CN115772492A

CN115772492A - Method for constructing pulmonary fibrosis disease model in vitro by using bleomycin

Info

Publication number: CN115772492A
Application number: CN202310101642.XA
Authority: CN
Inventors: 吴迪; 葛啸虎
Original assignee: Qijia Technology Tianjin Co ltd
Current assignee: Qijia Technology Suzhou Co ltd
Priority date: 2023-02-13
Filing date: 2023-02-13
Publication date: 2023-03-10
Anticipated expiration: 2043-02-13
Also published as: CN115772492B

Abstract

The invention provides a method for constructing a pulmonary fibrosis disease model in vitro by using bleomycin, wherein the model has a compact and complex lung structure, can tolerate the long-term induction of the general inducer bleomycin in vivo, and greatly prolongs the time window of fibrosis simulation and pharmacodynamic analysis. In addition to containing the major epithelial cell types of the lung, macrophages in the model are functional, faithfully restoring the pathological mechanisms of lung resident macrophages that promote collagen deposition by secreting TGF- β 1.

Description

Method for constructing pulmonary fibrosis disease model in vitro by using bleomycin

Technical Field

The invention relates to the field of stem cell biology and regenerative medicine, in particular to a method for constructing a pulmonary fibrosis disease model in vitro by using bleomycin.

Background

Idiopathic Pulmonary Fibrosis (IPF) is the most common Idiopathic interstitial lung disease with mortality higher than that of most tumors and median survival of patients of only 2-3 years. Over 20 years, more than 70 anti-fibrosis drugs have shown significant efficacy in preclinical models, but all have failed clinical trials. The insufficiency of the traditional model drug evaluation efficacy requires urgent establishment of a more accurate and reliable new model. With the rapid development of regenerative medicine, human organ-like technology has come to the fore to attract attention. Compared with the traditional cell model, the organoids show better physiological relevance in cell types, structural characteristics and functional characteristics; the humanization characteristic can well overcome the result deviation of animal models caused by species difference.

Currently, lung organoids can be induced to differentiate from human pluripotent stem cells (hpscs) or human adult stem cells (hascs). Whether of hPSC or hASC origin, are composed primarily of epithelial cells (e.g., alveolar cells, goblet cells, ciliated cells, etc.) and lack important cell types of pathological relevance, such as macrophages. A large number of researches indicate that in IPF, macrophages residing in the lung dominate a series of important pathological molecular events such as proinflammatory factor secretion, anti-inflammatory factor secretion, pre-fibrosis factor production and the like; in particular endogenous TGF- β 1 (central signal of fibrosis) produced by it, which directly leads to activation of the terminal signal of fibrosis, triggering collagen deposition and fibrotic scar accumulation. Thus, the current lack of macrophages in existing lung organoid models limits the ability to mimic fibrosis.

On the other hand, in addition to the problem of lineage loss, structural deficiencies of existing models limit the accuracy of fibrosis simulation. At present, bleomycin is a general inducer for pulmonary fibrosis modeling of animal models, and the induced fibrosis characteristics are similar to those of human bodies, so that bleomycin is widely accepted and adopted. However, since the structures of the current in vitro models are quite simple (e.g., monolayer cells, vesicle-like organoids, etc.), it is often difficult to tolerate the cytotoxicity of bleomycin, and the fibrotic phenotype has not been induced and is already largely killed. Therefore, there are few reports of the use of bleomycin in an in vitro model. Instead of the above, downstream signal molecules of fibrosis can be used only to directly trigger the fibrosis phenotype (such as TGF beta 1, IL-11, etc.), so that important molecular events occurring in many intermediate processes, such as impaired aging of lung epithelial cells, macrophage polarization, anti-inflammatory/pro-inflammatory regulation, etc., are ignored, and the real situation of the in vivo fibrosis development is seriously violated; this reveals to some extent why pharmacodynamic data based on such models differ greatly from clinical outcomes. Moreover, the formation of fibrosis is a long-lasting process which is day by day, and when the time for directly triggering fibrosis by using downstream signal molecules in the lower in vitro model is less than or equal to 48h (about 20 days for molding bleomycin in animals), the overall appearance and corresponding drug effect of the progress of fibrosis are difficult to capture.

In summary, the ability of current lung organoid models to mimic fibrosis is severely limited by the problems of 1) lack of functional macrophages and 2) excessive structural singleness. If a lung organoid with functional macrophages exists and has better structural property to resist the long-term modeling process of the general inducer bleomycin in vivo, the occurrence and development of a fibrosis pathology can be simulated in vitro from the beginning and accurately, and a reliable humanized evaluation tool is provided for an anti-fibrosis drug test.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for inducing pulmonary fibrosis by bleomycin based on a whole lung organoid, which has both functional macrophages and epithelial cells, has compact and complex lung structural features, and can tolerate the long-term induction of bleomycin. By means of the brand-new fibrosis disease model, the up-regulation of fibrosis marker genes/proteins is presented in vitro, and a mechanism that lung-resident macrophages promote collagen deposition by secreting TGF-beta 1 is faithfully restored, so that the model is suitable for being used as a new generation IPF in-vitro disease model and is used for whole-process simulation of fibrosis progress and accurate screening of anti-fibrosis drugs.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a method for constructing a pulmonary fibrosis disease model in vitro by using bleomycin comprises the following steps:

1) Construction of Whole Lung organoids

Inducing hPSC to differentiate into definitive endoderm by using culture medium containing GDF; continuously adding ATRA in the culture medium, and adding lung epithelial cell related induced differentiation factor, macrophage induced differentiation factor and EGM2 to induce and differentiate the lung epithelial cell into lung buds; finally, coating the lung buds with ECM containing type I collagen and type III collagen, adding lung epithelium to promote maturation factors to continue culturing, and obtaining the whole lung organoid with lung resident macrophages after 60-65 days;

2) Construction of pulmonary fibrosis disease model

I) Respectively incubating whole lung organoids with M1 and M2 polarization inducers to verify the ability of macrophages to polarize M1/M2 type macrophages;

II) treating the whole lung organoid obtained in the step 1) by using bleomycin with the concentration of 10-100 mu g/ml, inducing polarization of lung resident macrophages to M2 type macrophages, and secreting TGF-beta 1 from the M2 type macrophages obtained by polarization, so that the content of TGF-beta 1 in culture supernatant is increased and acts on the whole lung organoid to construct a pulmonary fibrosis disease model.

