Pulmonary fibrosis model, and screening method of prophylactic or therapeutic agent for pulmonary fibrosis

Abstract

A pulmonary fibrosis model that includes a co-culture system containing alveolar organoids and pulmonary fibroblasts that express a reporter gene when converted into myofibroblasts, wherein, when the alveolar organoids are injured, the pulmonary fibroblasts are converted into myofibroblasts and the reporter gene is expressed.

Description

Pulmonary fibrosis model and method for screening preventive or therapeutic agents for pulmonary fibrosis

[0001] The present invention relates to a pulmonary fibrosis model and a method for screening for a preventive or therapeutic agent for pulmonary fibrosis. This application claims priority to U.S. Provisional Application No. US 63 / 413,973 filed October 7, 2022, the contents of which are incorporated herein by reference.

[0002] In addition to the epithelium, the lungs contain tissue called the interstitium. Pathological conditions involving inflammation and fibrosis primarily occurring in the interstitium of the lungs are collectively referred to as interstitial pneumonia, and cases involving fibrosis of the alveolar interstitium are called pulmonary fibrosis. Pulmonary fibrosis is a disease with an extremely poor prognosis, with a high prevalence rate associated with aging, a lack of effective treatments, and an average survival time of 2-3 years after diagnosis (see, for example, Non-Patent Documents 1 and 2). To develop therapeutic agents for pulmonary fibrosis, an in vitro experimental system that can reproduce the molecular mechanisms that lead to lung fibrosis is required.

[0003] Conventionally, the most common pulmonary fibrosis model has been a drug-induced pulmonary fibrosis model in which mice are administered bleomycin to induce the onset of pulmonary fibrosis. Furthermore, cell culture models of pulmonary fibrosis have included experimental systems in which epithelial cells are cultured with bleomycin to examine the expression of myofibrogenic factors, and experimental systems in which fibroblasts are cultured in the presence of molecules with myofibrogenic potential, such as TGF-β, to examine their differentiation into myofibroblasts.

[0004] For example, Patent Document 1 describes an ex vivo pulmonary fibrosis model using co-cultured lung fibroblasts and pre-alveolar type 1 transitional state (PATS) cells for use in drug screening. Also, Patent Document 2 describes that lung blastoid organoids (LBOs) with mutations can be used to screen for drugs with potential therapeutic potential for pulmonary fibrosis.

[0005] International Publication No. 2021 / 062412 Special Publication No. 2020-511968

[0006] Travis WD, et al., An Official American Thoracic Society / European Respiratory Society Statement: Update of the International Multidisciplinary Classification of the Idiopathic Interstitial Pneumonias, Am J Respir Crit Care Med, 188, 6, 733-748, 2013.Fujisawa T., et al., Nationwide cloud-based integrated database of idiopathic interstitial pneumonias for multidisciplinary discussion, Eur Respir J, 53, 1802243, 2019.

[0007] However, animal experiments are costly and time-consuming, so simpler pulmonary fibrosis models are preferable. Furthermore, conventional pulmonary fibrosis models using cell culture systems are unable to model pulmonary fibrosis, whose pathogenesis is complex cell-cell interactions.

[0008] Epithelial cells are often used as cultured cell lines, which have properties different from those of living cells. It is necessary to develop a model that is closer to actual pulmonary fibrosis than conventional models by reproducing multicellular tissue using cells that are similar to those in living lungs.

[0009] An object of the present invention is to provide a pulmonary fibrosis model and a method for screening a preventive or therapeutic agent for pulmonary fibrosis.

