Inflammatory pulmonary epithelium injury treatment evaluation method based on alveolar organs

Abstract

The invention discloses an inflammatory pulmonary epithelium injury treatment evaluation method based on alveolar organs, and relates to the technical field of medical biology. The invention mainly aims at alveolar epithelium injury and repair in inflammatory lung injury, and establishes an organ-like model for simulating alveolar epithelium injury. By using the model, the efficacy of related drugs on alveolar epithelium injury repair can be detected in a targeted manner, and an efficient drug screening and evaluation platform and method are provided for pulmonary epithelium repair in inflammatory lung injury. According to the technical scheme, the method comprises the steps that mouse alveolar epithelial stem cells are separated through magnetic bead sorting, an alveolar organoid inflammatory lung injury model is established through LPS exposure, alveolar organoid is treated through drugs, the organoid generation number, the organoid formation rate, the epithelial cell proliferation rate and the AT2-AT1 differentiation efficiency are detected, and the drug treatment effect is evaluated.

Description

[0001] This application relates to the field of medical biotechnology, and particularly to a method for evaluating the treatment of inflammatory lung epithelial injury based on alveolar organoids. Background Art

[0002] The core function of the respiratory system is to conduct gas exchange and build a barrier between the organism and the external environment to prevent the circulating blood from being directly exposed to toxic substances and pathogens in the air. The outermost layer of the air-blood barrier is composed of alveolar epithelial cells, 99% of which are type I (AT1) and type II (AT2) epithelial cells. Acute lung injury caused by sepsis leads to the death of AT1 and AT2 cells, severely damages the epithelial barrier, and obstructs normal gas exchange in the lungs.

[0003] Traditional in vitro treatment evaluation models, due to their single cell type and single structure, cannot simulate the in vivo microenvironment and cannot well reproduce the processes of AT2 cell proliferation, differentiation, and repair of the alveolar epithelium in vivo, and thus cannot effectively evaluate drug treatment and analyze the mechanisms.

[0004] The repair of alveolar epithelial injury is mainly achieved by the proliferation of AT2 cells, which then differentiate into AT1 cells, thereby completing the renewal and repair of alveolar epithelial cells and forming a new and complete epithelial barrier. Alveolar organoids are mainly composed of AT2, AT1, and fibroblasts, and are ideal models for studying the repair of alveolar epithelial injury in vitro. They not only simulate the in vivo alveolar 3D structure but also can explore the role of drugs in promoting alveolar injury repair under the action of a single variable in vitro. Summary of the Invention

[0005] To solve the problems existing in the above-mentioned prior art, the object of the present invention is to provide a method for evaluating the treatment of inflammatory lung epithelial injury based on alveolar organoids.

[0006] To achieve the above object, the present invention provides the following solution:

[0007] A method for evaluating the treatment of inflammatory lung epithelial injury based on alveolar organoids, comprising the following steps: separating mouse alveolar epithelial stem cells by magnetic bead sorting, establishing an alveolar organoid inflammatory lung epithelial injury model by LPS exposure, treating the alveolar organoids with drugs, and detecting the number of organoids generated, the organoid formation rate, the epithelial cell proliferation rate, and the differentiation efficiency of AT2 to AT1 to evaluate the drug treatment effect.

[0008] Preferably, lipopolysaccharide (LPS) is used to poison the alveolar organoids to establish an evaluation model for the treatment of inflammatory lung epithelial injury of alveolar organoids.

[0009] Preferably, the polypeptide 4N1K is used to treat the organoids.

[0010] Preferably, the number of organoids is photographed and counted by a live cell workstation.

[0011] Preferably, the organoid formation rate is calculated by the number of organoids / the number of inoculated AT2 cells.

[0012] Preferably, AT2 cells are inoculated in a transwell to form a 3D culture system.

[0013] Preferably, the polycarbonate membrane under the transwell chamber and the organoid colloid thereon are removed with ophthalmic forceps, immersed in 4% paraformaldehyde for fixation, dehydration, paraffin embedding, sectioning, and immunofluorescence staining.

