Application of SPP1 and / or AREG as biomarker in preparation of product for evaluating curative effect of mesenchymal stem cells on alveolar proteinosis

Abstract

The invention provides application of SPP1 and / or AREG as a biomarker in preparation of a product for evaluating the curative effect of mesenchymal stem cells on alveolar proteinosis, and belongs to the technical field of biological medicine. The invention provides application of SPP1 and / or AREG as a biomarker in preparation of a product for evaluating the curative effect of mesenchymal stem cells on alveolar proteinosis. MSCs and GM-CSF treatment is carried out on aPAP model mice, and it is found that after MSCs treatment, SPP1 is positive, and the proportion of AREG positive macrophage subsets is decreased. Therefore, SPPI and AREG can be used as a biomarker for evaluating the curative effect of aPAP treated by MSC, a novel evaluation index is provided for development of clinical evaluation of the aPAP treatment curative effect, and a new treatment target and a theoretical basis are provided for clinical transformation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of SPP1 and / or AREG as a biomarker in preparation of a product for evaluating curative effect of mesenchymal stem cell treatment on pulmonary alveolar proteinosis. BACKGROUND

[0002] Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare interstitial lung disease mediated by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody, which is characterized by alveolar macrophage dysfunction and massive accumulation of surfactant, leading to progressive respiratory failure. Although GM-CSF antibody is the core of its diagnosis, the lung immune microenvironment of aPAP patients is significantly disordered, involving abnormal activation of multiple immune cells and aggregation of a large number of inflammatory cytokines, suggesting that its pathological mechanism is more complex, and there is a lack of biomarkers for evaluating the curative effect of aPAP treatment. SUMMARY

[0003] The application aims to provide application of SPP1 and / or AREG as a biomarker in preparation of a product for evaluating curative effect of mesenchymal stem cell treatment on pulmonary alveolar proteinosis.

[0004] The application provides application of SPP1 and / or AREG as a biomarker in preparation of a product for evaluating curative effect of mesenchymal stem cell treatment on pulmonary alveolar proteinosis.

[0005] Preferably, the expression level of SPP1 and / or AREG in the patient with pulmonary alveolar proteinosis is increased.

[0006] Preferably, the expression level of SPP1 and / or AREG in the macrophage of the patient with pulmonary alveolar proteinosis is increased.

[0007] The application also provides application of CD68-positive SPP1-positive macrophages and / or CD68-positive AREG-positive macrophages as a biomarker in preparation of a product for evaluating curative effect of mesenchymal stem cell treatment on pulmonary alveolar proteinosis.

[0008] Preferably, the proportion of the CD68-positive SPP1-positive macrophages and / or CD68-positive AREG-positive macrophages in the patient with pulmonary alveolar proteinosis is increased.

[0009] The application also provides a kit for evaluating curative effect of mesenchymal stem cell treatment on pulmonary alveolar proteinosis, comprising an anti-SPP1 specific antibody and / or an anti-AREG specific antibody.

[0010] The application also provides a method for evaluating curative effect of mesenchymal stem cell treatment on pulmonary alveolar proteinosis, comprising the following steps: collecting a patient sample; the sample includes lavage fluid and / or lung tissue; detecting the expression level of SPP1 and / or AREG in the patient sample; By comparing the changes of SPP1 and / or AREG expression levels before and after mesenchymal stem cell treatment in patients with pulmonary alveolar proteinosis, the evaluation results of mesenchymal stem cell efficacy are obtained; or, By comparing the expression levels of SPP1 and / or AREG in patients with pulmonary alveolar proteinosis after mesenchymal stem cell treatment with the expression levels of SPP1 and / or AREG in healthy control group, the evaluation results of mesenchymal stem cell efficacy are obtained.

[0011] The present application also provides a system for evaluating the efficacy of mesenchymal stem cell treatment for pulmonary alveolar proteinosis, comprising the following modules: The detection module is used for detecting the expression level of SPP1 and / or AREG in the patient sample; the sample includes lavage fluid and / or lung tissue; The analysis module is used for comparing the changes of SPP1 and / or AREG expression levels before and after mesenchymal stem cell treatment in patients with pulmonary alveolar proteinosis; or, comparing the expression levels of SPP1 and / or AREG in patients with pulmonary alveolar proteinosis after mesenchymal stem cell treatment with the expression levels of SPP1 and / or AREG in healthy control group; The result output module is used for obtaining the evaluation results of mesenchymal stem cell efficacy.

[0012] The present application also provides the use of mesenchymal stem cells in the preparation of a drug for treating pulmonary alveolar proteinosis.

[0013] The present application also provides the use of mesenchymal stem cells in the preparation of a drug for enhancing the phagocytosis of alveolar macrophages.

[0014] The present application provides the use of SPP1 and / or AREG as biomarkers in the preparation of products for evaluating the efficacy of mesenchymal stem cell treatment for pulmonary alveolar proteinosis. The present application reveals the immune mechanism of MSCs treatment of aPAP by analyzing the change rule of macrophage subpopulation after MSCs treatment of pulmonary alveolar proteinosis, and finds that the proportion of SPP1 positive and AREG positive macrophage subpopulation decreases after MSCs treatment. Therefore, SPPI and AREG can be used as a biomarker for evaluating the efficacy of MSCs treatment of aPAP, and a new evaluation index for clinical evaluation of aPAP treatment is developed, which provides a new treatment target and theoretical basis for clinical transformation.

