A method for enhancing the anti-fibrosis function of mesenchymal stem cells and application thereof

Abstract

The present application belongs to the field of cell therapy and biological medicine technology, and specifically relates to a method for enhancing the anti-fibrosis function of mesenchymal stem cells and application thereof. The method comprises pretreating mesenchymal stem cells with transforming growth factor beta 1 to obtain mesenchymal stem cells with enhanced function. The mesenchymal stem cells pretreated by the method of the present application exhibit significantly enhanced ability to inhibit the activation, migration and extracellular matrix deposition of fibroblasts in vivo and in vitro, can effectively improve the lung tissue structure, reduce collagen deposition and improve the survival rate of lung fibrosis model animals. The present application also relates to pretreated mesenchymal stem cells prepared by the method, related reagent applications, pharmaceutical compositions and their use in treating fibrotic diseases, especially pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy and biomedicine, specifically relating to a method for enhancing the anti-fibrotic function of mesenchymal stem cells and its application. Background Technology

[0002] Pulmonary fibrosis is a progressive, irreversible interstitial lung disease characterized by excessive deposition of extracellular matrix in the lung parenchyma, distortion of lung tissue structure, and impaired gas exchange, ultimately leading to respiratory failure. Currently approved antifibrotic drugs, such as nintedanib and pirfenidone, can only slow disease progression and cannot reverse existing fibrosis; they are also often accompanied by significant adverse reactions, limiting long-term use. Therefore, developing safer and more effective treatment strategies is an urgent clinical need.

[0003] Mesenchymal stem cells (MSCs), with their self-renewal, multi-lineage differentiation capabilities, and their ability to exert immunomodulatory and tissue repair effects through paracrine signaling, have become a highly promising new therapeutic option. Compared with traditional anti-fibrotic drugs, MSCs have shown superior potential in reducing extracellular matrix deposition and improving lung function, with a good safety profile. However, the therapeutic efficacy of MSC transplantation is severely affected by the pathological microenvironment of fibrotic lung tissue. This microenvironment is rich in pro-inflammatory and pro-fibrotic cytokines, which, while potentially inducing some immunomodulatory functions in MSCs, also limit their therapeutic potential.

[0004] Transforming growth factor-β1 (TGF-β1) is one of the most critical pro-fibrotic cytokines in the pulmonary fibrosis microenvironment. Conventionally, TGF-β1 in the microenvironment is primarily understood to drive the pathological effects of fibroblast activation and extracellular matrix deposition. Previous optimizations of mesenchymal stem cell therapy have largely focused on mitigating the adverse effects of the microenvironment, rather than actively utilizing its factors to enhance cell therapy function.

[0005] Growth differentiation factor 15 (GGF-15) belongs to the transforming growth factor-β superfamily and has been proven in recent years to be a core paracrine mediator for the anti-fibrotic effects of mesenchymal stem cells (MSCs), inhibiting fibroblast activation and extracellular matrix deposition. However, how the fibrotic microenvironment regulates the expression of GGF-15 in implanted MSCs, and how to actively increase the level of GGF-15 in MSCs to enhance their therapeutic function, remains an unsolved problem. Summary of the Invention

[0006] The purpose of this invention is to provide a simple and effective method for enhancing the anti-fibrotic function of mesenchymal stem cells, thereby solving the problem of the lack of effective means in the prior art to enhance the therapeutic potential of mesenchymal stem cells for pulmonary fibrosis.

[0007] This invention is based on a groundbreaking discovery: transforming growth factor-β1, a core pro-fibrotic factor in the pulmonary fibrosis microenvironment, not only does not weaken the function of mesenchymal stem cells, but can also serve as a beneficial "permission" signal. In vitro pretreatment of mesenchymal stem cells with transforming growth factor-β1 can significantly upregulate the expression and secretion of growth differentiation factor-15 by downregulating the transcriptional repressor RUNX1, thereby relieving its transcriptional inhibition of the potent anti-fibrotic factor RUNX1. This endows pretreated cells with a stronger ability to inhibit fibroblast activation and migration and reduce extracellular matrix deposition.

[0008] Based on this, the present invention provides the following technical solution.

