Pathological pulmonary fibrosis model and construction method and application thereof

Abstract

The invention relates to a pathological pulmonary fibrosis model and a construction method and application thereof. The pathological pulmonary fibrosis model provided by the invention is good in stability and controllability and high in maturity, not only can better simulate the function and structural characteristics of pulmonary alveoli-capillary barrier, but also has the pathological characteristics and immune microenvironment of pulmonary fibrosis lesions, has the advantage of high simulation degree, and has good application prospects in simulation and research of pulmonary fibrosis diseases. Good application prospects are realized in the aspects of drug detection, screening, evaluation, research and development and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pathological pulmonary fibrosis model and a construction method thereof, the use of the pathological pulmonary fibrosis model in drug detection, drug screening and / or detecting the effect of pathogen infection on the development process of lung tissue fibrosis, a culture medium combination, and a drug detection method, a drug screening method and / or a method for detecting the effect of pathogen infection on the development process of lung tissue fibrosis. Background Art

[0002] Pulmonary fibrosis is a type of end-stage interstitial lung disease. It is known that there are many factors that can cause pulmonary fibrosis, including toxicity, autoimmunity, drugs, infection, traumatic injury, etc. Pulmonary fibrosis is characterized by fibroblast proliferation, extracellular matrix aggregation, inflammatory damage, and destruction of tissue structure. Studies have shown that the median survival of patients with pulmonary fibrosis is only 2 to 5 years, and the 5-year survival rate is only 20%.

[0003] Previous studies on pulmonary fibrosis have mainly focused on in vitro 2D culture of fibroblasts and animal models. However, each of these aspects has its own drawbacks. Among them, when fibroblasts are cultured in culture dishes or slides (2D), the cell structure, adhesion, mechanical conduction and signal transduction of soluble cytokines cannot be well demonstrated, and the interaction between cells-cells / matrix in vivo cannot be simulated. For animal models, due to species differences and the fact that artificially induced fibrotic diseases can only simulate a certain stage in the course of fibrosis and cannot show the entire development process of the disease, most of the drugs that have been shown to alleviate fibrosis in animal models have failed in clinical Phase II / III trials.

[0004] Therefore, it is extremely urgent to find a new in vitro model of pulmonary fibrosis that can better simulate the in vivo lung microenvironment for the study of pulmonary fibrosis diseases and drug screening. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a pathological pulmonary fibrosis model. The pathological pulmonary fibrosis model constructed by this construction 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, so as to be used for the research of 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] Providing lung organoids and cell culture vessels derived from patients with pulmonary fibrosis, and optionally lung fibroblasts derived from patients with pulmonary fibrosis;

[0008] Inoculating the lung organoids on the upper layer of the cell culture container, optionally inoculating the lung fibroblasts on the upper layer of the cell culture container, and then adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container to culture the lung organoids and optionally the lung fibroblasts in a liquid-liquid mode;

[0009] Discarding the lung epithelial cell culture medium in the upper layer and the lower layer, keeping the upper layer dry, and adding fresh lung epithelial cell culture medium to the lower layer to perform a first-stage air-liquid mode culture of the lung organoids and optionally the lung fibroblasts;

[0010] Endothelial cells are inoculated in the lower layer of the cell culture container, and macrophages are inoculated in the upper layer of the cell culture container and culture is carried out in the second stage of air-liquid mode to obtain a pathological pulmonary fibrosis model.

[0011] In the present invention, the cell culture container is a culture container that can be divided into at least an upper layer and a lower layer (sometimes also referred to as an upper chamber and a lower chamber) culture unit, so as to achieve cell migration or to make different cells grow in different layers or even adhere to a membrane (e.g., a porous membrane) between the upper and lower layers according to needs. Accordingly, the resulting pathological pulmonary fibrosis model can also be divided into an upper layer (i.e., an upper layer of the model) and a lower layer (i.e., a 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. Wherein, when a cell culture chip is used, the resulting pathological pulmonary fibrosis model is also referred to as 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 202211680988.1, 202222130269.4 or 202222130382.2, and these application documents are incorporated herein by reference in their entirety.

[0012] In the present invention, the lung organoids may be lung organoids extracted from primary pulmonary fibrosis tissue from a patient with pulmonary fibrosis. The patient with pulmonary fibrosis may be any mammal suffering from pulmonary fibrosis, such as humans, domestic animals, farm animals, and zoo, sports field or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0013] In the present invention, the lung fibroblasts are 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 needs to be constructed with inoculation of the lung fibroblasts.

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

[0015] Providing lung organoids and cell culture vessels derived from patients with pulmonary fibrosis;

[0016] Inoculating the lung organoids on the upper layer of the cell culture container, and then adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container to culture the lung organoids in a liquid-liquid mode;

[0017] The lung epithelial cell culture medium in the upper layer and the lower layer is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to perform the first stage of culturing the lung organoid in an air-liquid mode;

[0018] Endothelial cells are inoculated in the lower layer of the cell culture container, and macrophages are inoculated in the upper layer of the cell culture container and culture is carried out in the second stage of air-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 comprises:

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

[0021] Inoculating the lung organoids and the lung fibroblasts in the upper layer of the cell culture container, then adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container to culture the lung organoids and the lung fibroblasts in a liquid-liquid mode;

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

[0023] Endothelial cells are inoculated in the lower layer of the cell culture container, and macrophages are inoculated in the upper layer of the cell culture container and culture is carried out in the second stage of air-liquid mode to obtain a pathological pulmonary fibrosis model.

[0024] In the present invention, "liquid-liquid mode 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 mode 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 the present invention, by extracting and culturing primary lung organoids and optionally lung fibroblasts from patients with pulmonary fibrosis, a pathological pulmonary fibrosis model can be constructed instead of traditional cell lines. By culturing lung organoids and optionally lung fibroblasts first in a liquid-liquid mode and then in an air-liquid mode (i.e., first-stage air-liquid mode culture), and then inoculating endothelial cells and macrophages in the order of culture, and macrophages in the second-stage air-liquid mode culture, the pathological pulmonary fibrosis model is made more mature and its simulation is improved.

[0026] In some embodiments, the lung organoids are treated with a pulmonary fibrosis primary tissue digestion solution and then inoculated on the upper layer of the cell culture container. In the present invention, the pulmonary fibrosis primary tissue digestion solution can be a commercial pulmonary primary tissue digestion solution. In some preferred embodiments, the pulmonary fibrosis primary tissue digestion solution comprises collagenase, hyaluronidase, Y27632 (a ROCK inhibitor) and culture medium.

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

[0028] In some more preferred embodiments, the final concentration of the collagenase in the primary lung fibrosis tissue digestion solution is 1-2 mg / mL.

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

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

[0031] In some more 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: Washing the pulmonary fibrosis tissue from the pulmonary fibrosis patient with a buffer solution to remove the surface mucosa; the buffer solution preferably contains penicillin-streptomycin and gentamicin;

[0034] (2) Disintegration: mince the tissue into minced meat, resuspend in buffer, let stand and discard the supernatant;

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

[0036] (4) Resuspension: The digested cell suspension is filtered through a 100 μm cell mesh, and the cells are 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 expansion medium (Cat. No. A01-001) produced by Biogen, or the lung organoid culture kit (Cat. No. KLU0201) produced by Avid.