[0015] The application also provides the use of mesenchymal stem cells in the preparation of a medicament for treating pulmonary alveolar proteinosis. MSCs can enhance the phagocytosis of alveolar macrophages, resulting in a decrease in intracellular surfactant deposition. BM-MSCs can reduce the accumulation of surfactant in the lungs, increase the survival rate of aPAP model mice, increase body weight, and reduce skin damage. MSCs have therapeutic potential and effectiveness in treating aPAP. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0017] Figure 1 Cell subpopulations and types in aPAP and healthy control alveolar lavage fluid; Figure 2 Figure 4 is a graph showing the proportion of cell subpopulations in aPAP and HCs alveolar lavage fluid; wherein, A: different cell types in aPAP and HC UMAP distribution; B: different cell types in aPAP and HC proportion; HC represents healthy control, PAP represents aPAP patient, A1, A2, A3 represent the proportion of cells in the alveolar lavage fluid of 3 healthy controls respectively, B1, B2, B3, C1 represent the proportion of cells in the alveolar lavage fluid of 4 aPAP patients respectively, summary represents the total proportion of each cell in the alveolar lavage fluid of 7 patients; Macrophages: macrophages; Neutrophils: neutrophils; CD8 / CD4 T cells: CD8 / CD4 T lymphocytes; Epithelial cells: epithelial cells; Mast cells: mast cells; Myeloid-derived suppressor cells: undifferentiated myeloid cells; B cells: B lymphocytes; Plasmacytoid dendritic cells: plasmacytoid dendritic cells; Conventional dendritic cells: conventional dendritic cells; Figure 3 Figure 5 is a graph showing the re-subpopulations of alveolar macrophages defined in aPAP and HCs; wherein, a: re-subpopulation (11 groups) of alveolar macrophages in 2 groups by using t-SNE integration; b: spatial distribution of aPAP and HCs in the above 11 groups of cells; c: developmental trajectory of 11 groups of cells is depicted with t-SNE as background combined with pseudo-time analysis; d: proportion of each alveolar macrophage subpopulation in aPAP and HCs; Figure 4 Figure for MSCs to improve the function of aPAP primary alveolar macrophages in vitro and in vivo; Wherein, A: phagocytosis analysis of mesenchymal stem cells (AT-MSC, UC-MSC, BM-MSC) after 3 days of indirect co-culture with aPAP macrophages by flow cytometry; B: phagocytosis analysis of mesenchymal stem cells (AT-MSC, UC-MSC, BM-MSC) after 6 days of indirect co-culture with aPAP macrophages by flow cytometry; C: phagocytosis analysis of mesenchymal stem cells (AT-MSC, UC-MSC, BM-MSC) after 9 days of indirect co-culture with aPAP macrophages by flow cytometry; D: indirect co-culture phagocytosis analysis at 6 days using aPAP serum treatment; E: indirect co-culture phagocytosis analysis at 6 days using aPAP bronchoalveolar lavage fluid treatment; F: phagocytosis analysis of mesenchymal stem cells (AT-MSC, UC-MSC, BM-MSC) after 3 days of direct co-culture with aPAP macrophages; G: phagocytosis analysis of mesenchymal stem cells (AT-MSC, UC-MSC, BM-MSC) after 6 days of direct co-culture with aPAP macrophages; H: using confocal laser scanning microscope, cell cytology examination was performed by periodic acid-schiff staining (PAS) and oil red O staining to analyze the phagocytosis of 6-day Transwell indirect co-culture; I: transmission electron micrograph of aPAP and PBS, GM-CSF and BM-MSCs primary alveolar macrophages; J: in vivo phagocytosis of aPAP mouse primary alveolar macrophages was evaluated using flow cytometry; K: BALF turbidity was measured by measuring absorbance at 450 nm; L: total protein of BALF was measured using BCA kit; M: FRCs were determined using mouse lung function test; N: Penh was measured by mouse WBP; O: survival rate was described by KM curve (saline n=11, GM-CSF and MSC n=12); P: body weight change of mice in each group; Q: skin lesion score of mice in different groups; Figure 5Immunofluorescence imaging of AREG+ alveolar macrophages in WT mice, aPAP mice PBS negative control, GM-CSF treated aPAP mice and MSC treated aPAP mice; Wherein, A: Immunofluorescence imaging of AREG+ alveolar macrophages in WT mice, aPAP mice PBS negative control, GM-CSF treated aPAP mice and MSC treated aPAP mice; DAPI: cell nucleus dye, blue fluorescence channel; CD68: red fluorescence channel; AREG: green fluorescence channel; B: Immunofluorescence imaging of SPP1+ alveolar macrophages in WT mice, aPAP mice PBS negative control, GM-CSF treated aPAP mice and MSC treated aPAP mice; DAPI: cell nucleus dye, blue fluorescence channel; CD68: red fluorescence channel; SPP1: green fluorescence channel; C: Cell counting of AREG / CD68 co-expression; D: Cell counting of SPP1 / CD68 co-expression; E: Evaluation of phagocytosis of macrophages co-cultured with mesenchymal stem cells or treated with GM-CSF by flow cytometry; F: Evaluation of phagocytosis of alveolar macrophages overexpressing SPP1 co-cultured with mesenchymal stem cells or treated with GM-CSF by flow cytometry. Figure 6 The results of the evaluation of the safety of the treatment of the mice in terms of their survival rate. DETAILED DESCRIPTION

[0018] The present application provides the use of SPP1 (osteopontin) and / or AREG (betacellulin) as a biomarker in the preparation of a product for evaluating the efficacy of mesenchymal stem cell treatment of pulmonary alveolar proteinosis.