[0009] In a first aspect, the present invention provides a method for enhancing the anti-fibrotic function of mesenchymal stem cells, comprising: pretreating mesenchymal stem cells with transforming growth factor-β1 to obtain pretreated mesenchymal stem cells with enhanced anti-fibrotic function.

[0010] In a preferred embodiment, the mesenchymal stem cells are selected from placental-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or induced pluripotent stem cell-derived mesenchymal stem cells. More preferably, the mesenchymal stem cells are placental-derived mesenchymal stem cells, and even more preferably, fetal-placental-derived mesenchymal stem cells.

[0011] This invention discovers that the transcription factor RUNX1 acts as a transcriptional repressor of the growth differentiation factor 15 (GDB15) gene in mesenchymal stem cells (MSCs). RUNX1 can directly bind to the promoter region of the GDB15 gene, negatively regulating its transcription. Treatment with transforming growth factor-β1 (TGF-β1) downregulates the expression level of RUNX1 in MSCs, thereby relieving RUNX1's transcriptional repression of GDB15, leading to a significant upregulation of GDB15 expression and secretion. This RUNX1-GDB15 regulatory axis is conserved in MSCs from different sources, particularly in placental and fetal-placental MSCs. Therefore, those skilled in the art can reasonably expect that the strategy of upregulating GDB15 and enhancing anti-fibrotic function through TGF-β1 pretreatment, as described in this invention, is also applicable to MSCs from other sources.

[0012] In a specific implementation, the pretreatment concentration of transforming growth factor-β1 is 1-100 ng / mL, preferably 10-100 ng / mL, and more preferably 50 ng / mL. The pretreatment time is 12-72 hours, preferably 24-48 hours. Within this concentration and time range, transforming growth factor-β1 can effectively induce the downregulation of RUNX1 expression and the upregulation of growth differentiation factor 15 expression and secretion in mesenchymal stem cells.

[0013] Furthermore, the pretreatment upregulates the expression and / or secretion of growth differentiation factor 15 in the mesenchymal stem cells. More preferably, the pretreatment also downregulates the expression of RUNX1 in the cells.

[0014] Secondly, the present invention provides pretreated mesenchymal stem cells prepared according to the above method. This cell product, compared to untreated mesenchymal stem cells, exhibits significantly enhanced ability to inhibit fibroblast activation and migration, and to inhibit extracellular matrix deposition. Preferably, the expression level of RUNX1 in the mesenchymal stem cells is downregulated compared to untreated mesenchymal stem cells, and the expression and secretion levels of growth differentiation factor 15 are upregulated compared to untreated mesenchymal stem cells.

[0015] Thirdly, the present invention provides the application of transforming growth factor-β1 in the preparation of a pretreatment reagent for enhancing the anti-fibrotic function of mesenchymal stem cells.

[0016] Fourthly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the pretreated mesenchymal stem cells and a pharmaceutically acceptable carrier.

[0017] Fifthly, the present invention provides the use of the pretreated mesenchymal stem cells or the pharmaceutical composition in the preparation of a medicament for treating fibrotic diseases. Preferably, the fibrotic disease is pulmonary fibrosis, more preferably idiopathic pulmonary fibrosis or fibrosis associated with other interstitial lung diseases.

[0018] Beneficial effects of the present invention

[0019] Compared with existing technologies, the contribution of this invention lies in breaking the cognitive barrier that factors in the fibrotic microenvironment only have negative effects. It creatively transforms transforming growth factor-β1 from a "pathogenic factor" into a "synergistic tool," using it as a pretreatment stimulus to specifically and significantly upregulate the expression and secretion of the key anti-fibrotic factor, growth differentiation factor 15, in mesenchymal stem cells. This method is simple to operate and has clear effects: in vitro and in vivo experimental data confirm that mesenchymal stem cells treated with this method have significantly enhanced abilities to inhibit fibroblast activation markers such as α-smooth muscle actin, type I collagen, and connective tissue growth factor, and significantly enhanced abilities to inhibit fibroblast migration. In animal models, it can effectively reduce collagen deposition in lung tissue, improve lung tissue structure, alleviate pulmonary edema, and significantly improve survival rates. Furthermore, this invention clarifies the mechanism by which RUNX1 negatively regulates the transcription factor of growth differentiation factor 15, providing a clear molecular basis for optimizing mesenchymal stem cell therapy. Attached Figure Description

[0020] Figure 1 Identification of isolated fetal-placental mesenchymal stem cells. A: Morphological observation of fetal-placental mesenchymal stem cells; B: Flow cytometry identification results; C: Trilineage differentiation experiment results.

