A reagent composition for identifying mouse fibroblast subpopulations and its use method

Abstract

The present invention discloses a reagent composition for identifying mouse fibroblast subpopulations and a method for using the same, relating to the field of cell technology. The reagent composition includes an antigen blocking solution, an allophycocyanin (APC)-labeled Ly6C antibody, a phycoerythrin (PE)-labeled CyclinD1 antibody, a fluorescein isothiocyanate (FITC)-labeled DCN antibody, a PBS phosphate buffer, a cell digestion solution, and an erythrocyte lysis solution. The present invention can identify and sort DCN-positive fibroblast subpopulations in the lung tissue of mice with acute lung injury, or identify and sort DCN-positive fibroblast subpopulations in the lung tissue of mice with acute lung injury that are undergoing tissue damage or repair, providing conditions for in-depth research on the role and mechanism of DCN-positive fibroblasts in ARDS, and exploring clinical treatment or prognostic targets.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, and in particular to a reagent composition for identifying mouse fibroblast subpopulations and a method for using the same. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is a heterogeneous disease characterized by increased alveolar-capillary permeability, increased lung water content, and persistent hypoxemia, resulting from various pathogenic factors such as infection, trauma, and shock. Its pathogenesis is complex and remains incompletely elucidated. Currently, the diagnosis of ARDS relies primarily on clinical guidelines and imaging studies, but existing diagnostic methods have poor specificity and accuracy. ARDS lacks specific treatments, and the biological heterogeneity of ARDS patients limits treatment to organ support. The mortality rate is as high as 40%, and its pathogenesis and diagnostic and therapeutic targets require extensive research. Therefore, the study of new targets or molecular markers and further exploration of their regulatory mechanisms in the immune response are of great significance for the clinical prevention and treatment of ARDS.

[0003] Fibroblasts play a key role in the pathogenesis of ARDS, playing a crucial role in inflammation, tissue repair, and alveolar structural remodeling. A link between early activation in ARDS and the development of pulmonary fibrosis has been established. Fibroblasts regulate the lung microenvironment by producing ECM-remodeling enzymes, such as ADAMTS4, influencing the infiltration and function of immune cells and, consequently, the course of ARDS. Furthermore, fibroblasts play a crucial role in lung repair after injury, and their abnormal activation is associated with poor patient prognosis. International studies have further explored the interplay between fibroblasts and immune cells and their synergistic effects in the progression of ARDS. With the advancement of single-cell sequencing technology, cell subpopulations have garnered significant research attention. The various cell subpopulations that differentiate from cell populations during disease progression have been shown to play a crucial role in disease development. However, the complexity of inducing these subpopulations through in vitro cell lines has hindered further investigation of their functions. The role of fibroblasts in acute lung injury and their potential as drug targets to alleviate lung inflammation and promote tissue repair warrant further investigation. A significant obstacle to clinical and translational research is the lack of methods for identifying and isolating fibroblast subpopulations highly relevant to ARDS. Cell membrane surface antigens serve as cell identification codes, enabling the classification of cells into types, groups, and subpopulations. Single-cell sequencing technology can help identify subpopulations within a cell population that uniquely express a specific antigen, paving the way for studying the relationship between these subpopulations and disease.

[0004] By performing single-cell sequencing on lung tissue from mice with lipopolysaccharide (LPS)-induced ARDS, the present inventors discovered a distinct population of fibroblasts in the lungs of ARDS mice, which is closely associated with lung tissue damage and repair in ARDS. Because this subpopulation uniquely expresses DCN on its cell membrane, we named it the DCN-positive fibroblast subpopulation. Currently, there are no methods for identifying and isolating DCN-positive fibroblast subpopulations. Based on this, we propose a reagent composition for identifying mouse fibroblast subpopulations and methods for its use. Summary of the Invention

[0005] The present invention aims to provide a reagent composition for identifying mouse fibroblast subpopulations and a method for using the same, so as to solve the problem that there is currently no method for identifying and sorting DCN-positive fibroblast subpopulations.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A reagent composition for identifying mouse DCN-positive fibroblast subpopulations comprises 10-20 ml of antigen blocking solution, 30-50 μL of allophycocyanin (APC)-labeled Ly6C antibody, 30-50 μL of phycoerythrin (PE)-labeled CyclinD1 antibody, 30-50 μL of fluorescein isothiocyanate (FITC)-labeled DCN antibody, 50-80 ml of PBS phosphate buffer, 40-60 ml of cell digestion solution, and 50-80 ml of red blood cell lysis solution. All reagents are individually packaged in sterile sealed bottles.

