Factor combination for improving Anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing risk of pulmonary embolism, and use thereof

Abstract

Disclosed are a factor composition for improving the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary embolism, and use thereof. The factor composition comprises (I) interferon-γ, the action concentration of which is 0.5-5 ng / mL, and (II) interleukin-4 and / or tumor necrosis factor α, the action concentration of interleukin-4 being 1-20 ng / mL and the action concentration of tumor necrosis factor α being 1-25 ng / mL.

Description

Factor combinations and their applications for enhancing the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary thrombosis.

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese patent application No. 202510110931.5, filed on January 23, 2025, entitled "A combination of factors for improving the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary thrombosis and its application".

[0003] Priority is given to Chinese patent application No. 202511684765.6 filed with the China National Intellectual Property Administration on November 17, 2025, entitled “A combination of factors for improving the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary thrombosis and its application”, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This application relates to the field of biotechnology, and in particular to a combination of factors for enhancing the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary thrombosis, and their application. Background Technology

[0005] The following statements are provided only as background information in connection with this application and do not necessarily constitute prior art.

[0006] Mesenchymal stem cells (MSCs) are receiving increasing attention in the treatment of pulmonary fibrosis, a disease characterized by lung scarring and dysfunction, often leading to shortness of breath and a decline in quality of life. MSCs can secrete various cytokines and growth factors, promoting lung tissue repair and regeneration. Simultaneously, MSCs can regulate immune responses, which is also crucial for mitigating the progression of pulmonary fibrosis. Furthermore, MSCs can stimulate the regeneration of lung epithelial cells, inhibit the activation and transformation of lung epithelial cells and fibroblasts into fibroblasts, thereby reducing collagen deposition and improving alveolar structure and function. Due to their immunomodulatory and regenerative capabilities, MSCs have been used in numerous trials to treat various lung diseases. However, CD142 (tissue factor, TF) expression in MSCs may have some potential negative effects, such as increasing the risk of thrombosis, thus exacerbating the pathological condition of lung diseases.

[0007] In view of the above, this application is hereby submitted. Summary of the Invention

[0008] The purpose of this application is to provide a factor composition for improving the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary thrombosis, and its application, so as to alleviate the above-mentioned technical problems.

[0009] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0010] In a first aspect, a factor composition is provided for enhancing the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary thrombosis, the factor composition comprising (I) and (II);

[0011] (I) Interferon γ, wherein the effective concentration of interferon γ is 0.5–5 ng / mL;

[0012] (II) Interleukin-4 and / or tumor necrosis factor-α, wherein the concentration of interleukin-4 is 1 to 20 ng / mL and the concentration of tumor necrosis factor-α is 1 to 25 ng / mL.

[0013] In a second aspect, the use of the factor composition described in the first aspect in any one of (i) to (v):

[0014] (i) Enhancing the anti-pulmonary fibrosis ability of mesenchymal stem cells for non-disease treatment purposes, and / or reducing the risk of pulmonary embolism caused by mesenchymal stem cells for non-disease treatment purposes.

[0015] (ii) Prepare products for improving the anti-pulmonary fibrosis ability of mesenchymal stem cells and / or reducing the risk of pulmonary embolism caused by mesenchymal stem cells;

[0016] (iii) Enhance the ability of mesenchymal stem cells to promote Treg cell proliferation;

[0017] (iv) Prepare products to enhance the ability of mesenchymal stem cells to promote the proliferation of Treg cells;

[0018] (v) Prepare a culture medium for culturing mesenchymal stem cells;

[0019] (vi) Increase the expression of at least one of indoleamine-2,3-dioxygenase, hepatocyte growth factor, and PD-L1 in mesenchymal stem cells;

[0020] (vii) Prepare a reagent for increasing the expression of at least one of indoleamine-2,3-dioxygenase, hepatocyte growth factor and PD-L1 in mesenchymal stem cells;

[0021] (ⅷ) Reduce the expression of CD142, a molecule on the surface of mesenchymal stem cells;

[0022] (ⅸ) Prepare a reagent for reducing the expression of CD142, a surface molecule of mesenchymal stem cells.

[0023] Thirdly, a mesenchymal stem cell culture medium is provided, which comprises a culture medium and the factor composition described in the first aspect.

[0024] Fourthly, a method for processing mesenchymal stem cells is also provided, the method comprising contacting the mesenchymal stem cells with the factor composition described in the first aspect.

[0025] Fifthly, a pretreated mesenchymal stem cell is also provided, wherein the pretreated mesenchymal stem cell is a mesenchymal stem cell obtained by the treatment method described in the fourth aspect.

[0026] In a sixth aspect, a pharmaceutical composition is provided comprising the pretreated mesenchymal stem cells described in the fifth aspect.

[0027] Compared with the prior art, this application has the following beneficial effects:

[0028] Studies have found that treating mesenchymal stem cells with the factor composition provided in this application can enhance the anti-pulmonary fibrosis ability of mesenchymal stem cells through the synergistic effect of various factors, regulate the expression of CD142 in MSCs, reduce the risk of pulmonary thrombosis of mesenchymal stem cells, and does not promote the occurrence of inflammation. This helps to improve the treatment effect and prognosis of patients with lung diseases and provides new ideas for the clinical application of MSCs in lung diseases. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 shows the IDO content in the culture supernatant after treating hUC-MSCs with different IFN-γ concentrations and treatment times in Example 1.

