Preparation method and application of Foxp1 protein-enriched mesenchymal stem cell exosome with immune organ targeting property

Abstract

The invention discloses a preparation method and application of a mesenchymal stem cell exosome which is enriched with Foxp1 protein and has immune organ targeting property. The method comprises the following steps: acquiring a single-cell suspension of the mesenchymal stem cells, performing suspension culture on the single-cell suspension of the mesenchymal stem cells in an exosome removal culture medium to induce the mesenchymal stem cells to aggregate to form a compact cell cluster, and then collecting the exosome through differential centrifugation. According to the method disclosed by the invention, the mesenchymal stem cells are induced to aggregate through a suspension culture technology, so that the exosome with high yield and immune targeting is obtained. The Foxp1 protein is specifically enriched in the exosome, the exosome has the characteristics of high yield, immune organ targeting and strong immune regulation capability, Treg cell differentiation is regulated through the Foxp1 / STAT5 / Foxp3 axis, the limitation in the application of the existing exosome is solved, and a new strategy is provided for the treatment of autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to a method for preparing mesenchymal stem cell exosomes enriched with Foxp1 protein and having immune organ targeting capabilities, and its application. Background Technology

[0002] MSC-derived exosomes (MSC-exos) are extracellular vesicles secreted by mesenchymal stem cells (MSCs) with important biological functions and great potential applications in the biomedical field. MSC-exos exhibit significant immunomodulatory effects, inhibiting the proliferation and activation of T cells and B cells, and regulating the function of macrophages and dendritic cells. In tissue repair, they can promote the proliferation and differentiation of vascular endothelial cells, osteoblasts, chondrocytes, and neural stem cells, and can also secrete neurotrophic factors. Clinically, they have shown positive effects in the treatment of cardiovascular, nervous system, and inflammatory diseases, and can also serve as drug delivery carriers. Therefore, MSC-exos have been extensively studied due to their potential immunomodulatory capabilities. However, the limited production of these exosomes, their lack of specificity in vivo distribution, and their limited immunomodulatory capacity restrict their application in clinical treatment. Summary of the Invention

[0003] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the prior art and to provide a method for preparing mesenchymal stem cell exosomes. This method induces the aggregation of mesenchymal stem cells through suspension culture to obtain exosomes with high yield, immune targeting, and the ability to regulate Treg differentiation.

[0004] A second objective of this invention is to provide mesenchymal stem cell exosomes prepared by the above method.

[0005] A third objective of this invention is to provide the application of the aforementioned mesenchymal stem cell exosomes.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] The present invention first provides a method for preparing mesenchymal stem cell exosomes, including obtaining a single-cell suspension of mesenchymal stem cells, suspending the single-cell suspension of mesenchymal stem cells in an exosome removal culture medium to induce mesenchymal stem cells to aggregate into dense cell clusters, and then collecting the exosomes by differential centrifugation.

[0008] This invention utilizes suspension culture technology to induce mesenchymal stem cell (MSC) aggregation, significantly increasing the exosome yield produced by MSCs. Exosomes (Agg-exos) obtained from the aggregation of an equal amount of single MSC cells in suspension were significantly higher in both exosome yield and protein concentration than those obtained from MSC-attached culture (Adh-exos). Furthermore, using exosome labeling technology, it was observed for the first time that, compared to Adh-exos, Agg-exos, when systemically administered to a systemic lupus erythematosus (MRL / lpr) mouse model, accumulated in immune-related organs (spleen, thymus, and lymph nodes), improving the therapeutic effect on SLE. This indicates that Agg-exos exhibits targeting specific immune organs, enhancing therapeutic efficacy and specificity. Further protein sequencing of Agg-exos revealed a high enrichment of Foxp1 protein in Agg-exos, with Foxp1 expression significantly higher in Agg-exos than in Adh-exos. Mechanistic studies revealed that Agg-exos regulates Treg differentiation through the Foxp1 / STAT5 / Foxp3 axis. These results indicate that induced aggregation of mesenchymal stem cells (MSCs) via suspension culture significantly increases the production of exosomes; the exosomes obtained from MSC suspension-induced aggregation exhibit targeting specific immune organs, thereby improving therapeutic efficacy and specificity; and these exosomes are highly enriched in Foxp1 protein and promote Treg differentiation through the Foxp1 / STAT5 / Foxp3 axis, demonstrating strong therapeutic potential for autoimmune diseases.

[0009] Furthermore, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, dental pulp mesenchymal stem cells, or bone marrow mesenchymal stem cells.

[0010] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0011] Furthermore, the process of obtaining a single-cell suspension of mesenchymal stem cells involves first culturing mesenchymal stem cells until their growth density reaches 80-90%, and then obtaining the single-cell suspension of mesenchymal stem cells.

[0012] Preferably, the growth density is 90%.

[0013] Furthermore, the exosome removal medium is a medium after removing serum-derived exosomes from the serum medium.

[0014] Preferably, the serum in the serum culture medium is fetal bovine serum.

[0015] Preferably, the serum culture medium is an α-minimum essential medium (α-MEM) containing 20% ​​fetal bovine serum, L-glutamine, 2-mercaptoethanol and penicillin / streptomycin.

[0016] Preferably, the serum-derived exosomes in the serum removal culture medium are removed by centrifugation.

