbol ester, and inoculating the human mononuclear / macrophages into the culture holes; s4, putting the Transwell chamber inoculated with the human alveolar epithelial cells and the human umbilical vein endothelial cells into the culture hole inoculated with the human mononuclear / macrophage for co-culture; s5, lipopolysaccharide is adopted as an inducer, inflammatory factors in the multi-cell co-culture system are detected, and the inflammation model is obtained. The inflammation model provided by the invention is applied to research on respiratory system disease pathogenesis and drug action mechanism or screening of drugs for treating respiratory system diseases taking inflammatory reaction as main pathological reaction.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for establishing a multi-cellular co-culture inflammation model of the respiratory system. Background Art

[0002] Inflammatory responses in the respiratory system are seen in various respiratory diseases, such as chronic obstructive pulmonary disease (COPD), pneumonia, interstitial pulmonary fibrosis, etc. Inflammatory responses play an important role in the occurrence and development of these diseases and affect the prognosis of the diseases. The inflammatory response in the respiratory system involves multiple cells such as lung parenchymal cells and immune cells, as well as multiple inflammatory factors and signaling pathways. Pathogenic factors such as bacteria, harmful particles or gases stimulate immune cells such as macrophages and neutrophils to release inflammatory mediators, damage and stimulate lung parenchymal cells such as epithelial cells and vascular endothelial cells to release more inflammatory mediators, resulting in a cascade-like inflammatory response and promoting the progression of the disease.

[0003] It is difficult to simulate the complex microenvironment of multi-cellular interactions by single-cell culture in vitro. Multi-cellular co-culture is conducive to observing the interactions between cells and between cells and the environment, and can better simulate the complex lung microenvironment of multi-cellular interactions during the occurrence of inflammatory responses in the respiratory system. It is an important tool for studying the mechanism of drug action and developing new drugs. A cell culture model will be used in the study of simulating inflammatory responses in the respiratory system.

[0004] Currently, existing cell culture models for simulating the inflammatory microenvironment of the respiratory system include monolayer cell culture of cells such as alveolar epithelial cells, macrophages, airway epithelial cells, and vascular endothelial cells. Although the above three monolayer cell models are easy to implement, monolayer cell culture is difficult to simulate the microenvironment of multi-cellular interactions during the occurrence of respiratory diseases and cannot explore the interactions between cells. Summary of the Invention

[0005] To solve the problem that monolayer cell culture in the prior art is difficult to simulate the microenvironment of multi-cellular interactions during the occurrence of respiratory diseases and cannot explore the interactions between cells, the present invention aims to provide a method for establishing a multi-cellular co-culture inflammation model of the respiratory system, so as to provide a tool for studying the pathogenesis of respiratory diseases, the mechanism of drug action, and the development of new drugs.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for establishing a multi-cellular co-culture inflammation model of the respiratory system, comprising the following steps:

[0008] S1. Separately perform cell culture on human alveolar epithelial cells, human umbilical vein endothelial cells, and human monocytes / macrophages;

[0009] S2. Seed the human umbilical vein endothelial cells after the end of cultivation on the outer side of the bottom of the Transwell chamber at a density of 3 - 5×10 5 cells per well. Invert the Transwell chamber in the culture well and culture it for 5 - 7 h under the same culture conditions as in S1. After the human umbilical vein endothelial cells adhere to the wall, place the Transwell chamber upright in the culture well;

[0010] Seed the human alveolar epithelial cells after the end of cultivation on the inner side of the bottom of the Transwell chamber at a density of 3 - 5×10 5 cells per well and culture it for 11 - 13 h under the same culture conditions as in S1;

[0011] After inducing the human monocytes / macrophages after the end of cultivation with phorbol ester, seed them at a density of 3 - 5×10 5 cells per well in the culture well and culture it for 23 - 25 h under the same culture conditions as in S1;

[0012] S3. Place the Transwell chamber inoculated with human alveolar epithelial cells and human umbilical vein endothelial cells in the culture well inoculated with human monocytes / macrophages for co - culture to obtain a multi - cell co - culture system;

[0013] S4. Use an inflammation inducer to act on the multi - cell co - culture system after co - culture in S4 to obtain the said inflammation model.

[0014] Preferably, in S1, the human alveolar epithelial cells are cultured alone in a complete medium of DMEM / F12 containing 9 - 11% fetal bovine serum; the human umbilical vein endothelial cells are cultured alone in a complete medium of DMEM / F12 containing 9 - 11% fetal bovine serum and 1% endothelial cell growth factor; the human monocytes / macrophages are cultured alone in a complete medium of RPMI 1640 containing 9 - 11% fetal bovine serum and 0.05 - 0.15 mM / L β - mercaptoethanol.

[0015] Preferably, the cell culture conditions for culturing the human alveolar epithelial cells, human umbilical vein endothelial cells and human monocytes / macrophages alone are: static culture at 36 - 38°C and 4 - 6% CO2.

[0016] Preferably, in S2, the bottom of the Transwell chamber is made of a transparent polyester membrane with a pore size of 0.2 - 0.5 μm.

[0017] Preferably, in S3, the steps for preparing the co-culture medium for co-culture are as follows: Weigh L-glutamine, HEPES, penicillin, streptomycin, and β-mercaptoethanol respectively, add them to distilled water, and mix well to make their final concentrations 298 - 302 mg / L L-glutamine, 5956 - 5960 mg / L HEPES, 98 - 102 U / mL penicillin, 98 - 102 μg / mL streptomycin, 0.08 - 0.12 mM / L β-mercaptoethanol respectively. Then add 8 - 12 mL / L endothelial growth factor and 98 - 102 mL / L medicated serum, mix well, and sterilize to obtain the co-culture medium.

[0018] Preferably, the conditions for co-culture are: culture at 36 - 38 °C and 4 - 6% CO2 for 22 - 26 h.

[0019] Preferably, the inflammation inducer is lipopolysaccharide.

[0020] Preferably, in S4, the concentration of the lipopolysaccharide is 0.8 - 1.2 μg / mL.