[0014] Preferably, the epithelial cell proliferation rate is counted by immunofluorescently detecting the cell proliferation marker EdU.

[0015] Preferably, the number of AT1 and AT2 cells and the differentiation efficiency of AT2 to AT1 are calculated by immunofluorescently detecting SPC and T1a antibodies.

[0016] Preferably, alveolar organoids are established by the following method: S1: After anesthetizing the mouse, the lung tissue is washed through the pulmonary circulation with PBS; S2: Inject dispase into the lung from the main trachea using a syringe, and ligate the main trachea of the lung with a fine cotton thread to digest the lung epithelial cells; S3: Cut the lung tissue into pieces and filter to isolate single lung epithelial cells; S4: Magnetically sort lung epithelial stem cells (AT2 cells), mix them with lung fibroblasts and Matrigel, transfer them to a transwell, and add induction medium for culture.

[0017] Preferably, the induction medium contains the following components: DMEM-F12, double antibodies, HEPES, ITS, MEM, FBS, Y27632, EGF.

[0018] Preferably, the induction medium uses the following concentration components: Double antibodies 1x; HEPES 25 mM; ITS 1x; MEM 1x; FBS 10% EGF 10 ng / ml

[0019] Preferably, the alveolar epithelial repair detection includes: AT2 cell proliferation detection and AT2 to AT1 differentiation detection, and the organoid lung epithelial inflammatory injury model and drug treatment effect include: the number of organoids generated, the organoid formation rate, and the AT2 to AT1 differentiation efficiency detection. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 . Alveolar organoid inflammatory epithelial injury model and drug treatment detection. A: Images of the colony formation efficiency of alveolar organoids: Alveolar organoids were treated with LPS, the therapeutic drug 4N1K, and the 4N1K inhibitor, and the morphology of the organoids was observed. B: Statistical analysis of the total colony formation efficiency of alveolar organoids. C: Statistical analysis of the size of alveolar organoids. The scale bar is 400 µm. Data are presented as mean ± standard error, **p < 0.01.

[0022] Figure 2 . Detection of the cell proliferation rate of alveolar organoids. A: Results of immunofluorescence staining of the cell proliferation marker EdU in alveolar organoids (Alexa Fluor 555, red), and DAPI staining shows the cell nuclei (blue). B: Quantitative analysis of EdU-positive cells. The scale bar is 50 µm. Data are presented as mean ± standard error, **p < 0.01.

[0023] Figure 3 . Staining and statistics of AT1 and AT2 cells in alveolar organoids. A: Immunofluorescence staining of the cell differentiation markers T1a (Alexa Fluor 555, red) and the AT2 cell marker SPC (Alexa Fluor 488, green) in alveolar organoids, and DAPI staining shows the cell nuclei (blue). The merged image shows double-positive staining of T1a and SPC. B: Quantitative analysis of the percentage of T1a+ / SPC+ cells in the total number of SPC+ cells in alveolar organoids. The scale bar is 50 µm. Data are presented as mean ± standard error, **p < 0.01. Detailed Embodiments

[0024] In order to better enable those skilled in the art to understand the above technical solutions, the exemplary embodiments of the present invention are now described in detail. This description is only the preferred embodiment of this application and does not limit the present invention. Any equivalent replacement, modification, and improvement made within the design and principle of the present invention shall fall within the protection scope of the present invention.

[0025] The technical solutions in this application, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or are already publicly available.

[0026] Example 1 Separation steps of lung epithelial stem cells:

[0027] 1. Anesthetize the mouse, cut open the peritoneal cavity, open the thoracic cavity, fix the thoracic wall, and cut the descending aorta / inferior vena cava.

[0028] 2. Use a 10 ml syringe to draw 10 ml of PBS, insert the syringe into the right ventricle, and perfuse the lungs to make the lungs turn white.