[0017] Preferably, the centrifugation parameters are 100,000g for 6–12 hours.

[0018] Furthermore, the suspension culture is carried out using a culture dish with ultra-low adhesion.

[0019] Furthermore, the suspension culture time is 48–72 hours.

[0020] Preferably, the suspension culture time is 48 hours.

[0021] Preferably, the suspension culture is carried out at 37°C in a humid atmosphere with 5% CO2.

[0022] Furthermore, the differential centrifugation conditions are as follows: centrifuge at 200–400g for 10–15 minutes, centrifuge at 1800–2000g for 10–20 minutes, centrifuge at 8000–10000g for 30–60 minutes, centrifuge at 8000–100000g for 70–90 minutes, collect the precipitate, wash it, and centrifuge at 8000–100000g for 70–90 minutes.

[0023] Preferably, the differential centrifugation conditions are as follows: centrifuge at 300g for 10 minutes, centrifuge at 2000g for 10 minutes, centrifuge at 10000g for 30 minutes, centrifuge at 100000g for 70 minutes, collect the precipitate, wash it, and centrifuge at 100000g for 70 minutes.

[0024] This invention provides mesenchymal stem cell exosomes prepared by any of the methods described above. These mesenchymal stem cell exosomes are enriched with Foxp1 protein and exhibit immune organ targeting.

[0025] This invention provides the application of the above-described mesenchymal stem cell exosomes in the preparation of drugs for treating autoimmune diseases.

[0026] Furthermore, the autoimmune disease is systemic lupus erythematosus.

[0027] Furthermore, the drug is enriched with Foxp1 protein and regulates Treg differentiation through the Foxp1 / STAT5 / Foxp3 axis to treat autoimmune diseases.

[0028] The present invention also provides a medicine comprising the above-described mesenchymal stem cell exosomes and pharmaceutically acceptable excipients.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention provides a method for producing exosomes from mesenchymal stem cells. The method induces the aggregation of mesenchymal stem cells through suspension culture, yielding high-yield exosomes with immune targeting capabilities. These exosomes specifically enrich Foxp1 protein, exhibiting high yield, immune organ targeting, and potent immunomodulatory capabilities. Furthermore, they regulate Treg cell differentiation through the Foxp1 / STAT5 / Foxp3 axis, overcoming the limitations of existing exosome production, lack of specificity in vivo distribution, and limited immunomodulatory capacity. This provides a new strategy for the treatment of autoimmune diseases. Attached Figure Description

[0031] Figure 1 This study aims to characterize MSC aggregation induced by suspension culture and engineered exosomes. A shows a schematic diagram of MSC culture and exosome isolation methods; B shows the morphology of attached MSCs and the morphological changes of suspended MSCs at different time points observed under an optical microscope (scale bar: 200 μm); C shows the structure and immunofluorescence results of MSC aggregates cultured for 48 hours under low attachment conditions under a PH1 phase-contrast microscope (scale bar: 100 μm); D shows the results of nanoparticle tracking analysis (NTA); E shows the results of Pierce BCA protein analysis; F shows the structures of isolated Adh-exo and Agg-exo cells as displayed by TEM (scale bar: 200 nm); G and H show the particle size and mean potential results of Adh-exo and Agg-exo cells, respectively, obtained from NTA particle size analysis; I shows the results of nanoflow cytometry (NanoFCM); and J shows the results of Western blot. ns indicates no significant difference; *P<0.05; **P<0.01; ***P<0.001.

[0032] Figure 2 The therapeutic effects of Adh-exo and Agg-exo on SLE are shown below. A is a schematic diagram of the exosome treatment regimen and efficacy evaluation in MRL / lpr mice; B shows the effect of the Agg-exo and Adh-exo groups on spleen weight in MRL / lpr mice; C shows the effect of the Agg-exo and Adh-exo groups on serum ANA and BUN concentrations in mice; D shows the effect of the Agg-exo and Adh-exo groups on dsDNA (IgG) and dsDNA (IgM) concentrations in mice; E shows the confocal microscopy assay for IgG deposition in the kidneys and skin; F shows the H&E and PAS staining results; and G shows the effect of the Agg-exo and Adh-exo groups on CD4+ concentrations in the spleens of CBS- / -, CSE- / -, and MRL / lpr mice. + IFN-γ + Th1 cells and CD4 + IL-4 +The effect of Th2 cell ratio, H represents the effect of Agg-exo and Adh-exo groups on CD4 cell count in mouse spleen. + FoxP3 + Treg cells and CD4 + IL-17 + Effect of Th17 cell proportion; ns indicates no significant difference, *P<0.05; **P<0.01; ***P<0.001.

[0033] Figure 3 The distribution results of Adh-exo and Agg-exo in mice are shown. A represents the deposition results of Adh-exo and Agg-exo in the spleen, lymph nodes, and thymus as analyzed by IVIS; B represents the deposition results of Adh-exo and Agg-exo in the spleen, lymph nodes, and thymus as analyzed by immunofluorescence staining; and C represents the immunofluorescence analysis of PKH-26-labeled exosomes and CD4+ co-cultured in vitro. + PKH-26 fluorescence and CD4 in T cell exosomes 24 hours later + Colocalization results of T cells, D represents flow cytometry analysis of PKH-26-labeled exosomes and CD4+ in vitro co-culture. + The expression of PKH26 fluorescence in T cells in exosomes 24 hours later; ns indicates no significant difference, *P<0.05; **P<0.01; ***P<0.001.

