Pathological pulmonary fibrosis model, construction method therefor, and use thereof

Abstract

The present invention relates to a pathological pulmonary fibrosis model, a construction method therefor, and a use thereof. The pathological pulmonary fibrosis model provided by the present invention has good stability and controllability and high maturity, can better simulate the function and structural characteristics of the alveolar-capillary barrier, and has the pathological characteristics of pulmonary fibrosis lesions and the immune microenvironment. The model has the advantage of high simulation degree, and has good application prospects in the aspects of simulation and research of pulmonary fibrosis diseases, as well as drug detection, screening, assessment, and research and development, and the like.

Description

Pathological pulmonary fibrosis models, their construction methods and applications Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pathological pulmonary fibrosis model and its construction method, the use of the pathological pulmonary fibrosis model in drug detection, drug screening and / or detection of the influence of pathogen infection on the development of pulmonary fibrosis, a culture medium combination, and a drug detection method, a drug screening method and / or a method for detecting the influence of pathogen infection on the development of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a type of end-stage interstitial lung disease. Many factors are known to contribute to pulmonary fibrosis, including toxicity, autoimmune disorders, drug-induced fibrosis, infectious diseases, and traumatic injury. Pulmonary fibrosis is characterized by fibroblast proliferation, extracellular matrix accumulation, inflammatory damage, and destruction of tissue structure. Studies show that the median survival of patients with pulmonary fibrosis is only 2–5 years, with a 5-year survival rate of only 20%.

[0003] Previous research on pulmonary fibrosis has mainly focused on 2D in vitro culture of fibroblasts and animal models. However, these approaches each have their drawbacks. When fibroblasts are cultured in petri dishes or on slides (2D), they cannot adequately demonstrate cell structure, adhesion, mechanotransduction, and signal transduction of soluble cytokines, nor can they simulate cell-cell / matrix interactions in vivo. As for animal models, due to species differences and the fact that artificially induced fibrosis can only simulate a certain stage of the disease progression, failing to represent the entire development process, most drugs that have shown promise in alleviating fibrosis in animal models have failed in phase II / III clinical trials.

[0004] Therefore, it is extremely urgent to find new in vitro models of pulmonary fibrosis that can better simulate the lung microenvironment in vivo for research on pulmonary fibrosis and drug screening. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a pathological pulmonary fibrosis model. The pathological pulmonary fibrosis model constructed by this method can not only better simulate the function and structural characteristics of the alveolar-capillary barrier, but also have the pathological characteristics of pulmonary fibrosis lesions and the immune microenvironment, thereby being used for research on pulmonary fibrosis diseases, drug screening, etc.

[0006] Specifically, in a first aspect, the present invention provides a method for constructing a pathological pulmonary fibrosis model, comprising:

[0007] Provides lung organoids and cell culture containers derived from patients with pulmonary fibrosis, and optionally lung fibroblasts derived from patients with pulmonary fibrosis;

[0008] The lung organoids are seeded in the upper layer of the cell culture container, and optionally the lung fibroblasts are seeded in the upper layer of the cell culture container. Then, lung epithelial cell culture medium is added to the upper and lower layers of the cell culture container to culture the lung organoids and optionally the lung fibroblasts in liquid-liquid mode.

[0009] The lung epithelial cell culture medium in the upper and lower layers is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to perform a first-stage gas-liquid culture of the lung organoids and optionally the lung fibroblasts.

[0010] Endothelial cells were seeded in the lower layer of the cell culture container, and macrophages were seeded in the upper layer of the cell culture container and cultured in a second-stage gas-liquid mode to obtain a pathological pulmonary fibrosis model.

[0011] In this invention, the cell culture container is a culture container that can be divided into at least upper and lower (sometimes also referred to as upper and lower chambers) culture units, thereby enabling cell migration or allowing different cells to grow in different layers or even attach to a membrane (e.g., a porous membrane) located between the upper and lower layers as needed. Correspondingly, the resulting pathological pulmonary fibrosis model can also be divided into an upper layer (i.e., the upper layer of the model) and a lower layer (i.e., the lower layer of the model). In some embodiments, the cell culture container is a Transwell plate or a cell culture chip, such as a membrane chip or a barrier chip. When a cell culture chip is used, the resulting pathological pulmonary fibrosis model is also called a lung-on-a-chip (LOC) model. In some specific embodiments, the membrane chip is, for example, those disclosed in application numbers 202321349258.3, 202330709231.X, or 202222062961.8, and the barrier chip is, for example, those disclosed in application numbers 202211680988.1, 202222130269.4, or 202222130382.2, and these application documents are incorporated herein by reference in their entirety.

[0012] In this invention, the lung organoids can be primary lung organoids extracted from pulmonary fibrosis tissue derived from patients with pulmonary fibrosis. The patients with pulmonary fibrosis can be any mammal suffering from pulmonary fibrosis, such as humans, domestic animals, farm animals, and animals found in zoos, sports fields, or as pets, including dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0013] In this invention, the lung fibroblasts can be optionally used to construct the pathological pulmonary fibrosis model. Specifically, when the lung organoids are of passages P1-P3, the pathological pulmonary fibrosis model can be constructed without inoculating the lung fibroblasts; when the lung organoids are of passages P4 and later, the pathological pulmonary fibrosis model must be constructed with inoculation of the lung fibroblasts.

[0014] Therefore, specifically, the present invention provides a method for constructing a specific pathological pulmonary fibrosis model, which includes:

[0015] Provides lung organoids and cell culture containers derived from patients with pulmonary fibrosis;

[0016] The lung organoids were seeded into the upper layer of the cell culture container, and then lung epithelial cell culture medium was added to the upper and lower layers of the cell culture container to culture the lung organoids in liquid-liquid mode.

[0017] The lung epithelial cell culture medium in the upper and lower layers is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to carry out the first stage of gas-liquid mode culture of the lung organoids.

[0018] Endothelial cells were seeded in the lower layer of the cell culture container, and macrophages were seeded in the upper layer of the cell culture container and cultured in a second-stage gas-liquid mode to obtain a pathological pulmonary fibrosis model.

[0019] Another specific method for constructing a pathological pulmonary fibrosis model provided by the present invention includes:

[0020] Provides lung organoids and cell culture containers derived from patients with pulmonary fibrosis, as well as lung fibroblasts derived from patients with pulmonary fibrosis;

[0021] The lung organoids and the lung fibroblasts were seeded in the upper layer of the cell culture container, and then lung epithelial cell culture medium was added to the upper and lower layers of the cell culture container to culture the lung organoids and the lung fibroblasts in liquid-liquid mode.

[0022] The lung epithelial cell culture medium in the upper and lower layers is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to carry out the first stage gas-liquid mode culture of the lung organoids and the lung fibroblasts.

[0023] Endothelial cells were seeded in the lower layer of the cell culture container, and macrophages were seeded in the upper layer of the cell culture container and cultured in a second-stage gas-liquid mode to obtain a pathological pulmonary fibrosis model.

[0024] In this invention, "liquid-liquid culture" refers to a culture mode in which liquid (e.g., culture medium) is added to both the upper and lower layers of the cell culture container. "Gas-liquid culture" generally refers to a culture mode in which liquid (e.g., culture medium) is added only to the lower layer of the cell culture container, while the upper layer remains dry.

[0025] In the construction method of this invention, primary extraction and culture of lung organoids and optionally lung fibroblasts from patients with pulmonary fibrosis can replace traditional cell lines to construct a pathological pulmonary fibrosis model. By first culturing lung organoids and optionally lung fibroblasts in a liquid-liquid mode and then in a gas-liquid mode (i.e., the first stage of gas-liquid mode culture), and then seeding them in the order of endothelial cells and macrophages, and then culturing macrophages in the second stage of gas-liquid mode, it is beneficial to make the pathological pulmonary fibrosis model more mature and improve its simulation.

[0026] In some embodiments, the lung organoids are treated with a primary lung fibrosis tissue digestion solution before being seeded onto the upper layer of the cell culture vessel. In this invention, the primary lung fibrosis tissue digestion solution can be a commercially available primary lung tissue digestion solution. In some preferred embodiments, the primary lung fibrosis tissue digestion solution contains collagenase, hyaluronidase, Y27632 (a ROCK inhibitor), and culture medium.

[0027] In some preferred embodiments, the collagenase includes at least one of collagenase I, collagenase II, and collagenase IV.

[0028] In some preferred embodiments, the final concentration of the collagenase in the digestive fluid of the primary pulmonary fibrosis tissue is 1–2 mg / mL.

[0029] In some preferred embodiments, the final concentration of the hyaluronidase in the digestion fluid of the primary pulmonary fibrosis tissue is 0.5 to 1 mg / mL.

[0030] In some preferred embodiments, the final concentration of Y27632 in the digestion fluid of the primary pulmonary fibrosis tissue is 5–10 μM.

[0031] In some preferred embodiments, the culture medium is selected from DMEM or ADMEM / F12 (Advanced DMEM / F12).

[0032] In some embodiments, the lung organoids can be extracted using methods known in the art, preferably using the following steps:

[0033] (1) Washing: The pulmonary fibrosis tissue from patients with pulmonary fibrosis was washed with a buffer solution to remove the surface mucosa; the buffer solution preferably contained penicillin and streptomycin.