[0037] (5) Plating: Centrifuge, resuspend the lung organoids in extracellular matrix (Matrigel, BME, etc.), and plate. Add lung organoid culture medium and place in a 37°C incubator for culture.

[0038] In some embodiments, the lung fibroblasts are treated with a digestion solution of primary pulmonary fibrosis tissue and then inoculated into the upper layer of the cell culture container. The digestion solution of primary pulmonary fibrosis tissue is as described above and will not be described again 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: Washing the pulmonary fibrosis tissue from the pulmonary fibrosis patient with a buffer solution to remove the surface mucosa; the buffer solution preferably contains penicillin-streptomycin and gentamicin;

[0041] (2) Disintegration: mince the tissue into minced meat, resuspend in buffer, let stand and discard the supernatant;

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

[0043] (4) Resuspension: The digested cell suspension is filtered through a 70 μm cell mesh, and the cells are 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: Plate the lung fibroblasts into six-well plates, T25 cell culture flasks, or T75 cell culture flasks.

[0045] In some embodiments, the cell culture container may be pretreated before inoculating various cells to facilitate cell attachment. The pretreatment method may be those known in the art, for example, pretreatment with a solution including an extracellular matrix, wherein the extracellular matrix may be Matrigel, collagen, fibronectin, etc., which are not limited here.

[0046] In some embodiments, before inoculation, the lung organoids are digested with an organoid passage digestion solution (trypsin, Tryple or Accutase cell digestion solution, etc.) to form single cells, and then lung organoid culture medium is added to terminate the digestion, and then inoculation is performed.

[0047] In some embodiments, in the step of inoculating the lung organoids on the upper layer of the cell culture container, the lung organoids are inoculated with 1×10 5 ~10×10 5 cells / cm 2 .

[0048] In some embodiments, in the step of inoculating the lung fibroblasts on the upper layer of the cell culture container, the lung organoids are inoculated with 0.3×10 3 ~0.3×10 4 cells / cm 2 .

[0049] In some embodiments, in 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 commercial lung epithelial cell culture medium. The lung epithelial cell culture medium in the present invention includes a lung organoid culture medium. In some specific embodiments, the lung epithelial cell culture medium is, for example, a respiratory organoid expansion medium (item number A01-001) produced by Bogen, a lung organoid culture kit (item number KLU0201) produced by Avid, or an epithelial cell culture medium (PneumaCult 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 to 300 μL of lung epithelial cell culture medium is added to the upper layer, and 800 μL to 1 mL of lung epithelial cell culture medium is added to the lower layer.

[0051] In some embodiments, the liquid-liquid mode culture of the lung organoids and optionally the lung fibroblasts is 3 to 7 days, such as 3 days, 4 days, 5 days, 6 days, 7 days or any time within this range. In some specific embodiments, the temperature of the liquid-liquid mode culture is 37° C. In some specific embodiments, the liquid-liquid mode culture is replaced every 3 days.

[0052] In some embodiments, when the lung organoids overgrow the upper layer, the lung epithelial cell culture medium in the upper layer and the lower layer 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 mode culture of the lung organoid is 2 to 21 days, such as 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or any time within this range. In some specific embodiments, the temperature of the first stage of gas-liquid mode culture is 37°C.

[0055] In some embodiments, endothelial cells are seeded in the lower layer of the cell culture container, and the endothelial cells are seeded at 0.5×10 5 ~5×10 5 cells / cm 2 .

[0056] In some embodiments, the endothelial cells are derived from umbilical vein endothelial cells, primary lung endothelial cells, or a lung endothelial cell line.

[0057] In some embodiments, in the step of inoculating endothelial cells in 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, wherein 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; for example, the lung epithelial cell culture medium and the endothelial cell culture medium are mixed in a ratio 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 commercial culture medium, such as the endothelial cell culture medium (item number #1001) produced by Sciencell. Preferably, the endothelial cells are digested, resuspended with the first mixed culture medium, the cell density is adjusted, and then inoculated. 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 inoculating macrophages on the upper layer of the cell culture container and performing the second-stage gas-liquid mode culture, the macrophages are inoculated at 0.1×10 5 ~5×10 5 cells / cm 2 In some specific embodiments, the macrophages are resuspended in a macrophage culture medium and the cell density is adjusted. The macrophage culture medium can be selected according to the specific source of the macrophages, such as the macrophage culture medium (Cat. No. 1921) produced by ScienCell.

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

[0060] In some embodiments, in the step of inoculating macrophages in the upper layer of the cell culture container and performing the second-stage gas-liquid mode culture, the macrophages are resuspended in macrophage culture medium, inoculated in the upper layer of the cell culture container and adhered to the wall, then the culture medium of the upper layer and the lower layer is discarded and the upper layer is kept dry, and a fresh first mixed culture medium is added to the lower layer to perform the second-stage gas-liquid mode culture, wherein 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. In some specific embodiments, 600-700 μL of fresh first mixed culture medium is added to the lower layer.

[0061] In some embodiments, the second stage gas-liquid mode culture time of the lung organoid is 1 to 5 days, such as 1 day, 2 days, 3 days, 4 days, 5 days or any time within the range.

[0062] In some embodiments, after the macrophages are inoculated into the upper layer of the cell culture container and cultured in the second stage of the gas-liquid mode, the step of inoculating immune cells into the lower layer of the cell culture container and culturing them is also included, so that the constructed pathological pulmonary fibrosis model has immune function.

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

[0064] In some embodiments, the immune cells are seeded at 0.5×10 5 ~1×10 6 cells / cm 2 .

[0065] In some embodiments, in the step of inoculating immune cells in the lower layer of the cell culture container and culturing, the immune cells are resuspended with 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 in a gas-liquid mode, and the second mixed culture medium is obtained by mixing the immune cell culture medium and the first mixed culture medium in a volume ratio of (0.5-3.5):1; for example, the immune cell culture medium and the first mixed culture medium are mixed in a ratio of 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1 or 3.5:1. Wherein, the immune cell culture medium can be a commercially available immune cell culture medium, for example, it can be a PBMC culture medium, using a commercial PBMC culture medium, such as SuperCulture L100 lymphocyte serum-free culture medium (Da Kewei, catalog number #6911011) supplemented with 5-10% SuperGrow cell culture additives (Da Kewei, catalog number #6122011) and 1-5‰ IL-2.

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

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

[0068] In a third aspect, the present invention provides a culture medium combination, comprising a lung epithelial cell culture medium, a first mixed culture medium, and optionally a second mixed culture medium;

[0069] Among them, the first mixed culture medium is obtained by mixing a lung epithelial cell culture medium and an endothelial cell culture medium in a volume ratio of (0.5-3.5):1; the second mixed culture medium is obtained by mixing an immune cell culture medium and the first mixed culture medium in 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 for 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 air-liquid mode culture, second stage air-liquid mode, and optionally third stage air-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 model culture and / or the first stage air-liquid model 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 gas-liquid mode culture.

[0074] In a fourth aspect, the present invention provides a use of the 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 effect of pathogen infection on the development of pulmonary fibrosis.

[0077] In some embodiments, the drug includes a small molecule drug, an antibody drug, an ADC drug, or an immune drug.

[0078] In some embodiments, the pathogens include bacteria, viruses, fungi, parasites, and the like.