[0031] Figure 2 shows the IDO content in the culture supernatant of hUC-MSCs after treatment with different concentrations of IL-4 combined with IFN-γ in Example 2.

[0032] Figure 3 shows the expression level of α-SMA in MRC-5 cells, an in vitro lung fibrosis model, after treatment with different concentrations of IL-4 combined with IFN-γ in Example 2.

[0033] Figure 4 shows the expression level of COL1A1 in MRC-5 cells, an in vitro lung fibrosis model, after treating hUC-MSCs with different concentrations of TNF-α combined with IFN-γ / IFN-γ+IL-4 in Example 3.

[0034] Figure 5 shows the HGF content in the culture supernatant after hUC-MSCs were treated with IFN-γ in combination with other cytokines in Example 4;

[0035] Figure 6 shows the IDO content in the culture supernatant after hUC-MSCs were treated with IFN-γ in combination with other cytokines in Example 4;

[0036] Figure 7 shows the IL-1β content in the culture supernatant of RAW264.7 macrophages co-cultured after treatment with different combinations of factors for hUC-MSCs in Example 5.

[0037] Figure 8 shows the TNF-α content in the culture supernatant of RAW264.7 macrophages co-cultured after treatment with different combinations of factors for hUC-MSCs in Example 5.

[0038] Figure 9 shows the flow cytometry results of surface markers of A hUC-MSC in the experimental group of Example 6;

[0039] Figure 10 shows the flow cytometry results of surface markers of D hUC-MSCs in the experimental group in Example 6;

[0040] Figure 11 shows the flow cytometry results of the surface markers of F hUC-MSC in the experimental group of Example 6;

[0041] Figure 12 shows the flow cytometry results of the surface markers of G hUC-MSC in the experimental group in Example 6;

[0042] Figure 13 shows the flow cytometry results of hUC-MSC surface immune-related markers (PD-L1) in different factor treatments in Example 7;

[0043] Figure 14 shows the ELISA detection results of culture supernatant IDO after different factor treatments of hUC-MSC in Example 7;

[0044] Figure 15 shows the flow cytometry results of hUC-MSC surface tissue factor (CD142) after different factor treatments in Example 8.

[0045] Figure 16 shows the expression level of α-SMA in co-cultured lung fibroblasts (MRC-5) after different combinations of factors were treated with hUC-MSCs in Example 9.

[0046] Figure 17 shows the expression level of α-SMA in co-cultured lung epithelial cells (A549) after treatment with different combinations of factors for hUC-MSCs in Example 9.

[0047] Figure 18 shows the mRNA levels of the hUC-MSC surface immune-related marker (PD-L1) after different factor treatments in Example 10.

[0048] Figure 19 shows the ELISA detection results of culture supernatant IDO after different factor treatments of hUC-MSC in Example 10;

[0049] Figure 20 shows the α-SMA expression level in model cells co-cultured after treatment with different factor combinations of hUC-MSCs in Example 10;

[0050] Figure 21 shows the COL1A1 expression level in co-cultured model cells after treatment with different combinations of factors with hUC-MSCs in Example 10.

[0051] Figure 22 shows the mRNA levels of tissue factor (CD142) on the surface of hUC-MSCs after different factor treatments in Example 10.

[0052] Figure 23 shows the flow cytometry results of Treg cell proliferation in PBMC cells after co-culturing hUC-MSCs with PBMCs treated with different factors in Example 11.

[0053] Figure 24 shows the flow cytometry results of T cell proliferation in PBMC cells after co-culturing hUC-MSCs with PBMCs treated with different factors in Example 11.

[0054] Figure 25 shows the flow cytometry results of Th1 cell proliferation in PBMC cells after co-culturing hUC-MSCs with PBMCs treated with different factors in Example 11. Detailed Implementation

[0055] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0056] In this document, unless otherwise stated, any numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order or importance between these entities or behaviors, such as numbers (I), (II); or (i), (ii)...(eight).

[0057] In this document, the term “decrease” may be used interchangeably with “inhibit,” “reduce,” “silence,” “downregulate,” “repress,” and other similar terms, and includes any level of reduction, such as transcriptional levels or protein expression levels. The term “increase” may be used interchangeably with “promote,” “increase,” “upregulate,” and other similar terms, and includes any level of increase, such as transcriptional levels (mRNA content) or protein expression levels (protein content). Decrease and increase can be assessed by a reduction or increase in one or more of these variables compared to a control level. This control level can be any type of control level used in the art, such as baseline levels before administration or levels determined from untreated or controlled (e.g., buffer-only control or inert agent control) subjects, cells, or samples.

[0058] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0059] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0060] In this document, mesenchymal stem cells can be mesenchymal stem cells from any source known in the art, including but not limited to bone marrow, umbilical cord, adipose tissue, placenta, and other tissues. In optional embodiments, mesenchymal stem cells include umbilical cord mesenchymal stem cells. Mesenchymal stem cells can be mesenchymal stem cells derived from humans or other mammals. Such mammals include, for example, humans, monkeys, mice, rats, rabbits, donkeys, cattle, horses, pigs, or dogs.

[0061] In this document, the terms “patient,” “subject,” or “individual” are used interchangeably and include human or non-human animals, or cells or tissues derived from human or non-human animals, such as human, monkey, mouse, rat, rabbit, donkey, cow, horse, pig, or dog.