[0034] Figure 4 Organ distribution of Adh-exo and Agg-exo in MRL / lpr mice; where A is the IVIS analysis results of the distribution of Agg-exos and Adh-exos in the liver, lungs, heart, and kidneys, and B is the immunofluorescence staining analysis of frozen tissue sections of Agg-exos and Adh-exos in the liver, lungs, heart, and kidneys.

[0035] Figure 5 Adjust CD4 for Agg-exos + Results of T cell differentiation; where A represents flow cytometry analysis of CD4. + Following co-culture of T cells with exosomes, CD4+ levels were found in the spleens of CBS- / -, CSE- / -, and MRL / lpr mice. + IFN-γ + Th1 cells, CD4 + FoxP3 + Treg cells, CD4 + IL-4 + Th2 cells, CD4 + IL-17 +The proportion of Th17 cells, B represents the results of qRT-PCR assessment of Treg-related gene expression, and C represents CD4. + T cell immunofluorescence staining results, scale bars are 2μm, D represents flow cytometry analysis of exosomes and CD4+. + CD4 co-cultured T cells + Results of T cell expression of CD44 and Ki67, ns indicate no significant difference, *P<0.05, **P<0.01, ***P<0.001.

[0036] Figure 6 CD4 + Results of T cell in vitro culture and Treg induction; where A represents flow cytometry analysis of in vitro Treg-induced A5-7aad-CD4. + The proportion of T cells, B represents the Foxp3 concentration analyzed by flow cytometry after in vitro induction of Treg differentiation. + CD4 + The proportion of Treg cells, C represents the CD4 count in the exosome co-culture group analyzed by flow cytometry. + CD25 expression in T cells.

[0037] Figure 7 High-throughput proteomics analysis of Agg-exos; where A is the protein expression profile of exosomal markers in Agg-exos and Adh-exos samples, B is the proteins that are significantly upregulated and downregulated in Agg-exos and Adh-exos, C is the hierarchical clustering analysis of Agg-exos and Adh-exos, with rows representing individual genes and columns representing biological repeats, D is the GO enrichment analysis of the molecular function of proteins upregulated in Agg-exos, and E is the KEGG pathway analysis of differentially expressed proteins in Agg-exos annotated to specific pathway groups.

[0038] Figure 8 This presents the results of a study on the mechanism by which AgG-exos regulates Treg differentiation; where A represents differential expression through proteomics analysis, and B represents CD4 expression through Western blot analysis. + The total expression of p-STAT5 and STAT5 after T cells were co-cultured with exosomes. C represents the immunofluorescence analysis of CD4+ expression after exosome treatment. + Expression and fluorescence colocalization of STAT5 and Foxp3 in the nucleus of T cells. Scale bar: 2 μm. D: Pearson correlation coefficient. E: Immunofluorescence analysis of exosome-treated CD4. +The expression of Foxp1 in T cells and its co-localization with Foxp3 in the nucleus are shown in the figure. Scale bar is 2 μm. F represents the Pearson correlation coefficient. G represents the expression of Foxp1 gene in MSCs after silencing Foxp1 with RNAi, analyzed by qRT-PCR. H represents the expression of Foxp1 gene in MSCs and Agg-exos after silencing Foxp1 with RNAi, analyzed by Western blot. I represents the Treg differentiation induced by Agg-exo group and siFoxp1 Agg-exo group, analyzed by flow cytometry. J represents the expression of Treg-related genes, assessed by qRT-PCR. K represents the immunofluorescence analysis of CD4+ cells co-cultured with Agg-exo group and siFoxp1 Agg-exo group. + Expression of CD44, CD25, and Ki67 in T cells. Scale bar: 2 μm. L represents Western blot analysis of Agg-exo and siFOXP1-induced CD44 expression. + p-STAT5 level in T cells. M represents the expression levels of STAT5 and Foxp3 in the cell nucleus and the co-localization results of STAT5 and Foxp3 in the Agg-exo group and siFoxp1 Agg-exo group, respectively, with a scale bar of 2 μm. N represents the expression levels of Foxp1 and Foxp3 in the cell nucleus and the co-localization results of Foxp1 and Foxp3 in the Agg-exo group and siFoxp1 Agg-exo group, respectively, with a scale bar of 2 μm. ns indicates no significance. *P<0.05, **P<0.01, ***P<0.001.

[0039] Figure 9 The results show the expression of Foxp1 in Agg-exos and its regulation of Treg differentiation. A is the Western blot analysis of Foxp1 expression in Agg-exos and Adh-exos, and B is the flow cytometry analysis of CD4+. + The effects of Agg-exos and siFoxp1 treatments on the proportions of Th1, Th2, and Th17 cells in T cells. C represents the results of statistical analysis of the co-localization of Foxp3 and STAT5 in the cell nucleus in the Agg-exos and siFoxp1 group. D represents the results of statistical analysis of the co-localization of Foxp3 and Foxp1 in the cell nucleus in the Agg-exos and siFoxp1 group. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and 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.

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] Table 1 shows the sources of antibodies or reagents involved in the examples.