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

[0080] The pathological pulmonary fibrosis model provided by the present invention can be used for the efficacy detection and drug mechanism exploration of drugs for treating lung diseases (for example, drugs for treating pulmonary fibrosis diseases), and the response of the pathological pulmonary fibrosis model to drugs is evaluated by changes in cell marker expression, changes in cell (for example, alveolar cell) ratios, cell activity, inflammatory factor secretion, changes in immune cell typing, cell barrier function, etc., and then used for drug research and development, especially for the research and development of drugs for treating pulmonary fibrosis. For example, adding a drug to be tested to detect whether the drug will cause acceleration or inhibition of the development process of pulmonary fibrosis, thereby detecting and / or screening drugs for treating pulmonary fibrosis; after the pathogen infects the pathological pulmonary fibrosis model (upper or lower layer), the effect of the pathogen on the development process of pulmonary tissue fibrosis can be confirmed by indicator detection; after the pathological pulmonary fibrosis model is induced, the drug can be administered and the indicator detection can be performed to detect whether the drug will accelerate or inhibit the development process 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 of the present invention in (a) or (b). It should be understood that the applicable methods of the pathological pulmonary fibrosis model of the present invention are not limited to these:

[0081] (i) Changes in the expression of pulmonary fibrosis markers: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting changes in the expression of pulmonary fibrosis markers in the pathological pulmonary fibrosis model; wherein the pulmonary fibrosis markers are, for example, α-SMA, type I collagen (Collagen I), and fibronectin. In some specific embodiments, the method comprises: fixing the pathological pulmonary fibrosis model with 4% paraformaldehyde at room temperature for 30 minutes, removing the paraformaldehyde, washing the sample with PBS, permeabilizing the sample with 0.1% Triton-X-100 and blocking the sample with 5% BSA, respectively dropping primary antibodies α-SMA, type I collagen, and fibronectin, incubating at 4°C overnight, dropping secondary antibodies, incubating at room temperature for 1 hour, performing nuclear staining with DAPI for 15 minutes, washing with PBS, and imaging using a confocal microscope.

[0082] (ii) flow cytometry detection of immune cell typing changes: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the typing changes and / or dynamic immune response process of cells in the pathological pulmonary fibrosis model by flow cytometry; wherein the detection markers of the flow cytometry are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, CRTH2; the typing changes of the cells include the typing changes of macrophages and / or the typing changes of immune cells, and the typing changes of the macrophages are, for example, the typing changes of M1 macrophages and the typing changes of M2 macrophages; the typing changes of the immune cells are, for example, the typing changes of T cells, the typing changes of B cells, the typing changes of NK cells, and the typing changes of innate lymphocytes. In some specific embodiments, the flow cytometry includes: changes in the typing of M1 macrophages and M2 macrophages in the upper cells of the model, and the flow cytometry indicators include but are not limited to CD45, CD80, HLA-DR, CD163, CD68, CD206, etc.; or changes in the typing of immune cells such as T cells, B cells, NK cells, innate lymphocytes, etc. in the lower cells of the model, and the flow cytometry indicators include but are not limited to CD45, CD3, CD4, CXCR3, CCR4 (Th1, Th2 cells), CD19 (B cells), Lin, CD127, CD94, CD117, CRTH2 (ILC cells), etc. The operation steps are as follows: collect cells, digest into single cells with Tryple, Trypsin or Accutas, and resuspend cells in flow buffer. Use CD45, CD80, HLA-DR, CD163, CD3, CD4 and other antibodies for surface staining, 4°C, 30min. Add cell fixative and fix at room temperature in the dark for 10min. After centrifugation and washing with PBS, resuspend the cells with a membrane permeabilization reagent and incubate at room temperature for 10-15 minutes. Incubate the antibodies that need to be permeabilized, such as CD68 and CD206, at room temperature in the dark for 30 minutes. After incubation, wash twice with a membrane permeabilization reagent, add 200-500 μL of flow cytometry buffer to resuspend the cells, and detect using a flow cytometer.

[0083] (iii) detecting the transmembrane resistance of the model to characterize the cell barrier function of the model: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance of the pathological pulmonary fibrosis model; wherein the transmembrane resistance of a normal lung model is 200 to 1500 Ω·cm 2, the transmembrane resistance value of the pathological pulmonary fibrosis model will be lower than that of the normal lung model. Therefore, the effect of the test drug or pathogen infection on the development process of lung tissue fibrosis can be judged according to the change of the transmembrane resistance value. For example, when the test drug is added or infected by the pathogen, the transmembrane resistance value drops significantly to a statistically significant difference, then the test drug or pathogen infection will destroy the cell barrier; when the test drug is added, the transmembrane resistance value of the pathological pulmonary fibrosis model is significantly increased to a statistically significant difference compared with before the addition, then the test drug can repair the cell barrier; when the test drug is added or infected by the pathogen, the change in the transmembrane resistance value of the pathological pulmonary fibrosis model compared with the change before the addition of the test drug or infection by the pathogen is not statistically different, then the test drug or pathogen infection has no significant effect on the integrity of the cell barrier. In some specific implementation schemes, the following steps are included: taking out the transmembrane resistance meter, plugging in the power supply, assembling the monitoring device, placing the chopstick detection head in PBS for 15 minutes for equilibrium; after the instrument value stabilizes at 0Ω, filling the inside and outside of the model with PBS; inserting the chopstick detection head into the model, and recording the value after the display value stabilizes; data processing: TEER = TΩ × A cm 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 and characterizing the cell barrier function of the model: applying the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting changes in the barrier permeability of the pathological pulmonary fibrosis model. In some specific embodiments, it includes: adding fluorescent permeable molecules, such as FITC-Dextran (molecular weight 10kDa, 30KDa, 70KDa), etc., to the upper layer of the model, incubating at different time points, using an enzyme marker to detect the mass of the fluorescent molecules permeated in the lower layer of the model, and calculating the apparent permeability coefficient to reflect the changes in the barrier permeability of the model. The operation steps are as follows: discard all the culture medium in the well, add 50-100μM FITC-Dextran solution to the upper layer, add 400μL fresh culture medium to the lower layer, and incubate in a 37°C incubator; collect 200μL of the lower culture medium at 2h, 4h, 8h, and 24h of incubation respectively. 200 μL of culture medium was taken out each time, and 200 μL of culture medium was added to each well to continue culturing; the fluorescence value in the culture medium was detected using an ELISA instrument; and the following calculation was performed: Papp = (dQ / dt) / (A*C0); wherein Papp is the apparent permeability coefficient, A is the membrane area of ​​the membrane chip, C0 is the initial concentration of the fluorescent dye, and dQ / dt is the amount of fluorescent dye that passes through per unit time.

[0085] (v) Detecting the activity of model cells: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the 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. In some specific embodiments, the activity of model cells is detected using methods such as MTT, CCK8, and CTG. Taking CTG detection as an example: add an amount of CTG activity detection reagent stock solution equal to that of the culture medium to the model, mix by pipetting for 5 minutes, incubate at room temperature for 30 minutes, and use an enzyme marker to detect its chemiluminescence value.

[0086] (vi) Detecting cell apoptosis in the model: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting cell apoptosis in the pathological pulmonary fibrosis model; wherein the detection method is, for example, immunofluorescence or flow cytometry; and the markers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL.