[0021] Figure 2 TGF-β1 pretreatment enhances the anti-fibrotic ability of fetal placental mesenchymal stem cells. A: Western blot results and quantitative analysis; B: Scratch healing assay results (0h, 24h, 48h).

[0022] Figure 3 TGF-β1 pretreatment upregulated the expression and secretion of growth differentiation factor 15 (GDF15) in fetal placental mesenchymal stem cells. A: Western blot results and quantitative analysis; B: Real-time quantitative PCR (RT-qPCR) results; C: Enzyme-linked immunosorbent assay (ELISA) results.

[0023] Figure 4 TGF-β1 pretreatment downregulates RUNX1 expression in fetal placental mesenchymal stem cells. A: Western blot results and quantitative analysis; B: Immunofluorescence staining results.

[0024] Figure 5 The anti-fibrotic enhancement effect of growth differentiation factor 15 (GDF15) neutralizing antibody on TGF-β1 pretreated fetal placental mesenchymal stem cells. A: Western blot results and quantitative analysis; B: scratch healing assay results.

[0025] Figure 6RUNX1 directly inhibits the expression of GDF15 in fetal placental mesenchymal stem cells. A: ELISA results; B: Western blot results and quantitative analysis.

[0026] Figure 7 TGF-β1 pretreatment enhances the in vivo anti-fibrotic function of mesenchymal stem cells. A: Photographs of lung tissue appearance in each group of mice; B: Results of H&E staining and Masson's trichrome staining of lung tissue. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0028] Experimental materials

[0029] Unless otherwise stated, all chemical reagents were purchased from Sigma-Aldrich, Inc., USA; anti-RUNX1, anti-transforming growth factor-β1, anti-type I collagen, and anti-β-tubulin primary antibodies were purchased from Abogen Biosciences, Inc., UK; growth differentiation factor 15 neutralizing antibody was purchased from Protec Technologies, Inc., USA; and immunofluorescence reagents were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China.

[0030] Experimental methods

[0031] Cell line and culture: The human lung fibroblast cell line MRC-5 was purchased from Wuhan Pronosei Biotechnology Co., Ltd., China, and cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% carbon dioxide. Before the co-culture experiment, MRC-5 cells were stimulated with 5 ng / mL transforming growth factor-β1 for 24 hours to construct a fibrotic phenotype cell model.

[0032] Isolation and Culture of Fetal Placental Mesenchymal Stem Cells: With the approval of the Institutional Ethics Committee of Ningxia Medical University General Hospital (Approval No.: NXMU-2023-IRB-012) and after obtaining informed consent from the donors, full-term placental tissue from healthy mothers undergoing elective cesarean section was collected. Based on existing methods, fetal placental mesenchymal stem cells were isolated by slight modifications: fetal side tissue of the placenta was obtained, digested with collagenase A and deoxyribonuclease I, filtered, and centrifuged. The cell pellet was resuspended in a mesenchymal stem cell-specific culture medium containing glutamine supplement and gentamicin, and cultured at 37℃ in a 5% CO2 incubator. Cells from passages 3 to 5 were used in the experiment.

[0033] Flow cytometry analysis: Fourth-generation fetal placental mesenchymal stem cells were collected and stained with fluorescently labeled antibodies against CD14, CD34, CD45, human leukocyte antigen-DR (negative markers), and anti-CD73, CD90, and CD105 (positive markers). Detection was performed using a FACS Celesta flow cytometer (BD), and data were analyzed using FlowJo v10.8 software. ###2.5 Trilineage Differentiation Assay: Fetal placental mesenchymal stem cells were cultured for 21 days in adipogenic, osteogenic, and chondrogenic differentiation media. Their differentiation capacity was assessed using Oil Red O, Alizarin Red, and Alcian Blue staining, respectively.