[0029] 3. After perfusion, pass two independent sutures under the main trachea. Use ophthalmic scissors to make a small incision on the ventral surface of the trachea on the side close to the head of the two sutures, taking care not to cut it completely. Insert a flexible tube at the opening to the tracheal bifurcation, and tie one suture firmly.

[0030] 4. Slowly inject 1 ml of PBS with a 1 ml syringe and aspirate 800 μL to perfuse the lungs.

[0031] 5. Slowly inject 2 ml of dispase into the lung cavity with a 1 ml syringe.

[0032] 6. Use another suture to tie firmly at the proximal end of the trachea to prevent the leakage of dispase.

[0033] 7. Hold the suture with one hand and use ophthalmic scissors to separate the lung completely from the thoracic cavity with the other hand, taking care not to cut the lungs.

[0034] 8. Place the lung in a 15 ml centrifuge tube and add 3 ml of dispase solution (incubate at room temperature for 30 minutes).

[0035] 9. Place the lung in a petri dish and cut the lung back and forth with ophthalmic scissors until the tissue becomes thick soup / mud, which takes about 3 minutes.

[0036] 10. Add 3 ml of medium containing 0.05% DnaseI to the minced tissue, pipette up and down with a 5 ml pipette, and then filter the lung tissue through 70, 40, and 20 μm filters in sequence to harvest single lung cells.

[0037] 11. Centrifuge the filtrate at 130 g for 8 min at 4°C, resuspend the cells in 1 mL of 1X red blood cell lysis buffer, incubate at room temperature for 1 minute, add 10 ml of PBS to terminate the reaction, centrifuge at 300 g for 5 min at 4°C, and then resuspend with 5 ml of medium containing 10% FBS.

[0038] Example 2 Separation steps of lung epithelial stem cells (AT2 cells):

[0039] 1. Incubate CD45 / CD31 antibody in a 6 cm dish 24 hours in advance. The antibody is dissolved in PBS.

[0040] 2. Remove the antibody in the 6-cm dish, add the lung epithelial cells isolated in Example 1 to the culture dish, incubate in an incubator for 1 hour, collect the non-adherent cells, centrifuge at 300 g for 5 min at 4°C, and remove the supernatant.

[0041] 3. Resuspend the cells collected in the above steps with 500 μl of PBS (without calcium and magnesium) containing a final concentration of 2 mM EDTA and 5% BSA (Buffer #1), centrifuge at 300 g for 5 min at 4°C.

[0042] 4. Resuspend the cells with 100 μl of Buffer #1, add 10 μL / biotinylated anti-mouse CD326 antibody, and incubate at 4°C for 10 min.

[0043] 5. Add 2 ml of Buffer #1 to the cells in the above steps, mix well, centrifuge at 300 g for 5 min at 4°C.

[0044] 6. Remove the supernatant, resuspend the cells in the above steps in 100 μl of Buffer #1, add 20 μL / anti-biotin MicroBeads, mix well, and incubate at 4°C for 15 min.

[0045] 7. Add 2 ml of Buffer #1, mix well, centrifuge at 300 g for 5 min at 10°C.

[0046] 8. Remove the supernatant, resuspend the cells in 500 μl of Buffer #1, and mix well.

[0047] 9. Rinse a new LS column (Miltenyi Biotec) with 3 ml of Buffer #1, place the LS column on a magnetic stand, add the above cell suspension to the LS column, then add 3 ml of Buffer #1 for washing once, and then add 3 ml of PBS (without calcium and magnesium) containing 5% BSA (Buffer #2) for washing twice. Take out the LS column from the magnet device, quickly place it in a new 15-ml centrifuge tube, add 5 ml of Buffer #2 to the LS column, use a plunger to forcefully push out the liquid in the LS column, centrifuge at 300 g for 5 min, collect the AT2 cells and count.

[0048] Steps for inducing and culturing alveolar organoids in Example 3:

[0049] 1. Digest the pre-cultured lung fibroblast cell line MLG cells, resuspend and count.