[0034] (2) Crushing: mince the tissue into a paste-like consistency, resuspend it in buffer solution, let it stand, and then discard the supernatant.

[0035] (3) Digestion treatment: Add the digestion solution of primary pulmonary fibrosis tissue, mix well by pipetting, and then use a metal bath shaker at 37°C (other similar equipment can be used) to shake and digest for 30-60 minutes to digest into small cell clusters. Finally, add buffer solution to stop digestion.

[0036] (4) Resuspension: The digested cell suspension was filtered through a 100 μm cell sieve, and the cells were resuspended in lung organoid culture medium and counted; wherein, the lung organoid culture medium can be a commercial lung organoid culture medium, such as the respiratory organoid amplification medium (catalog number A01-001) produced by Bogeng Company, or the lung organoid culture kit (catalog number KLU0201) produced by Ivyde Company.

[0037] (5) Plating: Centrifuge, resuspend lung organoids in extracellular matrix (Matrigel, BME, etc.), and plate them. Add lung organoid culture medium and incubate at 37°C.

[0038] In some embodiments, the lung fibroblasts are treated with a digestive solution of primary pulmonary fibrosis tissue before being seeded onto the upper layer of the cell culture vessel. The digestive solution of primary pulmonary fibrosis tissue has been described above and will not be repeated here.

[0039] In some embodiments, the lung fibroblasts can be extracted using methods known in the art, preferably using the following steps:

[0040] (1) Washing: The pulmonary fibrosis tissue from patients with pulmonary fibrosis was washed with a buffer solution to remove the surface mucosa; the buffer solution preferably contained penicillin and streptomycin.

[0041] (2) Crushing: mince the tissue into a paste-like consistency, resuspend it in buffer solution, let it stand, and then discard the supernatant.

[0042] (3) Digestion treatment: Add the digestion solution of primary pulmonary fibrosis tissue, mix well by pipetting, and then use a metal bath shaker at 37°C (other similar equipment can be used) to shake and digest for 30-60 minutes until single cells are digested. Finally, add buffer solution to stop digestion.

[0043] (4) Resuspension: The digested cell suspension was filtered through a 70 μm cell sieve and the cells were resuspended in lung fibroblast medium; wherein, the lung fibroblast medium can be a commercial lung fibroblast medium, such as #AC-1001015 produced by EZEN Biotech.

[0044] (5) Plating: Plating lung fibroblasts onto six-well plates, T25 cell culture flasks or T75 cell culture flasks.

[0045] In some embodiments, the cell culture vessel may be pretreated before inoculating various types of cells to facilitate cell adhesion. Pretreatment methods may be those known in the art, such as pretreatment with a solution comprising an extracellular matrix, wherein the extracellular matrix may be Matrigel, collagen, fibronectin, etc., and is not limited thereto.

[0046] In some implementations, prior to inoculation, the lung organoids are digested using organoid passage digestion solutions (trypsin, Tryple, or Accutase cell digestion solutions, etc.) to form single cells. Then, lung organoid culture medium is added to terminate the digestion before inoculation.

[0047] In some embodiments, in the step of seeding the lung organoids onto the upper layer of the cell culture vessel, the lung organoids are seeded at a rate of 1 × 10⁻⁶ m² / g², calculated by the seeding area (e.g., the area of ​​the membrane in a cell culture chip). 5 ~10×10 5 cells / cm 2 .

[0048] In some embodiments, in the step of seeding the lung fibroblasts onto the upper layer of the cell culture container, the lung organoids are seeded at a rate of 0.3 × 10⁻⁶, calculated by the seeding area (e.g., the area of ​​the membrane in a cell culture chip). 3 ~0.3×10 4 cells / cm 2 .

[0049] In some embodiments, during the step of adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container, the lung epithelial cell culture medium may be a commercially available lung epithelial cell culture medium. In this invention, the lung epithelial cell culture medium includes lung organoid culture medium. In some specific embodiments, the lung epithelial cell culture medium is, for example, respiratory organoid amplification medium (catalog number A01-001) manufactured by Bogeng Pharmaceuticals, lung organoid culture kit (catalog number KLU0201) manufactured by Ivytec Pharmaceuticals, or epithelial cell culture medium (PneumaCult) manufactured by Stemcell Pharmaceuticals. TM -Ex Plus Medium).

[0050] In some embodiments, in the step of adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container, 200-300 μL of lung epithelial cell culture medium is added to the upper layer, and 800 μL-1 mL of lung epithelial cell culture medium is added to the lower layer.

[0051] In some embodiments, the liquid-liquid culture time for the lung organoids and optionally the lung fibroblasts is 3 to 7 days, for example, 3, 4, 5, 6, 7 days or any time within this range. In some specific embodiments, the liquid-liquid culture temperature is 37°C. In some specific embodiments, the medium is changed every 3 days during liquid-liquid culture.

[0052] In some implementations, when the lung organoids have grown to fill the upper layer, the culture medium for the lung epithelial cells in the upper and lower layers is discarded.

[0053] In some embodiments, in the step of adding fresh lung epithelial cell culture medium to the lower layer, 600-700 μL of lung epithelial cell culture medium is added to the lower layer.

[0054] In some embodiments, the first stage of gas-liquid culture of the lung organoids is carried out for 2 to 21 days, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or any time within this range. In some specific embodiments, the temperature for the first stage of gas-liquid culture is 37°C.

[0055] In some embodiments, endothelial cells are seeded in a lower step of the cell culture vessel, with the seeding area calculated as 0.5 × 10⁻⁶ cells / cm². 5 ~5×10 5 cells / cm 2 .

[0056] In some implementations, the endothelial cells are derived from umbilical vein endothelial cells, primary lung endothelial cells, or lung endothelial cell lines.

[0057] In some embodiments, during the step of seeding endothelial cells into the lower layer of the cell culture vessel, after the endothelial cells have adhered to the vessel, a first mixed culture medium is added to the lower layer. The first mixed culture medium is a mixture of lung epithelial cell culture medium and endothelial cell culture medium at a ratio of (0.5–3.5):1; for example, the lung epithelial cell culture medium and endothelial cell culture medium are mixed at ratios of 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, or 3.5:1. The endothelial cell culture medium can be a commercially available medium, such as endothelial cell culture medium (catalog number #1001) manufactured by Sciencell. Preferably, the endothelial cells are digested, resuspended in the first mixed culture medium, and their cell density is adjusted before seeding. In some specific embodiments, 600–700 μL of the first mixed culture medium is added to the lower layer.

[0058] In some embodiments, in the step of seeding macrophages onto the upper layer of the cell culture vessel and performing a second-stage gas-liquid culture, the macrophage seeding area is calculated as 0.1 × 10⁻⁶ m² / g². 5 ~5×10 5 cells / cm 2 In some specific implementations, the macrophages are resuspended and their density adjusted using macrophage culture medium. The macrophage culture medium can be selected according to the specific source of the macrophages, such as macrophage culture medium (catalog number #1921) manufactured by ScienCell.

[0059] In some implementations, the macrophages are derived from macrophages expanded from PBMCs, primary alveolar macrophages, passaged alveolar macrophages, or macrophages induced by the THP-1 cell line.

[0060] In some embodiments, in the step of seeding macrophages into the upper layer of the cell culture vessel and performing a second-stage gas-liquid culture, the macrophages are resuspended in macrophage culture medium, seeded into the upper layer of the cell culture vessel, and after adhering to the vessel wall, the culture media of the upper and lower layers are discarded and the upper layer is kept dry. Fresh first mixed culture medium is added to the lower layer for the second-stage gas-liquid culture. The first mixed culture medium is a mixture of lung epithelial cell culture medium and endothelial cell culture medium at a ratio of (0.5–3.5):1. In some specific embodiments, 600–700 μL of fresh first mixed culture medium is added to the lower layer.

[0061] In some implementations, the second-stage gas-liquid culture of the lung organoids is carried out for 1 to 5 days, such as 1 day, 2 days, 3 days, 4 days, 5 days, or any time within that range.

[0062] In some implementations, after the macrophages are seeded in the upper layer of the cell culture container and cultured in a second-stage gas-liquid mode, the method further includes the step of seeding immune cells in the lower layer of the cell culture container and culturing them, thereby enabling the constructed pathological pulmonary fibrosis model to have immune function.

[0063] In some implementations, the immune cells include PBMCs, T cells, B cells, and NK cells.

[0064] In some implementations, the inoculation area (e.g., the area of ​​the membrane in a cell culture chip) is calculated as 0.5 × 10⁻⁶. 5 ~1×10 6 cells / cm 2 .

[0065] In some embodiments, in the step of seeding immune cells into the lower layer of the cell culture container and culturing them, the immune cells are resuspended in a second mixed culture medium to obtain a suspension. The upper layer is kept dry, the culture medium in the lower layer is discarded, and then the suspension is added. A third-stage gas-liquid culture is then performed in a gas-liquid mode. The second mixed culture medium is obtained by mixing the immune cell culture medium and the first mixed culture medium at a volume ratio of (0.5 to 3.5):1; for example, the immune cell culture medium and the first mixed culture medium are mixed at a ratio of 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, or 3.5:1. The immune cell culture medium can be a commercially available immune cell culture medium, such as PBMC medium. A commercially available PBMC medium, such as SuperCulture L100 serum-free lymphocyte medium (Dakco, catalog number #6122011) supplemented with 5-10% SuperGrow cell culture additive and 1-5‰ IL-2, is used.