[0010] The present invention includes the following aspects. [1] A pulmonary fibrosis model, comprising a co-culture system containing alveolar organoids and pulmonary fibroblasts that express a reporter gene upon myofibrosis, wherein injury to the alveolar organoids causes the pulmonary fibroblasts to undergo myofibrosis and express the reporter gene. [2] The pulmonary fibrosis model described in [1], wherein the alveolar organoids are formed by three-dimensional culture of type II alveolar epithelial cells. [3] The pulmonary fibrosis model described in [1] or [2], wherein the reporter gene is formed by linking a gene encoding a fluorescent protein downstream of the ACTA2 gene promoter. [4] The pulmonary fibrosis model described in any of [1] to [3], wherein the injury is DNA damage. [5] The pulmonary fibrosis model described in [4], wherein the DNA damage is induced by the addition of bleomycin (CAS number: 11056-06-7). [6] A screening method for a preventive or therapeutic agent for pulmonary fibrosis, comprising the steps of culturing the pulmonary fibrosis model according to any one of [1] to [5] in the presence of a test substance, damaging the alveolar organoids, and detecting the expression of the reporter gene, wherein a significant decrease in the expression level of the reporter gene compared to that in the absence of the test substance indicates that the test substance is an active ingredient in a preventive or therapeutic agent for pulmonary fibrosis.

[0011] According to the present invention, a pulmonary fibrosis model and a method for screening a preventive or therapeutic agent for pulmonary fibrosis can be provided.

[0012] FIG. 1 is a representative microscopic image of alveolar organoids in Experimental Example 1. FIG. 2 is a representative microscopic image of alveolar organoids in Experimental Example 2. FIG. 3 is an image showing the results of Western blotting in Experimental Example 2. FIG. 4 is a schematic diagram showing the experimental schedule of Experimental Example 3. FIG. 5 is a representative microscopic image and graph showing the results of Experimental Example 3. FIG. 6 is a schematic diagram showing the experimental schedule of Experimental Example 4. FIG. 7 is a representative microscopic image and graph showing the results of Experimental Example 4. FIG. 8 is a representative microscopic image and graph showing the results of Experimental Example 6. FIG. 9 is a representative microscopic image of alveolar organoids in Experimental Example 8. FIG. 10 is a representative microscopic image and graph showing the results of Experimental Example 9.

[0013] [Notation of Gene Names and Protein Names] In this specification, human genes and human proteins are represented by capital letters. Mouse genes are represented by an initial capital letter followed by lowercase letters. Mouse proteins are represented by capital letters. However, in some cases, human genes, mouse genes, human proteins, and mouse proteins may be represented without strict distinction.

[0014] [Pulmonary fibrosis model] In one embodiment, the present invention provides a pulmonary fibrosis model comprising a co-culture system containing alveolar organoids and pulmonary fibroblasts that express a reporter gene upon myofibrosis, wherein injury to the alveolar organoids causes the pulmonary fibroblasts to undergo myofibrosis and express the reporter gene.

[0015] As described below in the Examples, the inventors have developed a co-culture system of alveolar organoids, which are epithelial multicellular tissues similar to those in vivo, and myofibrosis reporter lung fibroblasts, which visualize myofibrosis using a fluorescent reporter. The pulmonary fibrosis model of this embodiment can reproduce the epithelial-mesenchymal interaction that is characteristic of pulmonary fibrosis. The pulmonary fibrosis model of this embodiment is closer to actual pulmonary fibrosis than conventional models, and can reflect pulmonary fibrosis, the pathogenesis of which is complex cell-cell interactions.

[0016] In recent years, research has been actively conducted into "mini-organs" (organoids) that contain not only stem cells but also differentiated cells within artificially created cell masses, using a method of culturing stem and progenitor cells in three dimensions (organoid culture), and that partially retain the functions of the tissues from which they were derived. Cultured organoids are expected to be used in drug discovery applications such as drug screening, and as a source of information for regenerative medicine.

[0017] The alveolar organoids are preferably formed by three-dimensional culture of type II alveolar epithelial cells (AT2 cells), which are alveolar epithelial stem cells. The AT2 cells are preferably derived from mammals. Examples of mammals include humans, monkeys, mice, rats, and pigs.