Further, bleomycin treatment was carried out for 8 to 30 days.

Further, the GDF is any one of GDF3, GDF5, GDF7 and GDF8, preferably GDF8, and the use amount of GDF is 20-500ng/ml.

Further, the using amount of ATRA is 0.01-10 mu M, the macrophage induced differentiation factor comprises EGF, VEGF and bFGF, wherein the using amount of EGF is 1-50ng/ml, the using amount of VEGF is 0.5-100ng/ml, and the using amount of bFGF is 0.5-50ng/ml; the volume percentage of EGM2 added in each stage is 5-60%.

Further, the lung epithelial cell related induced differentiation factors comprise GSK-3 inhibitor, BMP4, FGF10, KGF and ATRA; wherein, the usage amount of the GSK-3 inhibitor is 0.1-10 MuM, the usage amount of the BMP4 is 1-50ng/ml, the usage amount of the FGF10 is 1-50ng/ml, the usage amount of the KGF is 1-50ng/ml, and the usage amount of the ATRA is 0.01-10 MuM.

Further, the lung epithelium maturation-promoting factors include dexamethasone, PDE inhibitors, and PKA activators.

Compared with the prior art, the method for constructing the pulmonary fibrosis disease model in vitro by using bleomycin has the following advantages:

according to the method for constructing the pulmonary fibrosis disease model in vitro by using bleomycin, the lung organoid not only contains various epithelial cell types, but also has functional macrophages residing in the lung; on the basis, a lung organoid fibrosis model is constructed, the lung fibrosis model can simulate the dynamic change and function (such as M1/M2 type polarization, proinflammatory/anti-inflammatory factor secretion and the like) of lung resident macrophages in the fibrosis process, meanwhile, the model reproduces different pathological representations in the fibrosis progress in multiple aspects, faithfully presents the pathology of IPF, and proves the key role of the lung resident macrophages in the IPF progress. In addition, the model is more suitable for researching the anti-fibrosis drug effect evaluation for reducing TGF-beta 1 signals by inhibiting M2 macrophage differentiation, shortens the drug development period and improves the success rate.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing the results of Day60 at stage S3; wherein, A is a Day60 whole lung organoid morphological map; the B picture is acTUB ⁺ Ciliated cell marker, panel C is CC10 ⁺ Rod cell marker, panel D is CHGA ⁺ Neuroendocrine cell marker, MUC5AC as Panel E ⁺ Goblet cell marker, panel F is P63 ⁺ Basal cell marker, map G is PDPN ⁺ Type I alveolar cell marker, and pattern H is SP-B ⁺ Type II alveolar cell marker, panel I CD68 present in airway-like structures ⁺ Macrophage marker, panel J is CD68 present in distal alveolar-like structures ⁺ A macrophage marker;

FIG. 2 shows the results of flow cytometry detection of macrophage markers; wherein, A picture is macrophage marker CD68 ⁺ The B picture is macrophage marker CD14 ⁺ Flow cytometry detection of (a); CD68 ⁺ And CD14 ⁺ Is a human macrophage universal marker;

FIG. 3 is an AF594 fluorescent latex bead phagocytosis assay; a is CD68 ⁺ A dyeing result graph; b is a graph marked with red fluorescent AF 594; c is DAPI staining result graph; d is a stacked graph of dyeing results; CD68 ⁺ Human macrophage universal marker; AF594 latex beads, using AF594 fluorescent-labeled latex beads;

FIG. 4 is a Dil-Ac-LDL uptake assay; FIGS. A-D show the CD68 ⁺ Dil-Ac-L was used as macrophageGraph of fluorescence staining results for DL incubation; FIGS. E-H show CD68 depletion ⁺ Graph of fluorescent staining after macrophages incubated with Dil-Ac-LDL; CD68 ⁺ Human macrophage universal marker; dil-Ac-LDL, acetylated low density lipoprotein fluorescently labeled with Dil;

FIG. 5 shows the results of flow cytometry assays performed after incubation with M1/M2-type macrophage polarization inducing agent; panel A shows the results of M1-type macrophages before polarization; panel B is the results after M1-type macrophages were polarized; panel C is the results before M2-type macrophages are polarized; d is the result after M2 type macrophage polarization; CD86, M1-type macrophage marker; CD206, an M2-type macrophage marker;

FIG. 6 is a comparison of cell viability rates of lung organoids versus other models after 10 days of treatment with different concentrations of bleomycin;

FIG. 7 is a comparison of the morphology of the control group and the molding group before and after molding; before the model is made, after the model is made, before the model is made, and after the model is made, for the comparison group, for the diagram A and the comparison group;

FIG. 8 shows the expression levels of mRNA, a marker associated with fibrosis in the control and model groups; * ,p＜0.05；**，pless than 0.01; technique repetition n =3; biological repeats n =4; a is the expression level of the marker FN, B is the expression level of the marker COL1A, C is the expression level of the marker COL3A, D is the expression level of the marker VIM, E is the expression level of the marker DES, and F is the expression level of the marker THY 1; g is the expression level of the marker SLUG, H is the expression level of the marker SNAIL, and I is the expression level of the marker TWIST;

FIG. 9 shows the immunofluorescence of fibrosis markers of control group and modeling group and the quantitative result thereof; * ***,pless than 0.0001; fluorescence quantification n =18 sections per group, derived from 3 organoids; panel A-C shows the expression of marker α -SMA, panel D-F shows the expression of marker FN, panel G-I shows the expression of marker PDGFR α, and panel J-L shows the expression of marker PDGFR β;

FIG. 10 shows Masson staining results for control and modeling groups;

FIG. 11 is a graph showing the change in proportion of M2-type macrophages during fibrosis modeling;

FIG. 12 shows the results of ELISA assay of culture supernatants. Technique repetition n =3; biological repeats n =4;

FIG. 13 shows the M2-type macrophage marker CD206 before/after modeling after macrophage scavenger was used ⁺ Performing immunofluorescence; before molding, the pattern A is shown, and after molding, the pattern B is shown;

FIG. 14 shows the results of flow cytometry using macrophage scavenger, the M2-type macrophage marker CD206 after modeling;

FIG. 15 is the TGF-. Beta.1 content in the supernatant after molding with or without the use of a macrophage scavenger;

FIG. 16 shows the results of immunofluorescence of COL3A, a fibrosis marker after molding, with or without macrophage scavenger; panel A shows the use of macrophage scavenger, and panel B shows the use of macrophage scavenger;

FIG. 17 shows the results of fluorescent quantitation of marker COL 3A; n =18 sections, derived from 3 lung organoids; * *,P＜0.01。

Detailed Description

It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

Definitive endoderm: definitive endoderm (definitive endoderm) refers to the primary site of development of cells that are the major constituents of the internal organs, such as the lung, liver, small intestine, and large intestine, during early embryonic development.