[0062] In a first aspect, a factor composition is provided for enhancing the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary thrombosis, the factor composition comprising (I) and (II);

[0063] (I) Interferon γ, wherein the effective concentration of interferon γ is 0.5 to 5 ng / mL, for example, but not limited to 0.5, 1, 2, 3, 4 or 5 ng / mL;

[0064] (II) Interleukin-4 and / or tumor necrosis factor-α, wherein the effective concentration of interleukin-4 is 1 to 20 ng / mL, for example, but not limited to 1, 2, 5, 8, 10, 12, 15, 18 or 20 ng / mL; and the effective concentration of tumor necrosis factor-α is 1 to 25 ng / mL, for example, but not limited to 1, 2, 5, 8, 10, 12, 15, 17, 20, 22 or 25 ng / mL.

[0065] In an optional embodiment, the factor composition comprises interferon γ, interleukin 4, and tumor necrosis factor α.

[0066] In an optional embodiment, the effective concentration of interferon γ in the factor composition is 5 ng / mL.

[0067] In an optional embodiment, the effective concentration of interleukin-4 in the factor composition is 20 ng / mL.

[0068] In an optional embodiment, the effective concentration of the tumor necrosis factor α in the factor composition is 1 ng / mL.

[0069] In an optional embodiment, the factor composition comprises interferon γ, interleukin 4 and tumor necrosis factor α, wherein the effective concentration of interferon γ is 5 ng / mL, the effective concentration of interleukin 4 is 20 ng / mL, and the effective concentration of tumor necrosis factor α is 1 ng / mL.

[0070] Further optimization and adjustment of the concentrations of interferon-γ, interleukin-4, and tumor necrosis factor-α in the factor composition have improved its ability to improve mesenchymal stem cells.

[0071] Secondly, the application of the factor composition of the first aspect in any one of (i) to (v):

[0072] (i) Enhancing the anti-pulmonary fibrosis ability of mesenchymal stem cells for non-disease treatment purposes, and / or reducing the risk of pulmonary embolism caused by mesenchymal stem cells for non-disease treatment purposes.

[0073] (ii) Prepare products for improving the anti-pulmonary fibrosis ability of mesenchymal stem cells and / or reducing the risk of pulmonary embolism caused by mesenchymal stem cells;

[0074] (iii) Enhance the ability of mesenchymal stem cells to promote Treg cell proliferation;

[0075] (iv) Prepare products to enhance the ability of mesenchymal stem cells to promote the proliferation of Treg cells;

[0076] (v) Prepare a culture medium for culturing mesenchymal stem cells;

[0077] (vi) Increase the expression of at least one of indoleamine-2,3-dioxygenase (IDO), hepatocyte growth factor (HGF) and PD-L1 in mesenchymal stem cells;

[0078] (vii) Prepare a reagent for increasing the expression of at least one of indoleamine-2,3-dioxygenase (IDO), hepatocyte growth factor (HGF) and PD-L1 in mesenchymal stem cells;

[0079] (ⅷ) Reduce the expression of CD142, a molecule on the surface of mesenchymal stem cells;

[0080] (ⅸ) Prepare a reagent for reducing the expression of CD142, a surface molecule of mesenchymal stem cells.

[0081] In an optional implementation, (i), (iii), (vi) and (vii) in the above applications are independently for non-diagnostic and non-therapeutic purposes.

[0082] In an optional implementation, the mesenchymal stem cells in any one of (i) to (e) include umbilical cord mesenchymal stem cells.

[0083] Thirdly, a mesenchymal stem cell culture medium is provided, the mesenchymal stem cell culture medium comprising a culture medium and the factor composition described in the first aspect.

[0084] In an optional embodiment, the mesenchymal stem cell culture medium is an umbilical cord mesenchymal stem cell culture medium.

[0085] Using this culture medium to culture mesenchymal stem cells can enhance the anti-pulmonary fibrosis ability of umbilical cord mesenchymal stem cells and reduce the risk of pulmonary embolism.

[0086] It should be noted that this application does not impose specific restrictions on the culture medium; any culture medium known to those skilled in the art that can be used for mesenchymal stem cell culture may be used.

[0087] Fourthly, a method for processing mesenchymal stem cells is provided, the method comprising contacting the mesenchymal stem cells with the factor composition described in the first aspect. The mesenchymal stem cells obtained by this method exhibit strong anti-pulmonary fibrosis ability and a low risk of pulmonary embolism.

[0088] In an optional implementation, the processing time in the processing method is 2 to 72 hours, for example, but not limited to 2 hours, 6 hours, 24 hours, 36 hours or 72 hours, preferably 24 hours.

[0089] In an optional embodiment, the treatment method includes contacting mesenchymal stem cells with the factor composition in a culture medium or buffer solution.

[0090] In an optional embodiment, the treatment method includes bringing the factor composition to an effective concentration in a solvent, and placing the mesenchymal stem cells in the solvent to achieve sufficient contact between the mesenchymal stem cells and the factor composition. Optionally, the solvent includes a culture medium or a buffer solution.

[0091] In an optional embodiment, the processing method includes culturing mesenchymal stem cells in the culture medium described in the third aspect to obtain processed mesenchymal stem cells.

[0092] In an optional embodiment, the mesenchymal stem cells include umbilical cord mesenchymal stem cells.

[0093] Fifthly, a pretreated mesenchymal stem cell is provided, wherein the pretreated mesenchymal stem cell is obtained using the treatment method described in the fourth aspect. This pretreated mesenchymal stem cell exhibits strong anti-pulmonary fibrosis ability and a low risk of pulmonary embolism.