[0043]

[0044]

[0045]

[0046] Example 1: Obtaining and Identifying Aggregated Exosomes

[0047] 1. Experimental Methods

[0048] 1.1 Cell Culture

[0049] Mesenchymal stem cells (MSCs) were cultured. When the MSC growth density reached 90%, a single-cell suspension of MSCs was obtained and seeded into ultra-low adhesion culture dishes at a density of 1×10^6 / 4 mL for suspension culture. After 48 hours, dense MSC cell clusters were formed. The specific method is as follows:

[0050] Human umbilical cord mesenchymal stem cells (UMSCs) were cultured until 90% adherence was achieved. Single-cell suspensions were then seeded into ultra-low adhesion culture dishes (i.e., suspension culture) and replaced with exosome-removing medium. This exosome-removing medium contained 20% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Invitrogen), 55 mM 2-mercaptoethanol (Invitrogen), and 1% penicillin / streptomycin (Invitrogen) in an α-minimum essential medium (α-MEM, Invitrogen). The medium was then centrifuged overnight at 100,000 g to remove FBS-derived exosomes. The single-cell suspensions were seeded into 10 cm ultra-low adhesion culture dishes (Corning) and cultured at 37°C in a humidified atmosphere with 5% CO2. Within 48 hours, the cells formed mesenchymal stem cell aggregates.

[0051] In addition, a traditional attachment culture condition group was set up: the culture method was basically the same, the only difference being that the 10cm ultra-low adhesion culture dish was replaced with a 10cm culture flask (Corning, Cambridge, MA, USA) for attachment culture.

[0052] 1.2 Isolation of exosomes

[0053] Following the above method, an equal number of mesenchymal stem cells (MSCs) were cultured in an exosome-removing medium for 48 hours to obtain cell culture systems from which exosomes were isolated. Exosomes were separated from the culture supernatant by differential centrifugation, sequentially centrifuging at 300g for 10 minutes and then at 2,000g for 10 minutes to remove cell debris. The supernatant was then centrifuged at 10,000g for 30 minutes to remove medium-sized vesicles, followed by centrifugation at 100,000g for 70 minutes. The precipitate was collected, washed with PBS, and further purified by centrifugation at 100,000g for 70 minutes. The precipitate was collected as exosomes, yielding exosomes Adh-exo from the attached cell culture system and exosomes Agg-exo from the suspension cell culture system.

[0054] 1.3 Protein Blotting

[0055] 20 μg of protein was used for electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking for 1 h, the membrane was incubated overnight at 4 °C with the corresponding primary antibody and then incubated for 1 h with HRP-conjugated secondary antibody. Finally, the membrane was visualized using a chemiluminescent substrate (Thermo Fisher) and analyzed via ChemiDoc. TM MP imaging system (Bio-Rad, CA, USA) scan.

[0056] 1.4 Exosome Characterization

[0057] After each exosome preparation, characterization was performed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), nanoflow cytometry (nFCM), and Western blot analysis. TEM (JEOL, Tokyo, Japan) was used to evaluate the morphology and ultrastructure of exosomes. Protein concentrations of exosomes were determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). NTA was performed using a ZetaView PMX120 (Particle Metrix, Germany) to measure the number, size, and zeta potential of exosomes. Furthermore, nFCM was used to assess the expression levels of exosome markers CD63, CD9, and CD81. Specifically, exosomes were incubated with PE-labeled antibodies against CD63, CD9, and CD81 at 37°C for 30 minutes, followed by detection using nFCM (NanoFCM, Xiamen, China). Purified exosomes were characterized by Western blot analysis using antibodies including anti-CD63, TSG101, Alix, and Calnexin antibodies.

[0058] Exosome labeling was performed using DiR (Invitrogen, USA) and PKH-26 (Sigma), following the manufacturer's instructions.

[0059] 2. Experimental Results

[0060] The same number of mesenchymal stem cells (MSCs) were cultured under both suspension and conventional attachment culture conditions. Exosomes were then isolated from the culture medium using differential centrifugation. Figure 1 (A). In suspension culture, MSC aggregates begin to appear after 12 hours of culture, forming stable, dense cell aggregates after 48 hours, eventually leading to multicellular spheroids. In attachment culture, MSCs spread out in a 2D plane, exhibiting spindle-shaped and whorled cell arrangements. Figure 1 (Middle B). Immunofluorescence staining of MSC spheroids revealed tight cell adhesion and significantly elevated expression of the exosome marker CD63. Figure 1 (C)

[0061] Exosomes were collected from equal volumes of MSC supernatant under two culture conditions and quantitatively analyzed by nanoparticle tracking analysis (NTA) and biuret assay (BCA). The results showed that suspension-induced aggregated exosomes (Agg-exos) had significantly higher exosome yield and protein concentration than attached-cultured exosomes (Adh-exos). Figure 1 (D-E). Transmission electron microscopy (TEM) and NTA further characterized the exosomes, showing that Agg-exos and Adh-exos were typical cup-shaped vesicles with a main diameter of less than 150 nm and average zeta potentials of -38.07 mV and -42.57 mV, respectively. Figure 1 (F~H). Nanoflow cytometry confirmed positive expression of exosome markers such as CD63, CD9, and CD81 in both exosome types. Figure 1 Western blot analysis showed that both Agg-exos and Adh-exos highly expressed transmembrane protein CD63, cytoplasmic protein TSG101, and Alix, while Calnexin expression was low. Figure 1 (J). These data indicate that high yields of AgG-exos were successfully isolated through suspension-induced aggregation culture.