[0066] In some embodiments, the step of seeding immune cells into the lower layer of the cell culture container and culturing them for 1 to 3 days, such as 1 day, 2 days, 3 days or any time within that range.

[0067] Secondly, the present invention provides a pathological pulmonary fibrosis model, which is constructed by the method for constructing the pathological pulmonary fibrosis model described above in the first aspect.

[0068] Thirdly, the present invention provides a culture medium composition comprising a lung epithelial cell culture medium, a first mixed culture medium, and optionally a second mixed culture medium;

[0069] The first mixed culture medium is obtained by mixing lung epithelial cell culture medium and endothelial cell culture medium at a volume ratio of (0.5-3.5):1; the second mixed culture medium is obtained by mixing immune cell culture medium and the first mixed culture medium at a volume ratio of (0.5-3.5):1; the lung epithelial cell culture medium, the endothelial cell culture medium and the immune cell culture medium are as described above, and will not be repeated here.

[0070] In some embodiments, the culture medium combination is used in the construction method provided by the present invention, or in the construction of the pathological pulmonary fibrosis model provided by the present invention, including for the culture of lung organoids and lung fibroblasts, liquid-liquid mode culture, first-stage gas-liquid mode culture, second-stage gas-liquid mode culture, and optionally third-stage gas-liquid mode culture.

[0071] In some preferred embodiments, the lung epithelial cell culture medium is used for the culture of the lung organoids and the lung fibroblasts, the liquid-liquid mode culture and / or the first stage gas-liquid mode culture.

[0072] In some preferred embodiments, the first mixed culture medium is used in the second stage gas-liquid mode.

[0073] In some preferred embodiments, the second mixed culture medium is used for the third stage of gas-liquid mode culture.

[0074] Fourthly, the present invention provides the use of a pathological pulmonary fibrosis model constructed by the construction method provided in the first aspect, or the pathological pulmonary fibrosis model described in the second aspect, in the following (a) or (b);

[0075] (a) Drug testing and / or drug screening;

[0076] (b) Detect the impact of pathogen infection on the progression of pulmonary fibrosis.

[0077] In some implementations, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs.

[0078] In some implementations, the pathogens include bacteria, viruses, fungi, parasites, etc.

[0079] In some implementations, the drug testing and / or drug screening includes drug testing and / or drug screening for the treatment of pulmonary fibrosis.

[0080] The pathological pulmonary fibrosis model provided by this invention can be used for the efficacy testing and mechanism exploration of drugs for treating lung diseases (e.g., drugs for treating pulmonary fibrosis). It assesses the response of the pathological pulmonary fibrosis model to drugs by evaluating changes in cell marker expression, cell (e.g., alveolar cell) ratio, cell activity, inflammatory factor secretion, immune cell typing, and cell barrier function. This data can then be used for drug research and development, particularly for drugs treating pulmonary fibrosis. For example, by adding a test drug, it can be tested to determine whether the drug accelerates or inhibits the progression of pulmonary fibrosis, thereby detecting and / or screening drugs for treating pulmonary fibrosis. Furthermore, after pathogen infection of the pathological pulmonary fibrosis model (upper or lower layer), the influence of pathogens on the progression of pulmonary fibrosis can be confirmed through indicator detection. Additionally, after inducing a pathological pulmonary fibrosis model, drugs can be administered and indicator detection performed to determine whether the drug accelerates or inhibits the progression of pulmonary fibrosis, thereby detecting and / or screening drugs for treating pulmonary fibrosis. The following are some examples (i) to (xii) of the use of the pathological pulmonary fibrosis model described in (a) or (b) of the present invention. It should be understood that the application of the pathological pulmonary fibrosis model described in the present invention is not limited to these:

[0081] (i) Changes in the expression of pulmonary fibrosis markers: The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and the changes in the expression of pulmonary fibrosis markers in the pathological pulmonary fibrosis model are detected; wherein, the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin. In some specific embodiments, the method includes: fixing the pathological pulmonary fibrosis model with 4% paraformaldehyde at room temperature for 30 min, removing the paraformaldehyde, washing the sample with PBS, permeabilizing with 0.1% Triton-X-100 and blocking the sample with 5% BSA, adding primary antibodies against α-SMA, type I collagen, and fibronectin respectively, incubating overnight at 4°C, adding secondary antibodies, incubating at room temperature for 1 h, staining the nuclear surface with DAPI for 15 min, washing with PBS, and imaging using confocal microscopy.

[0082] (ii) Flow cytometry detection of changes in immune cell typing: The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model. Flow cytometry is used to detect changes in cell typing and / or dynamic immune response processes in the pathological pulmonary fibrosis model. The biomarkers detected by the flow cytometry include, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, and CRTH2. The changes in cell typing include changes in macrophage typing and / or changes in immune cell typing. The changes in macrophage typing include, for example, changes in M1 macrophage typing and M2 macrophage typing. The changes in immune cell typing include, for example, changes in T cell typing, changes in B cell typing, changes in NK cell typing, and changes in intrinsic lymphocyte typing. In some specific implementation schemes, the following methods are included: flow cytometry analysis of changes in M1 and M2 macrophage typing in the upper layer of cells of the model, with flow cytometry indicators including, but not limited to, CD45, CD80, HLA-DR, CD163, CD68, and CD206; or flow cytometry analysis of changes in immune cell typing (T cells, B cells, NK cells, and intrinsic lymphocytes) in the lower layer of cells of the model, with flow cytometry indicators including, but not limited to, CD45, CD3, CD4, CXCR3, CCR4 (Th1 and Th2 cells), CD19 (B cells), Lin, CD127, CD94, CD117, and CRTH2 (ILC cells). The procedure is as follows: collect cells, digest them into single cells using Tryple, Trypsin, or Accutas, and resuspend the cells in flow cytometry buffer. Stain the surface with antibodies against CD45, CD80, HLA-DR, CD163, CD3, and CD4 at 4°C for 30 minutes. Add cell fixation solution and fix at room temperature in the dark for 10 minutes. After centrifugation and washing with PBS, resuspend the cells using a cell permeation reagent and incubate at room temperature for 10-15 minutes. Incubate the cells with the required cell permeation antibodies, such as CD68 and CD206, at room temperature in the dark for 30 minutes. After incubation, wash twice with a cell permeation reagent, resuspend the cells in 200-500 μL of flow cytometry buffer, and perform analysis using a flow cytometer.

[0083] (iii) Detecting the transmembrane resistance value of the model to characterize the cell barrier function of the model: The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is infected into the pathological pulmonary fibrosis model, and the transmembrane resistance value of the pathological pulmonary fibrosis model is detected; wherein, the transmembrane resistance value of the normal lung model is 200–1500 Ω·cm. 2The transmembrane resistance value of a pathological pulmonary fibrosis model is lower than that of a normal lung model. Therefore, changes in transmembrane resistance can be used to determine the impact of a test drug or pathogen infection on the progression of pulmonary fibrosis. For example, if the transmembrane resistance value decreases significantly to a statistically significant degree after the addition of a test drug or infection with a pathogen, then the test drug or pathogen infection damages the cell barrier; if the transmembrane resistance value of the pathological pulmonary fibrosis model increases significantly to a statistically significant degree after the addition of a test drug compared to before the addition, then the test drug can repair the cell barrier; if the change in the transmembrane resistance value of the pathological pulmonary fibrosis model after the addition of a test drug or infection with a pathogen is not statistically significant compared to before the addition of the test drug or infection with a pathogen, then the test drug or pathogen infection has no significant impact on the integrity of the cell barrier. In some specific implementation schemes, the following steps are included: Remove the transmembrane resistance meter, plug in the power supply, assemble the monitoring device, and place the chopstick probe in PBS for equilibration for 15 minutes; after the instrument reading stabilizes at 0Ω, fill the model inside and out with PBS; insert the chopstick probe into the model, and record the reading after the display stabilizes; data processing: TEER = TΩ × Acm 2 Where T is the resistance value and A is the area of ​​the pathological pulmonary fibrosis model.

[0084] (iv) Detecting changes in barrier permeability to characterize the cell barrier function of the model: The test drug is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and changes in the barrier permeability of the pathological pulmonary fibrosis model are detected. In some specific implementations, this includes: adding fluorescent permeating molecules, such as FITC-Dextran (molecular weight 10kDa, 30kDa, 70kDa), to the upper layer of the model, incubating at different time points, and using a microplate reader to detect the mass of the fluorescent molecules permeating into the lower layer of the model. The apparent permeability coefficient is calculated to reflect changes in the barrier permeability of the model. The operation steps are as follows: Discard all the culture medium in the wells, add 50-100 μM FITC-Dextran solution to the upper layer, add 400 μL of fresh culture medium to the lower layer, and incubate at 37°C; collect 200 μL of the lower layer culture medium at 2 h, 4 h, 8 h, and 24 h of incubation, respectively. Take out 200 μL of culture medium each time, and add another 200 μL of culture medium to each well to continue culturing; use an ELISA reader to detect the fluorescence value in the culture medium; calculate: Papp=(dQ / dt) / (A*C0); where Papp is the apparent permeability coefficient, A is the membrane area of ​​the membrane chip, C0 is the initial concentration of fluorescent dye, and dQ / dt is the amount of fluorescent dye that permeates per unit time.