[0021] Preferably, in S4, after culturing for 22 - 26 h after adding lipopolysaccharide, collect the supernatant of the cell culture medium in the multi-cell co-culture system, centrifuge at 1000 rpm / min for 4 - 6 min, collect the supernatant, and detect inflammatory factors.

[0022] Preferably, the inflammatory factors include IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IL-13, TNF-α, IFN-γ.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention provides a method for establishing a multi-cell co-culture inflammation model of the respiratory system. The present invention establishes an alveolar epithelial cell / macrophage / vascular endothelial cell multi-cell co-culture inflammation model that simulates the inflammatory microenvironment of the respiratory system, providing a tool for studying the pathogenesis of respiratory diseases, the mechanism of drug action, and the development of new drugs. The multi-cell co-culture inflammation model of the respiratory system established by the present invention has the following advantages:

[0025] Traditional single-layer cell culture cannot simulate the microenvironment of multi-cell interaction in the pathological process of diseases, and the disease mechanisms that can be studied are single. The present invention uses co-culture of three kinds of cells to construct an in vitro inflammation model, which can solve multiple problems with one model and has a wide range of uses.

[0026] 2. The multi - cell co - culture inflammation model of the respiratory system established in the present invention is: an alveolar epithelial cell / macrophage / vascular endothelial cell co - culture inflammation model. This model can provide a tool for studying the mechanisms of respiratory system diseases with inflammatory reactions as the main pathological changes, the mechanisms of drug action, drug screening, and new drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shows the effects of LPS acting for different times on inflammatory factors in A549 / HUVECs / THP - 1 multi - cell co - culture cells in the present invention. Among them, compared with the blank group, *p < 0.05, **p < 0.01;

[0028] Figure 2 Shows the effects of LPS induced for 24 h on inflammatory factors in monolayer cell culture (Comparative Examples 1 - 3) and co - culture cells (Example 1) in the present invention. Among them, CT is the co - culture normal group, and LPS is the co - culture LPS model group; compared with the blank group, *p < 0.05, **p < 0.01;

[0029] Figure 3 Shows the effects of LPS on the gene expression of inflammatory factors in A549 / HUVECs / THP - 1 multi - cell co - culture cells in the present invention. Among them, CT is the co - culture normal group, and LPS is the co - culture LPS model group; compared with the blank group, *p < 0.05, **p < 0.01;

[0030] Figure 4 Shows the effects of LPS on the surface molecule expression of THP - 1 cells co - cultured with A549 and HUVECs in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The present invention will be described in detail below with reference to the drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well - known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0032] Example 1

[0033] A method for establishing a multi - cell co - culture inflammation model of the respiratory system, comprising the following steps:

[0034] 1. Preparation of lipopolysaccharide

[0035] Prepare a stock solution of lipopolysaccharide (LPS) with a concentration of 1 mg / mL using 1×PBS.

[0036] 2. Sub - culture of cells

[0037] Resuscitate and subculture A549 cells (human alveolar epithelial cells) with DMEM / F12 complete medium (Gibco) containing 10% fetal bovine serum, and the culture conditions are static culture at 37°C and 5% CO2.

[0038] Resuscitate and subculture HUVECs cells (human umbilical vein endothelial cells, vascular endothelial cells) with DMEM / F12 complete medium (referred to as: medium 1) containing 10% fetal bovine serum and 1% endothelial cell growth factor (ScienCell), and the culture conditions are static culture at 37°C and 5% CO2.

[0039] Resuscitate and subculture THP-1 cells (human monocytes / macrophages) with RPMI 1640 complete medium (Gibco) containing 10% fetal bovine serum and 0.1 mM / L β-mercaptoethanol (referred to as: medium 2), and the culture conditions are static culture at 37°C and 5% CO2.

[0040] The above three types of cells, A549 cells, HUVECs cells and THP-1 cells, are all purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0041] 3. Cell seeding and grouping treatment

[0042] After the three types of cells, A549 cells, HUVECs cells and THP-1 cells, are each passaged 3 times respectively, co-culture is carried out. The co-culture medium for co-culture is obtained by mixing the above medium 1 and medium 2 according to a mass ratio of 1:1. The steps for preparing the co-culture medium are as follows: Weigh L-glutamine, HEPES, penicillin, streptomycin, and β-mercaptoethanol respectively, add distilled water, mix well, so that their final concentrations are 300 mg / L L-glutamine, 5958 mg / L HEPES, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.1 mM / L β-mercaptoethanol, then add 10 mL / L endothelial growth factor and 100 mL / L drug-containing serum, mix well, and sterilize to obtain the co-culture medium.

[0043] Before co-culture, a co-culture normal group and a co-culture LPS model group are set up respectively.

[0044] Among them, the cell seeding and treatment of the co-culture normal group are as follows:

[0045] Take the HUVECs cells subcultured in a 100 mm culture dish above, digest them with 1 mL of trypsin digestion solution (Solarbio, T1320) containing 0.25% EDTA, count them, and then 5A number of HUVECs cells were seeded on the outer side of the PET membrane (with a pore size of 0.4 μm) at the bottom of the Transwell chamber. The Transwell chamber was inverted in the culture well of a 12-well cell culture plate. After 6 h, when the HUVECs cells adhered to the wall, the Transwell chamber was placed upright in the culture well of the above-mentioned cell culture plate with the corresponding pore size; The A549 cells subcultured in a 100 mm culture dish were digested with 1 mL of trypsin digestion solution containing 0.25% EDTA and then counted. 5 A number of A549 cells were seeded on the inner side of the PET membrane at the bottom of the above-mentioned Transwell chamber seeded with HUVECs cells; The 2 1×10 6 THP-1 cells in a 75 cm 5 culture flask were induced with 20 ng / mL phorbol 12-myristate 13-acetate (PMA). A number of 4×10 2 induced THP-1 cells were seeded in the culture well of the cell culture plate. The Transwell chamber seeded with A549 cells and HUVECs cells was placed in the culture well of the cell culture plate seeded with THP-1 cells for co-culture to obtain a multi-cell co-culture system. The culture conditions for co-culture were: static culture in an incubator at 37 °C and 5% CO