[0087] (vii) Detecting cytokine content: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting changes in the content of inflammatory cytokines in the pathological pulmonary fibrosis model; wherein the inflammatory factors are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18. In some specific embodiments, detection is performed using methods such as ELISA, CBA multifactor detection, and Luminex multifactor detection. Take ELISA detection of inflammatory factor TNF-α content as an example: prepare 0, 11.7, 23.4, 46.9, 93.8, 187.5, 375 and 750pg / mL standard solutions, add 100μL standard solution and sample solution to each well, incubate at room temperature for 1h, wash each well with 300μL cleaning solution 4 times, 2min each time, add 100μL antibody of inflammatory cytokine to be tested to each well, incubate at room temperature for 30min, wash each well with 300μL cleaning solution 4 times, 2min each time, add 100μL color development solution to each well, incubate at room temperature for 20min, add 50μL stop solution to each well, and detect the OD value at 450nm within 10min.

[0088] (viii) Detecting cell types and expressions related to lung tissue: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the cell phenotype or cell content related to lung tissue in the pathological pulmonary fibrosis model; wherein the cell phenotype is, for example, α-tubulin (ciliary 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) Detecting the level of oxidative stress: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathogen to the pathological pulmonary fibrosis model, and detecting the level of cellular oxidative stress in the pathological pulmonary fibrosis model. In some specific embodiments, the ROS oxidative stress detection kit is used to detect the level of ROS / RNS and evaluate the regulation of the drug on oxidative stress. For example, using Invitrogen TM The CellROX kit is used to test the model. CellROX is a fluorescent probe that can measure generalized oxidative stress in cells using fluorescence microscopy, high-content imaging systems, microplate fluorometers, or flow cytometry platforms. The dye is non-fluorescent in the reduced state and emits bright green, orange, or deep red fluorescence when oxidized.

[0090] (x) Detecting the expression of genes related to the development of pulmonary fibrosis: administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the expression changes of pulmonary fibrosis-related genes in the pathological pulmonary fibrosis model; 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 included to detect the changes in the transcription levels of pulmonary fibrosis-related genes.

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

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

[0093] In a fifth aspect, 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) applying the drug to be tested to the pathological pulmonary fibrosis model or infecting the 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. In some embodiments, the detection method is an immunofluorescence detection method; preferably, the detection method comprises: after fixing, washing, and blocking the pathological pulmonary fibrosis model, adding a primary antibody of a pulmonary fibrosis marker and incubating, then adding a secondary antibody and incubating, and obtaining the expression change results of the pulmonary fibrosis marker by staining and imaging;

[0095] (ii) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the phenotypic changes and / or dynamic immune response process of cells in the pathological pulmonary fibrosis model by flow cytometry. In some embodiments, the detection markers of the 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 typing changes of the cells include typing changes of macrophages and / or typing changes of immune cells, and the typing changes of the macrophages are, for example, typing changes of M1 macrophages and typing changes of M2 macrophages; the typing changes of the immune cells are, for example, typing changes of T cells, typing changes of B cells, typing changes of NK cells, and typing changes of innate lymphocytes; preferably, the detection method comprises: collecting cells in the pathological pulmonary fibrosis model and resuspending them with a flow buffer, staining the surface with an antibody for the detection marker, fixing, centrifuging, and washing, adding a flow buffer to resuspend the cells, and detecting the fluorescence signal by a flow cytometer;

[0096] (iii) applying the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance value of the pathological pulmonary fibrosis model; the effect of the drug to be tested or the pathogen infection on the development process of pulmonary tissue fibrosis can be judged according to the change in the transmembrane resistance value. When the drug to be tested is added or infected by the pathogen, the transmembrane resistance value significantly decreases to a statistically significant difference, then the drug to be tested or the pathogen infection can destroy the cell barrier; when the drug to be tested is added, the transmembrane resistance value of the pathological pulmonary fibrosis model is significantly increased to a statistically significant difference compared with before the addition, then the drug to be tested can repair the cell barrier; when the drug to be tested is added or infected by the pathogen, the transmembrane resistance value of the pathological pulmonary fibrosis model is not statistically different from the change before the addition of the drug to be tested or the pathogen infection, then the drug to be tested or the 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) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the 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 comprises: 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 permeated in the lower layer of the pathological pulmonary fibrosis model, and calculating the apparent permeability coefficient;

[0098] (v) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the 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 comprises: adding MTT, CCK8 or CTG activity detection reagent to the pathological pulmonary fibrosis model and incubating, and detecting the chemiluminescence value;

[0099] (vi) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting apoptosis in the pathological pulmonary fibrosis model. In some embodiments, the detection method is, for example, immunofluorescence or flow cytometry; the markers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, TUNEL;

[0100] (vii) Applying the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting changes in the content of inflammatory cytokines in the pathological pulmonary fibrosis model. In some embodiments, the inflammatory factors 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: preparing a standard solution of inflammatory cytokines with gradient concentrations, adding the standard solution and sample culture medium to a culture container, incubating and washing, adding an antibody to the inflammatory cytokine to be tested, and then incubating and washing, adding a color developing solution and incubating, and then adding a stop solution and detecting the OD value;

[0101] (viii) administering the drug to be tested to a pathological pulmonary fibrosis model or infecting the pathogen to the pathological pulmonary fibrosis model, and detecting the cell phenotype or cell content associated with lung tissue in the pathological pulmonary fibrosis model. In some embodiments, the cell phenotype is, for example, α-tubulin, MUC5AC, AQP5, SFTPC, CC10, or KRT5;

[0102] (ix) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the level of cellular oxidative stress in the pathological pulmonary fibrosis model;

[0103] (x) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the expression changes of pulmonary fibrosis-related genes in the pathological pulmonary fibrosis model. 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) allowing the pathogen to infect a pathological pulmonary fibrosis model, and detecting changes in the expression of receptors associated with the pathogen infection in the pathological pulmonary fibrosis model;

[0105] (xii) allowing the pathogen to infect a pathological pulmonary fibrosis model, then administering the drug to be tested to the pathological pulmonary fibrosis model, and detecting the inhibitory effect of the drug to be tested on the pathogen;

[0106] Wherein, the pathological pulmonary fibrosis model is 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 embodiments, the drug includes a small molecule drug, an antibody drug, an ADC drug, or an immune drug.

[0108] First, the present invention can replace traditional cell lines to construct a pathological pulmonary fibrosis model by extracting and culturing primary lung organoids and optionally lung fibroblasts from patients with pulmonary fibrosis. Compared with animal models, the present invention has a shorter experimental cycle, which is beneficial to reducing experimental costs, while simplifying the difficulty of operation and enhancing model stability and controllability. Secondly, the construction method provided by the present invention is to inoculate lung organoids (and optionally lung fibroblasts), endothelial cells, macrophages and optionally immune cells in sequence, and obtain a pathological pulmonary fibrosis model by first culturing in a liquid-liquid mode and then in a gas-liquid mode (including a first stage gas-liquid mode culture, a second stage gas-liquid mode culture and optionally a third stage gas-liquid mode culture). This is not only beneficial to making the pathological pulmonary fibrosis model more mature, but also can better simulate the function and structural characteristics of the alveolar-capillary barrier, and has the pathological characteristics and immune microenvironment of pulmonary fibrosis lesions, with a high degree of simulation. In addition, the pathological pulmonary fibrosis model provided by the present invention comprises lung organoids, endothelial cells, macrophages, and optionally lung fibroblasts and immune cells, which can well simulate the microenvironment associated with these cells and the cell-cell interactions, and also has the advantages of being able to be passaged multiple times and maintaining the phenotype in vitro for a long time, providing a good platform for the simulation and research of pulmonary fibrosis diseases, and the detection, screening, evaluation and development of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 The bright field image of the construction process of the pathological pulmonary fibrosis model in Example 1 of the present invention is shown.