Foregut tube derivative (or gut tube anterior derivative): gut tube (gut tube) refers to a stringy tissue developed from the definitive endoderm early in embryonic development. The in vivo research shows that the front end and the back end of the human body consist of different progenitor cells which can develop into different visceral organs respectively; among these, the lung develops from a progenitor cell population near the anterior end of the gut tube, which is developmentally called the anterior gut tube or the anterior part of the gut tube.

The foregut tube derivative (or anterior gut tube derivative) in the present patent refers to a cell population that is SOX2 positive and has the potential for lung differentiation.

The invention provides a lung organoid, which is constructed by a method comprising the following steps:

s1, inducing hPSC to differentiate into definitive endoderm by using a culture medium containing GDF;

s2, inducing the differentiation of definitive endoderm into lung buds, comprising two stages: an early stage and a later stage;

formation of lung progenitor cell spheres in the early stage, during which ATRA is continuously added; during the later stage, saccular lung buds are formed, and common factors for inducing differentiation of vascular endothelial cells and macrophages are added on the basis of using lung epithelial cell related differentiation inducing factors to perform lung directional differentiation; and EGM2 is added into the culture medium of the early stage and the later stage;

s3, inducing the lung buds to differentiate into lung organoids;

coating the lung buds by using ECM (extracellular matrix) containing type I collagen and type III collagen, wherein the ECM is used for simulating a three-dimensional microenvironment for cell growth; and then adding a common factor for inducing differentiation of vascular endothelial cells and macrophages and EGM2 into the culture medium to continuously culture to obtain the whole lung organoids.

Specifically, the culture medium used in S1 comprises a culture medium A and a culture medium B, wherein the culture medium A is added with GDF and GSK-3 inhibitor, the culture medium B is added with GDF, FGF2 and vitamin C, hPSC is cultured in the culture medium A and the culture medium B in sequence,

GDF may be used in an amount of 20-500ng/ml, preferably 50-200ng/ml, or more preferably 100-150ng/ml, within which the object of the present invention can be achieved, and specifically 100ng/ml may be used; the GSK-3 inhibitor may be used in an amount of 0.1 to 10 μ M, preferably 0.5 to 5 μ M, or more preferably 2 to 5 μ M, within which range the object of the present invention can be achieved, and specifically 3 μ M; FGF2 may be used in an amount of 1-100ng/ml, preferably 2.5-30ng/ml, or more preferably 5-20ng/ml, within which range the object of the invention can be achieved, in particular 10ng/ml; the amount of vitamin C (AA) used may be 5 to 300. Mu.g/ml, preferably 10 to 200. Mu.g/ml, or more preferably 20 to 100. Mu.g/ml, within which the object of the present invention can be achieved, and particularly 50. Mu.g/ml may be used.

Wherein, the GDF is Nodal signal activator, and can be any one of GDF3, GDF5, GDF7 and GDF8, preferably GDF8; GDF8 makes cell growth more homogeneous and lays the foundation for increasing SOX2 expression and sufficient lung progenitor cell balls in the S2 stage at a later stage;

the GSK-3 inhibitor can be CHIR99021 and its salt, BIO, SB216763, AT7519, CHIR-98014, TWS119, tideglusib, but not always limited thereto, and specifically can be CHIR99021;

FGF2 can be replaced by FGF4, FGF10, preferably FGF2;

culturing in culture medium A and culture medium B for 1-3 days, preferably 1 day.

ATRA is added continuously during the early stages of S2 and kept at a concentration of 0.01-10 μ M, preferably 0.25-5 μ M, or more preferably 0.5-2 μ M, and in particular may be 1 μ M, to ensure that sufficient lung progenitor cell spheres can be generated in suspension for collection and continued differentiation. At the later stage, common factors EGF, VEGF and bFGF for inducing differentiation of vascular endothelial cells and macrophages are added, and EGM2 is added in the culture medium at the whole S2 stage, so that the expression of endothelial cells and immune cell markers is facilitated.

The percentage by volume of EGM2 added at each stage is between 5% and 60%, preferably between 10% and 50%, or more preferably between 15% and 30%.

Before the early stage of S2, the culture medium may be first cultured in a medium containing a BMP inhibitor and a TGF β inhibitor, and EGM2 may be added to the medium. The percentage by volume of EGM2 added is between 5% and 60%, preferably 23.5%.

The pre-EGM 2 was added to generate mesodermal progenitors, which are precursors to macrophages and vascular endothelial cells.

The BMP inhibitor is any one of NOG, CC, LDN193189, DMH1, LDN-212854, UK-383367 and K02288, and NOG is preferred; the TGF beta inhibitor is any one of SB431542, repsox, A83-01, galunesertib, vactosertib, R-268712, ML347, SD-208, R-268712, LY2109761, LY-364947, AZ12601011, LY3200882 and GW788388, preferably SB431542.

Specifically, the culture medium used in S2 includes a culture medium C, a culture medium D, a culture medium E, and a culture medium F, the culture medium D-E being used in an early stage, the culture medium F being used in a later stage; the culture medium C is added with a BMP inhibitor and a TGF beta inhibitor, the culture medium D is added with a Wnt inhibitor, a TGF beta inhibitor and ATRA, the culture medium E is added with lung epithelial cell related induced differentiation factors, and the lung epithelial cell related induced differentiation factors are GSK-3 inhibitors, BMP4, keratinocyte growth factors KGF, FGF10 and ATRA; and the culture medium F increases EGF, VEGF and bFGF on the basis of the culture medium E, and reduces the dosage of ATRA.