[0008] Furthermore, the antigen blocking solution is prepared into a liquid with a mass / concentration of 1% using bovine serum albumin and PBS phosphate buffer, and then filtered through a 0.22 micron filter membrane and then packaged.

[0009] Furthermore, the PBS phosphate buffer, cell digestion solution and red blood cell lysis solution were all stored at -4°C, and the antigen blocking solution (10 ml), allophycocyanin (APC)-labeled Ly6C antibody A, phycoerythrin (PE)-labeled CyclinD1 antibody B and fluorescein isothiocyanate (FITC)-labeled DCN antibody C were all stored at -20°C.

[0010] A method for using a reagent composition for identifying mouse DCN-positive fibroblast subpopulations, which identifies and sorts fibroblast subpopulations in lung tissue of ARDS mice, comprising the following steps:

[0011] S1. Remove lung tissue from mice;

[0012] S2. Use PBS phosphate buffer to rinse and remove blood from the surface of mouse lung tissue;

[0013] S3. Mince the mouse lung tissue and place it in the center of a cell sieve. Add cell digestion solution and grind the mouse lung tissue until it cannot be ground anymore. Collect the tissue and prepare a cell suspension.

[0014] S4. Add cell digestion solution to the cell suspension again and digest at 40 rpm for 45 min at 37°C.

[0015] S5. Centrifuge the cell suspension at 1000-2000g for 10 min at room temperature, collect the cell pellet, and wash once with 2 mL of phosphate buffer;

[0016] S6. Discard the supernatant, add red blood cell lysis buffer, lyse the red blood cells at 4°C for 15 minutes, collect the cells by centrifugation, and resuspend them in PBS phosphate buffer to prepare a cell resuspension;

[0017] S7. Take 1-1.5 mL of cell resuspension and block with 100-150 μL of antigen blocking solution at room temperature for 30 minutes, mixing by inversion every 10 minutes.

[0018] S8. Dilute allophycocyanin (APC)-labeled Ly6C antibody and fluorescein isothiocyanate (FITC)-labeled DCN antibody in antigen blocking solution at a ratio of 1:50 to prepare antibody working solution. Add 100-150 μL of the prepared working solution to the cell resuspension and stain at 4°C in the dark for 1 h.

[0019] S9. Centrifuge the cell suspension at 2000 rpm for 10 min, discard the supernatant, wash once with PBS phosphate buffer, and finally resuspend in 1-1.5 mL of PBS phosphate buffer to prepare a sample test solution. Analyze by flow cytometry, using allophycocyanin (APC)-labeled Ly6C antibody as a fibroblast population marker, and using fluorescein isothiocyanate (FITC)-labeled DCN antibody as a fibroblast subpopulation marker;

[0020] S10. Using Ly6C antibody and DCN antibody positivity as markers, collect Ly6C antibody and DCN antibody-positive cells using a flow cytometry collection tube to collect the DCN-positive fibroblast subpopulation.

[0021] Furthermore, the steps for identifying and sorting lung tissue fibroblast subpopulations in ARDS mice in damaged and repair states differ in that, in step S8, allophycocyanin (APC)-labeled Ly6C antibody, phycoerythrin (PE)-labeled CyclinD1 antibody, and fluorescein isothiocyanate (FITC)-labeled DCN antibody are diluted with antigen blocking solution at a ratio of 1:50 to prepare an antibody working solution.

[0022] Furthermore, the steps for identifying and sorting fibroblast subpopulations in the lung tissue of ARDS mice in the damaged and repair states are different in that, in step S9, an allophycocyanin (APC)-labeled Ly6C antibody is used as a fibroblast population marker, a phycoerythrin (PE)-labeled CyclinD1 antibody is used as a damage and repair marker, and a fluorescein isothiocyanate (FITC)-labeled DCN antibody is used as a fibroblast subpopulation marker.