[0019] As an embodiment, the pulmonary alveolar proteinosis is autoimmune pulmonary alveolar proteinosis.

[0020] As an embodiment, the expression level of SPP1 and / or AREG in the patient with pulmonary alveolar proteinosis is increased.

[0021] As an embodiment, the expression level of SPP1 and / or AREG in the macrophages of the patient with pulmonary alveolar proteinosis is increased.

[0022] The present application also provides the use of CD68 positive SPP1 positive macrophages and / or CD68 positive AREG positive macrophages as a biomarker in the preparation of a product for evaluating the efficacy of mesenchymal stem cell treatment of pulmonary alveolar proteinosis.

[0023] As an embodiment, the proportion of CD68 positive SPP1 positive macrophages and / or CD68 positive AREG positive macrophages in the patient with pulmonary alveolar proteinosis is increased.

[0024] Compared with healthy people, the proportion of CD68-positive SPP1-positive macrophages and / or CD68-positive AREG-positive macrophages in patients with pulmonary alveolar proteinosis is increased.

[0025] The present application also provides a kit for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary alveolar proteinosis, comprising an anti-SPP1 specific antibody and / or an anti-AREG specific antibody.

[0026] As an embodiment, the anti-SPP1 specific antibody and / or the anti-AREG specific antibody is a monoclonal antibody or a polyclonal antibody.

[0027] As an embodiment, the kit comprises an immunofluorescence kit.

[0028] The present application also provides a method for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary alveolar proteinosis, comprising the following steps: Collecting a patient sample; the sample comprises lavage fluid and / or lung tissue; Detecting the expression level of SPP1 and / or AREG in the patient sample; By comparing the changes in the expression levels of SPP1 and / or AREG in patients with pulmonary alveolar proteinosis before and after mesenchymal stem cell treatment, the evaluation results of the therapeutic effect of mesenchymal stem cells are obtained; or, By comparing the expression levels of SPP1 and / or AREG in patients with pulmonary alveolar proteinosis after mesenchymal stem cell treatment with the expression levels of SPP1 and / or AREG in healthy controls, the evaluation results of the therapeutic effect of mesenchymal stem cells are obtained.

[0029] As an embodiment, the expression level of SPP1 and / or AREG comprises the expression level of SPP1 and / or AREG in alveolar macrophages.

[0030] As an embodiment, the detection method for detecting the expression level of SPP1 and / or AREG in the patient sample comprises immunofluorescence.

[0031] In the present application, after mesenchymal stem cell treatment, the cell percentage of CD68+AREG+ macrophages decreases by 0.91, the total number of macrophages decreases by 1925.88, the cell percentage of CD68+SPP1+ macrophages decreases by 0.20, and the total number of macrophages decreases by 579.63.

[0032] The present application also provides a system for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary alveolar proteinosis, comprising the following modules: A detection module for detecting the expression level of SPP1 and / or AREG in a patient sample; the sample comprises lavage fluid and / or lung tissue; The analysis module is configured to compare the expression level changes of SPP1 and / or AREG before and after the mesenchymal stem cell treatment of the patient with pulmonary alveolar proteinosis, or compare the expression level of SPP1 and / or AREG after the mesenchymal stem cell treatment of the patient with pulmonary alveolar proteinosis with the expression level of SPP1 and / or AREG of the healthy control group. The result output module is configured to obtain an evaluation result of the mesenchymal stem cell treatment effect.

[0033] The application further provides an application of the mesenchymal stem cell in the preparation of a drug for treating pulmonary alveolar proteinosis.

[0034] The MSCs can improve the phagocytosis function of the primary alveolar macrophages of the aPAP patient through indirect co-culture, and show good safety and treatment effect in the aPAP model mice. The MSCs can significantly reduce the intracellular surface active agent and cell volume of the alveolar macrophages, improve the functional residual capacity (FRC) and Penh value, and have a protective effect on the inflammatory damage.

[0035] The application further provides an application of the mesenchymal stem cell in the preparation of a drug for enhancing the phagocytosis of the alveolar macrophages.

[0036] As an implementation form, the alveolar macrophages are primary alveolar macrophages.

[0037] As an implementation form, the mesenchymal stem cells include bone marrow mesenchymal stem cells.

[0038] In order to further illustrate the application, the application of SPP1 and / or AREG as biomarkers in the preparation of a product for evaluating the treatment effect of the mesenchymal stem cell on pulmonary alveolar proteinosis is described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the application.