[0046] The cell seeding and treatment of the co-culture LPS model group were as follows:

[0047] The HUVECs cells subcultured in a 100 mm culture dish were digested with 1 mL of trypsin digestion solution containing 0.25% EDTA and then counted. A number of 4×10 5 HUVECs cells were seeded on the outer side of the PET membrane (with a pore size of 0.4 μm) at the bottom of the Transwell chamber. The Transwell chamber was inverted in the culture well of a 12-well cell culture plate. After 6 h, when the HUVECs cells adhered to the wall, the Transwell chamber was placed upright in the culture well of the above-mentioned cell culture plate with the corresponding pore size; The A549 cells subcultured in a 100 mm culture dish were digested with 1 mL of trypsin digestion solution containing 0.25% EDTA and then counted. A number of 4×10 5 A549 cells were seeded on the inner side of the PET membrane at the bottom of the above-mentioned Transwell chamber seeded with HUVECs cells; The 2 1×10 6 THP-1 cells in a 75 cm 5An induced THP-1 cell was seeded into the culture well of a cell culture plate. A Transwell chamber seeded with A549 cells and HUVECs cells was placed into the culture well of the cell culture plate seeded with THP-1 cells for co-culture. Meanwhile, 1 μg / mL LPS was added to the culture plate and acted for 24 h. The culture conditions for co-culture were the same as those of the normal co-culture group.

[0048] 4. Inflammatory factor detection

[0049] To detect the inflammatory factors in the multi-cell co-culture system obtained by the method for establishing a respiratory multi-cell co-culture inflammation model provided by the present invention, we respectively collected the cell culture supernatant after 6 h, 12 h, 24 h and 48 h of co-culture for the normal co-culture group and the LPS co-culture model group, and used the Luminex detection technology and a multi-factor detection kit (BIO-RAD, 171304090M) to detect the levels of inflammatory factors IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, TNF-α and IFN-γ. The specific steps are as follows:

[0050] (1) Reagent preparation: Equilibrate the BIO-RAD multi-factor detection kit at room temperature for 30 min.

[0051] (2) Standard preparation: Dissolve the standard completely with 250 μL of Standard Diluent HB, let it stand on ice for 30 min, pipette 50 μL of the standard and add 150 μL of Standard Diluent HB, mix well. This is the highest concentration of the standard. Based on this concentration, serially dilute the standard by 4-fold, with a total of 7 concentration points and 1 blank well set. The concentrations are as follows: IL-1β: 1247, 311.75, 77.94, 19.48, 4.87, 1.22, 0.3, 0 pg / mL; IL-2: 4806, 1202, 300.38, 75.09, 18.77, 4.69, 1.17, 0 pg / mL; IL-4: 1261, 315.25, 78.81, 19.7, 4.93, 1.23, 0.31, 0 pg / mL; IL-6: 1456, 364, 91, 22.75, 1.42, 0.36, 0 pg / mL; IL-8: 3770, 942.5, 235.62, 58.91, 14.73, 3.68, 0.92, 0 pg / mL; IL-10: 4474, 1118, 279.62, 69.91, 17.48, 4.37, 1.09, 0 pg / mL; IL-12: 7663, 1916, 478.94, 119.73, 29.93, 7.48, 1.87, 0 pg / mL; TNF-α: 15596, 3899, 974.75, 243.69, 60.92, 15.23, 3.81, 0 pg / mL; IFN-γ: 4547, 1137, 284.19, 71.05, 17.76, 4.44, 1.11, 0 pg / mL.

[0052] (3) Beads preparation: Pipette 285 μL of the beads (20X) for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, TNF-α, and IFN-γ into 2850 μL of Assey Buffer respectively, and mix well.

[0053] (4) Add 50 μL of the Beads mixture to each well of the microplate, place the microplate on the magnetic stand for at least 1 min to ensure that the Beads are completely adsorbed, and discard the liquid.

[0054] (5) Add 100 μL of the washing solution to each well, wash 2 times, and discard the liquid.

[0055] (6) Add the corresponding 50 μL of the standard and cell culture supernatant to each well, cover with a sealing film, incubate with shaking at room temperature for 60 min, and set the shaker speed to 800 rpm.

[0056] (7) Within 10 minutes before the end of incubation, prepare the detection antibody: Pipette 150 μL of the detection antibody DetentionAntibodies into each well, add 1500 μL of Detention Antibody Diluent HB, and mix 10 detection antibodies DetentionAntibodies (20X) thoroughly to obtain an antibody mixture for later use.

[0057] (8) Place the microplate on the magnetic rack for at least 1 minute to ensure that the Beads are completely adsorbed, then discard the liquid. Add 100 μL of washing solution to each well and wash 3 times, discarding the liquid each time.

[0058] (9) Add 25 μL of the antibody mixture prepared in (7) to each well, cover with a sealing film, and incubate with shaking at room temperature for 30 minutes, setting the shaker speed to 800 rpm.

[0059] (10) Repeat step (8).

[0060] (11) Add 50 μL of diluted streptavidin-labeled PE to each well, cover with a sealing film, and incubate with shaking at room temperature for 30 minutes, setting the shaker speed to 800 rpm.

[0061] (12) Repeat step (8).

[0062] (13) Detection: Add 125 μL of Assay Buffer to each well, place the microplate on the shaker and shake for 30 seconds, then detect on the machine. Analyze and process the detection results, and the results are as Figure 1 shown.

[0063] It can be Figure 1 seen that the levels of IL-1β, TNF-α, IL-2, IL-6, IL-8, IL-12, and IFN-γ in the multi-cell co-culture system can be significantly increased after 24-hour treatment with 1 μg / mL LPS (P<0.05, P<0.01). There is no significant difference in the IL-13 level between the normal co-culture group and the LPS-induced co-culture model group. The IL-4 level is too low to be detected. IFN-γ and LPS can induce the M1-type differentiation of macrophages, and M1-type macrophages mainly secrete pro-inflammatory factors such as IL-1β and TNF-α and participate in the inflammatory response. IL-4 and IL-13 can induce the M2-type differentiation of macrophages. The IL-4 level in the multi-cell co-culture system is extremely low, and the IL-13 level shows no significant change. The results indicate that macrophages co-cultured with alveolar epithelial cells and vascular endothelial cells mainly differentiate into the M1 phenotype under LPS induction and mediate the inflammatory response.