[0085] (v) Detection of model cell viability: The test drug is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and changes in cell viability in the pathological pulmonary fibrosis model are detected; wherein, the cell viability is, for example, cell proliferation activity. In some specific embodiments, methods such as MTT, CCK8, and CTG are used to detect model cell viability. Taking CTG detection as an example: An equal volume of CTG activity detection reagent stock solution is added to the model, mixed by pipetting for 5 min, incubated at room temperature with shaking for 30 min, and its chemiluminescence value is detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0086] (vi) Detection of cell apoptosis in the model: The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and the cell apoptosis in the pathological pulmonary fibrosis model is detected; wherein the detection method is, for example, immunofluorescence or flow cytometry; the biomarkers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL.

[0087] (vii) Detection of cytokine levels: The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and changes in the levels of inflammatory cytokines in the pathological pulmonary fibrosis model are detected; wherein the inflammatory cytokines are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18. In some specific implementations, the detection is performed using methods such as ELISA, CBA multifactor assay, and Luminex multifactor assay. Taking the detection of inflammatory factor TNF-α content by ELISA as an example: Prepare standard solutions of 0, 11.7, 23.4, 46.9, 93.8, 187.5, 375 and 750 pg / mL. Add 100 μL of standard solution and sample solution to each well, and incubate at room temperature with shaking for 1 h. Wash each well 4 times with 300 μL of washing buffer for 2 min each time. Add 100 μL of antibody of the inflammatory cytokine to be tested to each well, and incubate at room temperature with shaking for 30 min. Wash each well 4 times with 300 μL of washing buffer for 2 min each time. Add 100 μL of chromogenic solution to each well, and incubate at room temperature with shaking for 20 min. Add 50 μL of stop solution to each well, and detect the OD value at 450 nm within 10 min.

[0088] (viii) Detection of lung tissue-related cell types and expression: The test drug is applied to the pathological pulmonary fibrosis model or the pathogen is infected into the pathological pulmonary fibrosis model, and the lung tissue-related cell phenotypes or cell contents in the pathological pulmonary fibrosis model are detected; wherein, the cell phenotypes are, for example, α-tubulin (ciliated cell marker), MUC5AC (goblet cell marker), AQP5 (AT1 cell marker), SFTPC (AT2 cell marker), CC10 (club cell marker), and KRT5 (basal cell marker).

[0089] (ix) Detection of oxidative stress levels: The drug to be tested is administered to the pathological pulmonary fibrosis model or the pathogen is introduced into the pathological pulmonary fibrosis model, and the cellular oxidative stress levels in the pathological pulmonary fibrosis model are detected. In some specific embodiments, this includes using a ROS oxidative stress assay kit to detect ROS / RNS levels and assess the drug's modulatory effect on oxidative stress. For example, using Invitrogen... TM The CellROX kit is used to analyze the model. CellROX is a fluorescent probe that can measure generalized oxidative stress within cells using fluorescence microscopy, high-content imaging systems, microplate fluorometers, or flow cytometry platforms. The dye is non-fluorescent in its reduced state and emits bright green, orange, or deep red fluorescence upon oxidation.

[0090] (x) Detection of the expression of genes related to the development of pulmonary fibrosis: The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and changes in the expression of pulmonary fibrosis-related genes in the pathological pulmonary fibrosis model are detected; wherein, the pulmonary fibrosis-related genes are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene. In some specific embodiments, RNA-seq or RT-PCR is used to detect changes in the transcriptional level of pulmonary fibrosis-related genes.

[0091] (xi) Detection of pathogen infection-related receptor expression: The pathological pulmonary fibrosis model is infected with the pathogen, and changes in the expression of pathogen infection-related receptors in the pathological pulmonary fibrosis model are detected. In some specific embodiments, this includes using an avian influenza virus to infect the pathological pulmonary fibrosis model and using a kit to detect the expression of influenza virus receptor α-2,6 linked to sialic acid.

[0092] (xii) Detecting the antipathogenic effect of the drug: The pathological pulmonary fibrosis model is infected with the pathogen, and then the test drug is applied to the pathological pulmonary fibrosis model. The inhibitory effect of the test drug on the pathogen is then detected. In some specific embodiments, this includes detecting the viral titer at different time points after infecting the pathological pulmonary fibrosis model with the pathogen to detect the antiviral effect of the test drug. Alternatively, RT-PCR is used to detect the viral RNA copy number after the model is infected with the virus, and the infection dose in 50% tissue culture is determined.

[0093] Fifthly, the present invention provides a drug detection method, a drug screening method, and / or a method for detecting the effect of pathogen infection on lung tissue, comprising:

[0094] (i) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and the expression changes of pulmonary fibrosis markers in the pathological pulmonary fibrosis model are detected; wherein, the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin. In some embodiments, the detection method is immunofluorescence detection; preferably, the detection method includes: fixing, washing, and blocking the pathological pulmonary fibrosis model, adding primary antibodies against pulmonary fibrosis markers and incubating, then adding secondary antibodies and incubating, and obtaining the expression changes of pulmonary fibrosis markers by staining and imaging;

[0095] (ii) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is infected into a pathological pulmonary fibrosis model, and the cell typing changes and / or dynamic immune response process in the pathological pulmonary fibrosis model are detected by flow cytometry. In some embodiments, the flow cytometry detection markers are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, and CRTH2; the cell typing changes include macrophage typing changes and / or immune cell typing changes, the macrophage typing changes being, for example, M1 type macrophage typing changes and M2 type macrophage typing changes; the immune cell typing changes being, for example, T cell typing changes, B cell typing changes, NK cell typing changes, and intrinsic lymphocyte typing changes; preferably, the detection method includes: collecting cells from a pathological pulmonary fibrosis model and resuspending them in flow cytometry buffer, staining the surface with antibodies against the detection markers, fixing, centrifuging, washing, resuspending the cells in flow cytometry buffer, and detecting fluorescence signals using a flow cytometer;

[0096] (iii) Applying the test drug to a pathological pulmonary fibrosis model or infecting a pathological pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance value of the pathological pulmonary fibrosis model; the change in transmembrane resistance value can determine the effect of the test drug or pathogen infection on the development of pulmonary fibrosis. When the transmembrane resistance value decreases significantly to a statistically significant level after the addition of the test drug or infection with the pathogen, the test drug or pathogen infection can disrupt the cell barrier; when the transmembrane resistance value of the pathological pulmonary fibrosis model increases significantly to a statistically significant level after the addition of the test drug compared to before the addition, the test drug can repair the cell barrier; when the change in the transmembrane resistance value of the pathological pulmonary fibrosis model after the addition of the test drug or infection with the pathogen is not statistically significant compared to before the addition of the test drug or infection with the pathogen, the test drug or pathogen infection has no significant effect on the integrity of the cell barrier. In some embodiments, the detection method includes: using a transmembrane resistance meter to detect the resistance value of the pathological pulmonary fibrosis model, using the formula TEER = TΩ × A cm. 2 Calculate the transmembrane resistance value, where T is the resistance value and A is the area of ​​the pathological pulmonary fibrosis model;

[0097] (iv) Applying the test drug to a pathological pulmonary fibrosis model or infecting a pathological pulmonary fibrosis model with the pathogen, and detecting changes in the barrier permeability of the pathological pulmonary fibrosis model. In some embodiments, the detection includes: adding a fluorescent molecule (e.g., FITC-Dextran) to the upper layer of the pathological pulmonary fibrosis model and incubating, detecting the amount of the fluorescent molecule permeating into the lower layer of the pathological pulmonary fibrosis model, and calculating the apparent permeability coefficient;

[0098] (v) Applying the test drug to a pathological pulmonary fibrosis model or infecting a pathological pulmonary fibrosis model with the pathogen, and detecting changes in cell activity in the pathological pulmonary fibrosis model. In some embodiments, the cell activity is, for example, cell proliferation activity; preferably, the detection method includes: adding an MTT, CCK8, or CTG activity detection reagent to the pathological pulmonary fibrosis model and incubating, and detecting the chemiluminescence value;

[0099] (vi) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and the apoptosis status in the pathological pulmonary fibrosis model is detected. In some embodiments, the detection method is, for example, immunofluorescence or flow cytometry; the biomarkers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL;

[0100] (vii) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and changes in the levels of inflammatory cytokines in the pathological pulmonary fibrosis model are detected. In some embodiments, the inflammatory cytokines are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18; the detection methods include ELISA, CBA multifactor assay, and Luminex multifactor assay; preferably, the detection method includes: preparing standard solutions of inflammatory cytokines with gradient concentrations, adding the standard solutions and sample culture medium to a culture vessel, incubating and washing, adding antibodies to the inflammatory cytokines to be tested, incubating and washing again, adding chromogenic solution and incubating, then adding stop solution and detecting the OD value;

[0101] (viii) The test drug is administered to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and the lung tissue-related cell phenotypes or cell contents in the pathological pulmonary fibrosis model are detected. In some embodiments, the cell phenotypes are, for example, α-tubulin, MUC5AC, AQP5, SFTPC, CC10, KRT5;

[0102] (ix) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is infected into a pathological pulmonary fibrosis model, and the level of cellular oxidative stress in the pathological pulmonary fibrosis model is detected.

