Mesenchymal stem cell secretome with high expression of il-10, preparation method thereof and application thereof in prevention and treatment of ischemic stroke

Abstract

The application relates to an IL-10 high-expression mesenchymal stem cell secretion component, a preparation method thereof and application thereof in prevention and treatment of ischemic cerebral stroke, and belongs to the technical field of cerebral stroke treatment. In order to solve the problems existing in the current stem cell clinical medication and administration method for ischemic cerebral stroke, the application provides a specially treated mesenchymal stem cell secretion component (hMSC-M), the secretion component is an IL-10 high-expression stem cell secretion component obtained by using a basic culture medium containing human IFN-gamma protein and TNF-alpha simulation peptide to induce human mesenchymal cells after expansion, and then harvesting culture and repeatedly freezing and thawing. The application finds that the hMSC-M has a significant effect on prevention and treatment of ischemic cerebral stroke through animal experiments, thereby opening up a new drug use for stem cell application, laying a foundation for developing high-efficiency related drugs for preventing and treating ischemic cerebral stroke injury, and providing a new application scheme and thought.

Description

Technical Field

[0001] This invention belongs to the field of stroke treatment technology, specifically relating to a mesenchymal stem cell secretory component with high IL-10 expression, its preparation method, and its application in the prevention and treatment of ischemic stroke. Background Technology

[0002] Stroke is the second leading cause of death and disability worldwide. Its incidence and mortality rates continue to rise, placing a significant burden on patients' families and society. According to the latest epidemiological studies, ischemic stroke accounts for as much as 69.6% of all stroke cases in my country. Ischemic stroke presents diverse pathological types and complex pathogenesis, involving the release of inflammatory mediators, the penetration of inflammatory cells, damage to the blood-brain barrier, and the generation of inflammatory factors. Although the treatment window for endovascular mechanical thrombectomy or thrombolysis in ischemic stroke has been extended, the narrow treatment time and the risk of hemorrhagic complications remain significant challenges for clinicians. Therefore, developing effective prevention and treatment measures is crucial for reducing the incidence of cerebral hemorrhage and improving patient prognosis.

[0003] Stem cell transplantation therapy is considered a novel treatment method to promote brain repair after stroke. Human umbilical cord mesenchymal stem cells have attracted widespread attention due to their ease of acquisition and fewer ethical concerns. However, the poor homing ability and viability of transplanted stem cells after homing reduce their therapeutic efficacy. Stem cell derivatives—exosomes and vesicles—can promote neurogenesis and reduce inflammation, thus mitigating these problems to some extent. However, their clinical application as cell-free therapy still requires further exploration. Furthermore, while intravenous injection is the most widely used route of exosome administration, it carries the risk of retention in the pulmonary capillary network, causing infusion problems. Therefore, effectively avoiding these dilemmas is a key issue that needs to be addressed in the current treatment of ischemic stroke using stem cells. Summary of the Invention

[0004] To address the shortcomings of current clinical drug use and administration methods for stem cells in ischemic stroke, this invention provides a specially treated mesenchymal stem cell secretory component (hMSC-M). This secretory component is obtained by inducing expanded human mesenchymal cells with a basal culture medium containing human IFN-γ mimic peptide and TNF-α mimic peptide, followed by harvesting culture and repeated freeze-thaw cycles, resulting in a stem cell secretory component with high IL-10 expression. This invention utilizes hMSC-M pretreatment in mice, revealing that pretreatment with hMSC-M increases the proportion and number of B cells in peripheral immune organ lymph nodes. Furthermore, a cerebral ischemia model demonstrates that pretreatment with stem cell secretory components significantly reduces cerebral ischemia volume and improves spleen weight reduction induced by cerebral ischemia. In addition, it was found that hMSC-M has a more significant therapeutic effect on cerebral ischemia compared to mesenchymal stem cell secretory supernatant (hMSC-L), improving pathological damage and blood-brain barrier leakage, inhibiting the expression of inflammatory factors, and promoting the expression of downstream SOCS1 / STAT6 / p-STAT6 pathways by activating the nuclear translocation of PPAR-γ / p-PPAR-γ, thus inducing neutrophil N2 reprogramming. Therefore, it has significant clinical application value.

[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution:

[0006] The first objective of this invention is to provide a method for preparing a mesenchymal stem cell secretory component with high IL-10 expression, the method comprising the following steps:

[0007] S1. Expansion culture: A sufficient amount of human mesenchymal stem cells are obtained through expansion culture as the first mesenchymal stem cells;

[0008] S2. Induction Culture: The first mesenchymal stem cells obtained in S1 were placed in an induction culture medium and cultured at 2-5% O2, 5% CO2, and 37°C for 12-24 hours to obtain the second mesenchymal stem cells. The induction culture medium was prepared by adding cytokines to the basal culture medium. The cytokines were human IFN-γ mimic peptide and TNF-α mimic peptide. The amino acid sequence of the human IFN-γ mimic peptide was as shown in SEQ ID NO.1, and the amino acid sequence of the TNF-α mimic peptide was NKHNRKI.

