In-vitro cell model for simulating particulate matter induced lung disease occurrence and application of in-vitro cell model

Abstract

The invention relates to an in-vitro cell model for simulating particulate matter induced lung disease occurrence and application. According to the cell culture device, different small chambers are formed, cells between adjacent small chambers cannot be in contact, but cell factors generated by the cells can penetrate through the small chambers along with flowing of a culture medium to affect the cells, and then interaction between the cells in a body is reduced; meanwhile, the segmentation assembly is removed before the particulate matter is added, but the cells are still reserved in the small chamber, and therefore the in-vivo interaction of the particulate matter and the cells can be simulated more truly. In addition, gel does not need to be used as a support, and the real effect of particulate matter and cells in the body can be better simulated only through installation and disassembly of the segmentation assembly. Therefore, the in-vitro cell model disclosed by the invention can really simulate the interaction between in-vivo cells and accurately predict the influence of the particulate matters on the cells, and the preparation method is simple and effective and provides a new thought for the establishment of the in-vitro cell model.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to an in vitro cell model for simulating particle-induced lung disease and application thereof. Background Art

[0002] Environmental particulate matter is solid or liquid granular matter dispersed in the air. Respiratory exposure is the main way for it to enter the body. The smaller the particle size, the deeper it reaches the human body. Exposure to environmental particulate matter is one of the biggest environmental risk factors affecting global human health. A large number of studies at home and abroad have shown that short-term or long-term exposure to environmental particulate matter can have adverse effects on the human respiratory system, cardiovascular system, central nervous system, etc. For example, when particulate matter enters the lung tissue, it will irritate and corrode the alveolar wall, damage lung function, and significantly increase the incidence of chronic respiratory diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease, and bronchial asthma; particles with a size of less than 2.5μm can break through the gas-blood barrier and directly enter the blood circulation, causing direct damage to the cardiovascular system; it may also affect the autonomic nervous system, causing arrhythmias or changes in coronary vascular tension, etc.

[0003] Pulmonary fibrosis is the most common disease caused by environmental particulate matter. Pulmonary fibrosis is a serious interstitial lung disease characterized by damage to alveolar epithelial cells, massive proliferation of pulmonary fibroblasts, and increased aggregation of extracellular matrix, accompanied by inflammatory damage and tissue structure destruction. Many patients with pulmonary fibrosis inhale asbestos, mineral dust, harmful gases, animal fur, moldy dead grass, etc. for a long time in the production environment, which increases the risk of lung inflammation and induces the occurrence of pulmonary fibrosis.

[0004] At present, people have tried to establish a variety of in vitro models to evaluate the effects of environmental particulate matter on pulmonary fibrosis. Among them, the most commonly used 2D cell line model is widely used to study the effects and mechanisms of asbestos, carbon nanotubes, etc. on pulmonary fibrosis; with the development of 3D cell culture technology, some studies have also constructed three-dimensional models of various lung cells for the evaluation of the effects of environmental particulate matter on pulmonary fibrosis; in addition, scaffold-free 3D lung microtissues and organoid models have also been gradually developed for the study of the effects of environmental particulate matter on pulmonary fibrosis. In the literature "A novel human 3D lung microtissue model fornanoparticle-induced cell-matrix alterations", researchers constructed 3D lung microtissues containing three types of cells: human lung fibroblasts, epithelial cells, and macrophages. After the microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles, or crocidolite fibers for 4 or 7 days, the cells were collected to characterize the microtissue viability, tissue morphology, and the expression of genes and selected proteins related to inflammation and extracellular matrix remodeling. In the paper "In vitro model alveoli from photodegradable microsphere templates.", the researchers developed an in vitro 3D model of alveolar epithelium, using photodegradable microspheres as sacrificial templates to form spherical multicellular structures with hollow interiors, cultured within tunable hydrogel scaffolds. This method allows for the modification of the matrix properties of the adhesion surface and allows for co-culture with cell types found on the basolateral side of the epithelium, such as fibroblasts and endothelial cells.