[0064] On this basis, in order to verify the levels of inflammatory factors in the co-culture system, we used ELISA to detect the levels of inflammatory factors with significant differences between the normal co-culture group and the co-culture LPS model group in Example 1: IL-1β, IL-6, and TNF-α levels. Both Luminex multi-factor detection technology and ELISA are common methods for measuring the levels of inflammatory factors. The multi-factor detection method is mainly used to screen key inflammatory factors. On this basis, we used ELISA for re-verification, and the specific steps are as follows:

[0065] (1) Standard preparation: Within two hours before use, use the serial dilution method to prepare 7 concentrations of IL-1β, IL-6, and TNF-α standards, and set a blank well. The standard concentrations are: IL-1β: 250, 125, 62.5, 31.125, 15.56, 7.78, 3.89, 0 pg / mL; IL-6: 1000, 500, 250, 125, 62.5, 31.125, 15.56, 0 pg / mL; TNF-α: 1000, 500, 250, 125, 62.5, 31.125, 15.56, 0 pg / mL.

[0066] (2) Sample dilution: According to the estimation of the content of each factor in the preliminary experiment, dilute the collected cell culture supernatant to an appropriate multiple. Dilute IL-1β 2-fold, IL-6 2-fold, and TNF-α 2-fold for detection, so that the concentration of the factor to be measured in the diluted cell culture supernatant is within the optimal detection range of the ELISA kit.

[0067] (3) Sample addition: Add the diluted cell culture supernatant and standards to the enzyme-linked immunosorbent assay (ELISA) plate, 100 μL / well. For the wells of the samples to be tested: set 3 replicates for each sample; for the standard wells: set 8 standard wells (add sample diluent to 1 well as the zero well).

[0068] (4) Incubation: Cover the ELISA plate with a sealing film and react at 37 °C for 90 min.

[0069] (5) Add biotinylated antibody working solution: Discard the liquid in the plate, pat dry on the absorbent paper, and add 100 μL / well of the antibody working solution against human IL-1β, IL-6, and TNF-α diluted 1 / 100.

[0070] (6) Incubation: Cover the ELISA plate with a sealing film and react at 37 °C for 60 min.

[0071] (7) Plate washing: Wash 3 times with 330 μL / well of 1× washing solution, soak for 1 min each time, and pat dry the washing solution in the wells as much as possible.

[0072] (8) Add enzyme conjugate working solution: Add 100 μL / well of the avidin-peroxidase complex working solution, cover with a sealing film, and react at 37 °C for 30 minutes.

[0073] (9) Plate washing: Wash with 330 μL / well of 1× washing solution for 5 times, soak for 1 min each time, and pat dry the washing solution in the wells.

[0074] (10) Color development: Add 90 μL / well of TMB color development solution equilibrated at 37 °C for 30 min, and react at 37 °C for 20 min.

[0075] (11) Terminate the reaction: When an obvious gradient blue color appears in the standard wells, add 100 μL / well of reaction termination solution to terminate the reaction, and at this time, the blue color immediately turns yellow.

[0076] (12) Detection: Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay reader. Set the TMB blank color development well as the control. After subtracting the absorbance of the control well from the absorbance of all standard products and samples, plot the standard curve with the absorbance as the abscissa and the concentration as the ordinate to obtain the standard curve equation, and calculate the sample concentration according to the absorbance and the equation. Analyze and process the detection results, and the results are as Figure 2 shown:

[0077] Compared with the co-culture normal group, the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the co-culture LPS group were significantly increased, indicating that the action of LPS can significantly induce the inflammatory response of co-cultured A549 cells, HUVECs cells, and THP-1 cells.

[0078] Based on the above results, after the co-culture ended, we respectively extracted the total RNA of A549 cells, HUVECs cells, and THP-1 cells in the multi-cell co-culture system from the co-culture normal group and the co-culture LPS model group, and detected the mRNA expression levels of inflammatory factors by qPCR. The specific steps are as follows:

[0079] (1) After the co-culture ended, wash the cells in the co-culture normal group and the co-culture LPS model group with 1× PBS once, and then extract the total RNA of A549 cells / HUVECs cells / THP-1 cells in the above two groups respectively.

[0080] (2) Add 1 mL of Trizol Reagent (Invitrogen, 15596026) to each type of cell, fully lyse at room temperature for 10 minutes, and transfer to a nuclease-free tube.

[0081] (3) Add 200 μL of chloroform to each tube, shake vigorously for 15 seconds, and place on ice for 5 min.

[0082] (4) Centrifuge at 12000 g at 4 °C for 15 minutes, and transfer the upper aqueous phase to a new nuclease-free tube.

[0083] (5) Add isopropanol with the same volume as the water, invert and mix well, and then react at room temperature for 10 minutes.

[0084] (6) Centrifuge at 12,000 g for 15 minutes at 4°C, discard the supernatant, add 1 mL of 75% ethanol, and resuspend the pellet.

[0085] (7) Centrifuge at 12,000 g for 5 minutes at 4°C, discard the supernatant, add 20 μL of DEPC water to dissolve the pellet, and incubate on ice for 30 min until fully dissolved to obtain the total RNA solution.

[0086] (8) Measure the concentration and purity of the total RNA using NanoDrop 2000.