[0103] (x) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and the expression changes of pulmonary fibrosis-related genes in the pathological pulmonary fibrosis model are detected. In some embodiments, the pulmonary fibrosis-related genes are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene;

[0104] (xi) Infect a pathological pulmonary fibrosis model with the pathogen and detect changes in the expression of receptors related to the pathogen infection in the pathological pulmonary fibrosis model;

[0105] (xii) Infect a pathological pulmonary fibrosis model with the pathogen, then apply the test drug to the pathological pulmonary fibrosis model, and detect the inhibitory effect of the test drug on the pathogen.

[0106] The pathological pulmonary fibrosis model is either the pathological pulmonary fibrosis model constructed by the construction method of the first aspect, or the pathological pulmonary fibrosis model described in the second aspect.

[0107] In some implementations, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs.

[0108] First, this invention utilizes primary extraction and culture of lung organoids derived from pulmonary fibrosis patients and, optionally, lung fibroblasts. This method can replace traditional cell lines for constructing pathological pulmonary fibrosis models, offering a shorter experimental cycle compared to animal models, reducing experimental costs, simplifying operations, and enhancing model stability and controllability. Second, the construction method provided by this invention involves sequentially seeding lung organoids (and optionally lung fibroblasts), endothelial cells, macrophages, and optionally immune cells. The pathological pulmonary fibrosis model is obtained through liquid-liquid culture followed by gas-liquid culture (including a first-stage gas-liquid culture, a second-stage gas-liquid culture, and an optional third-stage gas-liquid culture). This not only allows for a higher level of maturity in the pathological pulmonary fibrosis model but also better simulates the function and structural characteristics of the alveolar-capillary barrier, exhibiting the pathological features and immune microenvironment of pulmonary fibrosis, resulting in a high degree of simulation. Furthermore, the pathological pulmonary fibrosis model provided by this invention includes lung organoids, endothelial cells, macrophages, and optionally lung fibroblasts and immune cells, which can well simulate the cell-related microenvironment and intercellular interactions. It also has the advantages of being able to be passaged multiple times and maintaining the phenotype for a long time in vitro, providing a good platform for the simulation and research of pulmonary fibrosis, as well as the detection, screening, evaluation and development of drugs. Attached Figure Description

[0109] Figure 1 shows a bright field diagram of the process of constructing a pathological pulmonary fibrosis model in Embodiment 1 of the present invention.

[0110] Figure 2 shows the immunofluorescence images of α-SMA and Collagen I expression in the pathological pulmonary fibrosis model of Example 2 of the present invention.

[0111] Figure 3 shows the α-SMA and Collagen I expression of each group of models in Embodiment 3 of the present invention.

[0112] Figure 4 shows the expression of the inflammatory factor IFN-γ in each group of models in Example 4 of the present invention.

[0113] Figure 5 shows the lung tissue-related cell content of the pathological pulmonary fibrosis model in Example 5 of the present invention. Detailed Implementation

[0114] The materials, methods, and embodiments described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from this specification, the drawings, and the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0115] Example 1

[0116] This embodiment provides a detailed process for constructing a pathological pulmonary fibrosis model:

[0117] Primary extraction and culture of lung organoids derived from patients with pulmonary fibrosis:

[0118] (1) Cleaning: Primary tissue cleaning solution was used to clean pulmonary fibrosis tissue from patients with pulmonary fibrosis to remove the surface mucosa. The primary tissue cleaning solution was a buffer solution containing penicillin and streptomycin.

[0119] (2) Disintegration: Chop the tissue into a paste, resuspend it in buffer solution, let it stand, and then discard the supernatant.

[0120] (3) Digestion: Add the digestion solution for primary pulmonary fibrosis tissue, mix well by pipetting, and then use a metal bath shaker at 37°C for 30-60 minutes to digest into small cell clusters. Finally, add buffer solution to terminate the digestion. The digestion solution for primary pulmonary fibrosis tissue contains collagenase I at a final concentration of 1 mg / mL, hyaluronidase at a final concentration of 0.5 mg / mL, Y27632 (purchased from MCE, catalog number HY-10071) at a final concentration of 10 μM, and Ad-DMEM / F12 culture medium.

[0121] (4) Resuspension: The digested cell suspension was filtered through a 100μm cell sieve, and the cells were resuspended in lung organoid culture medium and counted; where the lung organoid culture medium was produced by Ivytech and named Lung Organoid Culture Kit, catalog number: KLU0201.

[0122] (5) Plate plating: After centrifugation to remove the supernatant, the lung organoids were resuspended in Matrigel extracellular matrix and plated. 30 μL of gel was dropped into each well of a 24-well plate. After gel formation, lung organoid culture medium was added, and the plates were incubated at 37°C for 7 days.

[0123] (6) Digesting lung organoids: After the lung organoids have grown for 7 days, use lung organoid passage digestion solution Tryple to digest the lung organoids into single cells, and add lung organoid culture medium to stop the digestion.

[0124] (7) Inoculation: Centrifuge to remove supernatant, resuspend in lung epithelial cell culture medium, and adjust density to 1×10⁶. 6 Cells / mL. The chip membrane area is 0.33 cm². 2 2×10 5 10 cells, with a cell density of 6 × 10⁶. 5 pcs / cm 2Lung organoids were seeded onto the upper layer of the membrane. 1 mL of lung epithelial cell culture medium (Borg Biogen Respiratory Organoid Expansion Medium (Catalog No. A01-001), Ivylink Lung Organoid Culture Kit (Catalog No. KLU0201), or Stemcell PneumaCultura Cell Culture Medium) was added to the lower layer. TM -Ex Plus Medium)) was cultured in a liquid-liquid incubator at 37°C.

[0125] (8) Change the medium every 2 days and culture for 4 days.

[0126] Gas-liquid culture mode:

[0127] (1) Once the lung organoids have grown to the upper membrane layer, discard the culture medium in both the upper and lower membrane layers, and add 600 μL of lung epithelial cell culture medium to the lower membrane layer.

[0128] (2) Cultured in gas-liquid mode for 2 days (first stage of gas-liquid mode culture).

[0129] Inoculation with endothelial cells:

[0130] (1) Digest the endothelial cells and resuspend them in a primary mixed culture medium (lung epithelial cell culture medium: endothelial cell culture medium volume ratio 2:1). Count the cells and adjust the endothelial cell density to 6 × 10⁶ cells / year. 5 Cells / mL. The chip membrane area is 0.33 cm². 2 3×10 4 10 endothelial cells, with a cell density of 0.9 × 10⁻⁶. 5 pcs / cm 2 The endothelial cell culture medium used was Sciencell (catalog number #1001).

[0131] (2) Place the chip in a 37°C incubator for 2 hours. After the cells adhere to the wall, place the chip upright and add 0.6 mL of the first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium are mixed at a volume ratio of 2:1) to the lower chamber.

[0132] Inoculation with macrophages:

[0133] (1) Collect macrophages and resuspend them in macrophage culture medium (ScienCell, #1921). Count the cells and adjust the cell density to 1×10⁶. 5 Cells / mL. The chip membrane area is 0.33 cm². 2 Administer 1×10 4 There are 10 macrophages, with a cell density of 0.3 × 10⁶. 5 pcs / cm 2 .

[0134] (2) Macrophages were cultured overnight, and then the macrophage culture medium on the membrane was discarded.

[0135] (3) Change the culture medium under the membrane (first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium are mixed at a volume ratio of 2:1)).

[0136] (4) Continue gas-liquid mode culture for 2 days (second stage gas-liquid mode culture).

[0137] Inoculate immune cells (this step can be omitted if immune function is not required):

[0138] (1) Collect PBMCs and resuspend them in a second mixed medium (immune cell culture medium and the first mixed medium mixed at a volume ratio of 2:1). Discard the submucosa medium. The bottom area of ​​the lower chamber of the chip should be 2 cm². 2 2×10 5 One PBMC, with a cell density of 1×10⁶ cells. 5 pcs / cm 2 For example, commercially available PBMC media can be used, such as SuperCulture L100 serum-free lymphocyte medium (Dakorway, catalog number #6911011) supplemented with 5-10% SuperGrow cell culture additive (Dakorway, catalog number #6122011) and 1-5‰ IL-2.

[0139] (2) Continue gas-liquid culture for 1 day (third stage gas-liquid culture) to obtain a pathological pulmonary fibrosis model.

[0140] High-content imaging system was used to photograph the model at different time points during construction, and the bright-field images are shown in Figure 1. As can be seen from Figure 1, when lung organoids were inoculated, the cells were evenly spread on the upper layer of the culture chamber membrane; during gas-liquid culture, the cells grew in sheets with vacuolar morphology in the middle; after inoculation with endothelial cells and macrophages, the macrophages were scattered among the lung epithelial cells and grew together with them.

[0141] Example 2

[0142] This embodiment provides a detailed process for constructing a pathological pulmonary fibrosis model:

[0143] Primary extraction and culture of lung organoids derived from patients with pulmonary fibrosis:

[0144] (1) Cleaning: Primary tissue cleaning solution was used to clean pulmonary fibrosis tissue from patients with pulmonary fibrosis to remove the surface mucosa. The primary tissue cleaning solution was a buffer solution containing penicillin and streptomycin.

[0145] (2) Disintegration: Chop the tissue into a paste, resuspend it in buffer solution, let it stand, and then discard the supernatant.