[0018] By culturing mammalian-derived AT2 cells in a three-dimensional support, it is possible to promote proliferation and self-organization, and to create alveolar organoids that reproduce the cellular composition and morphology of the alveolar epithelium, a multicellular tissue. While conventional two-dimensional cultures were not able to maintain or proliferate AT2 cells, the three-dimensional culture technique has made it possible to maintain and proliferate AT2 cells.

[0019] An extracellular matrix can be used as the three-dimensional support. The extracellular matrix is ​​a substance that serves as a scaffold for cells in cell culture. Commercially available extracellular matrices include Matrigel (registered trademark) and collagen.

[0020] Components of the extracellular matrix include, for example, components contained in basement membranes and glycoproteins present in intercellular spaces. Components contained in basement membranes include, for example, type IV collagen, laminin, heparan sulfate proteoglycan, entactin, etc. Glycoproteins present in intercellular spaces include collagen, laminin, entactin, fibronectin, fibrinogen, heparin sulfate, etc. These may be used alone or in combination of two or more.

[0021] The proliferated AT2 cell population forms hollow spherical bodies called spheres. After a certain culture period, type 1 alveolar epithelial cells (AT1 cells), differentiated from the AT2 cells, appear within the spheres, and they begin to exhibit a tissue structure similar to that of alveolar epithelium. Spheres that have progressed to the differentiated stage are called "alveolar organoids." Using alveolar organoid culture technology, it is possible to conduct highly reproducible experiments using alveolar cells that are similar to those in the body, even on a culture dish.

[0022] As described later in the Examples, pulmonary fibroblasts that express a reporter gene upon myofibrosis can be pulmonary fibroblasts in which a reporter gene has been linked downstream of the Actin Alpha 2, Smooth Muscle (ACTA2) gene promoter.

[0023] The pulmonary fibroblasts that express a reporter gene upon myofibrosis may be pulmonary fibroblasts into which a construct linking a reporter gene downstream of the ACTA2 gene promoter has been introduced as a transgene. Alternatively, the pulmonary fibroblasts may be pulmonary fibroblasts into which a reporter gene has been introduced into the ACTA2 locus by genome editing. For example, pulmonary fibroblasts may be pulmonary fibroblasts into which a gene fragment encoding a 2A self-cleaving peptide and a reporter gene have been introduced, in this order, downstream of the ACTA2 gene at the ACTA2 locus. Examples of 2A self-cleaving peptides include T2A, P2A, E2A, and F2A.

[0024] Examples of NCBI accession numbers for the cDNA of mouse ACTA2 gene include NM_007392, etc. Examples of NCBI accession numbers for the cDNA of human ACTA2 gene include NM_001141945.3 and NM_001320855.2, etc.

[0025] Examples of reporter genes include genes encoding luminescent enzymes such as luciferase, genes encoding chromogenic enzymes such as alkaline phosphatase, and genes encoding fluorescent proteins. Among these, genes encoding fluorescent proteins are preferably used. That is, the reporter gene may be a gene encoding a fluorescent protein linked downstream of the ACTA2 gene promoter.

[0026] The fluorescent protein is not particularly limited, and examples thereof include DsRed, which is a red fluorescent protein, GFP, which is a green fluorescent protein, EBFP, which is a blue fluorescent protein, and YFP, which is a yellow fluorescent protein.

[0027] As described later in Examples, in the pulmonary fibrosis model of this embodiment, when alveolar organoid is injured, pulmonary fibroblasts are transformed into myofibrosis, and reporter gene is expressed.The injury that is given to alveolar organoid can be, for example, DNA damage.

[0028] Methods for inflicting DNA damage on alveolar organoids include, but are not limited to, adding a DNA damage inducer to the culture medium of alveolar organoids. Examples of DNA damage inducers include, but are not limited to, bleomycin (CAS number: 11056-06-7). As described below in the Examples, adding bleomycin (CAS number: 11056-06-7) to the culture medium of alveolar organoids can inflict DNA damage on alveolar organoids.