[0062] Example 2

[0063] 1. Experimental Methods

[0064] 1.1 Cell Culture

[0065] Human umbilical cord mesenchymal stem cells (UMSCs) were cultured until 90% adherence was achieved, then a single-cell suspension was obtained and seeded into ultra-low adhesion culture dishes (i.e., suspension culture). The culture medium was then replaced with exosome-removing medium. This exosome-removing medium contained 20% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Invitrogen), 55 mM 2-mercaptoethanol (Invitrogen), and 1% penicillin / streptomycin (Invitrogen) in an α-minimum essential medium (α-MEM, Invitrogen). Subsequently, the medium was centrifuged overnight at 100,000 g to remove FBS-derived exosomes. The single-cell suspension was seeded into 10 cm ultra-low adhesion culture dishes (Corning) and cultured at 37°C in a humid atmosphere with 5% CO2. Within 72 hours, the cells formed mesenchymal stem cell aggregates.

[0066] 1.2 Isolation of exosomes

[0067] Following the above method, an equal number of mesenchymal stem cells (MSCs) were suspended and cultured in exosome-removing medium for 72 hours to obtain a cell culture system from which exosomes were isolated. Exosomes were separated from the culture supernatant by differential centrifugation, sequentially centrifuging at 200g for 15 minutes and 1,800g for 20 minutes to remove cell debris. The supernatant was then centrifuged at 8,000g for 60 minutes to remove medium-sized vesicles, followed by centrifugation at 80,000g for 90 minutes. The precipitate was collected, washed with PBS, and further purified by centrifugation at 80,000g for 90 minutes, with the precipitate collected as exosomes. This method yielded a high yield of mesenchymal stem cell exosomes, enriched with Foxp1 protein and exhibiting immune organ targeting.

[0068] Example 3

[0069] 1. Experimental Methods

[0070] 1.1 Cell Culture

[0071] After dental pulp mesenchymal stem cells (SHED) reached 90% adhesion in attachment culture, single-cell suspensions were obtained and seeded into ultra-low adhesion culture dishes (i.e., suspension culture). The culture medium was then replaced with exosome-removing medium. This exosome-removing medium contained 20% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Invitrogen), 55 mM 2-mercaptoethanol (Invitrogen), and 1% penicillin / streptomycin (Invitrogen) in an α-minimum essential medium (α-MEM, Invitrogen). Subsequently, the medium was centrifuged overnight at 100,000 g to remove FBS-derived exosomes. The single-cell suspensions were seeded into 10 cm ultra-low adhesion culture dishes (Corning) and cultured at 37°C in a humid atmosphere of 5% CO2. Within 72 hours, the cells formed mesenchymal stem cell aggregates.

[0072] 1.2 Isolation of exosomes

[0073] Following the method described above, an equal number of mesenchymal stem cells (MSCs) were suspended in exosome-removing medium for 72 hours to obtain a cell culture system from which exosomes were isolated. Exosomes were separated from the culture supernatant by differential centrifugation, sequentially centrifuging at 300g for 10 minutes and then at 2,000g for 10 minutes to remove cell debris. The supernatant was then centrifuged at 10,000g for 30 minutes to remove medium-sized vesicles, followed by centrifugation at 100,000g for 70 minutes. The precipitate was collected, washed with PBS, and further purified by centrifugation at 100,000g for 70 minutes, with the precipitate collected as exosomes. This method yielded a high yield of mesenchymal stem cell exosomes enriched with Foxp1 protein and exhibiting immune organ targeting.

[0074] Example 4

[0075] 1. Experimental Methods

[0076] 1.1 Cell Culture

[0077] After bone marrow mesenchymal stem cells (HBMSCs) reached 90% confluence in attachment culture, single-cell suspensions were obtained and seeded into ultra-low adhesion culture dishes (i.e., suspension culture). The culture medium was then replaced with exosome-removing medium. This exosome-removing medium contained 20% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Invitrogen), 55 mM 2-mercaptoethanol (Invitrogen), and 1% penicillin / streptomycin (Invitrogen) in an α-minimum essential medium (α-MEM, Invitrogen). Subsequently, the medium was centrifuged overnight at 100,000 g to remove FBS-derived exosomes. The single-cell suspensions were seeded into 10 cm ultra-low adhesion culture dishes (Corning) and cultured at 37°C in a humidified atmosphere of 5% CO2. Within 72 hours, the cells formed mesenchymal stem cell aggregates.

[0078] 1.2 Isolation of exosomes

[0079] Following the method described above, an equal number of mesenchymal stem cells (MSCs) were suspended in exosome-removing medium for 72 hours to obtain a cell culture system from which exosomes were isolated. Exosomes were separated from the culture supernatant by differential centrifugation, sequentially centrifuging at 300g for 10 minutes and then at 2,000g for 10 minutes to remove cell debris. The supernatant was then centrifuged at 10,000g for 30 minutes to remove medium-sized vesicles, followed by centrifugation at 100,000g for 70 minutes. The precipitate was collected, washed with PBS, and further purified by centrifugation at 100,000g for 70 minutes, with the precipitate collected as exosomes. This method yielded a high yield of mesenchymal stem cell exosomes enriched with Foxp1 protein and exhibiting immune organ targeting.