The BMP inhibitor may be used in an amount of 50-500ng/ml, preferably 100-400ng/ml, or more preferably 150-300ng/ml in the medium C, within which the object of the present invention can be achieved, and particularly preferably 200ng/ml; the TGF-beta inhibitor may be used in an amount of 1 to 50. Mu.M, preferably 5 to 30. Mu.M, or more preferably 5 to 10. Mu.M, within which the object of the present invention can be achieved, with 10. Mu.M being particularly preferred; culturing in the medium C for 1-3 days, preferably 1 day.

The Wnt inhibitor in the medium D may be IWP2, IWR-1, IWP-4, CCT251545, KY1220, but is not always limited thereto, and specifically may be preferably IWP2, and the amount thereof to be used may be 0.1 to 10. Mu.M, preferably 0.25 to 5. Mu.M, or more preferably 0.5 to 2. Mu.M, and specifically 1. Mu.M; the TGF beta inhibitor may be selected from a variety of agents as described above, and is particularly preferably SB431542, which may be used in an amount of 1-50. Mu.M, preferably 5-30. Mu.M, or more preferably 5-10. Mu.M, particularly preferably 10. Mu.M; culturing in the medium D for 1-3 days, preferably 1 day.

The GSK-3 inhibitor in the medium E can be selected from the above, specifically CHIR99021, and can be used in an amount of 0.1-10. Mu.M, preferably 1-7.5. Mu.M, or more preferably 2-5. Mu.M, specifically 3. Mu.M; BMP4 can be used in an amount of 1-50ng/ml, preferably 5-20ng/ml, and particularly preferably 10ng/ml; the amount of the keratinocyte growth factor KGF used can be 1-50ng/ml, preferably 5-20ng/ml, and particularly preferably 10ng/ml; FGF10 can be used in an amount of 1-50ng/ml, preferably 5-20ng/ml, and particularly preferably 10ng/ml; culturing in the medium E for 2-5 days, preferably 3 days.

The EGF in the medium F can be used in an amount ranging from 1 to 50ng/ml, preferably from 5 to 20ng/ml, and particularly preferably 10ng/ml; VEGF can be used in an amount of 0.5-100ng/ml, preferably 10-30ng/ml, and particularly preferably 15ng/ml; the amount of bFGF used may be 0.5-50ng/ml, preferably 2-10ng/ml, particularly preferably 5ng/ml; the use amount of ATRA is 0.01-0.5. Mu.M, preferably 0.05-0.2. Mu.M, and particularly preferably 0.1. Mu.M; culturing in the culture medium F for 8-12 days, preferably 10 days.

Specifically, the medium used in S3 is medium G, which is supplemented with a factor that promotes lung organoid maturation in addition to medium F.

Wherein the factors promoting the maturation of lung organoids are dexamethasone, PDE inhibitors, and PKA activators.

The PDE inhibitor is any one of IBMX, rolipram, sildenafil and Millinone, preferably IBMX; it may be used in an amount of 0.01 to 1mM, preferably 0.05 to 0.2mM, particularly preferably 0.1mM;

the PKA activator can be any one of cAMP and its salt, FSK, CW008, taxol, belinostat and its salt, preferably cAMP; it may be used in an amount of 0.01 to 1mM, preferably 0.05 to 0.2mM, particularly preferably 0.1mM;

dexamethasone MK125 may be used in an amount of 10-1000nM, preferably 25-100nM, particularly preferably 50nM.

In particular, the ECM is a three-layer structure, namely an a layer, a B layer and a C layer from bottom to top;

the layer A is 100% Matrigel;

the layer B is a lung bud formed by S2, matrigel with the volume ratio of 40%, type I collagen with the volume ratio of 40% and type III collagen with the volume ratio of 20%;

layer C was 100% Matrigel.

When in use, a layer A, B, C is added into the upper small chamber of the 12-hole transwell in sequence; standing for 10min, 60min, and 10min respectively to completely solidify. Thereafter, stage 3 induction medium was added to both the upper and lower chambers.

The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.

The reagents used in the examples are shown in tables 1 and 2, and the antibodies used are shown in table 3.

EXAMPLE 1 construction of Whole Lung organoids

(one) Generation of Whole Lung organoids

On the basis of the lung epithelial organoid induced differentiation factor, the human multi-lineage lung organoid induced differentiation of epithelial cells, endothelial cells and immune cells is realized through innovation in three aspects: 1) Basic culture medium: addition of medium suitable for growth of mesoderm lineage EGM2, 2) induction factor: adding EGF, VEGF, bFGF, 3) ECM (extracellular matrix) which is a common factor for inducing differentiation of vascular endothelial cells and macrophages: the addition of Collagen type I and Collagen type III promotes the maturation of multiple lineages.

The present embodiment is divided into 3 phases in a whole, lasting days:

s1 (stage 1): induction of differentiation of hPSC into definitive endoderm (Day 1-2)

Differentiation can be initiated when the degree of hPSC confluence is 40-70%. Specifically, in this example, after the degree of fusion of hPSC reaches 40%, the hPSC is added to culture medium A and culture medium B of S1 stage in sequence for induced differentiation.

S11（Day 1）：

Human pluripotent stem cells were cultured for 24h in medium A, RPMI-1640 medium supplemented with 100ng/ml rhGDF8, 3. Mu.M CHIR 99021.

S12（Day 2）：

Then, culture was continued for 24h in medium B supplemented with 100ng/ml rhGDF8, 10ng/ml rhFGF2, 50. Mu.g/ml vitamin C (AA), and medium B was RPMI-1640 containing 2% (by volume) SM 1.

S2 (stage 2): induction of differentiation of definitive endoderm into Lung bud (Day 3-17)

S21（Day 3）：

This stage was incubated for 24h with medium C supplemented with 200ng/ml NOG, 10. Mu.M SB431542 and medium C containing 0.5% (v/v) N2 (A), 1% (v/v) SM1, 75% (v/v) IMDM, 23.5% (v/v) EGM2 and 0.05% (v/v) BSA, 0.4. Mu.M MTG.

S22（Day 4）：

This stage was cultured for 24h with medium D supplemented with 1. Mu.M IWP2, 10. Mu.M SB431542, 1. Mu.M ATRA and containing 0.5% (vol.) N2 (A), 1% (vol.) SM1, 75% (vol.) IMDM, 23.5% (vol.) EGM2 and 0.05% (solid to liquid) BSA, 0.4. Mu.M MTG.