[0009] S3. Harvesting Culture: Discard the culture medium obtained in S2, wash the cells twice with physiological saline, harvest the cells by trypsin digestion, and then... 6 Inoculate the substrate with an inoculum of 1 / mL into an ultra-low adsorption 96U plate, centrifuge, add α-MEM medium containing 5-10% human AB serum, and incubate at 5% O2, 5% CO2, 37℃ for 12h.

[0010] S4. Repeated freeze-thaw cycles: Freeze the 96-well plate at -80°C for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate, which is the secretory component of mesenchymal stem cells with high IL-10 expression.

[0011] In one embodiment of the present invention, the expansion culture described in S1 involves reviving human mesenchymal stem cells in a 37°C water bath, then seeding them in a basal culture medium and culturing them at 5% O2, 5% CO2, and 37°C until the cell confluence reaches 70%–90%.

[0012] In one embodiment of the present invention, the amount of human IFN-γ mimic peptide added in S2 is 600 U / mL, and the amount of TNF-α mimic peptide added is 10 ng / mL.

[0013] In one embodiment of the present invention, the basal culture medium S2 is α-MEM culture medium containing double antibiotics and 10-20% FBS or double antibiotics and 10-20% human AB serum.

[0014] The second objective of this invention is to provide a mesenchymal stem cell secretory component with high IL-10 expression obtained by the above preparation method.

[0015] A third objective of this invention is to provide the application of the above-mentioned mesenchymal stem cell secretory components in increasing the proportion and number of B cells in lymph nodes of peripheral immune organs.

[0016] A fourth objective of this invention is to provide the use of the above-mentioned mesenchymal stem cell secretion components in the preparation of a medicament for the prevention of ischemic stroke.

[0017] A fifth objective of this invention is to provide the use of the above-mentioned mesenchymal stem cell secretion components in the preparation of a medicament for treating ischemic stroke.

[0018] In one embodiment of the present invention, the application includes at least one of the following:

[0019] (1) Reduce the volume of cerebral ischemia;

[0020] (2) Promotes the recovery of motor function;

[0021] (3) Improves the pathological damage of ischemic brain tissue;

[0022] (4) Inhibits the expression of inflammatory factors in the brain;

[0023] (5) Promotes the expression of N2 neutrophils that regulate immunity;

[0024] (6) Improves blood-brain barrier leakage;

[0025] (7) Promotes angiogenesis;

[0026] (8) Activate PPAR-γ nuclear translocation;

[0027] (9) Promotes N2 type neutrophil reprogramming.

[0028] The beneficial effects of this invention are:

[0029] This invention proposes a stem cell secretion component with high anti-inflammatory capacity induced by hypoxia in combination with human IFN-γ mimic peptide and TNF-α mimic peptide. Specifically, human mesenchymal cells that have been expanded are induced using a basal culture medium containing human IFN-γ mimic peptide and TNF-α mimic peptide, and then the stem cell secretion component with high IL-10 expression is obtained after harvesting culture and repeated freeze-thaw cycles.

[0030] This invention utilizes hMSC-M pretreatment in mice, revealing that pretreatment with hMSC-M increases the proportion and number of B cells in peripheral immune organ lymph nodes, while the number of innate immune cells and T lymphocytes remains largely unchanged. This indicates that the primary effector cells for hMSC-M in preventing ischemic stroke are B cells in the lymph nodes. Furthermore, a cerebral ischemia model demonstrates that pretreatment with hMSC-M significantly reduces cerebral ischemia volume and alleviates spleen weight loss induced by cerebral ischemia. In addition, preventative experiments show that subcutaneous administration is more advantageous and has a longer-lasting effect than intravenous administration. Specifically, even 7 days after discontinuing the administration of stem cell secretion components, subcutaneous administration still effectively reduces cerebral ischemia volume in a cerebral ischemia model. This invention, through the prevention of ischemic stroke using stem cell secretion components, not only avoids the risks of stem cell tumorigenesis and immune rejection but also maximizes the preventative effects of various factors within stem cells. This invention lays the theoretical foundation for the preventive application of stem cell secretory components, identifies the main effector cells of stem cell secretory components in preventive health care functions, proposes a more effective drug administration route, and provides new solutions and ideas for the preventive application of stem cells for various diseases, including ischemic stroke.

[0031] This invention discovers and confirms the therapeutic effect of stem cell secretion components on ischemic stroke. Specifically, studies using a middle cerebral artery occlusion (MCAO) model show that nasal administration of stem cell secretion components can reduce cerebral ischemia volume in mice, improve ischemia-induced brain tissue pathological damage, promote motor function recovery in stroke-affected mice, improve blood-brain barrier leakage, and promote immunomodulatory N2 neutrophil expression. Specifically, stem cell secretion components can activate PPAR-γ nuclear translocation and promote N2 neutrophil reprogramming, and the effect of stem cell secretion components on neutrophils is superior to that of stem cell supernatant. Furthermore, nasal administration not only achieves the same efficacy as other administration methods but also has high patient compliance.