[0034] Transwell co-culture: 5 × 10 4 MRC-5 cells were seeded in the lower chamber of a 24-well Transwell chamber. Fetal placental mesenchymal stem cells pretreated with 50 ng / mL transforming growth factor-β1 for 24 hours (or infected with RUNX1-overexpressing lentivirus / control lentivirus) were seeded at a concentration of 5 × 10⁶ cells / well. 4 MRC-5 cells were seeded per well in the upper chamber, and 5 ng / mL of transforming growth factor-β1 was added to the lower chamber to initiate co-culture. After 24 hours, MRC-5 cells were collected for further analysis.

[0035] Western blot assay for proteins: Cells were lysed by radioimmunoprecipitation, and proteins were extracted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene fluoride membranes and incubated sequentially with primary antibody and horseradish peroxidase-labeled secondary antibody. Protein signals were detected by enhanced chemiluminescence assay, with β-tubulin as an internal control. Quantitative analysis was performed using ImageJ software.

[0036] Scratch healing assay: MRC-5 cell monolayers that had fused growth were scratched with a sterile pipette tip, washed, and then cultured in serum-free medium. Scratch images were taken at 0, 24, and 48 hours, and cell migration ability was quantitatively analyzed using ImageJ software.

[0037] Messenger RNA sequencing: Total RNA was extracted from fetal placental mesenchymal stem cells treated with 50 ng / mL transforming growth factor-β1 for 48 hours and untreated cells. After constructing libraries, paired-end 150 bp sequencing was performed on the Ilumina NextSeq platform, and differential gene expression analysis was performed using DESeq2 software.

[0038] Real-time quantitative polymerase chain reaction: Total ribonucleic acid was reverse transcribed into complementary deoxyribonucleic acid, and real-time quantitative polymerase chain reaction was performed on the QuantStudio 5 system using SYBR Green dye. β-actin was used as an internal control, and gene expression levels were analyzed using the 2^(-ΔΔCT) method.

[0039] Enzyme-linked immunosorbent assay (ELISA): Fetal placental mesenchymal stem cells were treated with transforming growth factor-β1 at gradient concentrations of 0–50 ng / mL for 48 hours. The content of human growth differentiation factor-β1 in the cell supernatant was detected using the human growth differentiation factor-β1 ELISA kit from Posteco (USA).

[0040] Immunofluorescence staining: After fixing, permeabilizing, and blocking the cells, they were incubated with primary antibody and fluorescent secondary antibody in sequence. The cell nuclei were counterstained with 4',6-diamidindo-2-phenylindole, and fluorescence images were captured. The fluorescence intensity was quantified using ImageJ software.

[0041] Nuclease targeted cleavage and release assay: The nuclease targeted cleavage and release assay was performed according to the instructions of the reagent kit from Cell Signaling Technologies, Inc. (CSN). The binding of RUNX1 to the growth differentiation factor 15 promoter was detected. After sequencing the library, the data were analyzed using MACS2 software and the results were visualized using IGV software.

[0042] Lentiviral construction and infection: The human RUNX1 coding region sequence was cloned into the pLVX-Puro vector to construct a RUNX1 overexpressing lentivirus; fetal placental mesenchymal stem cells were infected with a multiplicity of infection of 50, and positive cells were screened with puromycin. The overexpression effect of RUNX1 was verified by Western blotting and real-time quantitative polymerase chain reaction.

[0043] Bleomycin-induced mouse pulmonary fibrosis model: 6-8 week old female C57BL / 6 mice were randomly divided into 4 groups (n=8 per group): blank control group (physiological saline), bleomycin group (2 mg / kg bleomycin administered intratracheally), bleomycin + fetal placental mesenchymal stem cell group (bleomycin + fetal placental mesenchymal stem cells pretreated with transforming growth factor-β1), and bleomycin + fetal placental mesenchymal stem cell-RUNX1 overexpression group (bleomycin + fetal placental mesenchymal stem cells pretreated with RUNX1 overexpression). The mice were administered via tail vein injection on day 9 after modeling and observed for 28 days.

[0044] Histological staining: Mouse lung tissue sections were taken and stained with hematoxylin-eosin (to assess lung tissue structure) and Masson's trichrome (to detect collagen deposition), respectively. Quantitative analysis was performed using ImageJ software.