[0094] In an optional embodiment, the mesenchymal stem cells include umbilical cord mesenchymal stem cells.

[0095] In a sixth aspect, a pharmaceutical composition is provided comprising the pretreated mesenchymal stem cells described in the fifth aspect. This pharmaceutical composition exhibits strong anti-pulmonary fibrosis activity and a low risk of pulmonary embolism.

[0096] In an optional embodiment, the mesenchymal stem cells include umbilical cord mesenchymal stem cells.

[0097] In an optional embodiment, the pharmaceutical composition further comprises the factor composition described in the first aspect, or the mesenchymal stem cell culture medium described in the third aspect; and in the pharmaceutical composition, the pretreated mesenchymal stem cells are in continuous contact with the factor composition, that is, when the pharmaceutical composition is administered to a subject, the mesenchymal stem cells in the pharmaceutical composition are administered in an environment in which the factor composition is present.

[0098] In an optional embodiment, the pharmaceutical composition comprises isolated pretreated mesenchymal stem cells, and the pharmaceutical composition does not contain the factor composition described in the first aspect, or does not contain the mesenchymal stem cell culture medium described in the third aspect.

[0099] In an optional embodiment, the pharmaceutical composition may further include pharmaceutically acceptable optional excipients, including but not limited to one or more of solvents, culture media, cryopreservation solutions, serum substitutes, extracellular matrix, antibiotics, and buffer components.

[0100] In an optional embodiment, the mesenchymal stem cells in any of the above embodiments include human mesenchymal stem cells.

[0101] The present application is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present application in any way.

[0102] In the following examples, the positive markers on the MSC surface include CD90, CD105, and CD73, and the negative markers include CD45, CD34, CD11b, CD19, and HLA-DR.

[0103] In the following examples, all factors were lyophilized recombinant proteins. After high-speed centrifugation, they were dissolved in sterile D-PBS, mixed by pipetting, and prepared as stock solutions, which were then aliquoted. Before use, they needed to be diluted with D-PBS and added to the culture medium to ensure their effective concentration conformed to the experimental design (Table 1).

[0104] Table 1. Factors and Working Concentrations

[0105] Example 1. Comparison of IFN-γ concentration and time of action

[0106] hUC-MSCs were revived in a water bath at 37–38°C, washed and resuspended in MEM medium supplemented with 5% human platelet lysate and 1% glutamine, and an appropriate amount of cell suspension was used for AO / PI counting. Complete medium was used according to the experimental grouping information (Table 2) and the counting results, at a concentration of 6000–8000 cells / cm³. 2 Cells were seeded in 6-well plates, and IFN-γ of corresponding concentrations was added to the experimental groups. Supernatants were collected after treatment for 2 h, 6 h, 24 h, and 72 h. The content of indoleamine-2,3-dioxygenase (IDO) secreted by MSCs was detected using an ELISA kit to assess the enhancement of MSC immunosuppressive capacity by different factor treatments.

[0107] Table 2. Experimental group information for the study of IFNγ concentration and treatment time

[0108] The results of this embodiment show that IDO secretion by MSCs increases with increasing IFN-γ concentration in a dose-dependent manner. Compared with untreated natural MSCs, the IDO secretion of MSCs was significantly increased after treatment with IFN-γ concentrations of 0.5–5 ng / mL for 2 h–72 h (Figure 1).

[0109] Example 2. Comparison of IL-4 concentration and time of action

[0110] hUC-MSC cells were divided into 6×10 4 / cm 2The cells were seeded into 6-well plates, and corresponding concentrations of IFN-γ and IL-4 were added sequentially (Table 3). After co-culturing for 24 hours, the supernatant was collected, and the IDO content secreted by MSCs was detected using an ELISA kit to assess the enhancement of the immunosuppressive capacity of MSCs by different factor treatments.

[0111] Table 3. Experimental group information for the study of IL-4 concentration of action (Part 1)

[0112] An in vitro pulmonary fibrosis model was established using mouse embryonic fibroblasts MRC-5, and the optimal concentration of IL-4 in combination with IFN-γ was further investigated in this model (Table 4). Specifically, the experiment was conducted using Transwell chambers for cell co-culture. MRC-5 cells were cultured in complete medium at a concentration of 2 × 10⁶ cells / mL. 5 ~3×10 5 hUC-MSCs were seeded per well in the lower chamber of a Transwell 6-well plate and starved for 4–8 h. The medium was then replaced with DMEM containing 1% FBS, and 5 ng / mL TGF-β1 was added for 24 h to induce pulmonary fibrosis. The control group did not receive TGF-β1. hUC-MSCs were seeded at the same density in the upper chamber of a Transwell 6-well plate and co-cultured for 24 h. Cells from the lower chamber were collected for RNA extraction. qPCR was used to detect α-smooth muscle actin (α-SMA), a gene crucial for the development and progression of pulmonary fibrosis, to evaluate the effect of different concentrations of IL-4 combined with IFN-γ on the anti-pulmonary fibrosis ability of MSCs.

[0113] Table 4. Experimental Group Information for the Study of IL-4 Effective Concentration (Part Two)

[0114] The results showed that IL-4 concentrations in the range of 1–20 ng / ml significantly increased IDO secretion in MSCs (Figure 2), and had no significant difference in effect on the anti-fibrotic ability of MSCs, meaning that both could significantly inhibit the activation of lung fibroblasts (Figure 3).