[0032] This invention opens up new drug applications for stem cell research, lays the foundation for developing highly effective drugs to prevent and treat ischemic stroke, and provides new application schemes and ideas. Attached Figure Description

[0033] Figure 1 The image shows the ELISA results of the concentration of the anti-inflammatory factor IL-10 in hMSCs-M obtained in Examples 1-3 and the hMSCs-M obtained in each comparative example; where, hypoxia- indicates a normoxic culture environment of 20% O2, hypoxia+ indicates a hypoxic culture environment of 5% O2, and ①②③ indicate the addition of human IFN-γ mimic peptide and TNF-α mimic peptide according to the addition amounts in Examples 1, 2 and 3, respectively.

[0034] Figure 2 The figure shows the effect of pre-administration of hMSCs-M obtained in Example 2 via IV or IH on the proportion and number of B cells and T cells in mouse lymph nodes and bone marrow; where LN represents lymph nodes, BM represents bone marrow; ns indicates no statistical significance, **P<0.01, ***P<0.001, ****P<0.0001;

[0035] Figure 3 The figure shows the effect of pre-administration of hMSCs-M obtained in Example 2 via IV or IH on the proportion and number of innate immune cells in mouse lymph nodes (LN) and bone marrow (BM); where, Figure 3 In this context, A represents NK1.1 in lymph nodes and bone marrow. + The effect of NK cell proportion and number on the results is shown in the figure. Figure 3 In this context, B represents Ly6C in bone marrow and peripheral blood (PB). + The effect of the proportion of monocytes on the results. Figure 3 C in the figure represents the effect of Ly6G in bone marrow and peripheral blood. + The effect of the proportion of neutrophils on the results is shown in the figure. Figure 3 D in the figure represents F4 / 80 in bone marrow and peripheral blood. + The effect of macrophage proportion on the result; ns indicates no statistical significance, **P<0.01, ***P<0.001;

[0036] Figure 4 The figure shows the effect of pre-administration of hMSCs-M obtained in Example 2 via IV or IH on the cerebral ischemia volume in a mouse MCAO model; where pre7+0, pre7+3, and pre7+7 represent modeling performed 7 days after pre-administration, 3 days after pre-administration, and 7 days after pre-administration, respectively; *P<0.05;

[0037] Figure 5 The figure shows the effect of pre-administration of hMSCs-M obtained in Example 2 via IV or IH on spleen weight in a mouse MCAO model; where pre7+0, pre7+3, and pre7+7 represent modeling performed 7 days after pre-administration, 3 days after 7 days after pre-administration, and 7 days after 7 days after pre-administration, respectively; **P<0.01, ##P<0.01;

[0038] Figure 6 The figure shows the effects of hMSC-L and hMSC-M treatments on cerebral ischemia volume and neurological dysfunction in mice; among them, Figure 6 Figure A in the figure shows the results of TTC staining to detect cerebral ischemic volume in mice after ischemic stroke following different treatments with hMSC-L or hMSC-M. Figure 6 In the figure, B represents the statistical results of cerebral ischemic volume after ischemic stroke in the hMSCs-L group mice. Figure 6 In the figure, C represents the neurological function impairment score of the hMSCs-L group mice. Figure 6 The figure shows the statistical results of cerebral ischemia volume after ischemic stroke in the hMSCs-M group mice. Figure 6 E in the figure represents the neurological dysfunction score of the hMSCs-M group mice; ns indicates no statistical significance, *P<0.05, **P<0.01, ***P<0.001;

[0039] Figure 7 The figure shows the results of evaluating the effects of hMSC-L and hMSC-M treatment on motor function recovery in stroke mice using the angle-turning test and body-lifting test; among them, Figure 7 Figure A in the diagram represents the results of the cornering experiment in the hMSCs-L group mice. Figure 7 Figure B in the diagram represents the results of the body elevation experiment in the hMSCs-L group mice. Figure 7 In the figure, C represents the results of the angle-turning experiment in the hMSCs-M group mice. Figure 7 In the figure, D represents the results of the body elevation experiment in the hMSCs-M group mice; ns indicates no statistical significance, *P<0.05, **P<0.01, ***P<0.001;

[0040] Figure 8 The figure shows the results of detecting the effects of HE staining and Nissl staining on the pathological damage of hMSCs-M treatment to the brain tissue of ischemic mice; among them, Figure 8 Image A in the image represents a representative HE-stained image of the cortical region (200x magnification, scale bar 50μm). Figure 8 Image B in the image represents a representative image of Nissl staining in the cortical region (200x magnification, scale bar 50μm). Figure 8 In the figure, C represents the statistical results of HE staining. Figure 8 D in the figure represents the statistical results of Nissl staining; ***P<0.001, ##P<0.01, ###P<0.001;