[0045] Immunohistochemistry: After incubating paraffin sections of lung tissue with primary antibody, horseradish peroxidase-labeled secondary antibody and diaminobenzidine chromogenic agent were added for color development. The expression of α-smooth muscle actin, type I collagen, and connective tissue growth factor was detected, and the average optical density value was quantified using ImageJ software.

[0046] Lung index and wet-to-dry weight ratio of lung tissue: Mouse lung tissue was taken, rinsed twice with physiological saline, dissected, surface moisture was blotted with filter paper, and weighed to calculate the lung index (lung index = (lung weight / body weight) × 100%). The middle lobe of the lung was taken and weighed immediately to obtain the wet weight, placed in a 60℃ oven for 72 hours to constant weight, and weighed to obtain the dry weight. The wet-to-dry weight ratio was calculated (wet-to-dry weight ratio = wet weight / dry weight).

[0047] Statistical analysis: Experimental data are expressed as mean ± standard deviation. Independent samples t-test or one-way ANOVA combined with Durkheim post-hoc test was used for inter-group comparisons. Log-rank test was used to analyze mouse survival rate. P < 0.05 was considered statistically significant.

[0048] Example 1: Isolation, culture and identification of fetal placental mesenchymal stem cells

[0049] With the approval of the Institutional Ethics Committee of Ningxia Medical University General Hospital (Approval No.: NXMU-2023-IRB-012) and informed consent from the donors, placental tissue was collected from full-term healthy women undergoing elective cesarean section. Placental tissue from the fetal side was digested with collagenase A and deoxyribonuclease I, filtered, centrifuged, and the cell pellet was collected. The cell pellet was resuspended in mesenchymal stem cell culture medium containing glutamine supplementation and gentamicin, and cultured adherently in a 37℃, 5% CO2 incubator. Cells from passages 3 to 5 were used in the experiment.

[0050] Fourth-generation fetal placental mesenchymal stem cells were collected for identification. For example... Figure 1 The results shown in Figure A indicate that the cells exhibit the typical spindle-shaped morphology of mesenchymal stem cells. Flow cytometry analysis confirmed this. Figure 1 B), cells express classic mesenchymal stem cell surface markers such as CD73, CD90, and CD105, but do not express hematopoietic markers such as CD14, CD34, CD45, and human leukocyte antigen-DR. After culturing for 21 days in adipogenic, osteogenic, and chondrogenic induction differentiation media, they were positive for Oil Red O, Alizarin Red, and Alcian Blue staining, respectively. Figure 1 (C) This confirms that the cells possess trilineage differentiation capacity. The above results indicate that the obtained cells are fetal placental mesenchymal stem cells that meet the standards of the International Cell Therapy Association.

[0051] Example 2: Transforming growth factor-β1 pretreatment enhances the in vitro anti-fibrotic function of fetal placental mesenchymal stem cells.

[0052] Fourth-generation fetal placental mesenchymal stem cells were pretreated with 50 ng / mL human recombinant transforming growth factor-β1 for 24 hours, while an untreated control group was set up. Human lung fibroblasts MRC-5 were stimulated with 5 ng / mL transforming growth factor-β1 for 24 hours to construct a fibrotic phenotype cell model.

[0053] The antifibrotic function of pretreated cells was detected using a Transwell co-culture system: 5 × 10⁶ cells were cultured in the Transwell co-culture system. 4 The above-mentioned MRC-5 cells were seeded in the lower chamber of a 24-well Transwell chamber, with 5 × 10⁶ cells per cell. 4 Fetal placental mesenchymal stem cells, pretreated or untreated with transforming growth factor-β1, were seeded in the upper chamber, and co-culture was initiated by adding 5 ng / mL of transforming growth factor-β1 to the lower chamber. MRC-5 cells from the lower chamber were collected for analysis after 24 hours.