S23（Day 5-7）：

The culture was continued for 3 days using medium E, and the medium was changed every 24 hours. Medium E was supplemented with 3. Mu.M CHIR99021, 10ng/ml rhBMP4, 10ng/ml rhKGF, 10ng/ml rhFGF10, 1. Mu.M ATRA, and medium E contained 0.5% (vol.) N2 (A), 1% (vol.) SM1, 75% (vol.) IMDM, 23.5% (vol.) EGM2 and 0.05% (solid to liquid) BSA, 0.4. Mu.M MTG.

By Day7 (Day 7), a large amount of SOX2 appeared on the upper layer of 2D cells ⁺ 、NKX2.1 ⁺ Solid spheres of lung progenitor cells (hereinafter referred to as lung progenitor cell spheres) were collected and transferred to ultra-low sorption plates and cultured continuously using stage S24 medium.

S24（Day 8-17）：

The culture was carried out for 10 days using medium F, and the medium was changed every 24 to 48 hours. Medium F containing 0.5% (vol/vol) N2 (A), 1% (vol/vol) SM1, 75% (vol/vol) IMDM, 23.5% (vol/vol) EGM2 and 0.05% (vol/vol) BSA, 0.4. Mu.M MTG was supplemented with 3. Mu.M CHIR99021, 10ng/ml rhBMP4, 10ng/ml rhKGF, 10ng/ml rhFGF10, 10ng/ml rhEGF, 15ng/ml rhVEGF, 5ng/ml bFGF, 0.1. Mu.M ATRA.

By Day17 (Day 17), the lung progenitor cell spheres will differentiate to express EpCAM ⁺ 、NKX2.1 ⁺ 、FOXA1 ⁺ 、SOX9 ⁺ Saccular lung bud, entering stage S3.

S3: inducing differentiation of the lung bud into Lung organoids (Day 18-60)

ECM preparation and lung bud coating:

ECM was divided into three layers, each layer consisting of:

layer A: 100% Matrigel;

layer B: day17 lung bud +40% Matrigel +40% Collagen I (2.0 mg/ml) +20% Collagen III (2.0 mg/ml);

layer C: 100% Matrigel.

Adding a layer A, B, C into the upper chamber of the 12-hole transwell in sequence; standing for 10min, 60min, and 10min respectively to completely solidify. Thereafter, the S2-stage medium G was added to both the upper and lower chambers.

Day 18-60：

Finally, the culture medium G is used for culturing for 43 days, and the culture medium is replaced every 24-72 h. Medium G was supplemented with 3. Mu.M CHIR99021, 10ng/ml rhBMP4, 10ng/ml rhKGF, 10ng/ml rhFGF10, 10ng/ml rhEGF, 15ng/ml rhVEGF, 5ng/ml bFGF, 0.1. Mu.M ATRA, 50nM MK125, 0.1mM IBMX, 0.1mM cAMP, medium G contained 0.5% (by volume) N2 (A), 1% (by volume) SM1, 75% (by volume) AECGM, 23.5% (by volume) EGM2 and 0.05% (by volume) BSA, 0.4. Mu.M MTG.

By Day60 (Day 60), human multi-lung organoids with both proximal and distal structures were available.

(II) organoid phenotype detection and tissue-resident macrophage function detection

The organoid phenotype and the function of tissue resident macrophages were examined in combination using the following criteria:

1) Morphological characterization of organoids under bright field;

2) Detecting the expression of the cell specific marker by using a frozen slice immunofluorescence and panoramic immunofluorescence technology;

3) Detecting macrophage proportion by flow cytometry;

4) The phagocytosis and uptake functions of macrophages are inspected through the phagocytosis of the fluorescence-labeled latex beads and the uptake of the fluorescence-labeled acetylated low-density lipoprotein;

5) Removing tissue resident macrophages by using a macrophage scavenger disodium clodronate liposome, and analyzing macrophage residues by combining the analysis method;

6) The polarization of the tissue resident macrophages to M1 type macrophages is induced by 1 mug/ml LPS +100ng/ml IFN-gamma, and the polarization of the tissue resident macrophages to M2 type macrophages is induced by 50ng/ml IL-4+50ng/ml IL-13, so that the macrophages have the capacity of polarizing to the M1/M2 type macrophages.

Example 2 bleomycin resistance comparative experiment

The lung organoid models obtained in example 1 were treated with bleomycin at 0. Mu.g/ml, 10. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml and 500. Mu.g/ml for 10 days, respectively, and cell viability was compared.

And then 1 lung organoid model and 2 common in-vitro efficacy models (both widely used for efficacy tests of diseases such as pulmonary fibrosis) are selected to be compared with the model of the invention. Wherein the lung organoid model is An organoid recently published by Kim JH for simulating pulmonary fibrosis (Kim J H, an G H, kim J Y, et al. Human pluripotent stem Cell-derived organic ligands for modulating pulmonary fibrosis [ J ]. Cell death discovery, 2021, 7 (1): 1-12.); the cell model comprises human lung epithelial cells A549 and mink lung epithelial cells Mv.1.Lu, and the cell viability of each group is compared after the cells are treated for 10 days by using the bleomycin with different concentrations.

Example 3 pulmonary organoid-based fibrosis modeling

Fiberization molding

Setting a model building group/a control group, and continuously incubating organoids for 20 days by using 50 mu g/ml of bleomycin/PBS respectively; the medium was changed every 48h during the period.

(II) verification of fibrosis pathology-related indexes

The fibrosis pathology was assessed comprehensively using the following indices:

1) Morphological characterization of organoids under bright field;

2) Detecting the expression level of the fibrosis related marker mRNA of the modeling/control group by a fluorescent quantitative PCR (Q-PCR) technology;

3) Detecting the protein expression condition of the fibrosis related marker of the modeling/control group by using a frozen section and an immunofluorescence technique, and carrying out quantitative analysis of fluorescence area by using ImageJ software (related antibodies are shown in an accessory list);

4) Evaluating the collagen deposition condition of the modeling/control group by a paraffin section and Masson staining technology;

5) The research on the fibrosis mechanism of the model comprises the following steps: before and after the macrophage scavenger is used, the secretion level of fibrosis-related secreted protein of the model/control group is detected by an ELISA technology; changes in the proportion of cells associated with molding/control fibrosis were analyzed by flow cytometry.