[0041] Figure 9 This image shows the results of detecting the levels of major pro-inflammatory cytokine genes in the brains of mice after ischemic stroke; among them, Figure 9 Figure A shows the results of qPCR detection of IFN-γ mRNA expression levels in the brain after cerebral ischemia. Figure 9 Figure B in the diagram shows the results of qPCR detection of TNF-α mRNA expression levels in the brain after cerebral ischemia. Figure 9 Figure C represents the results of qPCR detection of IL-1β mRNA expression levels in the brain after cerebral ischemia; *P<0.05, **P<0.01, ***P<0.001;

[0042] Figure 10 hMSC-L and hMSC-M therapy for immunomodulatory N2 neutrophils (CD45) high CD11b + Ly6G + CD206 + The effect of expression on the results is shown in the figure; ##P<0.01, ***P<0.001;

[0043] Figure 11 The figure shows the effect of hMSC-L and hMSC-M treatment on blood-brain barrier leakage after stroke in mice; among them, Figure 11 Image A in the figure represents the results of immunofluorescence staining detection of occludin expression (magnified 400 times, scale bar 50 μm). Figure 11 In the image, B represents the result of the EB method for detecting the integrity of the blood-brain barrier. Figure 11 Figure C in the graph represents the results of a statistical analysis of the integrity of the blood-brain barrier. Figure 11 In the figure, D represents the results of statistical analysis of immunofluorescence staining; *P<0.05, **P<0.01, ***P<0.001;

[0044] Figure 12 The figure shows the effect of hMSC-L and hMSC-M treatment on blood-brain barrier permeability; among them, Figure 12 In the image, A represents the results of immunofluorescence staining (400×) detecting the expression of the blood-brain barrier tight junction-related protein ZO-1. The scale bar is 50 μm. Figure 12 B in Figure 12 The statistical results for A in the figure are shown in the figure, **P<0.01;

[0045] Figure 13The figure shows the effects of hMSC-L and hMSC-M treatment on PPAR-γ nuclear translocation; among them, Figure 13 Figure A in the diagram represents the results of Western blotting (WB) detection of nuclear PPAR-γ protein expression levels. Figure 13 In the figure, B represents the statistical results of nuclear PPAR-γ protein expression levels. Figure 13 Figure C in the diagram shows the results of Western blotting (WB) detection of cytoplasmic PPAR-γ, total PPAR-γ, and p-PPAR-γ protein expression levels. Figure 13 In the figure, D represents the statistical results of cytoplasmic PPAR-γ, total PPAR-γ, and P-PPAR-γ protein expression levels; vs PMA, **P<0.01, ***P<0.001; PMA+L+ inhibitor vs PMA+L, ##P<0.01, ###P<0.001; PMA+M+ inhibitor vs PMA+M, $P<0.01, $$P<0.001;

[0046] Figure 14 Figure 1 shows the effects of hMSC-L and hMSC-M treatments on N2 neutrophil reprogramming; Figure 14 In the diagram, A represents the flow cytometry detection of Ly6G in neutrophils. + CD206 + and Ly6G + iNOS + The results of the expression level, Figure 14 B in the text represents Ly6G in neutrophils. + CD206 + and Ly6G + iNOS + The results of statistical analysis of expression levels are shown in the figure; compared with PMA, *P<0.05, **P<0.01, ***P<0.001; PMA+L+ inhibition vs PMA+L, ###P<0.001, PMA+M+ inhibition vs PMA+M, $$P<0.001. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The embodiments described in this invention are not all embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, and instruments used are all conventional materials, reagents, and instruments in the art, which can be obtained commercially by those skilled in the art unless otherwise specified.

[0048] The amino acid sequence of the human IFN-γ mimic peptide used in this invention is LTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQM (SEQ ID NO.1); the amino acid sequence of the TNF-α mimic peptide used in this invention is NKHNRKI.

[0049] The experimental data of this invention were statistically analyzed using GraphPad Prism 9.0 software. Unpaired t-tests were used for comparisons between two groups of samples; one-way ANOVA was used for comparisons between three or more groups of samples. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

[0050] Example 1: A method for preparing a mesenchymal stem cell secretory component with high IL-10 expression.

[0051] In this embodiment, the induction medium used is based on the basal medium with the addition of human IFN-γ mimic peptide and TNF-α mimic peptide. The specific composition of the induction medium is α-MEM medium, double antibiotics, 10% FBS, 300 U / mL human IFN-γ mimic peptide, and 10 ng / mL TNF-α mimic peptide.

[0052] The specific method for preparing IL-10-overexpressing mesenchymal stem cell secretory components using the above-mentioned induction culture medium is as follows:

[0053] S1. Expansion culture: Human umbilical cord mesenchymal stem cells were revived in a 37°C water bath and then seeded in α-MEM medium containing 10% FBS and double antibiotics. The cells were cultured at 37°C under conditions of 5% O2, 5% CO2, to obtain a sufficient amount of human mesenchymal stem cells as the first mesenchymal stem cells.