[0146] (3) Digestion: Add the digestion solution for primary pulmonary fibrosis tissue, mix well by pipetting, and then use a metal bath shaker at 37°C for 30-60 minutes to digest into small cell clusters. Finally, add buffer solution to terminate the digestion. The digestion solution for primary pulmonary fibrosis tissue contains collagenase I at a final concentration of 1 mg / mL, hyaluronidase at a final concentration of 0.5 mg / mL, Y27632 (purchased from MCE, catalog number HY-10071) at a final concentration of 10 μM, and Ad-DMEM / F12 culture medium.

[0147] (4) Resuspension: The digested cell suspension was filtered through a 100μm cell sieve, and the cells were resuspended in lung organoid culture medium and counted; where the lung organoid culture medium was produced by Ivytech and named Lung Organoid Culture Kit, catalog number: KLU0201.

[0148] (5) Plate plating: After centrifugation to remove the supernatant, the lung organoids were resuspended in Matrigel extracellular matrix and plated. 30 μL of gel was dropped into each well of a 24-well plate. After gel formation, lung organoid culture medium was added, and the plates were incubated at 37°C for 7 days.

[0149] Passaging of lung organoids derived from patients with pulmonary fibrosis:

[0150] (1) Collection: After primary culture of lung organoids for 7 days, remove the lung organoid culture medium, add pre-cooled PBS buffer, pipette Matrigel to resuspend the lung organoids, and collect them into centrifuge tubes.

[0151] (2) Digestion: Centrifuge to remove supernatant, add Tryple to digest organoids to break them down into small cell clusters. Add PBS buffer to stop digestion.

[0152] (3) Plate plating: After centrifugation to remove the supernatant, the lung organoids were resuspended in Matrigel extracellular matrix and plated. 30 μL of gel was dropped into each well of a 24-well plate. After gel formation, lung organoid culture medium was added, and the plates were incubated at 37°C for 7 days.

[0153] Primary extraction and culture of lung fibroblasts derived from patients with pulmonary fibrosis:

[0154] (1) Cleaning: The pulmonary fibrosis tissue from patients with pulmonary fibrosis was cleaned with primary tissue cleaning solution to remove the surface mucosa. The primary tissue cleaning solution was a buffer solution to which penicillin and streptomycin were added.

[0155] (2) Crushing: mince the tissue into a paste-like consistency, resuspend it in buffer solution, let it stand, and then discard the supernatant.

[0156] (3) Digestion: Add the digestion solution of primary pulmonary fibrosis tissue, mix well by pipetting, and then use a metal bath shaker at 37°C for 30-60 min to digest into single cells. Finally, add buffer solution to stop the digestion. The digestion solution of primary pulmonary fibrosis tissue contains collagenase I at a final concentration of 1 mg / mL, hyaluronidase at a final concentration of 0.5 mg / mL, Y27632 (purchased from MCE, catalog number HY-10071) at a final concentration of 10 μM, and Ad-DMEM / F12 culture medium.

[0157] (4) Resuspension: The digested cell suspension was filtered through a 70μm cell sieve and the cells were resuspended in lung fibroblast culture medium; the lung fibroblast culture medium was #AC-1001015 produced by EZEN Biotechnology Co., Ltd.

[0158] (5) Plating: Perform cell counting and adjust the cell density to 1×10⁻⁶. 6 Lung fibroblasts were plated onto T25 cell culture flasks, with 1 mL of cells and 5 mL of lung fibroblast culture medium added to each flask. The flasks were then incubated at 37°C for 7 days.

[0159] Inoculation with lung organoids and lung fibroblasts:

[0160] (1) Digesting lung organoids: Take lung organoids from the 5th generation and digest them with lung organoid passage digestion solution Tryple until they are digested into single cells. Add lung organoid culture medium to stop digestion.

[0161] (2) Digestion of lung fibroblasts: lung fibroblasts were digested using Tryple, a fibroblast passage digestion solution, to break them down into single cells. DMEM containing 10% FBS was added to terminate the digestion.

[0162] (3) Inoculation: Resuspend lung organoids and lung fibroblasts in lung epithelial cell culture medium, and adjust the lung organoid cell density to 1×10⁻⁶. 6 lung fibroblasts density was adjusted to 5 × 10⁶ cells / mL. 4 Cells / mL. The chip membrane area is 0.33 cm². 2 2×10 lung organoids were inoculated. 5 There are 6 × 10⁶ cells, with a cell density of 6 × 10⁶. 5 pcs / cm 2 ; Inoculate with 5×10 lung fibroblasts 3 There were 1.5 × 10⁶ cells, with a cell density of 1.5 × 10⁶ 4 pcs / cm 2 Lung organoids and lung fibroblasts were seeded onto the upper layer of the membrane. 1 mL of lung epithelial cell culture medium was added to the lower layer, and the cells were incubated in liquid-liquid mode at 37°C.

[0163] (4) Change the medium every 2 days and culture for 4 days.

[0164] Gas-liquid culture mode:

[0165] (1) Once the lung organoids have grown to the upper membrane layer, discard the culture medium in both the upper and lower membrane layers, and add 600 μL of lung epithelial cell culture medium to the lower membrane layer.

[0166] (2) Cultured in gas-liquid mode for 2 days (first stage of gas-liquid mode culture).

[0167] Inoculation with endothelial cells:

[0168] (1) Digest the endothelial cells and resuspend them in a primary mixed culture medium (lung epithelial cell culture medium: endothelial cell culture medium volume ratio 2:1). Count the cells and adjust the endothelial cell density to 6 × 10⁶ cells / year. 5 Cells / mL. The chip membrane area is 0.33 cm². 2 3×10 4 10 endothelial cells, with a cell density of 0.9 × 10⁻⁶. 5 pcs / cm 2 The endothelial cell culture medium used was Sciencell (catalog number #1001).

[0169] (2) Place the chip in a 37°C incubator for 2 hours. After the cells adhere to the wall, place the chip upright and add 0.6 mL of the first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium are mixed at a volume ratio of 2:1) to the lower chamber.

[0170] Inoculation with macrophages:

[0171] (1) Collect macrophages and resuspend them in macrophage culture medium (ScienCell, #1921). Count the cells and adjust the cell density to 1×10⁶. 5 Cells / mL. The chip membrane area is 0.33 cm². 2 Administer 1×10 4 There are 10 macrophages, with a cell density of 0.3 × 10⁶. 5 pcs / cm 2 .

[0172] (2) Macrophages were cultured overnight, and then the macrophage culture medium on the membrane was discarded.

[0173] (3) Change the culture medium under the membrane (first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium are mixed at a volume ratio of 2:1)).

[0174] (4) Continue gas-liquid mode culture for 2 days (second stage gas-liquid mode culture).

[0175] Inoculate immune cells (this step can be omitted if immune function is not required):

[0176] (1) Collect PBMCs and resuspend them in a second mixed medium (immune cell culture medium and the first mixed medium mixed at a volume ratio of 2:1). Discard the submucosa medium. The bottom area of ​​the lower chamber of the chip should be 2 cm². 2 2×10 5 One PBMC, with a cell density of 1×10⁶ cells. 5 pcs / cm 2 For example, commercially available PBMC media can be used, such as SuperCulture L100 serum-free lymphocyte medium (Dakorway, catalog number #6911011) supplemented with 5-10% SuperGrow cell culture additive (Dakorway, catalog number #6122011) and 1-5‰ IL-2.

[0177] (2) Continue gas-liquid culture for 1 day (third stage gas-liquid culture) to obtain a pathological pulmonary fibrosis model.

[0178] Immunofluorescence experiments were performed on the model:

[0179] (1) After the model is constructed, add 4% paraformaldehyde for fixation for 30 min;

[0180] (2) Discard the fixative and wash three times with PBS. Add breakthrough buffer (0.3% Triton-100) and break through for 15 min;

[0181] (3) Add 5% BSA blocking solution to block the sample for 1 hour.

[0182] (4) Add primary antibody α-SMA and Collagen I, incubate overnight at 4°C, add secondary antibody, incubate at room temperature for 1 h, perform nuclear staining with DAPI, wash with PBS, and image with confocal microscope.

[0183] The immunofluorescence results in Figure 2 show that α-SMA is labeled in green, Collagen I is labeled in red, and cell nuclei are labeled in blue, indicating that the constructed pathological pulmonary fibrosis model has the characteristics of pulmonary fibrosis.

[0184] Example 3

[0185] This embodiment provides a pathological pulmonary fibrosis model for drug testing:

[0186] (1) A pathological pulmonary fibrosis model was constructed according to the method in Example 1. A pulmonary fibrosis patient group and a nintedanib group were set up. The pulmonary fibrosis patient group was an in vitro lung model with a second mixed culture medium added to the lower layer of the chip; the nintedanib group had a second mixed culture medium containing 10 μM nintedanib added to the lower layer of the in vitro lung model.

[0187] (2) Drug treatment for 3 days.

[0188] (3) After the drug treatment was completed, immunofluorescence was used to detect whether there was a difference in the expression of α-SMA and Collagen I in the pulmonary fibrosis model.