[0087] (9) Calculate the required sample volume based on a concentration of 100 μg / mL, and perform RNA reverse transcription using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR reverse transcription kit (Invitrogen, 4368814), and qPCR using the SYBR Green kit (Invitrogen, 11746500). The primers for human TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-12, IL-10, IL-8, and IL-13 were designed and synthesized by Shanghai Jierui Biotechnology Co., Ltd. The gene primer information is shown in Table 1:

[0088] Table 1 Gene primer information

[0089]

[0090]

[0091] (10) The RNA reverse transcription reaction system is shown in Table 2:

[0092] Table 2 Reverse transcription system (μL)

[0093] Reagent Volume 10×RT Buffer 2.0 25×dNTP Mix(100mM) 2.0 10×RT Random Primers 2.0 MultiSribe Reverse Transcrptase 1.0 RNase Inhibitor 1.0 <![CDATA[Nuclease-free H2O]]> 3.2 Total volume per reaction 10.0

[0094] (11) The RNA reverse transcription reaction conditions are shown in Table 3. After RNA reverse transcription, cDNA is obtained.

[0095] Table 3 RNA reverse transcription reaction conditions

[0096] Step Reaction temperature Reaction time 1 25℃ 10min 2 37℃ 120min 3 85℃ 5min 4 4℃ ∞

[0097] (12) The qPCR reaction system is shown in Table 4:

[0098] Table 4 qPCR reaction system (384-well plate)

[0099] Reagent Volume DEPC water 0.2μL Primer 0.3μL SYBR green 2.5μL cDNA 2.0μL Total volume 5.0μL

[0100] (13) The qPCR procedure and reaction conditions are shown in Table 5 as follows:

[0101] Table 5 qPCR procedure and reaction conditions

[0102]

[0103] After the reaction, according to the formula F (relative gene expression level) = 2-△△Ct, calculate the relative gene expression level of F. △△Ct = (average ct of the target gene to be detected - average ct of the internal reference gene to be detected) - (average ct of the normal target gene - average ct of the normal internal reference gene). Analyze the test results as Figure 3 shown:

[0104] The expression levels of inflammatory factor genes in co-cultured A549 cells / HUVECs cells / THP-1 cells are as Figure 3 shown. The results show that in the co-culture system, A549 cells mainly secrete: IFN-γ, IL-2, HUVECs cells mainly secrete: TNF-α, IFN-γ, IL-2, THP-1 cells mainly secrete: IL-1β, IL-6, IFN-γ, IL-12, suggesting that in the co-culture system, IFN-γ secreted by A549 cells and HUVECs cells can promote the M1 phenotype differentiation of THP-1 cells, secrete more inflammatory factors, and amplify the inflammatory response.

[0105] Based on the above results, after the co-culture, we detected the expression of cell surface molecules CD68, CD86, and CD206 on the surfaces of A549 cells, HUVECs cells, and THP-1 cells in the co-culture normal group and the co-culture LPS model group respectively by immunofluorescence technique. The specific steps are as follows:

[0106] The reagents and consumables used in the immunofluorescence technique detection process are as follows: 12-well Transwell (Corning, 3460), 12-well cell slides (Thermo Fisher, 12-545-100), primary antibodies: B7-2 antibody (D-6) CD86 (Santa cruz, sc-28347), CD206 / MRC1 (E6T5J) XP Rabbit mAb (CST, 24595S), CD68 Monoclonal Antibody (FA-11) (Thermo, 14-0681-82); secondary antibodies: Goat Anti-Mouse lgG(H+L) Fluor 488-conjugated (Affinity, S0017), Goat Anti-Rabbit laG(H+) Fluor594-coniugated (Affinity, S0006), Anti-rat lgG(H+L) (Alexa Fluor@647 Conjugate) (CST, 4418S); 1×PBS (Solarbio, P1020), 4% paraformaldehyde (Solarbio, P1110), TritonX-100 (Solarbio, T8200), DAPI solution (Solarbio, 20220613), anti-fluorescence quenching mounting medium (Solarbio, S2100).

[0107] (1) After the co-culture is completed, wash the THP-1 cells in the co-culture normal group and the co-culture LPS model group with 1×PBS twice, 5 minutes each time; at room temperature, fix with 4% paraformaldehyde for 15 minutes; after fixation, wash with PBS twice, 5 minutes each time.

[0108] (2) Incubate with 0.3% TritonX-100 (prepared with 1×PBS) at room temperature for 30 minutes, and wash with 1×PBS twice, 5 minutes each time.

[0109] (3) Blocking: Block with the blocking solution (prepared with 10% NGS, 0.3% TritonX-100, 1×PBS) at room temperature for 2 hours.

[0110] (4) Prepare the primary antibody working solution with 1×PBS: Dilute all primary antibodies at a ratio of 1:800. Add 0.5 μl of B7-2 antibody (D-6) CD86, CD206 / MRC1 (E6T5J) XP Rabbit mAb, and CD68 Monoclonal Antibody to 400 μl of 1×PBS respectively, mix well, add 100 μl of the primary antibody working solution to each well to ensure complete coverage of the bottom of the plate, and incubate at 4°C overnight (≥12 hours).

[0111] (5) Let it stand at room temperature for 1 h (to make the antibody binding firmer), wash it 3 times with 1×PBS, 5 min each time.

[0112] (6) Prepare the working solution of fluorescent secondary antibody with 1×PBS. The preparation method is as follows: Dilute the secondary antibody at a ratio of 1:200. Add 4 μl of Goat Anti-Mouse lgG(H+L)Fluor488-conjugated, GoatAnti-RabbitlaG(H+L)Fluor594-coniugated, and Anti-rat lgG(H+L)(Alexa Fluor@647Conjugate) to 800 μl of 1×PBS respectively, mix well. Add 200 μl of the secondary antibody working solution to each well and incubate at room temperature in the dark for 2 h.

[0113] (7) Wash it 3 times with 1×PBS, 5 min each time. Wash it once with 500 μl of ddH2O for 5 min.

[0114] (8) Counterstain the cell nuclei with 200 μl of 100 nM DAPI for 2 min.

[0115] (9) Wash it once with 500 μl of ddH2O and mount the slides with 5 μl of anti-fluorescence quenching mounting medium. Use the confocal fluorescence microscope imaging system OLYMPUS to randomly capture 6 fields of view for each group of cells under a 1000× objective lens.