[0054] S2, Induction Culture: The first mesenchymal stem cells obtained in S1 were placed in induction culture medium and cultured at 5% O2, 5% CO2, and 37℃ for 24 h to obtain the second mesenchymal stem cells;

[0055] S3. Harvesting Culture: Discard the culture medium obtained in S2, wash the cells twice with 10 mL of physiological saline, harvest the cells by trypsin digestion, and then... 6 Inoculate the cells / mL into the ultra-low adsorption 96U substrate, centrifuge at 300g for 3min, add 100μL of α-MEM medium containing 10% human AB serum to each well, and incubate at 5% O2, 5% CO2, 37℃ for 12h.

[0056] S4. Repeated freeze-thaw cycle: Freeze the 96-well plate at -80°C for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate to obtain the IL-10-overexpressing mesenchymal stem cell secretory fraction (hMSC-M).

[0057] The method for preparing mesenchymal stem cell supernatant (hMSCs-L) is as follows: after step S3, a portion of the cells are centrifuged to obtain the mesenchymal stem cell supernatant.

[0058] Example 2: A method for preparing a mesenchymal stem cell secretory component with high IL-10 expression.

[0059] In this embodiment, the induction medium used is based on the basal medium with the addition of human IFN-γ mimic peptide and TNF-α mimic peptide. The specific composition of the induction medium is α-MEM medium, double antibiotics, 10% FBS, 1000 U / mL human IFN-γ mimic peptide, and 5 ng / mL TNF-α mimic peptide.

[0060] The specific method for preparing IL-10-overexpressing mesenchymal stem cell secretory components using the above-mentioned induction culture medium is as follows:

[0061] S1. Expansion culture: Human umbilical cord mesenchymal stem cells were revived in a 37°C water bath and then seeded in α-MEM medium containing 10% FBS and double antibiotics. The cells were cultured at 37°C under conditions of 5% O2, 5% CO2, to obtain a sufficient amount of human mesenchymal stem cells as the first mesenchymal stem cells.

[0062] S2, Induction Culture: The first mesenchymal stem cells obtained in S1 were placed in induction culture medium and cultured at 5% O2, 5% CO2, and 37℃ for 24 h to obtain the second mesenchymal stem cells;

[0063] S3. Harvesting Culture: Discard the culture medium obtained in S2, wash the cells twice with 10 mL of physiological saline, harvest the cells by trypsin digestion, and then... 6 Inoculate the cells / mL into the ultra-low adsorption 96U substrate, centrifuge at 300g for 3min, add 100μL of α-MEM medium containing 10% human AB serum to each well, and incubate at 5% O2, 5% CO2, 37℃ for 12h.

[0064] S4. Repeated freeze-thaw cycle: Freeze the 96-well plate at -80°C for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate to obtain the IL-10-overexpressing mesenchymal stem cell secretory fraction (hMSC-M).

[0065] The method for preparing mesenchymal stem cell supernatant (hMSCs-L) is as follows: after step S3, a portion of the cells are centrifuged to obtain the mesenchymal stem cell supernatant.

[0066] Example 3: A method for preparing a mesenchymal stem cell secretory component with high IL-10 expression.

[0067] In this embodiment, the induction medium used is based on the basal medium with the addition of human IFN-γ mimic peptide and TNF-α mimic peptide. The specific composition of the induction medium is α-MEM medium, double antibiotics, 10% FBS, 600 U / mL human IFN-γ mimic peptide, and 10 ng / mL TNF-α mimic peptide.

[0068] The specific method for preparing IL-10-overexpressing mesenchymal stem cell secretory components using the above-mentioned induction culture medium is as follows:

[0069] S1. Expansion culture: Human umbilical cord mesenchymal stem cells were revived in a 37°C water bath and then seeded in α-MEM medium containing 10% FBS and double antibiotics. The cells were cultured at 37°C under conditions of 5% O2, 5% CO2, to obtain a sufficient amount of human mesenchymal stem cells as the first mesenchymal stem cells.

[0070] S2, Induction Culture: The first mesenchymal stem cells obtained in S1 were placed in induction culture medium and cultured at 5% O2, 5% CO2, and 37℃ for 24 h to obtain the second mesenchymal stem cells;

[0071] S3. Harvesting Culture: Discard the culture medium obtained in S2, wash the cells twice with 10 mL of physiological saline, harvest the cells by trypsin digestion, and then... 6 Inoculate the cells / mL into the ultra-low adsorption 96U substrate, centrifuge at 300g for 3min, add 100μL of α-MEM medium containing 10% human AB serum to each well, and incubate at 5% O2, 5% CO2, 37℃ for 12h.

[0072] S4. Repeated freeze-thaw cycle: Freeze the 96-well plate at -80°C for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate to obtain the IL-10-overexpressing mesenchymal stem cell secretory fraction (hMSC-M).

[0073] The method for preparing mesenchymal stem cell supernatant (hMSCs-L) is as follows: after step S3, a portion of the cells are centrifuged to obtain the mesenchymal stem cell supernatant.