[0080] Example 5: Aggregated exosomes significantly alleviated symptoms of systemic lupus erythematosus.

[0081] The effects of exosomes Adh-exo from the attached culture cell system and Agg-exo from the suspension culture cell system prepared in Example 1 on systemic lupus erythematosus were investigated.

[0082] 1. Experimental Methods

[0083] 1.1 Western blot

[0084] Total protein extraction was performed using a protein extraction kit (Thermo, Rockford, IL, USA) according to the manufacturer's instructions. 20 μg of protein extracted from each sample was loaded into an SDS-PAGE gel and then transferred to a PVDF (Millipore) membrane. After transfer, the membrane was blocked at room temperature for 1 hour and then incubated overnight with primary antibody at 4°C. Next, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 hour. Finally, the gel was developed using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher) and detected using a gel imaging system.

[0085] 1.2 Immunofluorescence and Immunohistochemistry

[0086] Frozen sections and cell samples were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes at room temperature. Samples were then stained with primary antibodies followed by secondary antibody treatment. Finally, samples were mounted with DAPI and placed on gel-coated microscope slides for imaging. Frozen kidney and skin tissue sections were stained with Alexa Fluor488-labeled anti-mouse IgG, and IgG deposition was assessed using fluorescence intensity.

[0087] For histological evaluation, kidney tissues from different groups were fixed overnight in 4% paraformaldehyde (Sigma-Aldrich). Subsequently, paraffin sections were prepared and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS), respectively, to assess kidney damage.

[0088] 1.3 Flow cytometry

[0089] For intracellular cytokine staining, cells were stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA, 50 ng / mL), iomycin (1 μg / mL), and monosine (GolgiStop; 1 μg / mL), respectively. After surface marker staining, cells were permeabilized using a FoxP3 / transcription factor staining kit (Thermo Fisher, CA, USA), followed by intracellular staining with the following antibodies: APC anti-mouse CD4, FITC anti-mouse IFN-γ, PE anti-mouse IL-4, PE anti-mouse IL-17, and PE anti-mouse Foxp3. To assess apoptosis rate, cells were stained with FITC anti-Annexin V and PE-Cy7 anti-7AAD for 15 minutes at room temperature. Subsequently, flow cytometry analysis was performed using a NovoCyte instrument (Agilent Technologies, CA, USA).

[0090] 2. Experimental Results

[0091] Previous studies have shown that MSC-derived exosomes (MSC-exos) effectively alleviate systemic lupus erythematosus (SLE) symptoms through their immunomodulatory properties. MRL / lpr mice are a commonly used spontaneous SLE mouse model with severe lupus nephritis. Engineered Agg-exos and Adh-exos were systemically injected into 12–16-week-old female MRL / lpr mice via tail vein injection. Pathological evaluation was performed one month after treatment. Figure 2 (A). The results showed that, compared with the Adh-exos group, the Agg-exos treatment group had significantly lower spleen weight, and lower serum ANA, BUN, and anti-dsDNA levels. Figure 2 (B-D). Furthermore, compared to the Adh-exos group, the Agg-exos group showed reduced IgG deposition in the spleen and skin (B-D). Figure 2 Histopathological analysis of kidney tissue stained with HE and PAS showed that the Agg-exos group had reduced tubular basement membrane disorder and decreased mesangial cell proliferation. Figure 2 (F). Furthermore, both exosome treatment groups showed lower proportions of Th17 and Th1 cells, while exhibiting higher proportions of Treg and Th2 cells. Figure 2 (G~H). The proportion of Treg cells in the Agg-exos group was significantly higher than that in the Adh-exos group (G~H). Figure 2 These data indicate that systemic injection of Agg-exos provides significant therapeutic benefit in improving SLE pathology in MRL / lpr mice, and its effect is superior to that of the conventional Adh-exos group.

[0092] Example 6: Distribution of aggregated exosomes to target immune organs in MRL / lpr mice

[0093] The distribution of exosomes Adh-exo from the attached culture cell system and Agg-exo from the suspension culture cell system prepared in Example 1 was investigated to target immune organ distribution in mice.

[0094] 1. Experimental Methods

[0095] 1.1 Small animal live imaging

[0096] The in vivo distribution of exosomes was analyzed using IVIS imaging. After injection of DiR-labeled exosomes (the labeling procedure was the same as in Example 3), mice were sacrificed 24 hours later, and their organs were collected for IVIS analysis. The accumulation level of exosomes in various organs was assessed by measuring fluorescence intensity.

[0097] 1.2 Immunofluorescence

[0098] The specific experimental procedure is the same as in Experiment Example 5.

[0099] 1.3CD4 + T cell isolation and culture

[0100] Using mouse initial CD4 + The T-cell isolation kit (BD) isolates naïve CD4 cells from the spleen via positive selection, according to the manufacturer's protocol. + T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0101] 1.4 Co-culture of aggregated exosomes and MSCs

[0102] The isolated exosomes were fluorescently labeled using a PKH26(Sigma) kit at a concentration of 1×10⁻⁶. 8 The target concentration of / mL was added to the target cell culture medium for co-culture, and harvested after 48 hours for subsequent experimental detection.