[0039] All reagents and experimental consumables used in the following examples are as follows: Table 1 Experimental reagents

[0040] Table 2 Experimental instruments

[0041] Example 1 Revealing the distribution and function of immune cells in aPAP through single cell sequencing 1. Experimental samples BALF samples of 4 aPAP patients and 3 healthy people (all patients signed informed consent) were selected, after isolation, capture, reverse transcription, establishment of gene library and sequencing, and then through screening and dimension reduction, a total of 33028 effective cells were obtained, including 10 cell types of CD4+T lymphocytes, CD8+T lymphocytes, macrophages, epithelial cells, neutrophils, monocytes, mast cells, undifferentiated myeloid cells, B cells, dendritic cells, etc.

[0042] Sequencing was completed by a third party.

[0043] 2. Experimental method: 1) Single cell data analysis (1) Single cell data quality control RawData (unprocessed data generated directly by the sequencing instrument) obtained by sequencing was subjected to sequencing quality filtering (removal of low sequencing quality data), annotation (database alignment using GRCh38), and expression matrix was obtained. R3.6.1 and Rstudio tools were used for data visualization.

[0044] (2) Single cell data downstream analysis Seurat, Scater and Tidyverse tools were used for single cell data quality control and downstream analysis. The total number of UMI was corrected by normalization to make it comparable. Broken or dead cells, empty droplets that did not capture cells, and doublets that captured more than 2 cells were removed. Red blood cell genes were removed, and mitochondrial gene numbers were appropriately adjusted. Linear dimension reduction was performed using PCA, and the first 20 principal components were analyzed. Nonlinear dimension reduction was performed using TSNE, and cell clustering results were obtained. FindAllMarker was used to find marker genes for each cell group, and then the cell groups were annotated according to different cell markers. Finally, the macrophages were further analyzed for subgroups and differential genes. The results are shown in Figure 1 . As shown in Figure 1 , after isolation, capture, reverse transcription, establishment of gene library and sequencing, and then through screening and dimension reduction, a total of 33028 effective cells were obtained, including 10 cell types of CD4+T lymphocytes, CD8+T lymphocytes, macrophages, epithelial cells, neutrophils, monocytes, mast cells, undifferentiated myeloid cells, B cells, dendritic cells, etc. UMAP distribution of aPAP and HC different cell types was used.

[0045] 2) Pseudo-time analysis Temporal analysis of cells in different states, analysis of the transformation of cells in different microenvironments over time. This embodiment uses the Monocle tool to perform pseudo-temporal analysis on the cell clusters generated by Seurat. Based on Seurat clustering, the Monocle tool is used to search for the optimal cell ordering in high-dimensional and low-dimensional space, so as to fit the cell development or differentiation curve trajectory graph. The tree structure trajectory graph intuitively shows the position of each cell cluster in the development process. Cells with similar differentiation states are clustered together, and each branching point may represent a cell fate decision point. These branches have no fixed directionality, and the specific development and differentiation path needs to be judged according to the experimental background and the characteristics of the cell cluster. After determining the cell development trajectory, the dynamic changes of life processes on the trajectory are revealed by analyzing the changes in gene expression. The characteristic genes of each branch obtained by Monocle analysis can further be displayed by expression distribution graph to show their dynamic expression with pseudo-time value. Genes with similar expression trends will be clustered together to form different cell clusters. The pseudo-time expression clustering heat map of the characteristic genes of each branch obtained by Monocle analysis. See Figure 2 and Figure 3 . It can be seen from Figure 2 that there are differences in immune cells between aPAP patients and healthy people, the proportion of alveolar macrophages in aPAP samples is significantly decreased, the proportion of lymphoid cells is significantly increased, and the proportion of epithelial cells is also different. It can be seen from Figure 3 that the alveolar macrophages are further divided into 11 subgroups according to different marker genes, and it is found that AREG-positive, CCL4-positive, and SPPI-positive macrophages are enriched in aPAP patients among the above different subtypes of macrophages.

[0046] Example 2 MSCs improve the function of aPAP primary alveolar macrophages in vitro and in vivo 1. Experimental scheme: 1) Co-culture of alveolar macrophages and MSCs After recovering the MSCs, they were inoculated into a 24-well plate and cultured in MSC culture medium (DMEM + 10% FBS + double antibody) for 24 h. Alveolar macrophages were obtained by isolating and purifying alveolar lavage fluid from patients with pulmonary alveolar proteinosis, and resuspended in macrophage culture medium (RPMI-1640 medium + 10% autologous serum + double antibody). The ratio of alveolar macrophages to mesenchymal stem cells was about 1:2~1:3, and they were inoculated into the upper layer of the Transwell. The co-culture day was defined as the first day, and the co-culture lasted for 9 days.

[0047] 2) Phagocytic ability On day 3, day 6, day 9 of indirect co-culture, red fluorescent beads were added to the experimental wells of phagocytosis detection group, and incubated for 2 hours. The alveolar macrophages in the upper well of transwell were harvested, and the number of cells phagocytosing beads was detected by flow cytometry. By distinguishing the number of phagocytosed beads, alveolar macrophages were divided into strong phagocytosis cells and weak phagocytosis cells according to the size of phagocytosis ability: phagocytosis of 1-2 beads was defined as weak phagocytosis cells, and phagocytosis of more than 3 beads was defined as strong phagocytosis cells. On day 6 of co-culture, red fluorescent beads were added to the experimental wells of phagocytosis detection group, and incubated for 2 hours. Green fluorescent CD68 was used to label alveolar macrophage cytoplasm. The phagocytosis of red fluorescent beads by alveolar macrophages was observed under a laser confocal microscope.