[0023] Furthermore, the steps for identifying and sorting fibroblast subpopulations in the lung tissue of ARDS mice in the damaged and repaired states are different in that, in step S10, Ly6C antibody, CyclinD1 antibody and DCN antibody positivity are used as markers, and cells positive for Ly6C antibody, CyclinD1 antibody and DCN antibody are collected using a flow cytometer collection tube.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention constructs a fibroblast identification and sorting reagent composition by combining an allophycocyanin (APC)-labeled Ly6C antibody, a phycoerythrin (PE)-labeled CyclinD1 antibody, and a fluorescein isothiocyanate (FITC)-labeled DCN antibody. This reagent composition can identify and sort DCN-positive fibroblast subpopulations in the lung tissue of mice with acute lung injury, or identify and sort DCN-positive fibroblast subpopulations in the lung tissue of mice with acute lung injury that are undergoing tissue damage or repair. This provides conditions for in-depth research on the role and mechanism of DCN-positive fibroblasts in ARDS, as well as for exploring clinical treatment or prognostic targets.

[0026] 2. The reagent and antibody combination provided by the present invention can relatively simplify the identification steps, and quickly, conveniently and efficiently identify and sort DCN-positive fibroblasts.

[0027] 3. The present invention can: 1. identify the status of lung damage in ARDS mice; 2. identify the presence of DCN-positive fibroblasts in the lungs of ARDS mice, indicating whether tissue repair is progressing; 3. isolate and obtain DCN-positive fibroblasts; and 4. isolate and obtain subpopulations of DCN-positive fibroblasts undergoing repair. This has significant implications for research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the structure of a reagent composition for identifying mouse DCN-positive fibroblast subpopulations;

[0029] Figure 2 Schematic diagram of flow cytometry identification of DCN-positive fibroblast subpopulations in lung tissue of ARDS mice;

[0030] Figure 3 Schematic diagram of flow cytometry identification of DCN fibroblast subpopulations in damaged and repaired states in the lung tissue of ARDS mice. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:

[0032] Example 1

[0033] A reagent composition for identifying mouse DCN-positive fibroblast subpopulations comprises 10 ml of antigen blocking solution, 30 μL of allophycocyanin (APC)-labeled Ly6C antibody, 30 μL of phycoerythrin (PE)-labeled CyclinD1 antibody, 30 μL of fluorescein isothiocyanate (FITC)-labeled DCN antibody, 50 ml of 10× PBS phosphate buffer, 40 ml of cell digestion solution, and 50 ml of erythrocyte lysis solution, all of which are individually packaged in sterile sealed bottles.

[0034] Example 2

[0035] like Figure 1 As shown, a method for using a reagent composition for identifying a mouse DCN-positive fibroblast subpopulation, which comprises the following steps in identifying and sorting a DCN-positive fibroblast subpopulation in lung tissue of ARDS mice:

[0036] S1. ARDS mice were induced with mild lung injury by airway perfusion of LPS at a concentration of 0.3 mg / kg, and ARDS mice were induced with severe lung injury by airway perfusion of LPS at a concentration of 1.25 mg / kg. Untreated healthy mice were used as controls. Mice were euthanized by asphyxiation. Experiments were performed on a sterile clean bench exposed to UV light for 30 minutes. The skin of the mice was disinfected with 75% ethanol, and the chest cavity was opened with sterile surgical scissors, and the airway and lung tissue were completely removed.

[0037] S2. Use PBS phosphate buffer to rinse and remove blood from the surface of mouse lung tissue;

[0038] S3. Mince the mouse lung tissue with dissecting scissors and place it in the center of a cell sieve. Add 1 mL of cell digestion solution and grind the tissue with a syringe stopper until it can no longer be ground (3-5 minutes). Collect the tissue into a 15 mL centrifuge tube to prepare a cell suspension.

[0039] S4. Add 2 mL of cell digestion solution to the cell suspension again and digest at 40 rpm for 45 min at 37°C.