[0029] [Method for screening preventive or therapeutic agents for pulmonary fibrosis] In one embodiment, the present invention provides a method for screening preventive or therapeutic agents for pulmonary fibrosis, comprising the steps of culturing the above-mentioned pulmonary fibrosis model in the presence of a test substance, damaging the alveolar organoid, and detecting the expression of the reporter gene, wherein the expression level of the reporter gene is significantly reduced compared to that in the absence of the test substance, indicating that the test substance is an active ingredient for a preventive or therapeutic agent for pulmonary fibrosis.By the screening method of this embodiment, it is possible to screen preventive or therapeutic agents for pulmonary fibrosis.

[0030] In the screening method of this embodiment, the pulmonary fibrosis model, alveolar organoid, pulmonary fibroblasts that express a reporter gene upon myofibrosis, and the like are the same as those described above.

[0031] The test substance is not particularly limited, and examples thereof include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, antibody libraries, peptide libraries, and the like.

[0032] The step of damaging alveolar organoid can be the step of causing DNA damage to alveolar organoid.The method of causing DNA damage to alveolar organoid is not particularly limited, but for example, can be added DNA damage inducer to the culture medium of alveolar organoid.As DNA damage inducer, for example, can be bleomycin.

[0033] The step of damaging alveolar organoids and the step of culturing pulmonary fibrosis model in the presence of test substance can be carried out in any order.For example, after culturing alveolar organoids in the presence of test substance for several days, the alveolar organoids can be damaged, and then co-cultured with pulmonary fibroblasts that express a reporter gene when they become myofibrotic.Alternatively, the alveolar organoids can be damaged, and then co-cultured with pulmonary fibroblasts that express a reporter gene when they become myofibrotic, and then co-cultured with pulmonary fibroblasts that express a reporter gene when they become myofibrotic, and then add the test substance after the reporter gene is expressed.

[0034] Next, the expression of the reporter gene is detected. The detection of the expression of the reporter gene may be performed by a method appropriate for the reporter gene used. For example, if the reporter gene is a fluorescent protein, the lung fibroblasts may be irradiated with excitation light and the fluorescence may be measured. Alternatively, if the reporter gene is a luminescent enzyme, a luminescent substrate may be added and the luminescence may be measured. Alternatively, if the reporter gene is a chromogenic enzyme, a chromogenic substrate may be added and the color may be measured.

[0035] If the expression level of the reporter gene is significantly reduced compared to that in the absence of the test substance, the test substance is considered to be a candidate for a preventive or therapeutic agent for pulmonary fibrosis.

[0036] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0037] [Experimental Example 1] (Preparation of Mouse Alveolar Organoids) Mouse alveolar organoids were prepared as follows. ＣｒｅＥＲＴ２ Mice (Jackson Laboratory) and Rosa26 ｍＴｍＧ Mice (Jackson Laboratory) were bred and Sftpc ＣｒｅＥＲＴ２ :Rosa26 ｍＴｍＧ Mice were obtained. Sftpc is a marker for type II alveolar epithelial cells (AT2 cells). ＣｒｅＥＲＴ２ :Rosa26 ｍＴｍＧ Mouse AT2 cells express green fluorescent protein (GFP).

[0038] Sftpc ＣｒｅＥＲＴ２ :Rosa26 ｍＴｍＧ Mice were euthanized and their lungs were removed. The removed lungs were minced and placed in 4 mL of protease solution, placed on a rotator, and incubated at 37°C for 30 minutes. The tissue / cells were then dissociated by pipetting, and phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) was added. The cells were then filtered through 100 μm and 40 μm strainers. The cells were then suspended in PBS containing 3% FBS and centrifuged at 400 × g for 5 minutes.

[0039] Next, GFP ＋Cells (AT2 cells) were sorted and collected. The collected AT2 cells were suspended in culture medium and mixed with an equal volume of growth factor-reduced Matrigel (registered trademark, Corning). Subsequently, 20 μL droplets containing 20,000 cells were placed on the bottom of a 24-well plate. The droplets were incubated at 37°C for 20 minutes to solidify the gel. Subsequently, 400 μL of culture medium was added to each well. The composition of the culture medium is shown in Table 1 below. In Table 1, "F.C." indicates the final concentration.