[0074] Comparative Example 1: Preparation method of secretory components from mesenchymal stem cells

[0075] The induction medium used in this comparative example is a basal medium supplemented with commercially available IFN-γ and TNF-α. The specific composition of this induction medium is α-MEM medium, double antibiotics, 10% FBS, IFN-γ 300U / mL, and TNF-α 10ng / mL.

[0076] The specific method for preparing mesenchymal stem cell secretory components using the above-mentioned induction culture medium is as follows:

[0077] S1. Expansion culture: Human umbilical cord mesenchymal stem cells were revived in a 37°C water bath and then seeded in α-MEM medium containing 10% FBS and double antibiotics. The cells were cultured at 37°C under conditions of 5% O2, 5% CO2, to obtain a sufficient amount of human mesenchymal stem cells as the first mesenchymal stem cells.

[0078] S2, Induction Culture: The first mesenchymal stem cells obtained in S1 were placed in induction culture medium and cultured at 5% O2, 5% CO2, and 37℃ for 24 h to obtain the second mesenchymal stem cells;

[0079] S3. Harvesting Culture: Discard the culture medium obtained in S2, wash the cells twice with 10 mL of physiological saline, harvest the cells by trypsin digestion, and then... 6 Inoculate the cells / mL into the ultra-low adsorption 96U substrate, centrifuge at 300g for 3min, add 100μL of α-MEM medium containing 10% human AB serum to each well, and incubate at 5% O2, 5% CO2, 37℃ for 12h.

[0080] S4. Repeated freeze-thaw cycles: Freeze the 96-well plate at -80°C for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate to obtain the mesenchymal stem cell secretory component hMSCs-M.

[0081] This invention detected the IL-10 content in hMSCs-M obtained in Examples 1-3 and Comparative Example 1. Simultaneously, hMSCs-M obtained under normoxic conditions without any cytokine addition, hMSCs-M obtained under hypoxic conditions without any cytokine addition, and hMSCs-M obtained under normoxic conditions with the addition of IFN-γ and TNF-α (at the same amounts as in Example 1) served as controls. The IL-10 content detection results are shown below. Figure 1 ,Depend on Figure 1 It can be seen that the IL-10 concentration in hMSCs-M obtained in Examples 1-3 was significantly increased compared with various controls. The above results indicate that the induction medium and the method for preparing mesenchymal stem cell secretory components provided by the present invention can significantly increase the IL-10 secretion of mesenchymal stem cells.

[0082] Example 4: Application of IL-10-overexpressing mesenchymal stem cell secretion components in increasing the proportion and number of B cells in peripheral immune organ lymph nodes

[0083] Male C57B / 6 mice weighing 20-22g were randomly divided into three groups: a sham group, an intravenous (iv) administration group, and a subcutaneous (ih) administration group, with five mice in each group. The sham group received no administration. The other groups were pretreated for 7 consecutive days with IL-10-overexpressing mesenchymal stem cell secretory fraction (hMSCs-M) obtained in Example 2, at a concentration of 2 mg / mL, with each mouse receiving 100 μg. After administration, lymph nodes and bone marrow tissues were collected from the mice for flow cytometry analysis to assess the proportion of different immune cell subsets and changes in cell count.

[0084] The results are as follows Figure 2 As described above, after mice were administered hMSCs-M obtained in Example 2 via intravenous (IV) or intrahual (IH) administration for 7 consecutive days, the proportion and number of B cells in the lymph nodes of mice treated with IH administration were increased, as were CD4 cells. + T and CD8 + The proportion of T cells decreased, but the number did not change significantly; B cells and CD4+ in the bone marrow... + T and CD8 + The proportion and number of T cells did not change significantly; the number of B cells and CD4+ cells in the lymph nodes of mice treated with intravenous administration was significantly increased. + T and CD8 + The proportion and number of T cells did not change significantly, while only the proportion of B cells in the bone marrow decreased, with no significant difference in their numbers.

[0085] The hMSCs-M obtained in Example 2 were administered to mice via intravenous or intrahepatic administration for 7 consecutive days prior to the test, followed by detection of innate immune cells (NK cells) in the lymph nodes and bone marrow of the mice. + NK cells, Ly6C + Monocytes, Ly6G + Neutrophils, F4 / 80 + The results showed a decrease in the proportion of macrophages, but no significant change in the number of NK cells. For the other administration routes, whether IV or IH, the proportion of innate immune cells did not change significantly (see...). Figure 3 ).

[0086] It is evident that subcutaneous administration of IL-10-overexpressing mesenchymal stem cell secretion components to mice can increase the proportion and number of B cells in the lymph nodes of peripheral immune organs, but the number of innate immune cells and T lymphocytes does not change significantly.

[0087] Example 5: Application of IL-10-overexpressing mesenchymal stem cell secretion components in the prevention of ischemic stroke

[0088] Mice were divided into three groups: the Sham group (no drug administration), the intravenous (iv) drug administration group, and the subcutaneous (ih) drug administration group, with 5 mice in each group. They were pretreated for 7 consecutive days with the mesenchymal stem cell secretory fraction (hMSCs-M) with high IL-10 expression obtained in Example 2, at a concentration of 2 mg / mL, with 100 μg per mouse.