[0189] The immunofluorescence results in Figure 3 show that α-SMA is marked in green, Collagen I is marked in red, and cell nuclei are marked in blue. It can be seen that there are obvious fibroblasts and collagen deposition in the constructed pulmonary fibrosis patient group. The expression of fibroblasts in nintedanib component is weakened and collagen deposition is reduced.

[0190] Example 4

[0191] This embodiment provides a pathological pulmonary fibrosis model for drug testing:

[0192] (1) A pathological pulmonary fibrosis model was constructed according to the method in Example 2. A pulmonary fibrosis patient group and a nintedanib group were set up. The pulmonary fibrosis patient group was an in vitro lung model, and a second mixed culture medium was added to the lower layer of the chip. The nintedanib group was a lung in vitro model with a second mixed culture medium containing 5 μM nintedanib added to the lower layer of the chip.

[0193] (2) Drug treatment for 3 days.

[0194] (3) After the drug treatment, the content of inflammatory factor IFN-γ in the pulmonary fibrosis model was detected by ELISA kit, and the expression of inflammatory cytokines in the pulmonary fibrosis patient group (IPF) and the drug treatment group (5μM Nintedanib) was compared to see if there was a difference.

[0195] Figure 4 shows the ELISA results, indicating that in the constructed pulmonary fibrosis patient group, the IFN-γ secretion level was approximately 25 pg / mL, while in the nintedanib group, the IFN-γ secretion level was approximately 12 pg / mL. Treatment with the drug nintedanib significantly reduced IFN-γ secretion, demonstrating that nintedanib can inhibit the secretion of inflammatory factors.

[0196] Example 5

[0197] This embodiment provides a pathological pulmonary fibrosis model for detecting lung tissue-related cell phenotypes and content:

[0198] (1) A pathological pulmonary fibrosis model was constructed according to the method in Example 1. Flow cytometry was performed after the model was constructed.

[0199] (2) Use Tryple to digest lung epithelial cells into single cells.

[0200] (3) Staining with lung tissue-associated cell antibodies, including MUC5AC (goblet cell marker), PDPN (AT1 cell marker), SFTPB (AT2 cell marker), CC10 (club cell marker), and KRT5 (basal cell marker).

[0201] (4) Detection was performed using a flow cytometer.

[0202] The flow cytometry results in Figure 5 show that the constructed pathological pulmonary fibrosis model contains major lung tissue cells, of which cells expressing KRT5 account for 28.3% of the total number of cells, cells expressing PDPN account for 50.6% of the total number of cells, cells expressing SFTPB account for 34.1% of the total number of cells, cells expressing CC10 account for 38.8% of the total number of cells, and cells expressing MUC5AC account for 18.4% of the total number of cells.

Claims

1. A method for constructing a pathological pulmonary fibrosis model, comprising: Provides lung organoids and cell culture containers derived from patients with pulmonary fibrosis, and optionally lung fibroblasts derived from patients with pulmonary fibrosis; The lung organoids are seeded in the upper layer of the cell culture container, and optionally the lung fibroblasts are seeded in the upper layer of the cell culture container. Then, lung epithelial cell culture medium is added to the upper and lower layers of the cell culture container to culture the lung organoids and optionally the lung fibroblasts in liquid-liquid mode. The lung epithelial cell culture medium in the upper and lower layers is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to perform a first-stage gas-liquid culture of the lung organoids and optionally the lung fibroblasts. Endothelial cells were seeded in the lower layer of the cell culture container, and macrophages were seeded in the upper layer of the cell culture container and cultured in a second-stage gas-liquid mode to obtain a pathological pulmonary fibrosis model.

2. The construction method according to claim 1, wherein, The lung organoids, after being treated with a digestive solution of primary pulmonary fibrosis tissue, are seeded onto the upper layer of the cell culture vessel; wherein the digestive solution of primary pulmonary fibrosis tissue contains collagenase, hyaluronidase, Y27632, and culture medium; preferably, the collagenase includes at least one of collagenase I, collagenase II, and collagenase IV; preferably, the final concentration of the collagenase in the digestive solution of primary pulmonary fibrosis tissue is 1–2 mg / mL; preferably, the final concentration of the hyaluronidase in the digestive solution of primary pulmonary fibrosis tissue is 0.5–1 mg / mL; preferably, the final concentration of Y27632 in the digestive solution of primary pulmonary fibrosis tissue is 5–10 μM; preferably, the culture medium is selected from DMEM or ADMEM / F12; and / or The lung fibroblasts, after being treated with a digestive solution of primary lung fibrosis tissue, are seeded onto the upper layer of the cell culture vessel; wherein, the digestive solution of primary lung fibrosis tissue contains collagenase, hyaluronidase, Y27632, and culture medium; preferably, the collagenase includes at least one of collagenase I, collagenase II, and collagenase IV; preferably, the final concentration of the collagenase in the digestive solution of primary lung fibrosis tissue is 1–2 mg / mL; preferably, the final concentration of the hyaluronidase in the digestive solution of primary lung fibrosis tissue is 0.5–1 mg / mL; preferably, the final concentration of Y27632 in the digestive solution of primary lung fibrosis tissue is 5–10 μM; preferably, the culture medium is selected from DMEM or ADMEM / F12.

3. The construction method according to claim 1 or 2, wherein, The lung organoids were cultured in the liquid-liquid mode for 3 to 7 days. The first stage of gas-liquid culture of the lung organoids lasted for 2 to 21 days. The second stage of gas-liquid incubation lasts for 1 to 5 days; Based on the inoculation area, the lung organoid inoculation is 1×10 5 ~10×10 5 cells / cm 2 ; When inoculating the lung fibroblasts, the inoculation area is calculated as 0.3 × 10⁻⁶ cells / day. 3 ~0.3×10 4 cells / cm 2 ; Based on the inoculation area, the endothelial cells were inoculated at a rate of 0.5 × 10⁻⁶. 5 ~5×10 5 cells / cm 2 ; and / or Based on the inoculation area, the macrophage inoculation was 0.1 × 10⁻⁶. 5 ~5×10 5 cells / cm 2 ; Preferably, the endothelial cells are derived from umbilical vein endothelial cells, primary lung endothelial cells, or lung endothelial cell lines; Preferably, the macrophages are derived from macrophages expanded from PBMCs, primary alveolar macrophages, passaged alveolar macrophages, or macrophages induced by the THP-1 cell line.

4. The construction method according to any one of claims 1 to 3, wherein, In the step of seeding endothelial cells into the lower layer of the cell culture container, after the endothelial cells adhere to the wall, a first mixed culture medium is added to the lower layer. The first mixed culture medium is obtained by mixing the lung epithelial cell culture medium and the endothelial cell culture medium at a volume ratio of (0.5-3.5):

1. In the step of seeding macrophages into the upper layer of the cell culture container and performing the second stage of gas-liquid culture, after the macrophages adhere to the wall, the culture media of the upper and lower layers are discarded and the upper layer is kept dry. Fresh first mixed culture medium is added to the lower layer for the second stage of gas-liquid culture. The first mixed culture medium is obtained by mixing the lung epithelial cell culture medium and the endothelial cell culture medium in a ratio of (0.5-3.5):

1.

5. The construction method according to any one of claims 1 to 4, wherein, After seeding the macrophages into the upper layer of the cell culture container and performing a second-stage gas-liquid culture, the method further includes the step of seeding immune cells into the lower layer of the cell culture container and culturing them. Preferably, the immune cells include PBMCs, T cells, B cells, and NK cells; Preferably, the number of immune cells inoculated is 0.5 × 10⁻⁶, calculated based on the inoculation area. 5 ~1×10 6 cells / cm 2 ; Preferably, in the step of seeding immune cells into the lower layer of the cell culture container and culturing them, the immune cells are resuspended in a second mixed culture medium to obtain a suspension, the upper layer is kept dry, the culture medium in the lower layer is discarded, and then the suspension is added to perform a third-stage gas-liquid mode culture. The second mixed culture medium is obtained by mixing immune cell culture medium and the first mixed culture medium at a volume ratio of (0.5-3.5):

1. More preferably, the third stage gas-liquid mode culture time is 1 to 3 days.

6. The construction method according to any one of claims 1 to 5, wherein, The cell culture container is a Transwell or a cell culture chip, such as a membrane chip or a barrier chip.

7. A pathological pulmonary fibrosis model, which is constructed by the construction method according to any one of claims 1 to 6.

8. A culture medium assembly comprising a lung epithelial cell culture medium, a first mixed culture medium, and optionally a second mixed culture medium; in, The first mixed culture medium is obtained by mixing lung epithelial cell culture medium and endothelial cell culture medium at a volume ratio of (0.5-3.5):1; the second mixed culture medium is obtained by mixing immune cell culture medium and the first mixed culture medium at a volume ratio of (0.5-3.5):

1. Preferably, the culture medium combination is used in the construction method according to any one of claims 1 to 6, or in the construction of the pathological pulmonary fibrosis model according to claim 7, including culture of lung organoids, liquid-liquid mode culture, first-stage gas-liquid mode culture, second-stage gas-liquid mode culture, and optionally third-stage gas-liquid mode culture. More preferably, the lung epithelial cell culture medium is used for the culture of the lung organoids and the lung fibroblasts, the liquid-liquid culture mode and / or the first stage gas-liquid culture mode; The first mixed culture medium is used in the second stage gas-liquid mode; The second mixed culture medium is used for the third stage of gas-liquid mode culture.