[0040]

[0041] The medium was changed every 3 days. To increase cell viability, Y27632 (10 μM) was added to the medium for the first 3 days after sorting or passaging.

[0042] The left side of Figure 1 is a representative bright-field image of the obtained alveolar organoids. The scale bar is 1 mm. The center of Figure 1 is an image of GFP fluorescence captured in the same field as the left side of Figure 1. The scale bar is 1 mm. The right side of Figure 1 is a fluorescence microscope image showing the results of staining a section of alveolar organoids with anti-SFTPC antibody, anti-RAGE antibody, and DAPI. The scale bar is 20 μm.

[0043] [Experimental Example 2] (Bleomycin treatment of mouse alveolar organoids) Mouse alveolar organoids were incubated in the presence or absence of bleomycin (100 μM) for 24 hours. Subsequently, the expression of γH2AX, a marker of DNA double-strand breaks, was examined.

[0044] Figure 2 is a fluorescence microscope image showing the results of staining alveolar organoid sections with anti-γH2AX antibody and DAPI. The scale bar is 20 μm. Figure 3 is an image showing the results of examining the expression of γH2AX and GAPDH by Western blotting of alveolar organoids. In Figure 3, "BLM" indicates bleomycin, "+" indicates addition, and "-" indicates no addition.

[0045] The results showed that the majority of cells in bleomycin-treated alveolar organoids expressed γH2AX, indicating that bleomycin treatment caused DNA damage.

[0046] [Experimental Example 3] (Co-culture system of mouse alveolar organoids and pulmonary fibroblasts) To examine whether AT2 lineage cells can induce differentiation of fibroblasts into myofibroblasts, bleomycin-treated alveolar organoids were co-cultured with naive pulmonary fibroblasts.

[0047] 4 is a schematic diagram showing the experimental schedule of this experimental example. Alveolar organoids were prepared from AT2 cells derived from wild-type mice. EPCAM cells were isolated from the lungs of wild-type mice. ＋ MHC II ＋ CD31 － CD45 － Alveolar organoids were prepared in the same manner as in Experimental Example 1, except that the cells were sorted, collected, and used as AT2 cells.

[0048] Pulmonary fibroblasts were collected from Acta2-DsRed genetically modified mice (Jackson Laboratory). These pulmonary fibroblasts (hereinafter sometimes referred to as "Acta2-DsRed fibroblasts") express the red fluorescent protein DsRed when differentiated into myofibroblasts. Therefore, in the presence of fibrotic factors in the culture medium, they differentiate into myofibroblasts and emit red fluorescence.

[0049] Figure 5 shows representative microscopic images of (myo)fibroblasts co-cultured with alveolar organoids (scale bar 100 μm). ＋ Graph showing the proportion of myofibroblasts and DsRed ＋ 1 is a graph showing the size (relative value) of myofibroblasts.

[0050] As a result, increased DsRed fluorescence was observed in fibroblasts co-cultured with bleomycin-treated alveolar organoids, indicating that the fibroblasts differentiated into myofibroblasts.

[0051] DsRed derived from fibroblasts co-cultured with bleomycin-treated alveolar organoids ＋The myofibroblasts were larger than those co-cultured with control organoids (alveolar organoids not treated with bleomycin), indicating that differentiation of fibroblasts into myofibroblasts was induced by bleomycin-treated AT2 lineage cells.

[0052] [Experimental Example 4] (Study of the sensitivity of the Acta2-DsRed reporter) Figure 6 is a schematic diagram illustrating the experimental schedule of this experiment. 1 pg / mL to 1,000 pg / mL of TGF-β was added to the culture medium of Acta2-DsRed fibroblasts, and DsRed fluorescence was detected. A mixture of recombinant TGF-β1, TGF-β2, and TGF-β3 was used as TGF-β. TGF-β is a potent fibrosis-promoting factor.