Results and analysis:

1. continuously differentiating to Day60 to form mature human whole lung organoid, morphologically presenting complex tissue-like appearance, and volume up to 0.5cm ³ (Panel A in FIG. 1).

Immunofluorescence results after frozen section show that the lung organoid not only contains different types of lung epithelial cells, but also has lung resident macrophages: epithelial cells include ciliated cells (acubs) located primarily in the proximal end of the respiratory tract ⁺ FIG. 1, panel B), rod-shaped cells (CC 10) ⁺ C panel in FIG. 1), neuroendocrine Cells (CHGA) ⁺ D in FIG. 1), goblet cell (MUC 5 AC) ⁺ E in FIG. 1), basal cells (P63) ⁺ Graph F in fig. 1); and type I alveolar cells (PDPN) located primarily at the distal end of the respiratory tract ⁺ G in FIG. 1) and type II alveolar cells (SP-B) ⁺ H in fig. 1). On the other hand, lung resident macrophages are not only present in airway-like structures (CD 68) ⁺ I panel of fig. 1), was also detected in distal alveolar-like structures (CD 68) ⁺ And J in fig. 1), the dispersibility distribution is shown as a whole.

FIG. 2 flow cytometry results show that these macrophages account for about 10-11% (CD 68) ⁺ Or CD14 ⁺ ）。

2. To verify the phagocytic capacity of lung resident macrophages for foreign bodies, organoids were incubated with AF594 (red fluorescent) labeled 2 μm latex beads.

The results show that most of the red fluorescence and green fluorescence labeled CD68 ⁺ Cell overlap, as shown in panel D of figure 3, demonstrates that these macrophages are phagocytic.

3. To verify the ability of lung resident macrophages to take up low density lipoproteins, organoids were incubated with red fluorescently labeled acetylated low density lipoproteins (Dil-Ac-LDL).

The results show that most of the red fluorescence and green fluorescence labeled CD68 ⁺ Cells overlap as shown in panel D of FIG. 4. In contrast, CD68 was purified by macrophage specific scavenger (disodium clodronate liposome) ⁺ There was no red fluorescence signal after complete macrophage clearance by incubating with Dil-Ac-LDL, as shown in FIG. 4, panels E-H.

The results of this positive and negative validation demonstrated that these macrophages, residing in lung tissue, can take up Dil-Ac-LDL.

4. To verify the ability of lung resident macrophages to polarize to M1/M2 type macrophages, organoids were incubated with M1/M2 polarization inducers, respectively.

Flow cytometry results showed that M1-type macrophages increased from 0.62% to 7.05% (CD 86) and M2-type macrophages increased from 0.99% to 9.73% (CD 206), as shown in FIG. 5, indicating that these lung resident macrophages possess the ability to polarize both M1 and M2-type macrophages.

In conclusion, the invention establishes a set of multi-lineage lung organoid system with functional macrophages; these lung resident macrophages possess foreign body phagocytic capacity, ability to take up Ac-LDL, and ability to polarize to M1/M2 type macrophages.

5. Bleomycin is a general inducer for pulmonary fibrosis modeling of animal models, forms pulmonary fibrosis under the action of bleomycin, is similar to human body in characteristics, and is widely accepted and adopted. However, the in vitro model is difficult to tolerate due to its strong cytotoxicity, so there are few reports of using bleomycin in the in vitro model. Currently, in vitro fibrosis models are mostly modeled by using a fibrosis central signal molecule, TGF beta, so that a fibrosis effect is directly triggered. Although the method has the advantages of simplicity, rapidness and the like, the terminal signal (i.e. TGF (transforming growth factor) activation) of the pulmonary fibrosis is only considered, and important molecular events occurring in a plurality of intermediate processes, such as damaged and aged lung epithelial cells, macrophage polarization, release of inflammatory factors/pro-fibrosis factors and the like, are ignored and deviate from the occurrence and development of real fibrosis in vivo; this reveals to some extent why pharmacodynamic data based on such models differ greatly from clinical outcomes. In addition, the formation of fibrosis is a long-lasting process accumulating day and month, and the induction time of the in vitro model of TGF beta fibrosis is less than or equal to 48h, so the overall appearance of pathological progress and corresponding drug effect are difficult to capture.

In conclusion, if the in vitro model can also use bleomycin to induce pulmonary fibrosis like the animal model, even the induction time window is prolonged, the in vitro model is expected to simulate the occurrence and development of the fibrotic pathology ab initio and comprehensively.

To try to use bleomycin for in vitro modelling, the cell viability was first tested at different bleomycin concentrations and a moderate induction period was chosen: 10 days (animal models are typically around 20 days). Next, 3 other pulmonary fibrosis models were used in parallel comparisons.

As shown in fig. 6, compared with the recently reported lung organoids for simulating fibrosis and 2 commonly used in vitro pulmonary fibrosis pharmacodynamic models (human lung epithelial cells a549 and mink lung epithelial cells mv.1. Lu), the lung organoids obtained by the invention have significantly stronger bleomycin tolerance due to the compact tissue structure (near and far respiratory tract, such as a-J diagram in fig. 1).

Thereafter, to further open the induction time window to fit the real scene in vivo, a 50 μ g/ml dose was selected and a 20 day duration of continuous fibrosis induction was performed.

5. As shown in fig. 7, 20 days after molding with Bleomycin (BLM), the lung organoids were significantly smaller in volume and appeared in a collapsed state compared to before molding; the interior of the original alveolus-like structure is changed from hollow to partial solid; and a large number of mesenchymal cells appear around the original Epithelial-like structure, suggesting that an important pathological characterization of fibrosis occurrence-EMT (Epithelial-mesenchymal transition).

6. As shown in FIG. 8, the Q-PCR results show that: after BLM modeling, extracellular matrix-related markers (FN、COL1A、 COL3A) Mesenchymal-related marker (A)VIM、DES、THY1) And EMT occurrence-associated marker (SLUG、SNAIL、TWIST) The expression level is comprehensively up-regulated, and the occurrence and the development of fibrosis are prompted.

7. As shown in fig. 9, the immunofluorescence results indicated that: compared with a control group, 20-day BLM modeling enables large-area expression of fibrosis markers such as alpha-SMA, FN, PDGFR alpha, PDGFR beta and the like; the significance of these changes in the protein was confirmed by fluorescence area quantification, which confirmed the occurrence of fibrosis.