[0048] 3) Morphological detection On day 6 of co-culture, the alveolar macrophages in the upper well were harvested, and the macrophages were stained with PAS and oil red O respectively, and then observed under a microscope.

[0049] 4) Non-invasive lung function detection At the 4th week, 6th week and 8th week of the experiment, the airway reactivity of each group of mice was measured by whole body plethysmography system, and the enhanced expiratory pause (Penh) was recorded.

[0050] 5) Invasive lung function detection At the 8th week of the experiment, pentobarbital was injected intraperitoneally to anesthetize the mice, and the invasive lung function of all surviving mice in the experimental group was detected, and the lung capacity was evaluated by detecting functional residual capacity (FRC).

[0051] 6) Mouse alveolar lavage The mouse was anesthetized to death with pentobarbital sodium, placed in a supine position, fixed the limbs, fully exposed the neck, chest and abdomen, sprayed the surface skin of the mouse with 75% alcohol, and wet the fur to prevent the fur from falling off and affecting the field of view. The skin of the neck was cut with scissors, and the muscles of the neck were bluntly separated with forceps until the trachea was fully exposed. The trachea was cut in a "T" shape, and the tracheal cannula was inserted and fixed with surgical suture. 0.8 ml of 37°C preheated normal saline was injected into the tracheal cannula, and repeated 3 times. The BALF was stored at 4°C for standby. During the operation, the liquid was slowly injected into the lung to avoid breaking the lung tissue.

[0052] 7) BALF pathological examination The mouse BALF obtained by the above method was mixed well by vortex, 200 μl was directly added to a 96-well plate, 3 replicate wells were set for each sample, and the absorbance at 420 nm was read by a microplate reader to detect the turbidity of BALF. After centrifugation, the insoluble protein was removed, the supernatant was diluted 100 times with normal saline, and the BCA standard was gradient-diluted with normal saline. The diluted sample was added to the well plate, 3 replicate wells were set for each sample, 200 μl of mixed solution of A and B of BCA was added to each well, and the plate was incubated at 37°C for 20 min in the dark. The absorbance at 600 nm was read by a microplate reader to detect the total protein content of BALF.

[0053] 8) Mouse skin lesion score The mouse skin lesion score was re-evaluated according to the existing literature (Br J Dermatol. 2007; 157(3): 458-465. doi: 10.1111 / j.1365-2133.2007.07979.x). According to the hair loss of the mouse, the degree of skin swelling and the damage of the mouse skin, the mouse was scored from 0 to 4 to determine the skin lesion. See Table 3 for specific scoring criteria.

[0054] Table 3 Mouse skin lesion scoring criteria

[0055] Results are shown in Figure 4 , Figure 4 Results show that mesenchymal stem cells (MSCs) from three sources can more effectively enhance the phagocytosis of primary alveolar macrophages at days 3, 6 and 9 compared to granulocyte colony-stimulating factor (GM-CSF) (A, B, C in Figure 4 Even in the presence of GM-CSF autoantibody interference, MSCs are superior to GM-CSF in promoting macrophage phagocytosis (D and E in Figure 4 Direct co-culture experiments with pre-fluorescently labeled alveolar macrophages also show that the presence of MSCs significantly enhances phagocytosis (F and G in Figure 4 Bone marrow mesenchymal stem cells (BM-MSCs) have the most significant effect on phagocytosis. Confocal laser microscope observation results show that MSCs treatment significantly increases the number of phagocytosed fluorescent particles in alveolar macrophages compared to the GM-CSF group and the phosphate buffer solution (PBS) group (H in Figure 4

[0056] Compared to alveolar macrophages treated with PBS and GM-CSF, alveolar macrophages treated with MSCs have significantly reduced intracellular surfactant and cell volume (I in Figure 4 ​The H in the text is missing. Transmission electron microscopy was used to observe indirectly co-cultured alveolar macrophages, and the results were consistent with cytopathological findings. A significant reduction in surfactant deposition and a decrease in surfactant particle size were observed. Figure 4 In the I group, mice treated with BM-MSCs showed superior phagocytic capacity compared to the GM-CSF and saline groups. Figure 4 J in the middle.

[0057] like Figure 4 As shown in K, the turbidity of bronchoalveolar lavage fluid (BALF) in mice treated with bone marrow mesenchymal stem cells (BM-MSCs) every two weeks was reduced, indicating decreased surfactant deposition. Furthermore, the total BALF protein level in the every-two-week BM-MSCs group, as measured using the BCA kit, was lower than that in the granulocyte-macrophage colony-stimulating factor (GM-CSF) group and the saline group. Figure 4 L in the middle). Both GM-CSF and MSCs treatment improved functional residual capacity (FRC) and Penh value (L). Figure 4 (M and N in the text).