[0054] like Figure 2 As shown in Figure A, Western blotting results indicated that, compared to the untreated cell co-culture group, the expression levels of key fibrosis markers type I collagen, α-smooth muscle actin, and connective tissue growth factor were significantly downregulated in MRC-5 cells co-cultured with fetal placental mesenchymal stem cells pretreated with transforming growth factor-β1. Scratch healing assay results showed ( Figure 2 (B) Pretreatment of fetal placental mesenchymal stem cells significantly inhibited the migration ability of MRC-5 fibroblasts, and the scratch healing rate at 48 hours decreased by 62% compared with the control group (P<0.01). This confirms that pretreatment with transforming growth factor-β1 can significantly enhance the ability of fetal placental mesenchymal stem cells to inhibit fibroblast activation and migration in vitro.

[0055] Example 3: Pretreatment with transforming growth factor-β1 upregulates growth differentiation factor 15 and downregulates RUNX1 expression

[0056] Fourth-generation fetal placental mesenchymal stem cells were collected and treated with transforming growth factor-β1 at gradient concentrations of 0, 10, 25, and 50 ng / mL for 48 hours. Cells and culture supernatants were collected and subjected to the following assays.

[0057] Messenger RNA sequencing analysis revealed that after treatment with transforming growth factor-β1, 146 significantly upregulated genes and 159 significantly downregulated genes were identified in fetal placental mesenchymal stem cells (fold change > 1.2, P < 0.05). Growth differentiation factor 15 was one of the most significantly upregulated genes (logarithmic fold change ≈ 2.8) and was significantly enriched in extracellular regions related to gene ontology function annotation. Real-time quantitative polymerase chain reaction and Western blotting experiments further validated that transforming growth factor-β1 induced a concentration-dependent increase in the mRNA of growth differentiation factor 15 in fetal placental mesenchymal stem cells (…). Figure 3 B) and protein expression ( Figure 3 A) Upregulation. Enzyme-linked immunosorbent assay (ELISA) of cell supernatant confirmed ( Figure 3 C), the secretion of growth differentiation factor 15 was also upregulated in a concentration-dependent manner.

[0058] To screen upstream regulators of growth differentiation factor 15 (GGF15), this invention utilized the JASPAR database for bioinformatics analysis to predict transcription factors that may target and regulate GGF15. The intersection of these predictions with downregulated transcription factors in fetal placental mesenchymal stem cells treated with transforming growth factor-β1 revealed RUNX1 as a potential key inhibitor. Messenger RNA sequencing confirmed that RUNX1 expression was significantly downregulated after transforming growth factor-β1 treatment; Western blotting (WPBSA) further confirmed this. Figure 4 A) and immunofluorescence assay ( Figure 4 B) Further verification showed that the RUNX1 protein level decreased in a dependent manner with increasing transforming growth factor-β1 concentration, reaching its lowest expression level at a concentration of 50 ng / mL. These results indicate that transforming growth factor-β1 can downregulate the expression of the transcriptional repressor RUNX1 in fetal placental mesenchymal stem cells.

[0059] The above results indicate that transforming growth factor-β1 is a potent positive regulator of the expression and paracrine release of growth differentiation factor 15 in fetal placental mesenchymal stem cells, and can also downregulate the expression of the transcriptional repressor RUNX1.

[0060] Example 4: Growth differentiation factor 15 is a key effector molecule in pretreatment that enhances anti-fibrotic function.

[0061] Referring to the Transwell co-culture system of Example 2, a growth differentiation factor 15-specific neutralizing antibody was added to the co-culture system to block its function. The results showed that after adding the growth differentiation factor 15 neutralizing antibody, the inhibitory effect of fetal placental mesenchymal stem cells pretreated with transforming growth factor-β1 on the expression of type I collagen, α-smooth muscle actin, and connective tissue growth factor in MRC-5 cells was effectively reversed. Figure 5 A), its ability to inhibit MRC-5 cell migration is also lost ( Figure 5 B). This clearly confirms that growth differentiation factor 15 is a key paracrine mediator for the enhanced anti-fibrotic effect of fetal placental mesenchymal stem cells pretreated with transforming growth factor-β1.

[0062] Example 5: RUNX1 directly inhibits the expression of growth differentiation factor 15 in fetal placental mesenchymal stem cells.