[0089] Construction of a MCAO (Malignant Stroke Agenda): On days 1, 3, and 7 after pretreatment, 2% sodium pentobarbital solution was prepared and anesthetized male C57 / BL6 mice weighing 20-22g via intraperitoneal injection. Anesthetized mice were fixed in a supine position. After neck preparation and disinfection, the skin was incised along the midline of the neck using ophthalmic scissors. The neck muscles were bluntly dissected, and the right common carotid artery, external carotid artery, and internal carotid artery were carefully dissected. The common carotid artery and external carotid artery were ligated with sutures, and a slipknot was tied when ligating the internal carotid artery. A suture was inserted at the ligation site of the external carotid artery and at the branch of the common carotid artery, leaving a ligation suture plug. An incision was made between the two sutures of the external carotid artery, and the suture plug was slowly inserted into the common carotid artery, ligating the reserved suture. The ligation suture of the internal carotid artery was removed, and the suture plug was withdrawn to the bifurcation. The suture plug was gently reversed and inserted into the internal carotid artery, slowly pushed in until it blocked the origin of the cerebral artery, causing local ischemia in the brain tissue. Finally, the neck incision was sutured.

[0090] TTC staining:

[0091] On day 3 of MCAO construction, mice were sacrificed, and brain tissue was quickly extracted and placed in a pre-cooled brain slice mold. Using the accompanying blade, slices were cut into approximately 2mm sections. The sections were placed in a 2% TTC solution and incubated at 37°C for 30 minutes; the incubation time could be adjusted according to the degree of staining. After uniform staining, the brain slices were retrieved for photographic analysis.

[0092] Mouse spleen weight measurement:

[0093] Mice were sacrificed on day 3 of MCAO construction, and their spleens were quickly removed. Changes in spleen length were detected twice, and changes in spleen weight were detected by weighing.

[0094] TTC staining results showed that administration of hMSCs-M obtained in Example 2 to mice via either intravenous (IV) or intrahepatic (IH) administration reduced the volume of cerebral ischemia. Seven days after drug withdrawal, an MCAO model was constructed. IH administration was more effective than IV administration in reducing the volume of cerebral ischemia, indicating that pre-administration of hMSCs-M via IH has a longer-lasting immunomodulatory effect (see [link to original text]). Figure 4 ).

[0095] The results of spleen weight analysis in mice showed that, after administering hMSCs-M obtained in Example 2 to mice via intravenous or intrahepatic administration to construct an MCAO model, the pretreatment significantly increased the spleen weight compared to MCAO model mice. This indicates that hMSCs-M pretreatment can improve the inhibition of spleen function caused by cerebral ischemia (see [link to original text]). Figure 5 ).

[0096] Example 6: Application of IL-10-overexpressing mesenchymal stem cell secretion components in the treatment of ischemic stroke

[0097] Construction of a model for middle cerebral artery occlusion (MCAO):

[0098] Prepare a 2% sodium pentobarbital solution and anesthetize male C57 / BL6 mice weighing 20-22g via intraperitoneal injection. After anesthesia, fix the mice in a supine position, prepare and disinfect the neck area, and use ophthalmic scissors to make a midline incision in the neck. Bluntly dissect the neck muscles and carefully separate the right common carotid artery, external carotid artery, and internal carotid artery. Ligate the common carotid artery and external carotid artery with sutures, tying a slipknot when ligating the internal carotid artery. Insert a suture at the ligation site of the external carotid artery and at the branch of the common carotid artery, leaving a ligation plug. Make an incision between the two ligations of the external carotid artery, slowly insert the ligation plug into the common carotid artery, and ligate the reserved suture. Remove the ligation suture from the internal carotid artery, withdraw the ligation plug to the bifurcation, gently reverse the ligation plug and insert it into the internal carotid artery, slowly pushing the ligation plug until it blocks the origin of the cerebral artery, causing local cerebral ischemia. Finally, suture the neck incision.

[0099] Drug administration:

[0100] After the mice regained consciousness, they were treated with hMSC-L (mesenchymal stem cell supernatant) and hMSC-M (IL-10-overexpressing mesenchymal stem cell secretion fraction) obtained in Example 2 via intraperitoneal injection, tail vein injection, and nasal mucosal administration, respectively. The administration concentrations were 1 mg / mL and 2 mg / mL, and the administration volume was 100 μL per mouse, for 3 consecutive days.

[0101] The following experiments were used to evaluate the therapeutic effects of hMSC-L and hMSC-M:

[0102] TTC staining:

[0103] Mice were sacrificed on day 3 of MCAO, and brain tissue was quickly removed and placed in a pre-cooled brain slicing mold. The tissue was then sliced ​​into approximately 2mm sections using the accompanying blade. The sections were placed in a 2% TTC solution and incubated at 37°C for 30 minutes. The incubation time could be adjusted according to the degree of staining. After uniform staining, the brain slices were retrieved for photographing and statistical analysis.