[0005] In the above-mentioned in vitro model of pulmonary fibrosis, the 2D cultured cell line model only has cells of a single component, and the traditional cell co-culture model can only achieve co-culture between two types of cells, which is difficult to restore the true pathological state of cells in vivo, so its application has certain limitations; organoids will have problems such as uneven size and poor permeability of nanoparticles caused by extracellular matrix (necessary for organoid culture); the gel used in the 3D cell culture model has an adsorption effect on particulate matter, affecting the interaction between particulate matter and cells, and emerging technologies have high operation requirements, high resource and maintenance costs, and are difficult to standardize. Therefore, it is necessary to build an in vitro model that is fast and efficient, truly simulates in vivo conditions, and is relatively inexpensive. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the 3D cell culture model in the prior art requires the use of gel, which results in poor permeability of nanoparticles and is unable to truly simulate in vivo conditions.

[0007] To solve the above technical problems, the present invention provides an in vitro cell model simulating the occurrence of particulate matter-induced lung diseases and its application. The present invention uses a cell culture device to form different chambers, and the cells between adjacent chambers cannot contact each other, but the cytokines produced by the cells can pass through the chambers with the flow of the culture medium and affect the cells, thereby restoring the interaction between cells in vivo; at the same time, the present invention removes the segmentation component before adding the particulate matter, but the cells are still retained in the chamber, so that the interaction between the particulate matter and the cells in vivo can be more realistically simulated. In addition, when simulating the occurrence of pulmonary fibrosis in vivo, environmental particulate matter such as coal dust, asbestos, silica dust, organic dust, inorganic dust, etc. need to be used. Since gel is used in traditional cell co-culture technology, gel has an adsorption effect on these particulate matter, which affects the interaction between environmental particulate matter and cells. However, the present invention does not need to use gel as a scaffold, and only the installation and disassembly of the segmentation component can better simulate the real effect of particulate matter and cells in vivo. Therefore, the in vitro cell model of the present invention can truly simulate the interaction between cells in vivo, accurately predict the effect of particulate matter on cells, and the preparation method is simple and effective, which provides a new idea for the establishment of in vitro cell models.

[0008] The first object of the present invention is to provide a method for preparing an in vitro cell model for simulating the occurrence of particulate matter-induced lung disease, comprising the following steps:

[0009] S1. Inoculate cells in a cell culture device for co-culture, wherein the cell culture device comprises an upper chamber and a lower chamber, the upper chamber is enclosed by a cup body and a semipermeable membrane, the lower chamber is divided into an inner chamber and an outer chamber by a dividing component detachably connected to the lower chamber, the inner chamber and the outer chamber are both enclosed by the cup body and the dividing component, the upper chamber, the outer chamber and the inner chamber are interconnected, and the cells located between the upper chamber, the inner chamber and the outer chamber cannot contact each other, the cells inoculated between the upper chamber, the inner chamber and the outer chamber are different from each other, the cells grow by attaching to the cup body, and the cells cannot grow by attaching to the dividing component;

[0010] S2. removing the segmentation component, adding particles into the cell culture device and incubating the particles with the cells to obtain the in vitro cell model simulating the occurrence of lung diseases in vivo.

[0011] When cells are inoculated, they are cultured in separate chambers, and the cells adhere to the wall in a monolayer, which will not affect cell proliferation and material processing, as well as subsequent separation and analysis. In addition, each chamber is induced to fully incubate the cells and particles, and restore the body's true pathological process to the greatest extent.

[0012] Furthermore, before adding the particles to the cell culture device, the segmentation component is removed. Since the size of the particles is uncertain, when using large-sized particles, the presence of the segmentation component prevents the particles from fully contacting the cells in the upper chamber, inner chamber, and outer chamber, affecting the induction effect. At the same time, since the cup body is made of a material that allows cells to adhere to the wall for culture, after the cell culture is completed, the cells in the upper and lower chambers grow on the cup body and will not be taken out when the segmentation component is removed. Moreover, adding particles and co-incubating with cells after removing the segmentation component can better simulate the body's real pathological process.

[0013] Furthermore, the cup body is made of plastic or has been treated with polystyrene, and the segmentation component includes an Oxford cup. When the cup body is made of plastic or has been treated with polystyrene, the cells can be cultured on the cup body during the cell culture process. When the segmentation component is an Oxford cup, cells will not adhere to its surface, and during cell co-culture, the cells will grow on the inner wall of the cup body instead of on the segmentation component. After the co-culture is completed, when the segmentation component is removed, the cells will not be taken away after the segmentation component is removed. When particulate matter is added, the particulate matter can fully contact the cells, and the cytokines produced by different cells can also interact with each other, which can more realistically simulate the in vivo environment.