[0050] 2. Mix MLG and AT2 cells at a ratio of 4:1, centrifuge, and resuspend with alveolar organoid induction medium.

[0051] 3. Add Matrigel to the alveolar organoid induction medium in the above steps at a ratio of 1:1 and mix well. Place 400 μl (containing 5000 AT2 cells) of the mixture on the Transwell insert, and place the Transwell insert in a 24-well plate. Incubate in the incubator for 30 minutes until it solidifies.

[0052] 4. After the Matrigel solidifies, add 600 μl of alveolar organoid induction medium below the insert and change the medium every other day.

[0053] Example 4 Steps for inducing and treating lung epithelial injury of alveolar organoids:

[0054] 1. On the fourth day of culturing alveolar organoids, add 1 μg / ml LPS to the organoid medium and add the drug every other day to establish an inflammatory lung injury model of alveolar organoids.

[0055] 2. On the fourth day of culturing alveolar organoids, add 1 μg / ml LPS and 50 μM / L 4N1K to the organoid medium and add the drug every other day to establish a treatment model for inflammatory lung injury of alveolar organoids.

[0056] Example 5 Steps for evaluating the treatment of inflammatory lung injury of alveolar organoids:

[0057] 1. On the 4th day after drug treatment, collect some samples for cell proliferation detection. Take pictures with a live cell workstation on the 10th day after drug treatment, and use software to count the number of organoids and the formation efficiency. After taking pictures, collect the samples for immunofluorescence detection of AT1 and AT2 cells.

[0058] 2. Sample collection: Remove the Transwell insert, carefully remove the bottom polycarbonate membrane and the organoid colloid on it with ophthalmic forceps. The process should be fast to keep the membrane moist as much as possible. After removing the bottom membrane, immerse it in 4% paraformaldehyde for fixation at room temperature for 2 hours.

[0059] 3. Dehydration: Perform gradient dehydration with ethanol of different concentrations, 70% (1 h), 75% (1 h), 80% (1 h), 85% (1 h), 90% (30 min, 2 times), 100% ethanol (30 min, 2 times).

[0060] 4. Clearing: Immerse the samples in 50% xylene (prepared with 100% ethanol) for 2 minutes, remove the liquid, replace it with pure xylene and soak for 30 seconds to become clear, and then remove the liquid.

[0061] 5. Paraffin embedding: Immerse the samples in melted paraffin at 65 °C for 2 hours, change the paraffin once, and then soak at 60 °C overnight. The next day, place the fully infiltrated wax samples into a paraffin embedding cassette for embedding.

[0062] 6. Sectioning: Use a Leica paraffin slicer to section the trimmed paraffin sample with a thickness of 5 μm. After placing the sections on glass slides, dry them in a 42 °C drying oven for 1 hour.

[0063] 7. Deparaffinization and rehydration: Place the glass slides with samples on a 65 °C drying oven (30 min - 1 h), and then sequentially deparaffinize and rehydrate them with xylene (20 min, 2 times), 100% ethanol (10 min), 95% ethanol (10 min), 85% ethanol (10 min), 75% ethanol (10 min), and 50% ethanol (10 min).

[0064] 8. Antigen retrieval: Immerse the glass slides in a PH9.0 EDTA antigen retrieval solution, place them together with the container in a water bath, cover the lid, and heat to 100 °C and boil for 30 minutes.

[0065] 9. Permeabilization: Use 0.01% Triton X-100 (diluted with PBS) to permeabilize the samples for 8 minutes, and then wash with PBS.

[0066] 10. Immunofluorescence staining: After antigen retrieval, wash the glass slides with PBS. Then, use a histochemical pen to draw a circle with a diameter of 1 cm around the samples, add 3% BSA to block for 2 hours. After removing the blocking solution, add the primary antibody. After 12 - 18 hours, wash with PBS for 30 minutes, add the secondary antibody. After 2 hours, wash with PBS, add DAPI staining solution. After 5 minutes, add an anti-fluorescence quenching agent, cover with a coverslip, and take pictures using a live cell workstation.