[0058] In long-term efficacy assessments, the survival rate of the BM-MSCs group exceeded that of the saline group and the GM-CSF group. Figure 4 In addition, the body weight of mice treated with BM-MSCs was significantly higher than that of mice in the saline and GM-CSF groups (O). Figure 5 (P in the text). Throughout the observation period, some mice developed skin lesions, including hair or feather loss, redness, swelling, and ulceration. According to the skin lesion scoring system, mice in the BM-MSCs group had lower lesion scores, indicating a protective effect against inflammatory damage. Figure 5 (Q in the text). These results clearly demonstrate that MSCs enhance the phagocytic activity of alveolar macrophages, leading to a reduction in intracellular surfactant deposition. BM-MSCs can reduce surfactant re-accumulation in the lungs. Long-term efficacy evaluation further emphasizes the therapeutic potential of BM-MSCs, as evidenced by improved survival, weight gain, and reduced skin lesions in mice with aPAP. All of these results reveal the therapeutic potential and effectiveness of MSCs in treating aPAP.

[0059] Example 3 The therapeutic potential of MSCs on aPAP macrophage subsets Experimental protocol 1. Obtaining and processing mouse lung tissue 1) The mouse is fasted for 4 hours in advance, and a 4% sodium pentobarbital solution is prepared for intraperitoneal anesthesia of the mouse. The mouse is weighed, and the appropriate injection volume is selected according to the body weight. One hand holds the mouse's tail, and the other hand holds the mouse's neck and back. The mouse's body is turned over, and the mouse's chest and abdomen are fully exposed to the field of view. The injection site is selected, and the mouse is anesthetized in advance. The anesthetic effect is observed, and appropriate supplementary injection is performed to achieve a deep coma state; 2) The mouse is in a supine position, and the limbs are fixed with medical tape. The chest and abdomen are fully exposed, and the mouse's chest and abdomen are sprayed with 75% ethanol. The hair is wetted to prevent the hair from falling off during the skin preparation process and affecting the subsequent material taking; 3) Use scissors to make a skin incision from the mouse's abdomen to the neck. Use forceps to bluntly separate the mouse's skin, fully exposing the mouse's thoracic cage and neck; 4) Use ophthalmic scissors to cut the ribs along the lateral wall in turn. Turn up the anterior chest wall to fully expose the mouse's lungs and heart. Use tissue forceps to hold and fix the anterior chest wall. Use forceps to gently move the small intestine aside. Find the inferior vena cava and cut it. Use absorbent paper to absorb the blood to avoid affecting the field of view; 5) Gently lift the heart with forceps to expose the pulmonary artery. Use a 5ml syringe to draw normal saline. Insert the syringe into the root of the pulmonary artery and slowly inject normal saline. Repeat this step several times until the mouse's lungs turn from pink to white, indicating that the blood cells in the mouse's lungs have been completely washed out; 6) Use forceps to bluntly separate the neck muscles to fully expose the trachea. Pass a 10cm nylon thread through the trachea with a curved forceps. Use ophthalmic scissors to cut a "T" shaped incision along the upper end of the trachea. The incision should be as close to the upper end of the mouse's trachea as possible. Do not cut too wide to avoid directly cutting the mouse's trachea. Select an appropriate gavage needle and place a small gavage needle along the incision into the deep part of the trachea. Use a surgical knot to fix the trachea and the gavage needle with the nylon thread; 7) Use a 1ml syringe to draw 1ml of 4% paraformaldehyde. Slowly inject it into the mouse's lungs along the gavage needle. Avoid rapid injection to prevent damage to the alveolar septum. Determine whether multiple injections of 4% paraformaldehyde are needed based on the situation. Stop when the lung tissue is fully inflated; 8) Use another new 10cm nylon thread to fix and ligate the mouse's lung. Make sure the ligation is stable to prevent leakage of 4% paraformaldehyde. Note that this thread only fixes the trachea and does not fix the gavage needle; 9) Use ophthalmic scissors to slowly isolate the lung tissue along the mouse's spinal surface. Finally, cut off the mouse's trachea and remove the heart; 10) Place the lung tissue in a 4% paraformaldehyde solution for complete fixation. Make sure that the 4% paraformaldehyde solution is fully soaked into the tissue.

[0060] 2. Frozen section 1) Sucrose dehydration: Remove the fixed lung tissue from the 4% paraformaldehyde solution. Note that the lung tissue fixed in 4% paraformaldehyde should not be left for more than 2 days. Then, put it into the sucrose solution for gradient dehydration. Soak it in 10% sucrose solution for 3 days and 20% sucrose solution for 10 days to complete the dehydration. 2) Embedding: Embed the lung tissue with its largest surface area using OCT frozen section embedding medium. Since OCT embedding medium is relatively viscous and prone to air bubbles, avoid creating air bubbles near the tissue during embedding. Select an embedding cassette of appropriate size in advance, and evenly spread a layer of OCT embedding medium at the bottom of the cassette. Gently place the tissue on top of the OCT, and adjust the position of the lung tissue with toothless forceps. Then, fill the entire lung tissue with OCT embedding medium, ensuring it completely covers the tissue and extends beyond its highest point. Place the embedded tissue and embedding cassette together in dry ice for quick freezing. After quick freezing, store the tissue at -80℃ for long-term storage. 3) Sectioning: Pre-cool the microtome and take the tissue out of the -80℃ freezer and place it in the -20℃ freezer for slow freeze-thaw. Fix the frozen and thawed tissue OCT block on the microtome, fix the blade, adjust the section thickness to 8-10μm, and lift the wax strip with a brush while rotating the rotary wheel. Carefully lay the wax section flat on the glass slide with tweezers, mark it, and put it in the -20℃ freezer for short-term storage. For long-term storage, it needs to be transferred to the -80℃ freezer.