[0115] Example 3. Comparison of TNF-α concentration and time of action

[0116] An in vitro pulmonary fibrosis model was established using mouse embryonic fibroblasts MRC-5, and the optimal concentration of TNF-α in combination with IL-4 and / or IFN-γ was investigated in this model (Table 5).

[0117] The establishment of an in vitro pulmonary fibrosis model and the treatment method using MSCs were described in Example 2. After treatment, RNA was extracted from lower ventricular cells, and qPCR was used to detect collagen (COL1A1) to evaluate the ability of MSCs treated with different concentrations of TNF-α combined with IFN-γ / IFN-γ+IL-4 to enhance their anti-pulmonary fibrosis ability, thereby screening for the optimal TNF-α concentration. In fibrotic diseases, fibroblasts transform into myofibroblasts, and the proliferation and activation of myofibroblasts promote excessive collagen synthesis, leading to fibrosis. Therefore, the excessive synthesis of myofibroblasts and collagen plays a crucial role in fibrosis in multiple organs such as the lungs, liver, and heart, making it an important target for research on fibrotic diseases.

[0118] Table 5. Experimental grouping information for the study of TNF-α concentration.

[0119] The results showed that compared with natural MSCs, MSCs pretreated with IFN-γ combined with different concentrations of TNF-α, or pretreated with IFN-γ and IL-4 combined with different concentrations of TNF-α, had a better inhibitory effect on the activation of lung fibroblasts. That is, the anti-fibrotic ability of MSCs was significantly enhanced in the range of 1 to 25 ng / mL of TNF-α, and the efficacy was improved to some extent with the increase of the concentration used (Figure 4).

[0120] Example 4. Combination of IFNγ with other factors

[0121] To explore a richer and more effective combination of factors, this embodiment screened for factors other than IL-4 and TNF-α, such as IL-13, TGF-β1, and IL-6, focusing on IFN-γ. After resuscitation, washing, and resuspending in complete culture medium, hUC-MSCs were counted at 6000–8000 cells / cm³ according to AOPI results. 2 Cells were seeded in 6-well plates, and the various factors were added according to Table 6. After gentle shaking to mix, the plates were incubated for 24 hours. After 24 hours of incubation, the supernatant was collected, and the levels of IDO and hepatocyte growth factor (HGF) were detected by ELISA (Table 6). HGF is a pleiotropic factor with significant biological effects in enhancing lung tissue development, stimulating lung epithelial cell generation, repairing lung microvascular endothelial cells, inhibiting apoptosis caused by lung injury, and reducing the accumulation of myofibroblasts.

[0122] Table 6. Experimental grouping information for studies involving combinations of IFNγ and other factors

[0123] The results, as shown in Figures 5 and 6, indicate that treatment of MSCs with IL-13, TGF-β1, IL-6, and IFN-γ, respectively, can all increase the levels of HGF and IDO secreted by MSCs to varying degrees. When IFN-γ is used in combination with IL-13 and TGF-β1 individually, the effect on increasing HGF secretion by MSCs is significantly lower than that of IFN-γ and IL-4 combined. The effect of IFN-γ and IL-6 combined on increasing HGF secretion by MSCs is slightly weaker than that of IFN-γ and IL-4 combined. Regarding the effect on increasing IDO secretion by MSCs, the effect of IFN-γ combined with other factors is significantly lower than that of IFN-γ and IL-4 combined. These results demonstrate that the effect of IFN-γ and IL-4 combined is significantly better than that of IFN-γ combined with other factors.

[0124] Example 5. Pretreatment of the selected factors does not increase the risk of pro-inflammatory inflammation.

[0125] An in vitro inflammation model was established using mouse mononuclear macrophage leukemia cells (RAW264.7) induced with 10 ng / mL lipopolysaccharide (LPS) for 24 h, and the cells were seeded in the lower chamber of a 12-well Transwell. The upper chamber was inoculated at a concentration of 25,000–30,000 cells / cm². 2 hUC-MSCs were inoculated and various factors were added sequentially (Table 7). After culturing in a cell culture incubator for 24 hours, the supernatant from the lower chamber was collected, and inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β were detected by ELISA to assess the regulatory capacity of MSCs on macrophage inflammatory responses.

[0126] Table 7. Experimental grouping information for the study of the anti-inflammatory effect of factor pretreatment on hUC-MSCs

[0127] In the inflammatory environment of the lungs, the expression of TNF-α and IL-1β increases significantly, inducing the activation and recruitment of other immune cells, further promoting the inflammatory response. The results of this embodiment show that pretreatment with the selected factors does not increase the pro-inflammatory risk of MSCs, and compared with natural MSCs, factor-pretreated MSCs exhibit better anti-inflammatory capabilities to some extent (Figures 7 and 8).

[0128] Example 6. Effect of factor pretreatment on hUC-MSC surface markers

[0129] hUC-MSC cells were resuscitated, washed, counted, and seeded into 6-well cell culture plates using complete culture medium. After 6–12 h of incubation, the medium was replaced with one-third human platelet lysate, and the designed concentrations of each factor / factor group were added (Table 8). Cells were then cultured for 24 h and harvested. Flow cytometry was then used to detect hUC-MSC positive markers (CD90, CD105, CD73) and negative markers (CD45, CD34, CD11b, CD19, HLA-DR).

[0130] Table 8. Experimental group information for the study on the effect of factor pretreatment on hUC-MSC.