[0116] (10) Use Image J to count the number of cells (based on the number of cell nuclei) and fluorescence intensity (Integrateddensity), and compare the number of M1 and M2 macrophages in the two groups.

[0117] The results showed that in the normal co-culture group, a small number of macrophages expressed CD206+ and transformed into the M2 phenotype. In the LPS model co-culture group, the number of macrophages expressing CD206+ was significantly reduced, and the M1 phenotype differentiation showed an obvious advantage.

[0118] The macrophage phenotype results are as Figure 4 shown. According to the changes in the levels of various inflammatory factors in the co-culture system, I speculated that macrophages co-cultured with alveolar epithelial cells and vascular endothelial cells mainly differentiated into the M1 phenotype under the action of LPS. Therefore, we used immunofluorescence technology to detect the macrophage phenotype for verification. The results showed that the surface molecule CD86+ of macrophages in the LPS model group was significantly expressed, and the expression of CD206+ was weakened, indicating that macrophages co-cultured with alveolar epithelial cells and vascular endothelial cells mainly differentiated into the M1 phenotype under LPS induction and participated in the occurrence and development of the inflammatory response.

[0119] Comparative Example 1

[0120] A method for establishing an inflammatory model of monolayer cell culture in the respiratory system, comprising the following steps:

[0121] Resuscitate and subculture A549 cells (human alveolar epithelial cells) with DMEM / F12 complete medium (Gibco) containing 10% fetal bovine serum, and the culture conditions are static culture at 37 °C and 5% CO2; after the A549 cells are passaged 3 times, take the A549 cells subcultured in a 100 mm culture dish, digest them with 1 mL of trypsin digestion solution containing 0.25% EDTA, and then count. Transfer 4×10 5 A549 cells are inoculated into the culture wells of a 12-well cell culture plate and continue to be cultured. Set a normal group and an LPS model group. The LPS model group is added with 1 μg / mL LPS and acts for 24 h. The culture conditions are: static culture in a 37 °C, 5% CO2 incubator for 24 h; collect the cell culture supernatant, and detect the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the supernatant by ELISA method. The detection method is the same as that in Example 1, and the detection results are shown in Figure 2 .

[0122] The A549 cells were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0123] From Figure 2 The results showed that there was no significant difference in the levels of IL-1β, IL-6, and TNF-α between the LPS model group and the normal group, indicating that LPS acting for 24 h could not induce an inflammatory response in A549 cells.

[0124] Comparative Example 2

[0125] A method for establishing an inflammatory model of monolayer cell culture in the respiratory system, comprising the following steps:

[0126] Resuscitate and subculture HUVECs cells (human umbilical vein endothelial cells, vascular endothelial cells) with DMEM / F12 complete medium containing 10% fetal bovine serum and 1% endothelial cell growth factor (ScienCell), and the culture conditions are static culture at 37 °C and 5% CO2; after the HUVECs cells are passaged 3 times, take the HUVECs cells subcultured in a 100 mm culture dish, digest them with 1 mL of trypsin digestion solution containing 0.25% EDTA, and then count. Transfer 4×10 5 HUVECs cells are inoculated into the culture wells of a 12-well cell culture plate and continue to be cultured. Set a normal group and an LPS model group. The LPS model group is added with 1 μg / mL LPS and acts for 24 h. The culture conditions are: static culture in a 37 °C, 5% CO2 incubator for 24 h; collect the cell culture supernatant, and detect the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the supernatant by ELISA method. The detection method is the same as that in Example 1, and the detection results are shown in Figure 2。

[0127] HUVECs cells were purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0128] From Figure 2 The results showed that IL-6 in the LPS model group was significantly higher than that in the normal group, and there was no significant difference in the levels of IL-1β and TNF-α between the LPS model group and the normal group.

[0129] Comparative Example 3

[0130] A method for establishing an inflammatory model of monolayer cell culture of the respiratory system, comprising the following steps:

[0131] Resuscitate and passage THP-1 cells (human monocytes / macrophages) with RPMI 1640 complete medium (Gibco) containing 10% fetal bovine serum and 0.1 mM / L β-mercaptoethanol, and the culture conditions are static culture at 37 °C and 5% CO2; after THP-1 cells are passaged 3 times, take 1 × 106 THP-1 cells cultured in a 75 cm 2 1×106 THP-1 cells in the culture flask were induced with 20 ng / mL phorbol 12-myristate 13-acetate (PMA), and 4×10 5 induced THP-1 cells were seeded in the culture wells of a cell culture plate and continued to be cultured. A normal group and an LPS model group were set up. The LPS model group was added with 1 μg / mL LPS and acted for 24 h. The culture conditions were: static culture in a 37 °C and 5% CO2 incubator for 24 h; collect the cell culture supernatant, and detect the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the supernatant by ELISA method. The detection method is the same as that in Example 1, and the detection results are shown in Figure 2 。

[0132] THP-1 cells were purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0133] From Figure 2 The results showed that IL-1β, IL-6, and TNF-α in the LPS model group were significantly higher than those in the normal group. It shows that LPS acting for 24 h can induce an inflammatory response in THP-1 cells.

[0134] From Figure 2 It can be seen that under LPS induction, compared with the monolayer culture of A549 cells, HUVECs cells, and THP-1 cells, the levels of inflammatory factors IL-1β, IL-6, and TNF-α secreted by the co-cultured A549 cells / HUVECs cells / THP-1 cells were significantly increased. It shows that when the three cells are co-cultured, under the action of LPS, the interaction between cells promotes and amplifies the inflammatory response.

[0135] Comparative Examples 1 to 3 respectively established a monolayer cell culture inflammation model of the respiratory system. Alveolar epithelial cells, vascular endothelial cells, and macrophages are the main cells involved in the inflammatory response of respiratory diseases and are widely used in the establishment of in vitro models. However, in these three monolayer cell culture inflammation models of the respiratory system, each model contains only one type of cell, making it difficult to simulate the microenvironment of multicellular interactions during the occurrence of respiratory diseases and impossible to explore the interactions between cells.