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

[0111] Figure 3 The expression of α-SMA and Collagen I in each group of models in Example 3 of the present invention is shown.

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

[0113] Figure 5 The cell content associated with the lung tissue of the pathological pulmonary fibrosis model in Example 5 of the present invention is shown. DETAILED DESCRIPTION

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

[0115] Example 1

[0116] This example provides a specific construction process of a pathological pulmonary fibrosis model:

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

[0118] (1) Cleaning: Use primary tissue cleaning solution to clean the pulmonary fibrosis tissue from patients with pulmonary fibrosis and remove the surface mucosa, wherein the primary tissue cleaning solution is a buffer solution to which penicillin-streptomycin and gentamicin are added.

[0119] (2) Disintegration: Chop the tissue into minced meat, resuspend in buffer, let stand and discard the supernatant.

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

[0121] (4) Resuspension: The digested cell suspension is filtered through a 100 μm cell mesh, and the cells are resuspended in lung organoid culture medium and counted. The lung organoid culture medium is produced by Avid, and is called the lung organoid culture kit, with the catalog number KLU0201.

[0122] (5) Plating: Centrifuge and remove the supernatant, resuspend the lung organoids in extracellular matrix Matrigel, and plate them. Place 30 μL of gel droplets in each well and plate the lung organoids in a 24-well plate. After the gel is formed, add lung organoid culture medium and place in a 37°C incubator for 7 days.

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

[0124] (7) Inoculation: Centrifuge and remove the supernatant, resuspend in lung epithelial cell culture medium, and adjust the density to 1×10 6 / mL. The chip membrane area is 0.33cm 2 , inoculated with 2×10 5 cells, and the cell density was 6×10 5 Pieces / cm 2 . Inoculate the lung organoids on the upper layer of the membrane. Add 1 mL of lung epithelial cell culture medium (Respiratory Organoid Expansion Medium (Cat. No. A01-001) produced by Bogen, lung organoid culture kit (Cat. No. KLU0201) produced by Avid, or epithelial cell culture medium (PneumaCult TM -Ex Plus Medium)) and cultured in a 37°C incubator in liquid-liquid mode.

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

[0126] Air-liquid culture:

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

[0128] (2) Cultivation in the gas-liquid mode for 2 days (first stage gas-liquid mode cultivation).

[0129] Seeding of endothelial cells:

[0130] (1) Digest endothelial cells and resuspend the cells in the first mixed culture medium (lung epithelial cell culture medium: endothelial cell culture medium volume ratio 2:1). Count and adjust the endothelial cell density to 6×10 5 / mL. The chip membrane area is 0.33cm 2 , inoculated with 3×10 4 Endothelial cells, with a cell density of 0.9×10 5 Pieces / cm 2 The endothelial cell culture medium is the endothelial cell culture medium produced by Sciencell (Cat. No. #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 mixed in a volume ratio of 2:1) to the lower chamber.

[0132] Inoculation of macrophages:

[0133] (1) Collect macrophages and resuspend them in macrophage culture medium (ScienCell, #1921). Count and adjust the cell density to 1×10 5 / mL. The chip membrane area is 0.33cm 2 , inoculated with 1×104 Macrophages, cell density was 0.3×10 5 Pieces / cm 2 .

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

[0135] (3) The culture medium under the membrane is replaced (first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium mixed in a volume ratio of 2:1)).

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

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

[0138] (1) Collect PBMCs and resuspend them in the second mixed culture medium (immune cell culture medium and first mixed culture medium mixed in a volume ratio of 2:1). Discard the culture medium under the membrane. The bottom area of ​​the chip lower chamber is 2 cm 2 , inoculated with 2×10 5 PBMCs, with a cell density of 1×10 5 Pieces / cm 2 Among them, for example, a commercial PBMC culture medium is used, such as SuperCulture L100 lymphocyte serum-free culture medium (Dakoway, catalog number 6122011) supplemented with 5-10% SuperGrow cell culture supplement (Dakoway, catalog number 6122011) and 1-5‰ IL-2.

[0139] #6911011).

[0140] (2) The air-liquid mode culture was continued for 1 day (the third stage of air-liquid mode culture) to obtain a pathological pulmonary fibrosis model.

[0141] A high-content imaging system was used to take pictures at different time points during model construction. Bright field images are shown in the figure below. Figure 1 As shown. Figure 1 It can be seen that when lung organoids were inoculated, the cells were evenly spread on the upper layer of the culture chamber membrane; during air-liquid culture, the cells grew in sheets with vacuolar morphology in the middle; after inoculation of endothelial cells and macrophages, the macrophages were scattered among the lung epithelial cells and grew with them.

[0142] Example 2

[0143] This example provides a specific construction process of a pathological pulmonary fibrosis model:

[0144] Primary extraction and culture of lung organoids from patients with pulmonary fibrosis:

[0145] (1) Cleaning: Use primary tissue cleaning solution to clean the pulmonary fibrosis tissue from patients with pulmonary fibrosis and remove the surface mucosa, wherein the primary tissue cleaning solution is a buffer solution to which penicillin-streptomycin and gentamicin are added.

[0146] (2) Disintegration: Chop the tissue into minced meat, resuspend in buffer, let stand and discard the supernatant.

[0147] (3) Digestion: Add primary pulmonary fibrosis tissue digestion solution, mix by pipetting, and then use a metal bath shaker at 37°C for 30-60 minutes to digest into small cell clusters, and finally add buffer to terminate digestion. Primary pulmonary fibrosis tissue digestion solution contains collagenase I with a final concentration of 1 mg / mL, hyaluronidase with a final concentration of 0.5 mg / mL, Y27632 with a final concentration of 10 μM (purchased from MCE, catalog number HY-10071), and culture medium Ad-DMEM / F12.

[0148] (4) Resuspension: The digested cell suspension is filtered through a 100 μm cell mesh, and the cells are resuspended in lung organoid culture medium and counted. The lung organoid culture medium is produced by Avid, and is called the lung organoid culture kit, with the catalog number KLU0201.

[0149] (5) Plating: Centrifuge and remove the supernatant, resuspend the lung organoids in extracellular matrix Matrigel, and plate them. Place 30 μL of gel droplets in each well and plate the lung organoids in a 24-well plate. After the gel is formed, add lung organoid culture medium and place in a 37°C incubator for 7 days.

[0150] Subculture of lung organoids from patients with pulmonary fibrosis:

[0151] (1) Collection: After 7 days of primary culture of lung organoids, remove the lung organoid culture medium, add pre-cooled PBS buffer, pipette Matrigel, resuspend the lung organoids, and collect them in a centrifuge tube.

[0152] (2) Digestion: Centrifuge and remove the supernatant. Add Tryple to digest the organoids into small cell clusters. Add PBS buffer to terminate the digestion.

[0153] (3) Plating: Centrifuge and remove the supernatant, resuspend the lung organoids in extracellular matrix Matrigel, and plate them. Place 30 μL of gel droplets in each well and plate the lung organoids in a 24-well plate. After the gel is formed, add lung organoid culture medium and place in a 37°C incubator for 7 days.