[0103] 1.5 Cell Flow Cytometry

[0104] Specific experimental procedures, Example 5.

[0105] 2. Experimental Results

[0106] Exosomes were labeled using PKH-26 and DiR iodide probes, followed by tail vein injection, and their in vivo distribution was monitored 24 hours later. In vivo imaging system (IVIS) and fluorescence staining results of tissue sections showed that Agg-exos and Adh-exos were mainly concentrated in the liver, kidneys, and spleen, consistent with previous reports. Figure 3 China A, Figure 4 (A). However, Agg-exos showed significantly higher enrichment in immune-related organs (such as the spleen, lymph nodes, and thymus) than Adh-exos. Figure 3 (A-B). No significant differences were observed in the distribution of exosomes in other organs (such as liver, heart, and lungs). Figure 4 (A to B)

[0107] Considering CD4 + The crucial role of T cells in SLE progression; CD4 cells isolated from the spleen of MRL / lpr mice. + T cells were used to further validate the role of exosomes in immune-targeted distribution. After co-culturing with PKH26-labeled exosomes for 24 hours, immunofluorescence staining and flow cytometry confirmed that Agg-exos was more effectively targeted by CD4 than Adh-exos. + T cell capture ( Figure 3(C-D). These data indicate that systemic infusion of Agg-exos exhibits an in vivo distribution pattern targeting immune organs in MRL / lpr mice.

[0108] Example 7: Aggregated exosomes can promote Treg cell differentiation

[0109] The effects of exosomes Adh-exo from the attached culture cell system and Agg-exo from the suspension culture cell system prepared in Example 1 on Treg cell differentiation were investigated.

[0110] 1. Experimental Methods

[0111] 1.1 Treg cell induction

[0112] CD4 + The T cell isolation procedure was the same as in Experiment 6, and the isolated CD4 cells... + T cells were activated in RPMI 1640 medium coated with anti-mouse CD3 antibody (2 μg / mL) and anti-mouse CD28 antibody (2 μg / mL), supplemented with 10% FBS, 1 mL glutamine, 50 μM 2-mercaptoethanol, 100 U / mL penicillin, and 100 μg / mL streptomycin. To induce Treg cell differentiation, TGF-β1 (10 ng / mL; R&D Systems, USA) and IL-2 (100 U / mL; Peprotech, USA) were added to the cell culture to promote Treg cell transformation. Cells were replenished with fresh medium on day 3 and analyzed on day 5.

[0113] 2. Experimental Results

[0114] T-cell subtype dysregulation plays a central role in the pathogenesis of SLE due to its association with MHC proteins and its contribution to lupus-like symptoms in the MRL / lpr model. Treg cells are crucial for maintaining immune tolerance, and they are often absent or dysfunctional in mouse models and human studies of SLE. Therefore, this invention further incorporates Agg-exos and Adh-exos with spleen CD4 cells isolated from MRL / lpr mice. + T cells were co-cultured. Results showed that both types of exosomes induced CD4+ degeneration. + IFN-γ + Th1 and CD4 + IL-17 + The decrease in the proportion of Th17 cells, along with CD4 + IL-4 + Th2 and CD4 + FoxP3 +The proportion of Treg cells increased ( Figure 5 (A). Notably, Agg-exos significantly increased the proportion of Treg cells, which is consistent with previously observed increases in CD4+ cells in vivo. + The changes in T cells were consistent. Neither Agg-exos nor Adh-exos affected CD4. + T cell survival rate Figure 6 (A)

[0115] Subsequently, the present invention performed in vitro testing on CD4 + T cells were induced with Treg cells; flow cytometry analysis showed CD4+. + FoxP3 + Treg cells significantly increased ( Figure 6 Compared with Adh-exos, Agg-exos co-culture showed significantly higher expression levels of Treg differentiation genes, including forkhead box protein P3 (Foxp3), interleukin-2 receptor subunit α (IL2Ra), transforming growth factor-β (Tgfb1), and cytotoxic T lymphocyte-associated protein-4 (Ctla4). Figure 5 (See section B). Furthermore, this invention assessed Treg cell function using immunofluorescence and flow cytometry, focusing on the expression of three Treg activity markers: CD44, CD25, and Ki67. Compared to Adh-exos, these markers were expressed at higher levels in the Agg-exos co-culture group, indicating that Agg-exos enhances the functional activity of Treg cells. Figure 5 C to D and Figure 6 (C). These results highlight the superior ability of Agg-exos to drive Treg cell differentiation and functional activation.

[0116] Example 8: Aggregated exosomes regulate Treg cell differentiation via the Foxp1 / STAT5 / Foxp3 axis

[0117] 1. Experimental Methods

[0118] 1.1 siRNA transfection

[0119] For siRNA transfection, Foxp1 siRNA (Ribo, China) was used to transfect MSCs using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher, USA), following the manufacturer's instructions. A non-targeting control siRNA (Ribo, China) was used as a negative control. Transfection efficiency was assessed by immunoblotting.