[0014] Furthermore, the 3D cell culture device is a Transwell-24 system, and the segmentation component is an Oxford cup.

[0015] Furthermore, the lung disease includes pulmonary fibrosis.

[0016] Furthermore, when simulating the occurrence of pulmonary fibrosis in vivo, the cells in the upper chamber, the inner chamber and the outer chamber are independently selected from any one of pulmonary epithelial cells, pulmonary fibroblasts and pulmonary macrophages and are different from each other. The main function of pulmonary epithelial cells and pulmonary macrophages is to release verification factors under the stimulation of particulate matter, thereby stimulating the proliferation and differentiation of pulmonary fibroblasts. For example, pulmonary epithelial cells release TGF-beta cytokines, thereby promoting the occurrence of pulmonary fibrosis.

[0017] Furthermore, the inoculation ratio of the lung macrophages, the lung epithelial cells and the lung fibroblasts is (1-4): (1-4): (2-6). A higher ratio of fibroblasts is helpful to observe the occurrence of lung fibrosis.

[0018] Furthermore, the particulate matter includes multi-walled carbon nanotubes, M120 carbon black nanotubes, crocidolite fibers, and alumina particles.

[0019] Furthermore, the effective concentration of the particles is 30-70 μg / mL. The present invention has been tested through cytotoxicity pre-experiments and found that when the effective concentration of the particles is 30-70 μg / mL, both the cell survival rate and the induction effect of pulmonary fibrosis are guaranteed.

[0020] Furthermore, the components of the culture medium used for the cell co-culture in step S1 include glucose, amino acids, vitamins, minerals, trace elements, epithelial cell supplement factors, and fetal bovine serum.

[0021] Furthermore, the mass concentration of the epithelial cell additive factor is 1%; the mass concentration of the fetal bovine serum is 0.05%.

[0022] The second object of the present invention is to provide an in vitro cell model for simulating particulate-induced lung disease prepared by the above preparation method.

[0023] The third object of the present invention is to provide an application of the above-mentioned in vitro cell model simulating the occurrence of particulate matter-induced lung diseases in screening drugs for treating pulmonary fibrosis.

[0024] Beneficial effects of the present invention:

[0025] The present invention utilizes a cell culture device to form different chambers, and the cells between adjacent chambers cannot contact each other, but the cytokines produced by the cells can pass through the chambers with the flow of the culture medium and affect the cells, thereby restoring the interaction between cells in vivo; at the same time, the present invention removes the segmentation component before adding the particles, but the cells are still retained in the chamber, so it can more realistically simulate the interaction between particles and cells in vivo. In addition, the present invention does not need to use gel as a scaffold, and only the installation and disassembly of the segmentation component can better simulate the real effect of particles and cells in vivo. Therefore, the in vitro cell model of the present invention can truly simulate the interaction between cells in vivo, accurately predict the effect of particles on cells, and the preparation method is simple and effective, providing a new idea for the establishment of an in vitro cell model. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0027] Figure 1 is a schematic diagram of the Tranwell cell co-culture structure;

[0028] Figure 2 are the MTS test results of different treatment groups;

[0029] Figure 3This is the image of BrdU-labeled newborn lung fibroblasts (red) under a fluorescence microscope;

[0030] Figure 4 is the release of TGF-β in the bronchoalveolar lavage fluid of the mouse lung model after nanomaterial exposure;

[0031] Figure 5 This is Masson's staining of mouse lung tissue: collagen fibers are blue and muscle fibers are red. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0033] Example 1: Selection of Nanoparticles

[0034] 1. Reagents: TGF-β ELISA detection kit (mouse source) was purchased from RD.