[0154] Primary extraction and culture of lung fibroblasts from patients with pulmonary fibrosis:

[0155] (1) Washing: Wash the pulmonary fibrosis tissue from patients with pulmonary fibrosis with a primary tissue washing solution to remove the surface mucosa, wherein the primary tissue washing solution is a buffer solution to which penicillin-streptomycin and gentamicin are added;

[0156] (2) Disintegration: mince the tissue into minced meat, resuspend in buffer, let stand and discard the supernatant;

[0157] (3) Digestion: Add primary pulmonary fibrosis tissue digestion solution, mix by pipetting, and then use a metal bath shaker at 37°C for 30-60 min to digest into single cells, and finally add buffer to terminate digestion; primary pulmonary fibrosis tissue digestion solution contains collagenase I with a final concentration of 1 mg / mL, hyaluronidase with a final concentration of 0.5 mg / mL, Y27632 with a final concentration of 10 μM (purchased from MCE, catalog number HY-10071), and culture medium Ad-DMEM / F12;

[0158] (4) Resuspension: The digested cell suspension was filtered through a 70 μm cell mesh and resuspended in lung fibroblast culture medium; the lung fibroblast culture medium was #AC-1001015 produced by Ezen Biotech;

[0159] (5) Plating: Count the cells and adjust the cell density to 1×10 6 Lung fibroblasts were plated into T25 cell culture flasks, 1 mL of cells and 5 mL of lung fibroblast culture medium were added to each flask, and cultured in a 37°C incubator for 7 days.

[0160] Seeding of lung organoids and lung fibroblasts:

[0161] (1) Digestion of lung organoids: Take the 5th generation lung organoids and digest them with Tryple, a lung organoid digestion solution, into single cells. Add lung organoid culture medium to terminate the digestion.

[0162] (2) Digestion of lung fibroblasts: Use Tryple, a fibroblast cell culture digestion solution, to digest the lung fibroblasts into single cells, and add DMEM containing 10% FBS to terminate the digestion.

[0163] (3) Inoculation: Resuspend the lung organoids and lung fibroblast pellets in lung epithelial cell culture medium and adjust the cell density of the lung organoids to 1×10 6 The density of lung fibroblasts was adjusted to 5×10 4 / mL. The chip membrane area is 0.33cm 2 , inoculate lung organoids 2×10 5 The cell density was 6×10 5 Pieces / cm 2; Inoculate lung fibroblasts 5×10 3 The cell density was 1.5×10 4 Pieces / cm 2 Lung organoids and lung fibroblasts were seeded on the upper layer of the membrane. 1 mL of lung epithelial cell culture medium was added to the lower layer of the membrane and cultured in a 37°C incubator in a liquid-liquid mode.

[0164] (4) Change the medium every 2 days and culture for 4 days.

[0165] Air-liquid culture:

[0166] (1) When the lung organoids have grown all over the upper layer of the membrane, discard the culture medium on the upper and lower layers of the membrane and add 600 μL of lung epithelial cell culture medium to the lower layer of the membrane.

[0167] (2) Cultivation in the gas-liquid mode for 2 days (first stage gas-liquid mode cultivation).

[0168] Seeding of endothelial cells:

[0169] (1) Digest endothelial cells and resuspend the cells in the first mixed culture medium (lung epithelial cell culture medium: endothelial cell culture medium volume ratio 2:1). Count and adjust the endothelial cell density to 6×10 5 / mL. The chip membrane area is 0.33cm 2 , inoculated with 3×10 4 Endothelial cells, with a cell density of 0.9×10 5 Pieces / cm 2 The endothelial cell culture medium is the endothelial cell culture medium produced by Sciencell (Cat. No. #1001).

[0170] (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 mixed in a volume ratio of 2:1) to the lower chamber.

[0171] Inoculation of macrophages:

[0172] (1) Collect macrophages and resuspend them in macrophage culture medium (ScienCell, #1921). Count and adjust the cell density to 1×10 5 / mL. The chip membrane area is 0.33cm 2 , inoculated with 1×10 4 Macrophages, cell density was 0.3×10 5 Pieces / cm 2 .

[0173] (2) The macrophages were cultured overnight, and then the macrophage culture medium on the membrane was discarded.

[0174] (3) The culture medium under the membrane is replaced (first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium mixed in a volume ratio of 2:1)).

[0175] (4) Continue the gas-liquid mode culture for 2 days (second stage gas-liquid mode culture).

[0176] Vaccination of immune cells (if immune function is not required, this step can be omitted):

[0177] (1) Collect PBMCs and resuspend them in the second mixed culture medium (immune cell culture medium and first mixed culture medium mixed in a volume ratio of 2:1). Discard the culture medium under the membrane. The bottom area of ​​the chip lower chamber is 2 cm 2 , inoculated with 2×10 5 PBMCs, with a cell density of 1×10 5 Pieces / cm 2 Among them, for example, a commercial PBMC culture medium is used, such as SuperCulture L100 lymphocyte serum-free culture medium (Dakoway, catalog number 6122011) supplemented with 5-10% SuperGrow cell culture supplement (Dakoway, catalog number 6122011) and 1-5‰ IL-2.

[0178] #6911011).

[0179] (2) The air-liquid mode culture was continued for 1 day (the third stage of air-liquid mode culture) to obtain a pathological pulmonary fibrosis model.

[0180] Perform immunofluorescence experiments on the model:

[0181] (1) After the model was constructed, 4% paraformaldehyde was added for fixation for 30 min;

[0182] (2) Discard the fixative solution and wash three times with PBS. Add permeabilization solution (0.3% Triton-100) and permeabilize for 15 minutes;

[0183] (3) Add 5% BSA blocking solution to block the sample for 1 hour.

[0184] (4) Primary antibodies α-SMA and Collagen I were added and incubated at 4°C overnight. Secondary antibodies were added and incubated at room temperature for 1 h. Nuclear staining was performed with DAPI, and the cells were washed with PBS and imaged using a confocal microscope.

[0185] Figure 2 The immunofluorescence results showed that α-SMA was labeled in green, Collagen I was labeled in red, and cell nuclei were labeled in blue. It can be seen that the constructed pathological pulmonary fibrosis model has the characteristics of pulmonary fibrosis.

[0186] Example 3

[0187] This example provides a pathological pulmonary fibrosis model for drug testing:

[0188] (1) A pathological pulmonary fibrosis model was constructed according to the method of Example 1. A pulmonary fibrosis patient group and a nintedanib group were set up. The pulmonary fibrosis patient group was a lung in vitro model, and the lower layer of the chip was added with the second mixed culture medium; the nintedanib group was a lung in vitro model, and the lower layer of the chip was added with the second mixed culture medium containing 10 μM nintedanib.

[0189] (2) Drug treatment for 3 days.

[0190] (3) After drug treatment, immunofluorescence was used to detect whether there was any difference in the expression of α-SMA and Collagen I in the pulmonary fibrosis model.

[0191] Figure 3 The immunofluorescence results showed that α-SMA was labeled in green, Collagen I was labeled in red, and cell nuclei were labeled in blue. It can be seen that the constructed lung fibrosis patient group had obvious fibroblasts and collagen deposition, while the nintedanib group had weaker fibroblast expression and reduced collagen deposition.