[0131] The results of this experiment showed that the positive markers CD90, CD105, and CD73 of hUC-MSCs all reached over 98% in natural untreated MSCs, after pretreatment with different factors, and after pretreatment with different combinations of factors. Furthermore, the total expression of negative surface markers such as CD45, D34, CD11b, CD19, and HLA-DR did not exceed 1% (Table 9, Figures 9-12). This indicates that pretreatment of hUC-MSCs with different factors does not affect the expression of surface markers. CD90 participates in cell adhesion and signal transduction, indicating the stem cell characteristics of the cells; CD105 is related to cell proliferation and angiogenesis, indicating the characteristics and function of stem cells; CD73 has immunomodulatory effects and participates in cell metabolism and proliferation. Positive expression >95% indicates that the basic biological characteristics of MSCs are well maintained. Meanwhile, HLA-DR was almost not expressed. HLA-DR is a major histocompatibility complex (MHC) molecule related to immune responses; negative expression indicates that the treated MSCs still have low immunogenicity and are suitable for clinical use. Surface markers of hUC-MSCs are crucial for the identification of their characteristics and functions. According to the standards of the International Society for Stem Cell Research (ISCT), hUC-MSCs should express specific positive markers such as CD90, CD105, and CD73, but not negative markers such as CD45 (leukocyte marker), CD34 (marker of primitive hematopoietic progenitor cells and endothelial cells), CD11b (marker of monocytes and macrophages), CD19 (marker of B cells), HLA-DR, etc. These markers help to confirm the characteristics of hUC-MSCs and their safety and efficacy in clinical applications.

[0132] Table 9. Results of MSC surface marker dynamic testing

[0133] Example 7. Effects of factor pretreatment on PD-L1 expression and IDO secretion in hUC-MSCs

[0134] hUC-MSC cells were resuscitated, washed, counted, seeded, and pretreated with factors in the same manner as in Example 6 (Table 8). After factor pretreatment, the cell culture supernatant was collected, and the IDO content was detected by ELISA. The positivity rate of programmed death-ligand 1 (PD-L1) on the surface of hUC-MSCs was detected by flow cytometry.

[0135] The results of this embodiment show that, after pretreatment with different factors and different combinations of factors, the combined use of IFN-γ, IFN-γ and IL-4, IFN-γ and TNF-α, and IFN-γ, TNF-α and IL-4 can all significantly increase the expression of PD-L1, an immunosuppression-related surface marker of MSCs (Figure 13), and the expression of IDO, a cell secretion factor (Figure 14).

[0136] Factor pretreatment can enhance the immunomodulatory capacity of hUC-MSCs. PD-L1, by binding to the PD-1 receptor on T cells, can inhibit T cell activation and proliferation, thereby regulating the immune response. In pulmonary fibrosis, increased PD-L1 expression in MSCs helps suppress the inflammatory response, reduce lung tissue damage, and thus slow the progression of fibrosis. Meanwhile, IDO catalyzes tryptophan metabolism, and the resulting metabolites can inhibit T cell proliferation and induce the formation of regulatory T cells (Tregs), helping to reduce lung damage and fibrosis by regulating the immune response and suppressing inflammation.

[0137] Example 8. Effect of factor pretreatment on the expression of CD142 (tissue factor) on the surface of hUC-MSCs

[0138] hUC-MSC cell resuscitation, washing, counting, seeding, and factor pretreatment were performed in the same manner as in Example 6 (Table 8). Because the expression of CD142 (tissue factor) on the surface of hUC-MSCs may have some potential negative effects, such as increasing the risk of thrombosis and thus exacerbating the pathological condition of lung disease, after factor pretreatment, cells were harvested, and flow cytometry was used to detect CD142 expression to assess the reduction in embolic risk after factor pretreatment of MSCs.

[0139] The results of this embodiment show that, compared with natural untreated MSCs, the expression of CD142 on the surface of MSCs was significantly reduced after pretreatment with different factors and different combinations of factors (Figure 15), indicating a significant decrease in the risk of thrombosis and pulmonary embolism. The expression level of CD142 on the surface of MSCs is closely related to pulmonary embolism and thrombosis. CD142 mainly promotes coagulation by activating the extrinsic coagulation pathway, thus aggravating the pathological state of pulmonary embolism. High levels of CD142 expression may lead to stronger coagulation and inflammatory responses, affecting vascular endothelial function and tissue repair.

[0140] Example 9. Effects of factor pretreatment on the anti-pulmonary fibrosis ability of hUC-MSCs

[0141] In vitro pulmonary fibrosis models were constructed using human alveolar epithelial cells (A549) and human embryonic lung fibroblasts (MRC-5), respectively. Natural MSCs and factor-pretreated MSCs were then used to treat these models. The modeling procedures and treatment methods were the same as in Example 2. Experimental groups are shown in Table 10. Lower chamber cells were collected, RNA was extracted, and α-SMA gene expression was detected using qPCR.

[0142] Table 10. Effects of factor pretreatment on the anti-pulmonary fibrosis ability of hUC-MSCs. Experimental groups

[0143] The results of this embodiment show that, compared with the untreated MSC treatment group, factor-pretreated MSCs significantly reduced α-SMA expression in the fibrosis model (Figures 16 and 17). Increased α-SMA expression in fibrosis indicates that fibroblasts, epithelial cells, or other related cells are activated by the inflammatory environment and transform into myofibroblasts. The proliferation and activation of myofibroblasts promote excessive collagen synthesis, leading to fibrosis. The results of this embodiment show that factor pretreatment can inhibit the activation of lung epithelial cells and lung fibroblasts, thus exhibiting better anti-fibrotic ability.