8. Pathological section staining (Masson staining) results showed that: columnar epithelia arranged orderly in the original alveolus-like structure are subjected to BLM molding, and the tissue structure is broken, and a plurality of solid structures are formed respectively. The results of the local magnification comparison show that: similar to the pathological manifestations of patients with pulmonary fibrosis, on the one hand, the building block presents more fusiform mesenchymal-like cells, indicating the occurrence of EMT; on the other hand, a pronounced collagen deposition phenomenon occurred (fig. 10).

The results fully confirm that lung organoid fibrosis molding is successful after 20 days of BLM molding.

9. There is a large body of evidence that TGF- β 1 secreted by M2-type macrophages is an important regulator of the onset of fibrosis; the M2-type polarization of tissue-resident macrophages after stimulation by an injury signal is considered a prerequisite for the initiation of this process.

To explore the mechanisms behind this model fibrosis phenomenon, we hypothesized that macrophages also play a key role therein and developed a series of verifications:

flow cytometry results showed that the proportion of M2 macrophages was gradually increased from 1.84% to 15.05% during BLM modeling (fig. 11), demonstrating a persistent M2-type polarization, consistent with the pathological features of fibrosis. The ELISA results in FIG. 12 show that the TGF-. Beta.1 content of the culture supernatants also increased gradually during the molding process, suggesting that TGF-. Beta.1 was secreted by M2-type macrophages.

After using macrophage scavenger disodium clodronate lipid, and then using BLM for molding (fig. 13 and 14 verify that no M2 macrophages are present before and after molding), the ELISA results of fig. 15 show: compared with the non-cleared group, the TGF-beta 1 content in the supernatant is extremely obviously reduced, and the model proves that the TGF-beta 1 is mainly secreted by the M2 type macrophage. Accordingly, the immunofluorescence of fig. 16 and the quantitative results of fig. 17 indicate: the degree of fibrosis is very significantly reduced.

The above results demonstrate that: highly similar to in vivo pathology, macrophages in the in vitro model play a key role in lung fiber progression.

Unlike other in vitro fibrosis models which are widely used for direct modeling by using TGF-beta 1, the organoid model of the invention adopts the bleomycin which is commonly used in animal models, and the induction period is close to that of the animal models (about 20 days). The two models are mainly different in that the TGF-beta 1 modeling method forcibly activates TGF beta and downstream target molecules by directly adding TGF-beta 1 recombinant protein into a culture medium, so that the models tend to present pathological characterization; belongs to the direct activation of the central signal of fibrosis. In parallel to the above, bleomycin activates and simulates a series of molecular events from top to bottom in the fibrosis process, including simulating injury signals, inducing lung epithelial cells to age and die, and further converting to mesenchymal cells (EMT process), on one hand, macrophages tend to dynamically polarize and secrete profibrotic factors (especially TGF-beta 1 and PDGF), on the other hand, intracellular TGF beta signals of various cells are activated, and both the TGF beta signals and the profibrotic factors promote fibroblast to differentiate into myofibroblasts, thereby causing the generation of fibrosis scars. Therefore, on the premise that the biological model is sufficiently supported, the fibrosis pathological simulation of the bleomycin modeling method is more consistent with the real situation of a human body.

Therefore, the pulmonary fibrosis disease model is more suitable to be used as a new generation IPF in vitro disease model for accurate drug effect screening of anti-fibrosis drugs.

TABLE 1 list of reagents

Name of reagent consumable	Company (goods number)
		mTeSR1	STEMCELL (85850)
Y-27632	Sigma (SCM075)
		RPMI 1640	Gibco (31870082)
IMDM	Sigma (I2911)
		EGM2	Lonza (CC-3156 & CC-4176)
rhGDF8	R&D (6986-PG)
		rhFGF2	R&D (233-FB)
rhNOG	R&D (6057-NG)
		rhBMP4	R&D (314-BPE)
rhKGF	R&D (251-KG)
		rhFGF10	R&D (345-FG)
rhEGF	R&D (236-EG)
		rhVEGF	R&D (DVE00)
CHIR99021	Tocris (4423/10)
		SB431542	Tocris (1614)
IWP2	Tocris (3533)
		ATRA	Tocris (0695/50)
IBMX	Tocris (2845)
		MK125	Tocris (1126)
8-Br-cAMP	Tocris (1140/10)
		N2 (A)	STEMCELL (07152)
SM1	STEMCELL (05711)
		BSA	Sigma (A1933)
MTG	Sigma (M6145)
		Matrigel (GFR) (Matrigel)	Biocoat (356231)
Human Collagen I	Sigma (234138)
		Human Collagen III	Sigma (C4407)
Latex beads, carboxylate-modified polystyrene, fluorescent red	Sigma-Aldrich (L3030)
		Low sensitivity Lipoprotein from Human Plasma, esterified, alexa Fluor. 594 Conjugate (Low Density Lipoprotein in Human Plasma, acetyl) chemo-Alexa Fluor 594 conjugate	(Invitrogen, L35353)
Clodronate lipomes (Clodronate liposomes)	(Liposoma, C-005)
		LPS	Sigma-Aldrich (L2630)
IFN-γ	R&D (285-IF)
		IL-4	R&D (204-IL)
IL-13	R&D (213-ILB)
		Masson's Trichrome Stain Kit (Masson Trichrome Stain Kit)	Polysciences (25088-100)
Human/Mouse/Rat/Portine/Canine TGF-beta 1 Quantikine ELISA Kit (Human/Mouse/Rat/pig/dog TGF-beta 1 quantitative factor ELISA Kit)	R&D (DB100C)
		Evo M-MLV RT Kit with gDNA Clean for qPCR (in vitro reverse transcription Kit)	AG (AG11705)
SYBR Green Premix Pro Taq HS qPCR Kit (SYBR Green Premix Pro Taq HS qPCR Kit)	AG (AG11718)
		Human lung RNA	Clontech (636524)
Normal Donkey Serum (Normal Donkey Serum)	Jacksonlab (017-000-121)
		DAPI	Sigma (D9542)