9. The pathological pulmonary fibrosis model constructed by the construction method according to any one of claims 1 to 6, or the pathological pulmonary fibrosis model according to claim 7, is used in the following (a) or (b); (a) Drug detection and / or drug screening; preferably, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs; preferably, the drug detection and / or drug screening includes drug detection and / or drug screening for the treatment of pulmonary fibrosis; (b) Detect the impact of pathogen infection on the progression of pulmonary fibrosis.

10. The use according to claim 9, wherein, (a) and (b) include at least one of the following (i) to (xii): (i) The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is infected in the pathological pulmonary fibrosis model, and the expression changes of pulmonary fibrosis markers in the pathological pulmonary fibrosis model are detected; wherein, the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin. (ii) The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and the cell typing changes and / or dynamic immune response process in the pathological pulmonary fibrosis model are detected by flow cytometry; wherein, the biomarkers detected by flow cytometry are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, and CRTH2; the cell typing changes include macrophage typing changes and / or immune cell typing changes, the macrophage typing changes are, for example, M1 type macrophage typing changes and M2 type macrophage typing changes; the immune cell typing changes are, for example, T cell typing changes, B cell typing changes, NK cell typing changes, and intrinsic lymphocyte typing changes. (iii) Apply the test drug to the pathological pulmonary fibrosis model or infect the pathological pulmonary fibrosis model with the pathogen, and detect the transmembrane resistance value of the pathological pulmonary fibrosis model; wherein, if the transmembrane resistance value of the pathological pulmonary fibrosis model decreases significantly to a statistically significant degree after the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection can damage the cell barrier; if the transmembrane resistance value of the pathological pulmonary fibrosis model increases significantly to a statistically significant degree after the addition of the test drug compared with before the addition, then the test drug can repair the cell barrier; if the change in the transmembrane resistance value of the pathological pulmonary fibrosis model after the addition of the test drug or infection with the pathogen is not statistically significant compared with before the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection has no significant effect on the integrity of the cell barrier; (iv) Apply the test drug to the pathological pulmonary fibrosis model or infect the pathological pulmonary fibrosis model with the pathogen, and detect changes in the barrier permeability of the pathological pulmonary fibrosis model. (v) The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is infected into the pathological pulmonary fibrosis model, and changes in cell activity in the pathological pulmonary fibrosis model are detected; wherein, the cell activity is, for example, cell proliferation activity. (vi) The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and the cell apoptosis in the pathological pulmonary fibrosis model is detected; wherein the detection method is, for example, immunofluorescence or flow cytometry; the biomarkers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL; (vii) The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is used to infect the pathological pulmonary fibrosis model, and the changes in the content of inflammatory cytokines in the pathological pulmonary fibrosis model are detected; wherein the inflammatory cytokines are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18; (viii) The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is infected in the pathological pulmonary fibrosis model, and the cell phenotype or cell content related to lung tissue in the pathological pulmonary fibrosis model is detected; wherein the cell phenotype is, for example, a ciliated cell marker, a goblet cell marker, an AT1 cell marker, an AT2 cell marker, a club cell marker, or a basal cell marker. (ix) The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is infected in the pathological pulmonary fibrosis model, and the level of cellular oxidative stress in the pathological pulmonary fibrosis model is detected. (x) The drug to be tested is applied to the pathological pulmonary fibrosis model or the pathogen is infected into the pathological pulmonary fibrosis model, and the expression changes of genes related to the development of pulmonary fibrosis in the pathological pulmonary fibrosis model are detected; wherein, the related genes are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene. (xi) Infect the pathological pulmonary fibrosis model with the pathogen and detect the expression changes of the pathogen infection-related receptors in the pathological pulmonary fibrosis model; (xii) Infect the pathological pulmonary fibrosis model with the pathogen, then apply the test drug to the pathological pulmonary fibrosis model, and detect the inhibitory effect of the test drug on the pathogen.

11. A method for drug detection, drug screening, and / or a method for detecting the effect of pathogen infection on the progression of pulmonary fibrosis, comprising: (i) Applying the drug to be tested to a pathological pulmonary fibrosis model or infecting a pathological pulmonary fibrosis model with the pathogen, and detecting the expression changes of pulmonary fibrosis markers in the pathological pulmonary fibrosis model; wherein, the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin; preferably, the detection method is immunofluorescence detection; more preferably, the detection method includes: fixing, washing, and blocking the pathological pulmonary fibrosis model, adding primary antibodies against pulmonary fibrosis markers and incubating, then adding secondary antibodies and incubating, and obtaining the expression changes of pulmonary fibrosis markers by staining and imaging; (ii) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model. Flow cytometry is used to detect cell typing changes and / or dynamic immune response processes in the pathological pulmonary fibrosis model. The biomarkers detected by flow cytometry include, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, and CRTH2. The cell typing changes include macrophages. The cell typing changes and / or immune cell typing changes, wherein the macrophage typing changes are, for example, M1 type macrophage typing changes and M2 type macrophage typing changes; the immune cell typing changes are, for example, T cell typing changes, B cell typing changes, NK cell typing changes, and intrinsic lymphocyte typing changes; preferably, the detection method includes: collecting cells from a pathological pulmonary fibrosis model and resuspending them in flow cytometry buffer, staining with antibodies against detection markers, fixing, centrifuging, washing, resuspending the cells in flow cytometry buffer, and detecting fluorescence signals using a flow cytometer; (iii) Applying the test drug to a pathological pulmonary fibrosis model or infecting a pathological pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance value of the pathological pulmonary fibrosis model; wherein, if the transmembrane resistance value of the pathological pulmonary fibrosis model decreases significantly to a statistically significant level after the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection can damage the cell barrier; if the transmembrane resistance value of the pathological pulmonary fibrosis model increases significantly to a statistically significant level after the addition of the test drug compared to before the addition, then the test drug can repair the cell barrier; if the change in the transmembrane resistance value of the pathological pulmonary fibrosis model after the addition of the test drug or infection with the pathogen is not statistically significant compared to before the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection has no significant effect on the integrity of the cell barrier; preferably, the detection method includes: using a transmembrane resistance meter to detect the resistance value of the pathological pulmonary fibrosis model, using the formula TEER=TΩ×Acm 2 Calculate the transmembrane resistance value, where T is the resistance value and A is the area of ​​the pathological pulmonary fibrosis model; (iv) Apply the test drug to a pathological pulmonary fibrosis model or infect a pathological pulmonary fibrosis model with the pathogen, and detect changes in the barrier permeability of the pathological pulmonary fibrosis model; preferably, the detection includes: adding a fluorescent molecule (e.g., FITC-Dextran) to the upper layer of the pathological pulmonary fibrosis model and incubating it, detecting the amount of the fluorescent molecule permeating into the lower layer of the pathological pulmonary fibrosis model, and calculating the apparent permeability coefficient; (v) Applying the drug to be tested to a pathological pulmonary fibrosis model or infecting a pathological pulmonary fibrosis model with the pathogen, and detecting changes in cell activity in the pathological pulmonary fibrosis model; wherein, the cell activity is, for example, cell proliferation activity; preferably, the detection method includes: adding MTT, CCK8 or CTG activity detection reagents to the pathological pulmonary fibrosis model and incubating, and detecting chemiluminescence values; (vi) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and the cell apoptosis in the pathological pulmonary fibrosis model is detected; wherein the detection method is, for example, immunofluorescence or flow cytometry; the biomarkers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL. (vii) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and the changes in the content of inflammatory cytokines in the pathological pulmonary fibrosis model are detected; wherein the inflammatory cytokines are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18; the detection method includes ELISA, CBA multifactor detection, and Luminex multifactor detection; preferably, the detection method includes: adding sample culture medium to a culture container, incubating and washing, adding antibodies to the inflammatory cytokines to be tested, incubating and washing again, adding chromogenic solution and incubating, then adding stop solution and detecting OD value; (viii) The test drug is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and the lung tissue-related cell phenotypes or cell contents in the pathological pulmonary fibrosis model are detected; wherein the cell phenotypes are, for example, ciliated cell markers, goblet cell markers, AT1 cell markers, AT2 cell markers, club cell markers, and basal cell markers; the detection methods include: flow cytometry and immunofluorescence. (ix) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is infected into a pathological pulmonary fibrosis model, and the level of cellular oxidative stress in the pathological pulmonary fibrosis model is detected. (x) The drug to be tested is applied to a pathological pulmonary fibrosis model or the pathogen is used to infect a pathological pulmonary fibrosis model, and the expression changes of genes related to the development of pulmonary fibrosis in the pathological pulmonary fibrosis model are detected; wherein, the related genes are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene. (xi) Infect a pathological pulmonary fibrosis model with the pathogen and detect changes in the expression of receptors related to the pathogen infection in the pathological pulmonary fibrosis model; (xii) Infect a pathological pulmonary fibrosis model with the pathogen, then apply the test drug to the pathological pulmonary fibrosis model, and detect the inhibitory effect of the test drug on the pathogen. Wherein, the pathological pulmonary fibrosis model is the pathological pulmonary fibrosis model constructed by the construction method of any one of claims 1 to 6, or the pathological pulmonary fibrosis model of claim 7; Preferably, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs; Preferably, the drug detection method and drug screening method include drug detection and / or drug screening for the treatment of pulmonary fibrosis.

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