[0035] 2. Methods:

[0036] (1) Ultrasonication (32W, 10s) was performed on a 0.8 mg / mL mixed solution of Al2O3-1 and Fe2O3-1 nanomaterials diluted with PBS to make it uniformly dispersed;

[0037] (2) Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (200 mg / kg) with a total volume of 100 μL. After anesthesia, the mouse body was kept vertical to block the nasal breathing of the mouse. When the mouse opened its mouth to breathe, 50 μL of nanomaterials were dripped into the mouse lungs through the oropharynx (2 mg / kg). At the same time, blank and positive control groups were set up, and 50 μL PBS and 2 mg / kg Quartz were dripped respectively;

[0038] (3) Expose once a week for 3 consecutive weeks. After the instillation, keep the mice upright until they wake up and return them to the cage;

[0039] (4) On day 90, mice were killed by injection of an overdose of sodium pentobarbital (400 mg / kg);

[0040] (5) Fix the four legs of the mouse with a pin and then spray with medical alcohol for disinfection. Lift the mouse skin along the lower part with micro-pointed tweezers, cut the mouse abdominal cavity with scissors, cut the abdominal vein, and after the blood of the mouse is drained, cut along the chest cavity with scissors until the mouse throat is exposed, cut the muscles to expose the larynx completely, turn the mouse upside down, puncture a hole below the Adam's apple with a pointed needle, then replace it with a flat-headed needle and use a syringe to take 1mL of frozen PBS to rinse the alveoli three times, collect the alveolar cell lavage fluid in a 1.5mL EP tube, place it on ice, and detect the level of transforming growth factor (TGF-β) in the mouse alveolar lavage fluid by enzyme-linked immunosorbent assay (ELISA);

[0041] (6) Use a 5mL syringe (with a 1mL needle tip) to inject PBS into the lower left part of the heart, inflate the heart, flush the lung tissue until it turns white, cut the vein connecting the heart and lung with the needle tip, and clamp the two lungs with hemostats. Use a 2mL syringe with a flat-tip needle to take 0.8mL of 4% paraformaldehyde and inject it along the cut throat to expand half of the lung. Cut half of the lung and add it to a 1.5mL EP tube containing 1mL of 4% paraformaldehyde for Masson staining.

[0042] 3. Results:

[0043] (1) Figure 4 As shown, the release of TGF-β in the bronchoalveolar lavage fluid of mice exposed to Al2O3-1 increased 5 times compared with the control group, and the difference was statistically significant, while the release of TGF-β in the Fe2O3-1 treatment group did not change significantly.

[0044] (2) Al2O3-1 and Fe2O3-1 were exposed to mouse lungs at 2 mg / kg for three consecutive weeks, and the lung tissues of mice were obtained for Masson staining three months later. Figure 5 As shown in the figure, according to the Ashcroft semi-quantitative score, the lungs of mice treated with Al2O3-1 were severely fibrotic, while those of mice treated with Fe2O3-1 were non-fibrotic. Therefore, Al2O3-1 was selected as a positive control and Fe2O3-1 as a negative control.

[0045] Example 2: Construction of an in vitro model of human lung cells for early warning of pulmonary fibrosis

[0046] 1. Reagents: DMEM medium was purchased from Corning; epithelial cell supplement factors were purchased from Sciencell; fetal bovine serum was purchased from Biobond; trypsin was purchased from Bio-Tech; PMA was purchased from Sigma; PBS was purchased from Bio-Tech.

[0047] 2. Methods:

[0048] (1) Preparation of cell co-culture medium: Add 1% epithelial cell supplement factor and 0.05% fetal bovine serum to 500 mL DMEM, mix well and set aside.

[0049] (2) Cell inoculation: A Corning Transwell-24 system with a pore size of 0.4 μm was used for cell co-culture. The induced differentiated macrophage THP-1 cells were inoculated in the upper chamber, and an Oxford cup was placed in the center of the lower chamber of the Transwell to divide it into two parts, the inner and outer parts, which were inoculated with normal human lung epithelial cells BEAS-2B and human lung fibroblasts WI-38 / VA-13, respectively. The inoculation ratio of THP-1, BEAS-2B and WI-38 / VA-13 was 3:3:5. The specific operations are as follows:

[0050] (3) Inoculation of BEAS-2B cells: Take out the cells cultured to 80-90% confluence from the incubator, remove the culture medium in the culture dish, add 5 mL PBS to gently soak and wash the cells twice, then add 1.5 mL pancreatin for digestion; after the digested cells fall off the bottom of the plate, add 5 mL of co-culture medium to terminate the digestion, obtain the digested cells, centrifuge, discard the supernatant, and count the cells. Add 100 μL of cell suspension containing 30,000 BEAS-2B cells to the center of the Oxford cup.