[0192] Example 4

[0193] This example provides a pathological pulmonary fibrosis model for drug testing:

[0194] (1) A pathological pulmonary fibrosis model was constructed according to the method of Example 2. A pulmonary fibrosis patient group and a nintedanib group were set up. The pulmonary fibrosis patient group was a lung in vitro model, and the second mixed culture medium was added to the lower layer of the chip; the nintedanib group was a second mixed culture medium containing 5 μM nintedanib was added to the lower layer of the chip of the lung in vitro model.

[0195] (2) Drug treatment for 3 days.

[0196] (3) After drug treatment, the ELISA kit was used to detect the content of inflammatory factor IFN-γ in the pulmonary fibrosis model, and the expression of inflammatory cytokines in the pulmonary fibrosis patient group (IPF) and the drug treatment group (5 μM Nintedanib) was compared to see whether there was any difference.

[0197] Figure 4 The ELISA results showed that the secretion of IFN-γ in the constructed pulmonary fibrosis patient group was about 25pg / mL, and the secretion of IFN-γ in the nintedanib group was about 12pg / mL. After treatment with the drug nintedanib, the secretion of IFN-γ was significantly reduced, indicating that nintedanib can inhibit the secretion of inflammatory factors.

[0198] Example 5

[0199] This example provides a pathological pulmonary fibrosis model for detecting cell phenotypes and content related to lung tissue:

[0200] (1) A pathological pulmonary fibrosis model was constructed according to the method of Example 1. After the construction was completed, flow cytometry detection was performed.

[0201] (2) Lung epithelial cells were digested into single cells using Tryple.

[0202] (3) Staining was performed using lung tissue-related cell antibodies, including MUC5AC (goblet cell marker), PDPN (AT1 cell marker), SFTPB (AT2 cell marker), CC10 (club cell marker), and KRT5 (basal cell marker).

[0203] (4) Detection using flow cytometry.

[0204] Figure 5 The flow cytometry results showed that the constructed pathological pulmonary fibrosis model contained major lung tissue cells, among which cells expressing KRT5 accounted for 28.3% of the total cell number, cells expressing PDPN accounted for 50.6% of the total cell number, cells expressing SFTPB accounted for 34.1% of the total cell number, cells expressing CC10 accounted for 38.8% of the total cell number, and cells expressing MUC5AC accounted for 18.4% of the total cell number.

Claims

1. A method for constructing a pathological pulmonary fibrosis model, comprising: Providing lung organoids and cell culture vessels derived from patients with pulmonary fibrosis, and optionally lung fibroblasts derived from patients with pulmonary fibrosis; Inoculating the lung organoids on the upper layer of the cell culture container, optionally inoculating the lung fibroblasts on the upper layer of the cell culture container, and then adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container to culture the lung organoids and optionally the lung fibroblasts in a liquid-liquid mode; Discarding the lung epithelial cell culture medium in the upper layer and the lower layer, keeping the upper layer dry, and adding fresh lung epithelial cell culture medium to the lower layer to perform a first-stage air-liquid mode culture of the lung organoids and optionally the lung fibroblasts; Endothelial cells are inoculated in the lower layer of the cell culture container, and macrophages are inoculated in the upper layer of the cell culture container and culture is carried out in the second stage of air-liquid mode to obtain a pathological pulmonary fibrosis model.

2. The construction method according to claim 1, wherein: The lung organoids are treated with pulmonary fibrosis primary tissue digestion solution and then inoculated on the upper layer of the cell culture container; wherein the pulmonary fibrosis primary tissue digestion solution comprises collagenase, hyaluronidase, Y27632 and culture medium; preferably, the collagenase comprises at least one of collagenase I, collagenase II and collagenase IV; preferably, the final concentration of the collagenase in the pulmonary fibrosis primary tissue digestion solution is 1 to 2 mg / mL; preferably, the final concentration of the hyaluronidase in the pulmonary fibrosis primary tissue digestion solution is 0.5 to 1 mg / mL; preferably, the final concentration of the Y27632 in the pulmonary fibrosis primary tissue digestion solution is 5 to 10 μM; preferably, the culture medium is selected from DMEM or ADMEM / F12; and / or The lung fibroblasts are treated with pulmonary fibrosis primary tissue digestion fluid and then inoculated into the upper layer of the cell culture container; wherein the pulmonary fibrosis primary tissue digestion fluid 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 pulmonary fibrosis primary tissue digestion fluid is 1 to 2 mg / mL; preferably, the final concentration of the hyaluronidase in the pulmonary fibrosis primary tissue digestion fluid is 0.5 to 1 mg / mL; preferably, the final concentration of the Y27632 in the pulmonary fibrosis primary tissue digestion fluid is 5 to 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 liquid-liquid mode culture time of the lung organoid is 3 to 7 days; The time for culturing the lung organoids in the air-liquid mode in the first stage is 2 to 21 days; The duration of the second stage gas-liquid mode culture is 1 to 5 days; Based on the inoculation area, the lung organoids were inoculated with 1×10 5 ~10×10 5 cells / cm 2 ; When the lung fibroblasts are inoculated, the lung fibroblasts are inoculated with 0.3×10 3 ~0.3×10 4 cells / cm 2 ; Calculated by the inoculation area, the endothelial cells were inoculated with 0.5×10 5 ~5×10 5 cells / cm 2 ; and / or Calculated by the inoculation area, the macrophages were inoculated with 0.1×10 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 PBMC-expanded macrophages, primary alveolar macrophages, passaged alveolar macrophages, or macrophages induced by THP-1 cell line.

4. The construction method according to any one of claims 1 to 3, wherein: In the step of inoculating endothelial cells in 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, wherein the first mixed culture medium is obtained by mixing the lung epithelial cell culture medium and the endothelial cell culture medium in a volume ratio of (0.5-3.5):1; In the step of inoculating macrophages in the upper layer of the cell culture container and performing a second-stage gas-liquid mode culture, after the macrophages adhere to the wall, the culture medium in the upper layer and the lower layer is discarded and the upper layer is kept dry, and a fresh first mixed culture medium is added to the lower layer to perform a second-stage gas-liquid mode culture, wherein 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 the macrophages are inoculated in the upper layer of the cell culture container and cultured in the second stage of gas-liquid mode, the method further includes inoculating immune cells in the lower layer of the cell culture container and culturing them; Preferably, the immune cells include PBMC, T cells, B cells, and NK cells; Preferably, the immune cells are inoculated at 0.5×10 5 ~1×10 6 cells / cm 2 ; Preferably, in the step of inoculating the immune cells in the lower layer of the cell culture container and culturing, the immune cells are resuspended with 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 of gas-liquid mode culture, wherein the second mixed culture medium is obtained by mixing the immune cell culture medium and the first mixed culture medium in a volume ratio of (0.5-3.5):1; More preferably, the gas-liquid mode culture time of the third stage 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 constructed by the construction method according to any one of claims 1 to 6.

8. A culture medium combination 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 a lung epithelial cell culture medium and an endothelial cell culture medium in a volume ratio of (0.5-3.5):1; the second mixed culture medium is obtained by mixing an immune cell culture medium and the first mixed culture medium in a volume ratio of (0.5-3.5):1; Preferably, the culture medium combination is used for the construction method according to any one of claims 1 to 6, or for the construction of the pathological pulmonary fibrosis model according to claim 7, including culture for lung organoids, liquid-liquid mode culture, first stage air-liquid mode culture, second stage air-liquid mode, and optionally third stage air-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 mode culture and / or the first stage air-liquid mode culture; 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 gas-liquid mode culture.