[0136] Compared with Comparative Examples 1 to 3, the method for establishing a multicellular co-culture inflammation model of the respiratory system provided in Example 1 is to co-culture the three main cells involved in the inflammatory response of respiratory diseases in the same culture system, which can simulate the microenvironment of multicellular interactions during the occurrence of respiratory diseases, and the three types of cells can be separately detected to detect the mutual influence between different cells. In Comparative Examples 1 to 3, the same method was used to detect the levels of inflammatory factors secreted by cells cultured alone and co-cultured cells. The results showed that compared with the inflammatory factors secreted by cells cultured alone, the inflammatory factors secreted by co-cultured cells exceeded the sum of the inflammatory factors secreted by the three types of cells cultured alone, indicating that LPS induction can amplify the inflammatory response of co-cultured cells.

[0137] Comparative Example 4

[0138] A method for establishing a multicellular culture inflammation model of the respiratory system, which is different from Example 1 in that: the co-culture time is different. The co-culture time after adding 1 μg / mL LPS in the co-culture LPS model group is 6 h; the co-culture time of the co-culture normal group is 6 h, and the method includes the following steps:

[0139] The cell seeding and treatment of the co-culture normal group are as follows:

[0140] Take the HUVECs cells subcultured in a 100 mm culture dish, digest them with 1 mL of trypsin digestion solution containing 0.25% EDTA (Solarbio, T1320), count them, and inoculate 4×10 5 HUVECs cells on the outer side of the PET membrane (the pore size of the PET membrane is 0.4 μm) at the bottom of the Transwell chamber. The Transwell chamber is inverted in the culture well of a 12-well cell culture plate. After 6 h, when the HUVECs cells adhere to the wall, place the Transwell chamber upright in the culture well of the above-mentioned cell culture plate with the corresponding pore size; take the A549 cells subcultured in a 100 mm culture dish, digest them with 1 mL of trypsin digestion solution containing 0.25% EDTA, count them, and inoculate 4×10 5 A549 cells on the inner side of the PET membrane at the bottom of the above-mentioned Transwell chamber inoculated with HUVECs cells; take the above-mentioned cells subcultured in a 75 cm 21×10 in a culture flask 6 THP-1 cells were induced with 20 ng / mL phorbol 12-myristate 13-acetate (PMA), and 4×10 5 induced THP-1 cells were seeded into the culture wells of a cell culture plate. A Transwell chamber seeded with A549 cells and HUVECs was placed into the culture well of the cell culture plate seeded with THP-1 cells for co-culture, obtaining a multi-cell co-culture system. The co-culture conditions were: static culture for 6 h in an incubator at 37°C and 5% CO2.

[0141] The cell seeding and treatment of the co-culture LPS model group were as follows:

[0142] HUVECs cells sub-cultured in a 100 mm culture dish were digested with 1 mL of trypsin digestion solution containing 0.25% EDTA and then counted. 4×10 5 HUVECs cells were seeded on the outer side of the PET membrane (with a pore size of 0.4 μm) at the bottom of the Transwell chamber. The Transwell chamber was inverted in the culture well of a 12-well cell culture plate. After 6 h, when the HUVECs cells adhered to the wall, the Transwell chamber was placed upright in the culture well of the corresponding pore size of the above cell culture plate; A549 cells sub-cultured in a 100 mm culture dish were digested with 1 mL of trypsin digestion solution containing 0.25% EDTA, counted, and 4×10 5 A549 cells were seeded on the inner side of the PET membrane at the bottom of the above Transwell chamber seeded with HUVECs cells; 1×10 2 THP-1 cells in a 75 cm 6 culture flask were induced with 20 ng / mL phorbol 12-myristate 13-acetate (PMA), and 4×10 5 induced THP-1 cells were seeded into the culture wells of a cell culture plate. A Transwell chamber seeded with A549 cells and HUVECs was placed into the culture well of the cell culture plate seeded with THP-1 cells for co-culture. Meanwhile, 1 μg / mL LPS was added to the culture plate and acted for 6 h. The cell culture supernatant was collected, and the levels of inflammatory factors IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, TNF-α, and IFN-γ in the supernatant were detected by ELISA. The detection method was the same as in Example 1, and the detection results are shown in Figure 1 .

[0143] The three types of cells were purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0144] Comparative Example 5

[0145] A method for establishing a multi - cell culture inflammation model of the respiratory system, different from Comparative Example 4 in that: the co - culture time is different. After adding 1 μg / mL LPS to the co - culture LPS model group, the co - culture time is 12 h; the co - culture time of the co - culture normal group is 12 h, and the remaining steps are the same as those in Comparative Example 4. The test results are shown in Figure 1 .

[0146] Comparative Example 6

[0147] A method for establishing a multi - cell culture inflammation model of the respiratory system, different from Comparative Example 4 in that: the co - culture time is different. After adding 1 μg / mL LPS to the co - culture LPS model group, the co - culture time is 48 h; the co - culture time of the co - culture normal group is 48 h, and the remaining steps are the same as those in Comparative Example 4. The test results are shown in Figure 1 .

[0148] From Figure 1 the results, it can be seen that the levels of IL - 1β, IL - 6, TNF - α, IFN - γ, and IL - 10 gradually increase with the extension of time; when LPS acts for 24 h, the levels of IL - 1β, TNF - α, IL - 6, IL - 12, IFN - γ, and IL - 8 are significantly higher than those of the normal group; the levels of IL - 6, IL - 12, and IFN - γ reach the peak at 24 h. It shows that when LPS acts on the co - cultured A549 cells / HUVECs cells / THP - 1 cells for 24 h, the inflammatory reaction is the most significant, and 24 h is the best time point for establishing the model. It indicates that the co - culture time in the method for establishing the multi - cell culture inflammation model of the respiratory system provided by the present invention is the optimal time.