[0051] (4) WI-38 / VA-13 cells: After BEAS-2B cells were inoculated for about 12 hours, the cells were observed under a microscope. After most of them adhered to the wall, the WI-38 / VA-13 cells cultured to 80-9% confluence were taken out, the culture medium in the culture dish was aspirated, 5 mL of PBS was added to gently infiltrate and wash the cells twice, and then 1.5 mL of trypsin was added for digestion; after the digested cells fell off the bottom of the plate, 5 mL of co-culture medium was added to the inside to terminate the digestion, the digested cells were obtained, centrifuged, the supernatant was discarded, and the cells were counted. 300 μL of cell suspension containing 50,000 WI-38 / VA-13 cells was added to the outside of the Oxford cup.

[0052] (5) THP-1 cells: Take out cells that have grown to the stage where they can be subcultured, transfer the cell suspension completely to a 15 mL centrifuge tube, centrifuge, discard the supernatant, and count the cells. Add 100 μL of a cell suspension containing 30,000 THP-1 cells to the upper chamber, and add PMA at a ratio of 1:3000 to differentiate the cells.

[0053] (6) Construction of pulmonary fibrosis model:

[0054] (1) Preparation of co-culture medium containing nanomaterials:

[0055] ① Prepare quartz-containing pulmonary fibrosis induction culture medium: add Quartz to the cell co-culture medium, and the final drug concentration is 25 μg / mL.

[0056] ②Prepare Al2O3-1-containing culture medium: Add Al2O3-1 nanomaterials into the cell co-culture medium with a final concentration of 50 μg / mL.

[0057] ③Prepare culture medium containing Fe2O3-1: Add Fe2O3-1 nanomaterials into the cell co-culture medium with a final concentration of 50 μg / mL.

[0058] (2) After the cells were inoculated overnight, it was observed under a microscope that most of the three types of cells were attached to the wall. The Oxford cup in the lower chamber and the upper chamber were removed, the original culture medium was aspirated, and the cells were washed twice with PBS. One blank control group, one positive control group, and two experimental groups were set up:

[0059] The culture medium in the blank control group was replaced with fresh cell co-culture medium; the culture medium in the positive control group was replaced with Quartz-pulmonary fibrosis induction medium; the culture medium in experimental group 1 was replaced with Al2O3-1 medium; the culture medium in experimental group 2 was replaced with Fe2O3-1 medium. After the treatment, the upper chamber was gently placed on top of the lower chamber to avoid contact with the liquid surface to generate bubbles. The construction diagram is shown in the figure. Figure 1 shown.

[0060] (3) Place in a 37°C incubator and continue culturing for 48 hours.

[0061] Example 3: Detection of human lung fibroblast activity

[0062] 1. Reagents: MTS solution was purchased from Promega; phenol red-free DMEM medium was purchased from Promega; fetal bovine serum was purchased from Biobond; PBS was purchased from Bio-Tech.

[0063] 2. Methods:

[0064] (1) Prepare MTS detection solution: add MTS solution to DMEM medium without phenol red containing 10% fetal bovine serum at a ratio of 9:1, mix well and keep in dark for later use;

[0065] (2) Add MTS detection solution: discard the culture medium in the original lower chamber, add PBS to wash twice, and place an Oxford cup in the center. At this time, most BEAS-2B cells are located inside the Oxford cup, and WI-38 / VA-13 cells are located outside the Oxford cup. Therefore, 110 μL MTS detection solution is added to the outside of the Oxford cup and placed in a 37°C incubator for 2 hours;

[0066] (3) After incubation, take the supernatant and transfer 100 μL per well to a 96-well plate. Detect the absorbance at 490 nm using an ELISA reader.

[0067] 3. Results:

[0068] like Figure 2 As shown, in the wild-type cells, the survival rates of fibroblasts in the positive control group-Quartz treatment group and the experimental group 1-Al2O3-1 were higher than those in the Ctrl group, and the results were statistically significant. There was no significant difference between the experimental group 2-Fe2O3-1 and the Ctrl group, indicating that fibrosis-inducing nanoparticles may induce the generation of fibroblasts.

[0069] Example 4: BrdU labeling of human lung fibroblasts and fluorescence microscopy imaging

[0070] 1. Reagents: EdU-594 cell proliferation detection kit was purchased from Bio-Tech; 4% paraformaldehyde fixative was purchased from White Shark; PBS was purchased from Bio-Tech.