[0053] Figure 7 shows representative microscopic images of (myo)fibroblasts (scale bar 100 μm) and DsRed. ＋ 7 is a graph showing the area (relative value) of the region. In the microscopic images in Fig. 7, "Gel" indicates empty Matrigel (registered trademark). In the graph in Fig. 7, "**" indicates a significant difference at p<0.01, "***" indicates a significant difference at p<0.001, and "N.S." indicates no significant difference.

[0054] The results showed that Acta2-DsRed fibroblasts could detect 1 pg / mL of TGF-β, and that the addition of TGF-β in the range of 1 to 100 pg / mL increased DsRed fluorescence in a dose-dependent manner.

[0055] The conventional TGF-β detection method, commercially available ELISA, has a detection limit of 3 to 16 pg / mL, and the Acta2-DsRed reporter developed in this study was shown to be more sensitive than the conventional method.

[0056] Thus, we established a method for culturing alveolar organoids in a pulmonary fibrosis model that is sensitive enough to detect low doses of fibrotic factors, and that reflects inflammation-independent epithelial-mesenchymal profibrotic interactions.

[0057] [Experimental Example 5] (p53 signaling in AT2 lineage cells) To investigate the signaling pathway that enhances the fibrosis promotion of fibroblasts by AT2 lineage cells, we performed RNA-seq analysis of alveolar organoids incubated for 24 hours in the presence or absence of bleomycin (100 μM). Alveolar organoids were established independently from four mice.

[0058] Pathway analysis revealed that the p53 signaling pathway was significantly enriched in bleomycin-treated alveolar organoids.

[0059] [Experimental Example 6] (Bleomycin treatment activates the p53 pathway in alveolar organoids) We investigated whether AT2 lineage cells in which the p53 signaling pathway is activated directly communicate with pulmonary fibroblasts and induce differentiation into myofibroblasts.

[0060] Alveolar organoids were cultured for 24 hours in the presence of Nutlin-3a (2 μM), a compound that activates the p53 signaling pathway instead of bleomycin, and then co-cultured with Acta2-DsRed fibroblasts.

[0061] Figure 8 shows representative microscopic images of (myo)fibroblasts co-cultured with alveolar organoids (scale bar 1 mm), and the results of DsRed ＋ 8 is a graph showing the area (relative value) of the region. In the microscopic images of FIG. 8, "AOs" indicates Matrigel (registered trademark) containing alveolar organoids. In FIG. 8, "CTRL" indicates the result of a control not treated with a compound, "BLM" indicates the result of alveolar organoids treated with bleomycin (100 μM, 24 hours), and "Nutlin-3a" indicates the result of alveolar organoids treated with Nutlin-3a (2 μM, 24 hours). In the graph of FIG. 8, "*" indicates a significant difference at p<0.05, "**" indicates a significant difference at p<0.01, and "N.S." indicates no significant difference.

[0062] As a result, a significant increase in DsRed fluorescence was observed in fibroblasts co-cultured with Nutlin-3a-treated alveolar organoids, indicating that activation of the p53 signaling pathway is sufficient for the formation of AT2 lineage cells, which promote fibrosis in fibroblasts.

[0063] [Experimental Example 7] (Preparation of human alveolar organoids) HTII-280 was isolated from normal human lung tissue. ＋ Human AT2 cells were sorted and collected. Human normal lung tissue was collected from lung tissue surgically removed from an early stage lung cancer patient. Human alveolar organoids were prepared in the same manner as in Experimental Example 1, except that the collected human AT2 cells were used as AT2 cells.

[0064] [Experimental Example 8] (Bleomycin treatment of human alveolar organoids) Human alveolar organoids were incubated for 24 hours in the presence or absence of bleomycin (100 μM). Subsequently, sections of the alveolar organoids were incubated with anti-SFTPC antibody, anti-SFTPB antibody, anti-γH2AX antibody, anti-p53 antibody, anti-p21 antibody, and the like. ＷＡＦ１／ＣＩＦ１ Immunostaining was performed with antibodies.