[0144] Example 10. Study on the efficacy of factor pretreatment in treating hUC-MSCs

[0145] hUC-MSC cells were resuscitated, washed, counted, and seeded into 6-well plates. When cell confluence reached 85-90%, the original culture medium was discarded, and the cells were washed three times with basal medium. The medium was then replaced with 1% PL containing pretreatment factors, and the cells were cultured in a cell culture incubator for another 6 hours (Table 11). After 6 hours, the cell culture supernatant was collected, and IDO content was detected by ELISA. hUC-MSC cells were harvested, and RNA was extracted for qPCR detection of CD142 and PD-L1 mRNA levels.

[0146] In addition, hUC-MSC was prepared according to 1.2×10 5 Cells were seeded at a concentration of [number] cells / ml in the upper chamber of a Transwell culture medium and co-cultured for 6 h in medium containing the appropriate pretreatment factor and 1% PL (Table 11). After 6 h, the culture supernatant was discarded and replaced with complete medium. The lower chamber contained a fibrosis model established using MRC-5 lung fibroblasts. After 24 h of co-culture treatment, cells from the lower chamber were collected for RNA extraction, and qPCR was used to detect α-SMA and COL1A1 to evaluate the anti-pulmonary fibrosis ability.

[0147] Table 11. Experimental grouping information for the study on the effectiveness of factor pretreatment in hUC-MSC.

[0148] Experimental results:

[0149] 1. Factor pretreatment of hUC-MSCs has better immunomodulatory capacity. Experimental results showed that, after pretreatment with different factors and factor combinations, compared with natural untreated MSCs, the expression of the immunosuppression-related surface marker PD-L1 (Figure 18) and the expression of the cell secretion factor IDO (Figure 19) of hUC-MSCs were significantly increased.

[0150] 2. Factor-pretreated hUC-MSCs have better anti-fibrotic ability. Experimental results showed that compared with the natural untreated MSC treatment group, factor-pretreated MSCs significantly reduced the expression of α-SMA and COL1A1 in the fibrosis model and had better anti-fibrotic ability (Figures 20 and 21).

[0151] 3. Factor pretreatment of hUC-MSCs has a lower risk of pulmonary embolism. Experimental results showed that, compared with natural untreated MSCs, the expression of CD142 on the surface of MSCs after different factor pretreatment and different combinations of factor pretreatment was significantly reduced (Figure 22), that is, the risk of thrombosis and pulmonary embolism was significantly reduced.

[0152] Example 11. Effects of factor pretreatment on the immunomodulatory capacity of hUC-MSCs

[0153] hUC-MSC cells were resuscitated, washed, counted, and seeded into 12-well cell culture plates using complete culture medium. After culturing for 22–24 hours in a cell culture incubator, the medium was replaced with one-third human platelet lysate. Factors were added according to the treatment groups I and III in Table 11, and the cells were pretreated in the incubator for 6 hours. Then, 1×10⁻⁶ cells were added to each well. 6 One PBMC cell.

[0154] MSCs were added to PBMCs for Treg proliferation testing and co-cultured for 48 hours. Cell suspensions were collected and analyzed by flow cytometry. Four groups were established: a blank group, a live / dead group, an IgG isotype group, and a test group. The IgG group contained 2 μL IgG-APC, 2 μL IgG-FITC, and 1.3 μL IgG-PE, while the test group contained 3 μL CD4-APC, 3 μL CD25-FITC, and 3 μL CD127-PE. Cells were incubated at room temperature in the dark for 20-30 minutes, washed twice with flow cytometry buffer, and then loaded onto the flow cytometer. DAPI staining was added to all groups before loading to differentiate cell viability. The Treg proliferation promotion rate of MSCs was calculated as: (Treg proliferation rate in the test group - Treg proliferation rate in the negative control group) / (Treg proliferation rate in the negative control group) × 100%.

[0155] MSCs were added to the T-cell inhibition assay. Except for the negative control group, all other experimental groups were co-cultured with 5 μg / mL PMA stimulant for 4 days. Cell suspensions were then collected for flow cytometry analysis. The assay included a blank group, a Live / Dead group, an IgG isotype group, and a Test group. The IgG group received 2 μL of IgG-APC, and the Test group received 3 μL of CD3-APC. Cells were incubated at room temperature in the dark for 20-30 min, washed twice with flow cytometry buffer, and then loaded onto the flow cytometer. DAPI staining was added to all groups before loading to differentiate cell viability. The T-cell proliferation inhibition rate of MSCs was calculated as: (Proportion of daughter cells in the positive group - Proportion of daughter cells in the Tset group) / (Proportion of daughter cells in the positive group) × 100%.