[0071] 2. Methods:

[0072] (1) Wash the cells twice with PBS, add trypsin to digest, wait for the digested cells to fall off the bottom of the plate, add 5 mL of co-culture medium to terminate the digestion, obtain the digested cells, centrifuge, discard the supernatant, transfer the cells to a 96-well plate, and incubate at 37°C overnight;

[0073] (2) After overnight, discard the original culture medium, wash the cells twice with PBS, dilute EdU 2× EdU working solution at 1:500, add an equal volume of EdU and culture medium to the well plate to make the final concentration of the working solution 10 μM (1×), and incubate at 37°C for 2 hours for BrdU labeling;

[0074] (3) After incubation, remove the culture medium, add 4% paraformaldehyde fixative, and incubate at room temperature for 15 minutes;

[0075] (4) Remove the fixative solution, wash each well three times with washing solution (PBS containing 3% BSA), each time for 3 minutes, and then add permeabilization solution (PBS containing 0.3% Triton X-100) and incubate for 10 minutes;

[0076] (5) Remove the permeabilization solution and wash again with washing solution twice, each time for 3 minutes;

[0077] (6) Prepare the reaction solution (1.29 mL Click Reaction buffer + 60 μL CuSO4 + 3 μL Zinc 594 + 150 μL Click Additive Solution), 50 μL per well, incubate in the dark for 30 min, remove the reaction solution, wash with washing solution 4 times, and add 100 μL PBS to each well;

[0078] (7) Observe the cells under a fluorescence microscope and take images.

[0079] 3. Results: Figure 3 As shown, in the group treated with the fibrogenic material Al2O3-1, the proliferation rate of newly generated fibroblasts (red) was much greater than that in the Ctrl group, and there was no significant difference in the proliferation of new cells in the group treated with the non-fibrogenic material Fe2O3-1 and the Ctrl group, further proving that fibrogenic nanoparticles may induce the proliferation of lung fibroblasts and have a warning effect on the occurrence of pulmonary fibrosis.

[0080] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing an in vitro cell model for simulating particulate matter-induced lung disease, characterized in that: The following steps are involved: S1. Inoculate cells in a cell culture device for co-culture, wherein the cell culture device comprises an upper chamber and a lower chamber, the upper chamber is enclosed by a cup body and a semipermeable membrane, the lower chamber is divided into an inner chamber and an outer chamber by a dividing component detachably connected to the lower chamber, the inner chamber and the outer chamber are both enclosed by the cup body and the dividing component, the upper chamber, the outer chamber and the inner chamber are interconnected, and the cells located between the upper chamber, the inner chamber and the outer chamber cannot contact each other, the cells inoculated between the upper chamber, the inner chamber and the outer chamber are different from each other, the cells grow by attaching to the cup body, and the cells cannot grow by attaching to the dividing component; S2. removing the segmentation component, adding particles into the cell culture device and incubating the particles with the cells to obtain the in vitro cell model.

2. The preparation method according to claim 1, characterized in that: The lung diseases include pulmonary fibrosis.

3. The preparation method according to claim 2, characterized in that: When simulating the occurrence of pulmonary fibrosis in vivo, the cells in the upper chamber, the inner chamber and the outer chamber are independently selected from any one of lung epithelial cells, lung fibroblasts and lung macrophages and are different from each other.

4. The preparation method according to claim 3, characterized in that: The inoculation number ratio of the lung macrophages, the lung epithelial cells and the lung fibroblasts is (1-4): (1-4): (2-6).

5. The preparation method according to claim 1, characterized in that: The particles include multi-walled carbon nanotubes, M120 carbon black nanotubes, crocidolite fibers, and aluminum oxide particles.

6. The preparation method according to claim 1, characterized in that: The effective concentration of the particles is 30-70 μg / mL.

7. The preparation method according to claim 1, characterized in that: The components of the culture medium used for the cell co-culture in step S1 include glucose, amino acids, vitamins, minerals, trace elements, epithelial cell supplement factors, and fetal bovine serum.

8. The preparation method according to claim 7, characterized in that: The mass concentration of the epithelial cell additive factor is (0.5-1.5) %; the mass concentration of the fetal bovine serum is (0.03-0.07) %.

9. An in vitro cell model simulating particulate-induced lung disease prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the in vitro cell model for simulating particle-induced lung disease according to claim 9 in screening drugs for treating pulmonary fibrosis.

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