[0061] 3. In situ immunofluorescence staining 1) After removing the sections from the -80℃ freezer, immediately soak them in PBS solution for 5 minutes. Note that the PBS solution must completely cover the tissue block. 2) Soak the sections in 4% paraformaldehyde for 10-20 minutes. Note that the 4% paraformaldehyde must completely cover the tissue block. 3) Wash three times with 0.1% Triton-PBS solution, 5 minutes each time; 4) Soak the sections in 0.2% Triton-PBS solution for 20 minutes. Note that the 0.2% Triton-PBS solution must completely cover the tissue block. Preheat the antigen retrieval solution in advance. 5) Wash three times with 0.1% Triton-PBS solution, 5 minutes each time; 6) Place the slides into preheated sodium citrate antigen retrieval solution at 95°C for 7 minutes; 7) Remove the slides and slowly allow them to return to room temperature. Place the slides in the antigen retrieval solution to prevent them from drying out. 8) After cooling, wash the sections three times with 0.1% Triton-PBS solution, 5 min each time; 9) Blocking: After the drying of the section, 50-100 μl of 3% BSA was added to each tissue block to cover the surface of the tissue block. The section was placed in a wet box and incubated for 30-60 min. Note that the section should not be dried after this step. 10) The 3% BSA was removed, and the residual 3% BSA around the tissue was absorbed with a water-absorbing paper (note that the tissue should not be damaged by wiping). The tissue was circled with a histological pen. Note that the tissue should not be dried. 11) 50-100 μl of primary antibody was added to each tissue block. The section was placed in a wet box and incubated at 4°C overnight. Note that the primary antibody should completely cover the tissue, and the amount on each section should be consistent. 12) After the section returned to room temperature, the primary antibody was removed, and the section was soaked in a section box containing 0.1% Triton-PBS solution. Note that the 0.1% Triton-PBS should completely cover the tissue. The washing was performed 3 times, each for 5 min. 13) 50-100 μl of secondary antibody was added to each tissue block. The solution completely covered the tissue, and the section was placed in a wet box and incubated at room temperature for 60 min. Note that the incubation should be strictly protected from light. 14) The primary antibody was removed from the section, and the section was soaked in a section box containing 0.1% Triton-PBS solution. Note that the 0.1% Triton-PBS should completely cover the tissue. The washing was performed 3 times, each for 5 min. 15) DAPI was added, and the section was incubated in a wet box for 1 min. Note that the incubation should be strictly protected from light. 16) The DAPI was removed, and the section was washed with 0.1% Triton-PBS solution 3 times, each for 5 min. 17) After the section was dried, the tissue block around the section was wiped with a water-absorbing paper. 2-3 drops of anti-fluorescence quenching mounting agent were added to the center of the tissue block. A cover glass was placed on the section to avoid the generation of air bubbles. The cover glass was fixed with nail polish. Note that the whole process should be strictly protected from light. 18) The section was observed, photographed, and recorded using a TG panoramic tissue cell quantification-scanning system. The fluorescence quantification analysis of the section was performed using a TG panoramic tissue cell quantification-analysis system.

[0062] The results are shown in Figure 5 , which shows that DAPI is a cell nucleus dye, and the blue fluorescence channel; AREG is a red fluorescence channel; CD68 is a green fluorescence channel; and the scale bar is 50 μm. Figure 6Immunofluorescence imaging of SPP1+ alveolar macrophages in WT mice, aPAP mice PBS negative control, GM-CSF treated aPAP mice and MSC treated aPAP mice. DAPI: nuclear stain, blue fluorescence channel; SPP1: red fluorescence channel; CD68: green fluorescence channel; scale bar: 50 pm. Figure 6 B, E and F in FIG. 1.

[0063] The cell counts and percentages of CD68+SPP1+ macrophages, CD68+AREG+ macrophages in lung tissue samples of WT mice and aPAP mice were quantified by multiplex immunofluorescence. The counts of both macrophage subsets were reduced in aPAP mice compared to WT mice, and the counts of both CD68+SPP1+ macrophages and CD68+AREG+ macrophages were significantly reduced. This observation is consistent with the scRNA-seq results. In our study, we also performed in situ multiplex immunofluorescence on lung tissue of mice treated with MSCs. Our results showed that the proportion of SPP1+ macrophage subset decreased after MSCs treatment, similar to the distribution in healthy mice. This not only reinforces the results obtained by scRNA-seq, but also highlights the potential of MSCs to treat aPAP by regulating macrophage subsets.

[0064] Meanwhile, after treatment by mesenchymal stem cells, the percentage of CD68+AREG+ macrophages decreased by 0.91, the total number of macrophages decreased by 1925.88, the percentage of CD68+SPP1+ macrophages decreased by 0.20, and the total number of cells decreased by 579.63.

[0065] In summary, we found SPP1-positive, AREG-positive alveolar macrophages in aPAP model mice.

[0066] By treating aPAP model mice with MSCs and GM-CSF, we found that the proportion of SPP1-positive, AREG-positive macrophage subsets decreased after MSCs treatment. This not only confirms the results obtained by single-cell sequencing, but also highlights the potential of MSCs to treat aPAP by regulating macrophage subsets.