TABLE 2 reagent corresponding names

rhGDF8	Recombinant human myostatin 8
		CHIR99021	GSK-3 inhibitors
rhFGF2	Recombinant human fibroblast growth factor 2
		AA	Vitamin C
rhBMP4	Recombinant human bone morphogenetic protein 4
		rhKGF	Recombinant human keratinocyte growth factor
rhFGF10	Recombinant human fibroblast growth factor 10
		rhEGF	Recombinant human epithelial growth factor
rhVEGF	Recombinant human vascular endothelial cell growth factor
		bFGF	Basic fibroblast growth factor
rhNOG	Recombinant human noggin, BMP inhibitors
		SM1	Serum substitute with definite components
N2(A)	Neural differentiation additive
		BSA	Bovine serum albumin
MTG (1-Thioglycerol)	1-thioglycerol
		SB431542	TGF (transforming growth factor) beta inhibitor
IWP2	Wnt inhibitor
		Matrigel(GFR)	Matrigel (growth factor reducing type)
ATRA (All-trans-Retinoic acid)	All-trans retinoic acid, a natural agonist of RAR nuclear receptors; an inhibitor;
		IBMX	a broad spectrum Phosphodiesterase (PDE) inhibitor
cAMP	Cyclic AMP analogs, PKA activators
		MK125 (Dexamethasone)	Dexamethasone
IMDM (Iscove's Modified Dulbecco's Medium)	Serum-free basal medium suitable for epithelial cells or blood cells
		EGM2 (Endothelial Cell Growth Medium 2)	Serum-free medium suitable for endothelial growth
AECGM (Airway Epithelial Cell Growth Medium)	Serum-free culture medium suitable for lung epithelial cells
		DAPI	Nuclear dye
ECM (Extracellular matrix)	Extracellular matrix
		Collagen I	Type I collagen
Collagen III	Type III collagen
		MUC5AC (Mucin 5AC)	Mucin 5AC, goblet cell marker
CC10 (Clara cell 10 kDa proteins)	Clara cell 10-kDa protein, a rod cell marker
		acTUB (Acetylated α-tubulin)	Acetylated-alpha tubulin, a ciliated cell marker
CHGA (chromogranin A)	Human chromogranin A, a neuroendocrine cell marker
		P63 (Tumor protein p63)	Transcription factor p63, lung basal cell marker
PDPN (Podoplanin)	Podoprotein, a type I alveolar cell marker
		SP-B (Surfactant protein B)	Pulmonary surfactant protein B, type II alveolar cell marker
CD68	Differentiation antigenic Cluster 68, universal macrophage marker
		CD206	Differentiation of antigenic Cluster 206, M2-type macrophage marker
CD86	Differentiation antigen cluster 86, M1 type macrophage marker
		BLM (Bleomycin)	Bleomycin, a universal pulmonary fibrosis inducer for animals
FN (Fibronectin)	Fibronectin, a physiologically relevant marker of fibrosis (ECM-constituting protein)
		COL1A (Collagen 1A)	Type I collagen alpha chain, a marker of physiological relevance for fibrosis (ECM-constituting protein)
COL3A (Collagen 3A)	The alpha chain of the type III collagen,a marker (ECM constituting protein) related to fibrosis pathology
		VIM (Vimentin)	Vimentin, a marker associated with the fibrosis pathology (characterizing interstitial cells)
DES (Desmin)	Connexin, a marker associated with fibrosis pathology (characterizing interstitial cells)
		THY1 (Thy-1 cell surface antigen)	Glycoprotein, a marker of pathological relevance for fibrosis (characterisation of interstitial cells)
SLUG	Zinc finger protein transcription factor SLUG, a marker associated with fibrosis pathology (characterizing the occurrence of EMT)
		SNAIL	Zinc finger protein transcription factor, a marker associated with fibrosis pathology (characterizing the occurrence of EMT)
TWIST	Zinc finger protein transcription factor, a fibrosis pathology-associated marker (characterizing the occurrence of EMT)
		α-SMA (α-Smooth muscle actin)	Alpha-smooth muscle actin, a fibrotic pathological marker (characterizing myofibroblasts)
PDGFR-α(Platelet-derived growth factor receptor-α)	Platelet-derived growth factor receptor alpha, a fibrotic pathology-relatedMarker substance
		PDGFR-β(Platelet-derived growth factor receptor-β)	Platelet-derived growth factor receptor beta, a fibrosis pathology-related marker
TGF-β1 (Transforming growth factor beta 1)	Transforming growth factor-beta 1, a fibrotic central regulatory signal

TABLE 3 list of antibodies

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for constructing a pulmonary fibrosis disease model in vitro by using bleomycin is characterized by comprising the following steps: the method comprises the following steps:

1) Construction of Whole lung organoids

2) Construction of pulmonary fibrosis disease model

I) Respectively using M1 and M2 polarization inducers to incubate the whole lung organoid, and verifying that macrophages have the ability of polarizing M1/M2 type macrophages;

2. The method for in vitro construction of a pulmonary fibrosis disease model using bleomycin of claim 1, wherein: bleomycin was used for 8-30 days during which medium was changed every 48 h.

3. The method for in vitro construction of a pulmonary fibrosis disease model using bleomycin of claim 1, wherein: GDF is GDF8, and the dosage is 20-500ng/ml.

4. The method for in vitro construction of a pulmonary fibrosis disease model using bleomycin of claim 1, wherein: the using amount of ATRA is 0.01-10 mu M, the macrophage induced differentiation factor comprises EGF, VEGF and bFGF, wherein the using amount of EGF is 1-50ng/ml, the using amount of VEGF is 0.5-100ng/ml, and the using amount of bFGF is 0.5-50ng/ml; the volume percentage of EGM2 added in each stage is 5-60%.

5. The method for in vitro construction of a pulmonary fibrosis disease model using bleomycin of claim 1, wherein: the lung epithelial cell related induced differentiation factors comprise GSK-3 inhibitor, BMP4, FGF10, KGF and ATRA; wherein, the usage amount of the GSK-3 inhibitor is 0.1-10 MuM, the usage amount of the BMP4 is 1-50ng/ml, the usage amount of the FGF10 is 1-50ng/ml, the usage amount of the KGF is 1-50ng/ml, and the usage amount of the ATRA is 0.01-10 MuM.

6. The method for in vitro construction of a pulmonary fibrosis disease model using bleomycin of claim 1, wherein: the lung epithelium maturation-promoting factor includes dexamethasone, PDE inhibitors, and PKA activators.