[0065] 9 shows fluorescent microscopic images showing the results of immunostaining. The scale bar is 20 μm. As a result, in the bleomycin-treated human alveolar organoids, as in the mouse alveolar organoids, γH2AX, p53, and p21 ＷＡＦ１／ＣＩＦ１ This result indicates that bleomycin treatment caused DNA damage.

[0066] [Experimental Example 9] (Co-culture system of human alveolar organoids and pulmonary fibroblasts) Human alveolar organoids were incubated for 24 hours in the presence or absence of bleomycin (100 μM) and then co-cultured with pulmonary fibroblasts. Pulmonary fibroblasts (Acta2-DsRed fibroblasts) derived from Acta2-DsRed genetically modified mice (Jackson Laboratory) were used as pulmonary fibroblasts.

[0067] Figure 10 shows representative microscopic images of (myo)fibroblasts co-cultured with human alveolar organoids (scale bar 1 mm), and the results of DsRed ＋10 is a graph showing the area (relative value) of the region. In the microscopic images in Figure 10, "hAOs" indicates Matrigel (registered trademark) containing human alveolar organoids, "CTRL" indicates the result of a control not treated with bleomycin, and "BLM" indicates the result of human alveolar organoids treated with bleomycin. In the graph in Figure 10, "*" indicates a significant difference at p<0.05.

[0068] As a result, a significant increase in DsRed fluorescence was observed in fibroblasts co-cultured with bleomycin-treated human alveolar organoids, indicating that bleomycin-treated human alveolar organoids were able to activate the fluorescent reporter in mouse-derived Acta2-DsRed fibroblasts.

[0069] Experimental Example 10 Screening for preventive or therapeutic agents for pulmonary fibrosis Mouse alveolar epithelial stem cells, AT2 cells, were isolated and three-dimensionally cultured in a three-dimensional support for cells (e.g., gelled collagen). Spherical spheres (alveolar organoids) obtained on day 6 of culture were treated with bleomycin, and on day 7 of culture, pulmonary fibroblasts (Acta2-DsRed fibroblasts) were added and co-cultured in the presence or absence of a test substance.

[0070] After co-culture for a predetermined period, reporter expression in the Acta2-DsRed fibroblasts is observed. If the expression level of the fluorescent reporter is significantly reduced compared to that in the absence of the test substance, the test substance is considered to be a candidate for an active ingredient in an agent for preventing or treating pulmonary fibrosis.

[0071] According to the present invention, a pulmonary fibrosis model and a method for screening a preventive or therapeutic agent for pulmonary fibrosis can be provided.

Claims

1. A pulmonary fibrosis model, The system includes a co-culture system containing alveolar organoids and lung fibroblasts that express a reporter gene when they undergo myofibrosis. A pulmonary fibrosis model in which damage to the alveolar organoids causes the pulmonary fibroblasts to become myofibrillated and the reporter gene to be expressed.

2. The pulmonary fibrosis model according to claim 1, wherein the alveolar organoids are formed by three-dimensional culture of type II alveolar epithelial cells.

3. The pulmonary fibrosis model according to claim 1 or 2, wherein the reporter gene is a gene encoding a fluorescent protein linked downstream of the ACTA2 gene promoter.

4. The pulmonary fibrosis model according to claim 1 or 2, wherein the injury is DNA damage.

5. The pulmonary fibrosis model according to claim 4, wherein the DNA damage is induced by the addition of bleomycin (CAS number: 11056-06-7).

6. A screening method for agents that prevent or treat pulmonary fibrosis, A step of culturing the pulmonary fibrosis model according to claim 1 or 2 in the presence of a test substance, A step of damaging the aforementioned alveolar organoids, A step of detecting the expression of the reporter gene, Includes, A screening method in which the expression level of the reporter gene is significantly reduced compared to the absence of the test substance, thereby indicating that the test substance is an active ingredient in a preventive or therapeutic agent for pulmonary fibrosis.