[0067] From the above results, it can be seen that SPPI and AREG can be used as a biomarker for evaluating the efficacy of MSCs in treating aPAP, and a new evaluation index for clinical evaluation of aPAP treatment efficacy is developed.

[0068] Example 4 Safety evaluation 1. Experimental process 1) Age-matched aPAP mice were randomly divided into three groups: PBS group, MSC group, and GM-CSF group, with 16 mice in each group. 2) Both the PBS group and the MSC group were treated by injecting PBS and MSC suspensions into the mice via tail vein, respectively, while GM-CSF was treated by nebulized inhalation. 3) After treatment, the mice will be placed in a normal environment (non-SPF grade) for normal lighting, diet, and water. 4) Record the natural death time of mice in different groups, and then use GraphPad Prism to create survival curves.

[0069] Mouse administration A. Tail vein injection Use a cylindrical container to restrain the mouse's body. Hold the lower third of the tail with your left thumb and forefinger, straightening the tail and applying appropriate force to secure it. Place your middle finger on the tail cap of the restrainer to further stabilize the tail and prevent it from swaying. Wipe the tail with a cotton ball moistened with 75% alcohol to make the tail vein slightly varicose for clearer observation. Hold a 1ml syringe in your right hand, bevel facing upwards, and insert the needle into the mouse's tail vein at a 10°-20° angle. A noticeable feeling of resistance is felt upon insertion. The needle should penetrate approximately 2mm. When the needle is about halfway in, slowly advance it at a parallel angle for another 3-5mm. Inject 200μl of MSC cell suspension or PBS solution into each mouse's tail vein. After injection, apply pressure with a sterile cotton ball, observing for any oozing or bleeding. Avoid rapid injection to prevent embolism throughout the entire process. After injection, observe the mouse's condition, return it to its cage, and continue normal feeding.

[0070] B. Nebulized inhalation Based on the mice's respiratory rate, tidal volume, and nebulizer power, the GM-CSF concentration and drug volume inhaled by each mouse were calculated. The GM-CSF solution was prepared by diluting with ultrapure water. The nebulizer tube and chamber were connected, and the mice were separated into different cages to prevent clustering during nebulization, which could affect efficiency. Strict male and female mice were separated for nebulization. The nebulization frequency was once daily for four weeks. After nebulization, the mice were returned to their cages and raised as usual.

[0071] 2. Experimental Results See results ​ .Depend on ​ It can be seen that, compared with the GM-CSF and PBS groups, MSCs can significantly improve the survival rate and increase the survival time of aPAP model mice.

[0072] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained based on the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. Application of SPP1 and / or AREG as biomarkers in the preparation of products for evaluating the efficacy of mesenchymal stem cell therapy for pulmonary alveolar proteinosis.

2. The application according to claim 1, characterized in that, The expression levels of SPP1 and / or AREG are elevated in patients with pulmonary alveolar proteinosis.

3. The application according to claim 2, characterized in that, The expression levels of SPP1 and / or AREG are elevated in macrophages of patients with pulmonary alveolar proteinosis.

4. Application of CD68-positive SPP1-positive macrophages and / or CD68-positive AREG-positive macrophages as biomarkers in the preparation of products for evaluating the efficacy of mesenchymal stem cell therapy for pulmonary alveolar proteinosis.

5. The application according to claim 3, characterized in that, The proportion of CD68-positive SPP1-positive macrophages and / or CD68-positive AREG-positive macrophages is increased in patients with pulmonary alveolar proteinosis.

6. A kit for evaluating the efficacy of mesenchymal stem cell therapy in pulmonary alveolar proteinosis, characterized in that, This includes anti-SPP1 specific antibodies and / or anti-AREG specific antibodies.

7. A method for evaluating the efficacy of mesenchymal stem cell therapy for pulmonary alveolar proteinosis, characterized in that, Includes the following steps: Collect patient samples; the samples include lavage fluid and / or lung tissue; The expression levels of SPP1 and / or AREG in the patient samples were detected; The efficacy of mesenchymal stem cell therapy in patients with pulmonary alveolar proteinosis was evaluated by comparing changes in the expression levels of SPP1 and / or AREG before and after treatment; or, The efficacy of mesenchymal stem cell therapy was evaluated by comparing the expression levels of SPP1 and / or AREG in patients with pulmonary alveolar proteinosis after mesenchymal stem cell treatment with those in healthy controls.

8. A system for evaluating the efficacy of mesenchymal stem cell therapy for pulmonary alveolar proteinosis, characterized in that, Includes the following modules: A detection module for detecting the expression levels of SPP1 and / or AREG in patient samples, including lavage fluid and / or lung tissue; The analysis module is used to compare the changes in the expression levels of SPP1 and / or AREG in patients with alveolar proteinosis before and after mesenchymal stem cell treatment; or, to compare the expression levels of SPP1 and / or AREG in patients with alveolar proteinosis after mesenchymal stem cell treatment with the expression levels of SPP1 and / or AREG in healthy controls. The results output module is used to obtain evaluation results of the efficacy of mesenchymal stem cell therapy.

9. Application of mesenchymal stem cells in the preparation of drugs for the treatment of pulmonary alveolar proteinosis.

10. Application of mesenchymal stem cells in the preparation of drugs that enhance the phagocytic activity of alveolar macrophages.

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