[0040] S5. Centrifuge the cell suspension at 1000-2000g for 10 min at room temperature, collect the cell pellet, and wash once with 2 mL of phosphate buffer;

[0041] S6. Discard the supernatant, add 2 mL of red blood cell lysis buffer, lyse the red blood cells at 4°C for 15 min, collect the cells by centrifugation, and resuspend them in PBS phosphate buffer to prepare a cell resuspension;

[0042] S7. Take 1.5 mL of cell resuspension and place it in an EP tube. Block with 150 μL of antigen blocking solution at room temperature for 30 minutes, mixing by inversion every 10 minutes.

[0043] S8. Dilute allophycocyanin (APC)-labeled Ly6C antibody and fluorescein isothiocyanate (FITC)-labeled DCN antibody in antigen blocking solution at a ratio of 1:50 to prepare antibody working solution. Add 150 μL of the prepared working solution to the cell resuspension and stain at 4°C in the dark for 1 h.

[0044] S9. The cell suspension was centrifuged at 2000 rpm for 10 min, the supernatant was discarded, and the cells were washed once with PBS phosphate buffer. Finally, the cells were resuspended again with 1 mL of PBS phosphate buffer to prepare a sample test solution, and analyzed by flow cytometry, using allophycocyanin (APC)-labeled Ly6C antibody as a fibroblast population marker, and using fluorescein isothiocyanate (FITC)-labeled DCN antibody as a fibroblast subpopulation marker;

[0045] S10. Using healthy mice as negative controls, cell sorting and collection can be set up while identifying cells. Ly6C antibody and DCN antibody positivity are used as markers. Ly6C antibody and DCN antibody-positive cells are collected using flow cytometry collection tubes to collect DCN-positive fibroblast subpopulations.

[0046] like Figure 2 As shown, the number 1 is the vertical axis, which indicates the positive status of Ly6C antibody labeled with phycocyanin (APC), and the number of positive cells increases from bottom to top; the numbers 2, 3 and 4 are the horizontal axes, which are the DCN antibody labeled with fluorescein isothiocyanate (FITC) in healthy mice (2), DCN antibody labeled with fluorescein isothiocyanate (FITC) in mild lung injury ARDS mice (3), and DCN antibody labeled with fluorescein isothiocyanate (FITC) in severe lung injury ARDS mice (4), and the number of positive cells increases from left to right. Therefore, the black box in the upper right corner is the Ly6C antibody-positive and DCN antibody-positive cells, which are identified as DCN fibroblast subpopulations. It can be seen that the number of these cells is relatively small in healthy mice (2), but significantly increased in lung injury mice, and increased with the severity of the injury (3,4). Ly6C antibody-positive and DCN antibody-positive cells are collected using flow cytometry collection tubes to obtain DCN fibroblast subpopulations. This reagent composition can stain up to 2×10 7cells, and the DCN fibroblast subpopulation accounted for 34.1%, that is, a maximum of 0.68×10 7 DCN-positive fibroblast subsets.

[0047] Example 3

[0048] like Figure 1 As shown, a method for using a reagent composition for identifying mouse DCN-positive fibroblast subpopulations, which includes the following steps in identifying and sorting fibroblast subpopulations in lung tissue of ARDS mice in a damaged and repaired state:

[0049] S1. ARDS mice were induced with mild lung injury by airway perfusion of LPS at a concentration of 0.3 mg / kg, and ARDS mice were induced with severe lung injury by airway perfusion of LPS at a concentration of 1.25 mg / kg. Untreated healthy mice were used as controls. Mice were euthanized by asphyxiation. Experiments were performed on a sterile clean bench exposed to UV light for 30 minutes. The skin of the mice was disinfected with 75% ethanol, and the chest cavity was opened with sterile surgical scissors, and the airway and lung tissue were completely removed.

[0050] S2. Use PBS phosphate buffer to rinse and remove blood from the surface of mouse lung tissue;

[0051] S3. Mince the mouse lung tissue with dissecting scissors and place it in the center of a cell sieve. Add 1 mL of cell digestion solution and grind the tissue with a syringe stopper until it can no longer be ground (3-5 minutes). Collect the tissue into a 15 mL centrifuge tube to prepare a cell suspension.