[0067] 11. Counting of AT1 and AT2 cells: Immunofluorescent T1a-positive cells are AT1 cells, and immunofluorescent SPC-positive cells are AT2 cells, as Figure 3 .

[0068] 12. Counting of proliferating cells: Immunofluorescent EDU-positive cells are cells in the proliferative phase, as Figure 2 .

[0069] 13. Statistics of the number and formation efficiency of organoids: Take pictures using a live cell workstation before collecting the samples, and then use Image J software to count the number and diameter of organoids. Organoid formation efficiency = number of organoids / number of seeded AT2 cells, as Figure 1 .

[0070] In summary, we have successfully established alveolar organoids induced from alveolar epithelial stem cells. The three-dimensional structure of our organoid model is generated by the proliferation and differentiation of lung epithelial stem cells, which specifically mimics the normal development and repair processes of alveolar epithelium in vitro, and this cannot be achieved by two-dimensional cell models and other organoid models. This will provide an efficient in vitro drug screening and evaluation platform for the treatment of inflammatory lung epithelial injury.

[0071] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for evaluating the treatment of inflammatory lung epithelial injury based on alveolar organoids, characterized in that, It includes the following steps: Stimulate alveolar organoids with LPS (lipopolysaccharide) to establish an inflammatory epithelial injury model of alveolar organoids; treat the alveolar organoids with drugs; evaluate the treatment effect of the injury model based on the morphological changes and physiological index changes of the alveolar organoids.

2. The method for evaluating the treatment of inflammatory lung epithelial injury based on alveolar organoids according to claim 1, wherein The morphological changes of the alveolar organoids are specifically the changes in the organoid formation efficiency and diameter; the physiological index changes of the alveolar organoids include any one or more of the lung epithelial cell proliferation level, the number of type I lung epithelial cells, the number of type II lung epithelial cells, and the differentiation efficiency of type II lung epithelial cells into type I cells.

3. The method for evaluating the treatment of inflammatory lung epithelial injury based on alveolar organoids according to claim 1, wherein Detect the morphological changes and physiological indexes of the organoids using a live cell workstation. Use the ratio of the number of organoids to the initial number of seeded cells to represent the organoid formation efficiency. Stain the proliferating lung epithelial cells using an EDU staining kit. Detect the number of type I lung epithelial cells using a T1a antibody, and detect the number of type II lung epithelial cells using an SPC antibody.

4. The method for evaluating the treatment of inflammatory lung epithelial injury based on alveolar organoids according to claim 3, wherein Use ophthalmic forceps to separate the polycarbonate membrane under the transwell chamber together with the Matrigel attached to it, and fix, dehydrate, embed in paraffin, and section.

5. The method for evaluating the treatment of inflammatory lung epithelial injury based on alveolar organoids according to claim 1, characterized in that, Alveolar organoids are established by the following method: S1: After anesthetizing the mice, wash the lung tissue through the pulmonary circulation using PBS; S2: Inject dispase into the lungs through the main trachea using a syringe, and ligate the main trachea of the lungs with a fine cotton thread to digest the lung epithelial cells; S3: Cut the lung tissue into pieces and filter to separate the single lung epithelial cells; S4: Magnetically sort the lung epithelial stem cells, mix them with lung fibroblasts and Matrigel, transfer them to a transwell, and add induction medium for culture.

6. The therapeutic evaluation method for inflammatory lung epithelial injury based on alveolar organoids according to claim 5, characterized in that, The induction medium contains the following components: DMEM-F12, double antibody, HEPES, ITS, MEM, FBS, Y27632, EGF.

7. The method for evaluating the treatment of lung epithelial injury according to claim 6, wherein The induction medium uses the following concentration components: Double antibody 1x; HEPES 25 mM; ITS 1x; MEM 1x; FBS 10% EGF 10 ng / ml.

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