9. Use of 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 in the following (a) or (b); (a) drug detection and / or drug screening; preferably, the drug includes small molecule drugs, antibody drugs, ADC drugs, immune 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 effect of pathogen infection on the development of pulmonary fibrosis.

10. The use according to claim 9, wherein Said (a) and (b) include at least one of the following (i) to (xii): (i) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the 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; (ii) applying the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the typing changes and / or dynamic immune response process of cells in the pathological pulmonary fibrosis model by flow cytometry; wherein the detection markers of the flow cytometry are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, CRTH2; the typing changes of the cells include the typing changes of macrophages and / or the typing changes of immune cells, and the typing changes of the macrophages are, for example, the typing changes of M1 macrophages and the typing changes of M2 macrophages; the typing changes of the immune cells are, for example, the typing changes of T cells, the typing changes of B cells, the typing changes of NK cells, and the typing changes of innate lymphocytes; (iii) applying the drug to be tested to the pathological pulmonary fibrosis model or allowing the pathogen to infect the pathological pulmonary fibrosis model, and detecting the transmembrane resistance value of the pathological pulmonary fibrosis model; wherein, when the transmembrane resistance value of the pathological pulmonary fibrosis model significantly decreases to a statistically significant level after the drug to be tested is added or the pathogen is infected, the drug to be tested or the pathogen infection will damage the cell barrier; when the transmembrane resistance value of the pathological pulmonary fibrosis model significantly increases to a statistically significant level compared with that before the drug to be tested is added after the drug to be tested is added, the drug to be tested can repair the cell barrier; when the transmembrane resistance value of the pathological pulmonary fibrosis model does not have a statistically significant difference compared with that before the drug to be tested is added or the pathogen is infected after the drug to be tested is added or the pathogen is infected, the drug to be tested or the pathogen infection has no significant effect on the integrity of the cell barrier; (iv) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting changes in the barrier permeability of the pathological pulmonary fibrosis model; (v) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the 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; (vi) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting cell apoptosis in the pathological pulmonary fibrosis model; wherein the detection method is, for example, immunofluorescence or flow cytometry; and the markers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL; (vii) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting changes in the content of inflammatory cytokines in the pathological pulmonary fibrosis model; wherein the inflammatory factors are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18; (viii) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the cell phenotype or cell content related to the lung tissue in the pathological pulmonary fibrosis model; 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) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the level of cellular oxidative stress in the pathological pulmonary fibrosis model; (x) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the expression changes of genes related to the development of pulmonary fibrosis in the pathological pulmonary fibrosis model; wherein the related genes are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene; (xi) allowing the pathogen to infect the pathological pulmonary fibrosis model, and detecting changes in the expression of receptors associated with the pathogen infection in the pathological pulmonary fibrosis model; (xii) allowing the pathogen to infect the pathological pulmonary fibrosis model, then administering the drug to be tested to the pathological pulmonary fibrosis model, and detecting the inhibitory effect of the drug to be tested on the pathogen.

11. A method for drug detection, drug screening and / or detection of 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 the 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 an immunofluorescence detection method; more preferably, the detection method comprises: after fixing, washing, and blocking the pathological pulmonary fibrosis model, adding a primary antibody of a pulmonary fibrosis marker and incubating, then adding a secondary antibody and incubating, and obtaining the expression change results of the pulmonary fibrosis marker by staining and imaging; (ii) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the cell typing changes and / or dynamic immune response process in the pathological pulmonary fibrosis model by flow cytometry; wherein the detection markers of the flow cytometry are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, CRTH2; the cell typing changes include macrophage Typing changes of cells and / or typing changes of immune cells, the typing changes of macrophages are, for example, typing changes of M1 macrophages and typing changes of M2 macrophages; the typing changes of immune cells are, for example, typing changes of T cells, typing changes of B cells, typing changes of NK cells, and typing changes of innate lymphocytes; preferably, the detection method comprises: collecting cells in a pathological pulmonary fibrosis model and resuspending them with a flow cytometric buffer, staining with an antibody for detecting a marker, fixing, centrifuging, washing, adding a flow cytometric buffer to resuspend the cells, and detecting the fluorescence signal with a flow cytometer; (iii) applying the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance of the pathological pulmonary fibrosis model; wherein, when the transmembrane resistance of the pathological pulmonary fibrosis model significantly decreases to a statistically significant level after the drug to be tested is added or the pathogen is infected, the drug to be tested or the pathogen infection will damage the cell barrier; when the transmembrane resistance of the pathological pulmonary fibrosis model significantly increases to a statistically significant level compared with before the drug to be tested is added after the drug to be tested is added or the pathogen is infected, the drug to be tested can repair the cell barrier; when the transmembrane resistance of the pathological pulmonary fibrosis model does not have a statistically significant change compared with before the drug to be tested is added or the pathogen is infected after the drug to be tested is added or the pathogen is infected, the drug to be tested or the pathogen infection has no significant effect on the integrity of the cell barrier; preferably, the detection method comprises: using a transmembrane resistance meter to detect the resistance 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; (iv) administering the drug to be tested to the pathological pulmonary fibrosis model or allowing the pathogen to infect the pathological pulmonary fibrosis model, and detecting changes in the barrier permeability of the pathological pulmonary fibrosis model; preferably, the detection comprises: adding fluorescent molecules (such as FITC-Dextran) to the upper layer of the pathological pulmonary fibrosis model and incubating, detecting the amount of the fluorescent molecules permeated in the lower layer of the pathological pulmonary fibrosis model, and calculating the apparent permeability coefficient; (v) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the 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 comprises: adding MTT, CCK8 or CTG activity detection reagent to the pathological pulmonary fibrosis model and incubating, and detecting the chemiluminescence value; (vi) administering the drug to be tested to a pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting cell apoptosis in the pathological pulmonary fibrosis model; wherein the detection method is, for example, immunofluorescence or flow cytometry; and the markers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL; (vii) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting changes in the content of inflammatory cytokines in the pathological pulmonary fibrosis model; wherein the inflammatory factors 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, and then incubating and washing, adding a color developing solution and incubating, and then adding a stop solution and detecting the OD value; (viii) administering the drug to be tested to a pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the cell phenotype or cell content associated with the lung tissue in the pathological pulmonary fibrosis model; 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; and the detection method includes: flow cytometry, immunofluorescence; (ix) administering the drug to be tested to the pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the level of cellular oxidative stress in the pathological pulmonary fibrosis model; (x) administering the drug to be tested to a pathological pulmonary fibrosis model or infecting the pathological pulmonary fibrosis model with the pathogen, and detecting the expression changes of genes related to the development of pulmonary fibrosis in the pathological pulmonary fibrosis model; wherein the related genes are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene; (xi) allowing the pathogen to infect a pathological pulmonary fibrosis model, and detecting changes in the expression of receptors associated with the pathogen infection in the pathological pulmonary fibrosis model; (xii) allowing the pathogen to infect a pathological pulmonary fibrosis model, then administering the drug to be tested to the pathological pulmonary fibrosis model, and detecting the inhibitory effect of the drug to be tested on the pathogen; Wherein, the pathological pulmonary fibrosis model is 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; Preferably, the drugs include small molecule drugs, antibody drugs, ADC drugs, and immune drugs; Preferably, the drug detection method and drug screening method include drug detection and / or drug screening for treating pulmonary fibrosis.

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