[0052] S4. Add 2 mL of cell digestion solution to the cell suspension again and digest at 40 rpm for 45 min at 37°C.

[0053] S5. Centrifuge the cell suspension at 1000-2000g for 10 min at room temperature, collect the cell pellet, and wash once with 2 mL of phosphate buffer;

[0054] S6. Discard the supernatant, add 2 mL of red blood cell lysis buffer, lyse the red blood cells at 4°C for 15 min, collect the cells by centrifugation, and resuspend them in PBS phosphate buffer to prepare a cell resuspension;

[0055] S7. Take 1.5 mL of cell resuspension and place it in an EP tube. Block with 150 μL of antigen blocking solution at room temperature for 30 minutes, mixing by inversion every 10 minutes.

[0056] S8. Dilute allophycocyanin (APC)-labeled Ly6C antibody, phycoerythrin (PE)-labeled CyclinD1 antibody, and fluorescein isothiocyanate (FITC)-labeled DCN antibody in antigen blocking solution at a ratio of 1:50 to prepare antibody working solution. Add 150 μL of the prepared working solution to the cell resuspended solution and stain at 4°C in the dark for 1 h.

[0057] S9. The cell suspension was centrifuged at 2000 rpm for 10 min, the supernatant was discarded, and the cells were washed once with PBS phosphate buffer. Finally, the cells were resuspended in 1 mL of PBS phosphate buffer to prepare a sample test solution. The cells were analyzed by flow cytometry using allophycocyanin (APC)-labeled Ly6C antibody as a fibroblast population marker, phycoerythrin (PE)-labeled CyclinD1 antibody as a damage and repair marker, and fluorescein isothiocyanate (FITC)-labeled DCN antibody as a fibroblast subpopulation marker.

[0058] S10. Using healthy mice as negative controls, cell sorting and collection can be set up while identifying cells. Ly6C antibody, CyclinD1 antibody, and DCN antibody positivity are used as markers. Cells positive for Ly6C antibody, CyclinD1 antibody, and DCN antibody are collected using flow cytometry collection tubes to collect DCN-positive fibroblast subpopulations.

[0059] like Figure 3 As shown in the figure, the number 1 in the diagram is the vertical axis, which indicates the positive status of Ly6C antibody labeled with phycocyanin (APC), and the number of positive cells increases from bottom to top; the numbers 2, 3 and 4 are the horizontal axes, which are the CyclinD1 antibody labeled with phycoerythrin (PE) in healthy mice (2), the CyclinD1 antibody labeled with phycoerythrin (PE) in mice with mild lung injury ARDS (3), and the CyclinD1 antibody labeled with phycoerythrin (PE) in mice with severe lung injury ARDS (4), and the number of positive cells increases from left to right. Since the DCN cell subpopulation has been identified in the first labeling, the DCN cell subpopulation that is positive for damage and repair markers can be directly labeled in the second round of labeling. Therefore, the cells in the black box in the upper right corner are Ly6C antibody-positive, CyclinD1 antibody-positive, and DCN antibody-positive cells, which are identified as DCN fibroblast subpopulations in the damage and repair state. It can be seen that this cell is less in healthy mice (2), significantly increased in lung injury mice, and increased with the severity of the injury (3,4). Ly6C antibody-positive, CyclinD1 antibody-positive, and DCN antibody-positive cells are collected using flow cytometry collection tubes to obtain DCN fibroblasts in the damage and repair state. This reagent composition can sort out up to 0.68×10 7DCN-positive fibroblast subpopulations were obtained, of which 68% were DCN fibroblasts in the damaged and repaired state, that is, a maximum of 0.46×10 7 DCN fibroblasts in states of damage and repair.

[0060] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. An identification reagent composition for use in preparing a product for identifying and sorting DCN-positive fibroblast subpopulations in lung tissue of ARDS mice in a damaged and repaired state, characterized in that: The composition includes 10-20 ml of antigen blocking solution, 30-50 μL of allophycocyanin (APC)-labeled Ly6C antibody, 30-50 μL of phycoerythrin (PE)-labeled CyclinD1 antibody, 30-50 μL of fluorescein isothiocyanate (FITC)-labeled DCN antibody, 50-80 ml of PBS phosphate buffer, 40-60 ml of cell digestion solution and 50-80 ml of red blood cell lysis solution. All reagents are separately packed in sterile sealed bottles.