[0156] In the MSC-induced Th1 cell inhibition assay, PBMC cells were added and co-cultured in an incubator. After 42 hours of co-culture, except for the negative control group, all experimental groups were simultaneously treated with three stimulants: 0.1 μg / mL PMA, 4 μg / mL BFA, and 0.267 μg / mL BFA calcium ion carrier. After 6 hours of further culture, the cell suspension was collected and analyzed by flow cytometry. A blank group, an IgG isotype group, and a test group were also included. For the IgG group, add 2 μL IgG-FITC and 1.3 μL IgG-PE; for the Test group, add 3 μL CD3-FITC and 3 μL CD8-PE. Incubate at room temperature in the dark for 20-30 min. After washing once with flow cytometry buffer, fix with fixative at room temperature for 30 min. After washing once with flow cytometry buffer, wash once with permeabilization buffer, and resuspend the cells in permeabilization buffer. For the IgG group, add 2 μL IgG-APC; for the Test group, add 3 μL IFN-APC. Incubate at room temperature in the dark for 20-30 min, gently mixing the cells every 5 minutes to prevent cell adhesion. Analyze the cells. The MSC inhibition rate of Th1 cell proliferation = (proportion of Th1 cells in the positive group - proportion of Th1 cells in the Test group) / (proportion of Th1 cells in the positive group) × 100%.

[0157] Experimental results showed that MSCs pretreated with cytokines enhanced their ability to promote Treg cell proliferation by 4.5 times compared to natural cells (Table 12, Figure 23), but had no significant effect on inhibiting T cell and Th1 cell proliferation (Tables 13, 14, Figures 24, 25). Treg cells, or regulatory T cells, can suppress excessive immune responses, maintain immune tolerance, and secrete inhibitory cytokines. The enhanced ability of MSCs to promote Treg cells (regulatory T cells) indicates a more precise and enhanced regulation towards immune tolerance. This effect highlights the therapeutic potential of MSCs in suppressing excessive inflammation and restoring immune balance.

[0158] Table 12. Results of MSCs' effect on the proliferation promotion of Tregs

[0159] Table 13. Results of the inhibitory effect of MSCs on the proliferation of total T cells

[0160] Table 14. Results of the inhibition rate of MSCs on the proliferation of Th1 cells

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application. Industrial applicability

[0162] The factor combination provided in this application can enhance the anti-pulmonary fibrosis ability of mesenchymal stem cells, regulate the expression of CD142 in mesenchymal stem cells, reduce the risk of pulmonary thrombosis in mesenchymal stem cells, and does not promote the occurrence of inflammation. Therefore, this factor combination can help improve the treatment effect and prognosis of patients with lung diseases and has good application prospects.

Claims

1. A factor composition for enhancing the anti-pulmonary fibrosis ability of mesenchymal stem cells and reducing the risk of pulmonary thrombosis, characterized in that, Includes (Ⅰ) and (Ⅱ); (I) Interferon γ, wherein the effective concentration of interferon γ is 0.5–5 ng / mL; (II) Interleukin-4 and / or tumor necrosis factor-α, wherein the concentration of interleukin-4 is 1 to 20 ng / mL and the concentration of tumor necrosis factor-α is 1 to 25 ng / mL.

2. The factor composition according to claim 1, characterized in that, It contains interferon-γ, interleukin-4 and tumor necrosis factor-α; The effective concentration of interferon-γ is 5 ng / mL; and / or, the effective concentration of interleukin-4 is 20 ng / mL; and / or, the effective concentration of tumor necrosis factor-α is 1 ng / mL.

3. The use of the factor composition according to claim 1 or 2 in any one of (i) to (v): (i) Enhancing the anti-pulmonary fibrosis ability of mesenchymal stem cells for non-disease treatment purposes, and / or reducing the risk of pulmonary embolism caused by mesenchymal stem cells for non-disease treatment purposes. (ii) Prepare products for improving the anti-pulmonary fibrosis ability of mesenchymal stem cells and / or reducing the risk of pulmonary embolism caused by mesenchymal stem cells; (iii) Enhance the ability of mesenchymal stem cells to promote Treg cell proliferation; (iv) Prepare products to enhance the ability of mesenchymal stem cells to promote the proliferation of Treg cells; (v) Prepare a culture medium for culturing mesenchymal stem cells; (vi) Increase the expression of at least one of indoleamine-2,3-dioxygenase, hepatocyte growth factor, and PD-L1 in mesenchymal stem cells; (vii) Prepare a reagent for increasing the expression of at least one of indoleamine-2,3-dioxygenase, hepatocyte growth factor and PD-L1 in mesenchymal stem cells; (ⅷ) Reduce the expression of CD142, a molecule on the surface of mesenchymal stem cells; (ⅸ) Prepare a reagent for reducing the expression of CD142, a surface molecule of mesenchymal stem cells.

4. The application according to claim 3, characterized in that, Mesenchymal stem cells in any of (i) to (e) include umbilical cord mesenchymal stem cells.

5. A mesenchymal stem cell culture medium, characterized in that, It comprises a culture medium and the factor composition as described in claim 1 or 2.

6. A method for processing mesenchymal stem cells, characterized in that, This includes contacting mesenchymal stem cells with the factor composition of claim 1 or 2.

7. The processing method according to claim 6, characterized in that, This includes culturing mesenchymal stem cells in the culture medium described in claim 5 to obtain treated mesenchymal stem cells.

8. Pretreated mesenchymal stem cells, characterized in that, Mesenchymal stem cells obtained using the processing method described in claim 6 or 7.

9. A pharmaceutical composition, characterized in that, It includes the pretreated mesenchymal stem cells as described in claim 8.

10. The pharmaceutical composition according to claim 9, characterized in that, It also includes the factor composition of claim 1 or 2, or the mesenchymal stem cell culture medium of claim 5; Furthermore, the pretreated mesenchymal stem cells remain in continuous contact with the factor composition.

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