[0149] The present invention provides a method for establishing a multi-cell co-culture inflammation model of the respiratory system. A multi-cell co-culture inflammation model of the respiratory system is established by this method, which can be used to simulate the inflammatory microenvironment of the respiratory system. We used Luminex multi-factor detection technology to detect the levels of 9 inflammatory factors in the co-culture system. The results showed that macrophages co-cultured with alveolar epithelial cells and vascular endothelial cells mainly differentiated into the M1 phenotype under LPS induction, mediating the inflammatory response. On this basis, we screened out inflammatory factors IL-1β, IL-6, and TNF-α with high expression levels from 9 inflammatory factors, and there were significant differences between the model group and the normal group. The ELISA method was used for detection to verify the above results. The results showed that compared with the co-cultured normal group, the levels of IL-1β, IL-6, and TNF-α in the co-cultured LPS model group were significantly increased, indicating that LPS can induce the inflammatory response of co-cultured cells. At the same time, consistent results were obtained by using two different methods, Luminex multi-factor detection technology and ELISA, which demonstrated the feasibility of the co-culture model. The respiratory system multi-cell co-culture inflammation model established by the present invention can overcome the problems existing in monolayer cell culture and is used to simulate the microenvironment of multi-cell interaction when respiratory diseases occur.

[0150] In summary, the co-culture inflammation model constructed by the present invention can better simulate the microenvironment of multi-cell interaction when the disease occurs than the monolayer cell or two-cell co-culture model, and can provide a tool for the pathogenesis of respiratory diseases with inflammatory response as the main pathological mechanism, drug action mechanism, drug screening and new drug development.

[0151] The present invention provides a method for establishing a multi-cell co-culture inflammation model of the respiratory system. The present invention establishes an alveolar epithelial cell / macrophage / vascular endothelial cell co-culture inflammation model that simulates the respiratory system inflammation microenvironment, providing a tool for the study of the pathogenesis of respiratory diseases, the mechanism of drug action, and the development of new drugs.

[0152] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0153] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0154] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for establishing a multi - cell co - culture inflammation model of the respiratory system, characterized in that, It includes the following steps: S1. Separately perform cell culture on human alveolar epithelial cells, human umbilical vein endothelial cells, and human monocytes / macrophages; S2. Seed the human umbilical vein endothelial cells after the culture is completed on the outer side of the bottom of the Transwell chamber at a density of 3 - 5×10 5 cells / well. Invert the Transwell chamber and place it in the culture well, and culture for 5 - 7 h under the same culture conditions as in S1. After the human umbilical vein endothelial cells adhere to the wall, place the Transwell chamber upright in the culture well. After the cultivation is completed, human alveolar epithelial cells are inoculated at a density of 3-5×10 5 cells / well on the inner side of the bottom of the Transwell chamber and cultured for 11-13 h under the same culture conditions as S1; After culturing, human monocytes / macrophages were induced with phorbol ester and then seeded into culture wells at a density of 3 - 5×10 5 cells / well, and cultured under the same culture conditions as S1 for 23 - 25 h; S3. Place the Transwell chamber inoculated with human alveolar epithelial cells and human umbilical vein endothelial cells into the culture well inoculated with human monocytes / macrophages for co-culture to obtain a multi-cell co-culture system; S4. Use an inflammation inducer to act on the multi-cell co-culture system after co-culture in S4 to obtain the inflammation model.

2. The method for establishing a multi-cellular co-culture inflammation model of the respiratory system according to claim 1, characterized in that In S1, human alveolar epithelial cells are separately cultured in a complete medium of DMEM / F12 containing 9-11% fetal bovine serum; human umbilical vein endothelial cells are separately cultured in a complete medium of DMEM / F12 containing 9-11% fetal bovine serum and 1% endothelial cell growth factor; human monocytes / macrophages are separately cultured in a complete medium of RPMI 1640 containing 9-11% fetal bovine serum and 0.05-0.15 mM / L β-mercaptoethanol.

3. The method for establishing a multi-cellular co-culture inflammation model of the respiratory system according to claim 2, characterized in that, The cell culture conditions for separately culturing human alveolar epithelial cells, human umbilical vein endothelial cells, and human monocytes / macrophages are all: static culture at 36-38 °C and 4-6% CO2.

4. The method for establishing a multi-cellular co-culture inflammation model of the respiratory system according to claim 1, characterized in that, In S2, the bottom of the Transwell chamber is a structure made of a transparent polyester membrane, and the pore size of the transparent polyester membrane is 0.2-0.5 μm.

5. The method for establishing a multi-cellular co-culture inflammation model of the respiratory system according to claim 1, wherein In S3, the steps for preparing the co-culture medium for co-culture are as follows: Weigh L-glutamine, HEPES, penicillin, streptomycin, and β-mercaptoethanol respectively, add distilled water, and mix well to make their final concentrations 298-302 mg / L L-glutamine, 5956-5960 mg / L HEPES, 98-102 U / mL penicillin, 98-102 μg / mL streptomycin, 0.08-0.12 mM / L β-mercaptoethanol, then add 8-12 mL / L endothelial growth factor and 98-102 mL / L drug-containing serum, mix well, and sterilize to obtain the co-culture medium.

6. The method for establishing a multi-cellular co-culture inflammation model of the respiratory system according to claim 5, characterized in that, The co-culture conditions are: culture at 36-38 °C and 4-6% CO2 for 22-26 h.

7. The method for establishing a multi - cell co - culture inflammation model of the respiratory system according to claim 1, wherein, In S4, the inflammation inducer is lipopolysaccharide.

8. The method for establishing a multi - cell co - culture inflammation model of the respiratory system according to claim 7, wherein, The concentration of the lipopolysaccharide is 0.8-1.2 μg / mL.

9. The method for establishing a multi-cellular co-culture inflammation model of the respiratory system according to claim 1, characterized in that, In S4, after culturing for 22-26 h after adding lipopolysaccharide, collect the supernatant of the cell culture solution of the multi-cell co-culture system, centrifuge at 1000 rpm / min for 4-6 min, collect the supernatant, and detect inflammatory factors.

10. The method for establishing a multi-cellular co-culture inflammation model of the respiratory system according to claim 9, wherein, The inflammatory factors include IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IL-13, TNF-α, IFN-γ.

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