[0063] This invention further verifies the direct regulatory effect of RUNX1 on the transcription of growth differentiation factor 15. Nuclease-targeted cleavage and release assays showed that RUNX1 was significantly enriched in the promoter region of the growth differentiation factor 15 gene in fetal placental mesenchymal stem cells. To verify the function of this interaction, RUNX1 was overexpressed in fetal placental mesenchymal stem cells. The results showed that RUNX1 overexpression significantly reduced basal state and transforming growth factor-β1-induced secretion of growth differentiation factor 15 in cells. Figure 6 A) and protein expression levels ( Figure 6 B). The above results confirm that RUNX1 can directly bind to the promoter of growth differentiation factor 15 and is a transcriptional repressor of growth differentiation factor 15 in fetal placental mesenchymal stem cells.

[0064] Example 6: Transforming growth factor-β1 pretreatment enhances the in vivo anti-fibrotic function of mesenchymal stem cells.

[0065] A bleomycin-induced mouse model of pulmonary fibrosis was established: 6-8 week old female C57BL / 6 mice were randomly divided into four groups (n=8 per group): a blank control group (saline), a bleomycin model group (2 mg / kg bleomycin administered intratracheally), a treatment group (fetal placental mesenchymal stem cells pretreated with 50 ng / mL transforming growth factor-β1 for 24 hours via tail vein injection on day 9 after bleomycin modeling), and a validation group (fetal placental mesenchymal stem cells overexpressing RUNX1 and pretreated in the same manner via tail vein injection on day 9 after bleomycin modeling). The mice were observed for 28 days.

[0066] The results are as follows Figure 7 As shown in Figures A and B, lung organ photographs and histological staining results (hematoxylin-eosin staining and Masson's trichrome staining) indicate that infusion of fetal placental mesenchymal stem cells pretreated with transforming growth factor-β1 significantly improved bleomycin-induced lung injury in mice, manifested as improved lung tissue structure, reduced inflammatory infiltration, decreased collagen deposition (Mason's trichrome staining results), decreased lung index, and alleviated pulmonary edema (reduced wet-to-dry weight ratio), with a significantly improved mouse survival rate (approximately 40% in the bleomycin group vs. approximately 80% in the pretreated cell group). Conversely, infusion of fetal placental mesenchymal stem cells overexpressing RUNX1 failed to exert the above protective effects, and the pathological and physiological indicators of mouse lung tissue recovered to levels comparable to those in the bleomycin group.

[0067] The above in vivo experimental results strongly confirm that transforming growth factor-β1 pretreatment can significantly enhance the function of fetal placental mesenchymal stem cells in the treatment of pulmonary fibrosis in vivo, and this enhancement effect is achieved by downregulating RUNX1 and relieving its transcriptional repression of growth differentiation factor 15.

[0068] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for enhancing the anti-fibrotic function of mesenchymal stem cells, characterized in that, This includes pretreating mesenchymal stem cells with transforming growth factor-β1 to obtain pretreated mesenchymal stem cells with enhanced anti-fibrotic function.

2. The method according to claim 1, characterized in that, The mesenchymal stem cells are selected from placental-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or induced pluripotent stem cell-derived mesenchymal stem cells. Preferably, the mesenchymal stem cells are placental-derived mesenchymal stem cells, and more preferably fetal-placental-derived mesenchymal stem cells.

3. The method according to claim 1, characterized in that, The pretreatment concentration of transforming growth factor-β1 was 1-100 ng / mL, and the pretreatment time was 12-72 hours.

4. The method according to any one of claims 1-3, characterized in that, The pretreatment upregulated the expression and / or secretion of growth differentiation factor 15 in the mesenchymal stem cells.

5. A pretreated mesenchymal stem cell prepared by the method according to any one of claims 1-4.

6. The mesenchymal stem cells according to claim 5, characterized in that, The expression and secretion levels of growth differentiation factor 15 in the mesenchymal stem cells were upregulated compared to untreated mesenchymal stem cells.

7. Application of transforming growth factor-β1 in the preparation of pretreatment reagents for enhancing the anti-fibrotic function of mesenchymal stem cells.

8. A pharmaceutical composition, characterized in that, It contains a therapeutically effective amount of the pretreated mesenchymal stem cells according to claim 5, and a pharmaceutically acceptable carrier.

9. Use of the pretreated mesenchymal stem cells according to claim 5 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating fibrotic diseases.

10. The use according to claim 9, characterized in that, The fibrotic disease mentioned is pulmonary fibrosis.

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