[0120] 1.2 RNA extraction, reverse transcription, and RT-qPCR

[0121] RNA was extracted from cells using NucleoZOL reagent (Gene Company Limited) and subsequently reverse transcribed into complementary DNA (cDNA) using PrimeScript RT Master Mix (TaKaRa, Ltd., Osaka, Japan). Gene expression levels were quantified by real-time polymerase chain reaction (RT-qPCR) using Bio-Rad CFX96. TM The detection system (Roche, Sweden) was used in conjunction with Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). The primer sequences used for qPCR analysis are listed in Table 2.

[0122] Table 2 qPCR primer sequences

[0123]

[0124] 1.3 LC-MS / MS Analysis

[0125] Mass spectrometry analysis was performed using an Orbitrap Q Exactive HF-X mass spectrometer in conjunction with a UltiMate 3000LC system (Thermo Fisher). Samples were reconstituted in formic acid, loaded into a pre-column, and then connected to a microcapillary analytical column. High-energy collisional dissociation (HCD) tandem mass spectrometry (MS / MS) spectra were acquired in data-dependent mode using the top-20 method. The resulting raw data files were analyzed using pFind 3.0 (http: / / pfind.ict.ac.cn / software / pFind3 / index.htmL) and the UniProt standard database for mouse Mus musculus.

[0126] 2. Experimental Results

[0127] Proteomics results confirmed the expression of exosome marker proteins. Figure 7 The study revealed that, compared to Adh-exos, 1152 proteins were upregulated and 701 proteins were downregulated in Agg-exos. Figure 7 (B). GO enrichment analysis showed that proteins highly expressed in Agg-exos were concentrated in transcription-related pathways and nuclear processes (B). Figure 7 (C, D). Meanwhile, KEGG pathway analysis showed enrichment of RNA transcriptional regulatory pathways ( Figure 7 (E). Among differentially expressed proteins, Foxp1 was significantly more expressed in Agg-exos than in Adh-exos, a finding further confirmed by Western blot analysis. Figure 8 China A, Figure 9 (A)

[0128] On CD4 that has been processed by Agg-exos + In T cells, Western blot results showed a significant increase in p-STAT5 (phosphorylated STAT5) expression. Figure 8 (B). Immunofluorescence analysis further showed that STAT5 and Foxp3 were co-localized and increased in the cell nucleus. Figure 8 (C, D). Meanwhile, Foxp1 expression in the nucleus was also significantly increased, and it showed significant co-localization with Foxp3 (C, D). Figure 8 (E, F). These results indicate that Foxp1 not only indirectly regulates Foxp3 expression by promoting STAT5 activation and nuclear translocation, but also enhances Treg differentiation through direct interaction with Foxp3.

[0129] To confirm the key role of Foxp1, this invention used siRNA to silence Foxp1 in MSCs and generated siFoxp1 Agg-exos( Figure 8 (G, H). Compared with the Agg-exos treatment group, CD4 after processing siFoxp1 Agg-exos... + T cells showed a significant reduction in Treg cell differentiation. Figure 8 The number of Th1, Th2, or Th17 cells did not change significantly (I), while the number of Th1, Th2, or Th17 cells did not change significantly. Figure 9 (Middle B). The expression of genes related to Treg differentiation and the expression of Treg activity markers were also significantly decreased in the siFoxp1 Agg-exos group. Figure 8 (J, K). Furthermore, siFoxp1 Agg-exos failed to enhance CD4. + Expression of p-STAT5 in T cells ( Figure 8 (L), and the colocalization of STAT5 and Foxp3 in the cell nucleus was significantly reduced compared to Agg-exos (L). Figure 8 M, Figure 9 Similarly, the expression of Foxp1 and its nuclear co-localization with Foxp3 were also significantly reduced (C). Figure 8 N, Figure 9 (D).

Claims

1. A method for preparing mesenchymal stem cell exosomes, characterized in that, The process includes obtaining a single-cell suspension of mesenchymal stem cells, suspending the single-cell suspension in an exosome-removing culture medium to induce mesenchymal stem cells to aggregate into dense cell clusters, and then collecting the exosomes by differential centrifugation.

2. The preparation method according to claim 1, characterized in that, The mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, dental pulp mesenchymal stem cells, or bone marrow mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that, The exosome removal medium is the medium after removing serum-derived exosomes from the serum medium.

4. The preparation method according to claim 1, characterized in that, The suspension culture was carried out using ultra-low adhesion culture dishes.

5. The preparation method according to claim 1, characterized in that, The suspension culture time is 48–72 h.

6. The preparation method according to claim 1, characterized in that, The differential centrifugation conditions are as follows: centrifuge at 200–400g for 10–15 minutes, at 1800–2000g for 10–20 minutes, at 8000–10000g for 30–60 minutes, and at 8000–100000g for 70–90 minutes. After collecting the precipitate, wash it and centrifuge at 8000–100000g for 70–90 minutes.

7. Mesenchymal stem cell exosomes prepared by any of the preparation methods described in claims 1 to 6.

8. The use of the mesenchymal stem cell exosomes of claim 7 in the preparation of a medicament for treating autoimmune diseases.

9. The application according to claim 8, characterized in that, The autoimmune disease mentioned is systemic lupus erythematosus.

10. A drug, characterized in that, It contains the mesenchymal stem cell exosomes as described in claim 7 and pharmaceutically acceptable excipients.

Images