2. An identification reagent composition according to claim 1, characterized in that The antigen blocking solution is prepared with bovine serum albumin and PBS phosphate buffer to a liquid with a mass / concentration of 1%, and then filtered with a 0.22 micron filter membrane before being packaged.

3. An identification reagent composition according to claim 1, characterized in that The PBS phosphate buffer, cell digestion solution and red blood cell lysis solution were all stored at -4°C, and the antigen blocking solution, allophycocyanin (APC)-labeled Ly6C antibody A, phycoerythrin (PE)-labeled CyclinD1 antibody B and fluorescein isothiocyanate (FITC)-labeled DCN antibody C were all stored at -20°C.

4. A method for using the identification reagent composition according to claim 1, characterized in that: The method is a non-disease diagnosis and treatment method, comprising the following steps: PBS phosphate buffer was used to rinse to remove blood from the surface of mouse lung tissue; Cut the mouse lung tissue into small pieces and place it in the center of a cell sieve. Then add cell digestion solution and grind the mouse lung tissue until it can no longer be ground. Collect the tissue and prepare a cell suspension. Add cell digestion solution to the cell suspension again and digest at 40 rpm for 45 min at 37°C. The cell suspension was centrifuged at 1000-2000 g for 10 min at room temperature, the cell pellet was collected, and washed once with 2 mL of phosphate buffer; The supernatant was discarded, and red blood cell lysis buffer was added to lyse the red blood cells at 4°C for 15 min. The cells were collected by centrifugation and resuspended in PBS phosphate buffer to prepare a cell resuspension solution; Take 1-1.5 mL of cell resuspension and block with 100-150 μL of antigen blocking solution at room temperature for 30 minutes, inverting and mixing every 10 minutes; Dilute allophycocyanin (APC)-labeled Ly6C antibody and fluorescein isothiocyanate (FITC)-labeled DCN antibody in antigen blocking buffer at a ratio of 1:50 to prepare antibody working solution. Add 100-150 μL of the prepared working solution to the cell resuspension and stain at 4°C in the dark for 1 h. The cell resuspension was centrifuged at 2000 rpm for 10 min, the supernatant was discarded, and the cells were washed once with PBS phosphate buffer. Finally, the cells were resuspended again with 1-1.5 mL of PBS phosphate buffer to prepare a sample test solution. The cells were analyzed by flow cytometry using allophycocyanin (APC)-labeled Ly6C antibody as a fibroblast population marker and fluorescein isothiocyanate (FITC)-labeled DCN antibody as a fibroblast subpopulation marker. Using Ly6C antibody and DCN antibody positivity as markers, cells positive for Ly6C antibody and DCN antibody are collected using a flow cytometry collection tube to collect DCN-positive fibroblast subpopulations.

5. The method for using the identification reagent composition according to claim 4, wherein: The steps for further identifying and sorting fibroblast subsets in the lung tissue of ARDS mice in the damaged and repair states include diluting allophycocyanin (APC)-labeled Ly6C antibody, phycoerythrin (PE)-labeled CyclinD1 antibody, and fluorescein isothiocyanate (FITC)-labeled DCN antibody at a ratio of 1:50 using antigen blocking solution to prepare an antibody working solution.

6. The method for using the identification reagent composition according to claim 5, wherein: Allophycocyanin (APC)-labeled Ly6C antibody was used as a fibroblast population marker, phycoerythrin (PE)-labeled CyclinD1 antibody was used as a damage and repair marker, and fluorescein isothiocyanate (FITC)-labeled DCN antibody was used as a fibroblast subpopulation marker.

7. The method for using the identification reagent composition according to claim 5, wherein: Using Ly6C antibody, CyclinD1 antibody and DCN antibody positivity as markers, flow cytometer collection tubes were used to collect Ly6C antibody, CyclinD1 antibody and DCN antibody positive cells, and fibroblast subpopulations in the lung tissue of ARDS mice in the damaged and repair states were identified and sorted.

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