[0104] Neurological function score:

[0105] The Longa five-point scale is commonly used to assess the degree of neurological deficits in mouse models of ischemic stroke. The specific assessment criteria are as follows:

[0106] 0 points: No nerve damage, indicating that the experimental animal has no obvious abnormalities in neurological function;

[0107] 1 point: The contralateral forepaw cannot be fully extended: One of the forepaws of the experimental animal showed limited movement and could not be fully extended;

[0108] 2 points: Turning to the opposite side: This indicates that the experimental animal will lean towards the paralyzed side during movement;

[0109] 3 points: Leaning to the opposite side: The experimental animal lost its balance while walking and leaned to the paralyzed side;

[0110] 4 points: Unable to walk on their own, loss of consciousness.

[0111] Generally speaking, a higher score indicates more severe neurological damage in the experimental animal.

[0112] Cerebral edema detection:

[0113] Mice were sacrificed on day 3 of MCAO, and brain tissue was removed and its weight recorded. The brain tissue was then placed in a drying oven at 60℃-80℃ for 24 hours, with the specific drying time adjusted according to the dryness of the brain tissue. After drying, the brain tissue was weighed again and its weight recorded. The percentage of water content in the brain tissue was calculated as: (wet weight - dry weight) / wet weight × 100%.

[0114] Mouse behavioral experiments:

[0115] (1) Turning test: The experimental setup consists of two plates forming a 30° angle. The mouse is placed in the middle of the setup and allowed to move forward to the deepest part of the angle. Upon touching the plate wall, the mouse will stand up forward and then turn to the left or right. Only when the mouse's hind legs are fully upright before turning is it considered a single test. Each mouse is tested 10 times, with a 1-minute interval between each test. The score is calculated as: (Number of turns on the healthy side / Total number of turns) × 100%.

[0116] (2) Body elevation test: Lift the mouse by the tail, making it about 5 cm away from the plane. At this time, the mouse's head will rotate to the left or right. The counting standard is a unilateral rotation angle >100°. Repeat 20 times for each mouse, with a 1-minute interval between each time. Calculate: number of rotations on the affected side / total number of rotations × 100%.

[0117] Evans Blue staining:

[0118] 200 μL of 2% Evans blue staining solution was injected into mice via the tail vein. Two hours after injection, the mice were sacrificed, their thoracic cavities were opened, and the hearts were perfused with PBS until the outflowing fluid became clear. Brain tissue was then removed, photographed, and statistically analyzed.

[0119] HE staining:

[0120] (1) Remove the frozen slices and let them sit at room temperature for 5 minutes;

[0121] (2) Fix the slices in cold acetone for 15-30 minutes;

[0122] (3) Wash the pathological sections with PBS three times, for three minutes each time;

[0123] (4) Shake off the surface liquid and use a histological pen to circle the tissue;

[0124] (5) Immerse the slices in hematoxylin staining solution for 3 minutes;

[0125] (6) Use pathological PBS to wash away excess staining solution;

[0126] (7) Place the differentiation solution on the tissue surface and wash with pathological PBS after 1 minute;

[0127] (8) Immerse the slide in eosin staining solution for 2 minutes;

[0128] (9) Use pathological PBS to wash away excess staining solution;

[0129] (10) The sections were immersed in 80%, 90% and 100% ethanol in sequence to dehydrate, then immersed in xylene to clear, mounted with neutral resin, and photographed under a microscope.

[0130] Nissl staining:

[0131] (1) Remove the frozen slices and let them sit at room temperature for 5 minutes;

[0132] (2) Fix the slices in cold acetone for 15-30 minutes;

[0133] (3) Wash the pathological sections with PBS three times, for three minutes each time;

[0134] (4) Shake off the surface liquid and use a histological pen to circle the tissue;

[0135] (5) Immerse the slices in Nissl staining solution at 37°C for 10 minutes;

[0136] (6) Use pathological PBS to wash away excess staining solution;

[0137] (7) 95% ethanol rapidly differentiates, and anhydrous ethanol rapidly dehydrates;

[0138] (8) After the xylene is cleared, the slide is sealed with neutral resin and photographed under a microscope.

[0139] Proteins were extracted from tissues and cells, and protein concentration was measured using BCA assay.

[0140] Extraction of tissue protein samples: Blood was collected from mouse eyeballs, and the heart was perfused with PBS until the liver tissue turned white. The required brain tissue was then removed and placed in 1.5 mL of EP containing lysis buffer. The tissue was sonicated and then vortexed 6 times, with 5-minute intervals between each vortex. Centrifugation was then performed at 12000 rpm, 4°C, for 15 minutes. The supernatant was transferred to a new EP tube and stored at -80°C for long-term storage. In a 96-well plate, standards were added sequentially to plot a standard curve. 19.5 μL of NaCl and 0.5 μL of sample were added to each well. BCA mixture was prepared according to the manufacturer's instructions, and 200 μL of the mixture was added to each well. The plates were incubated at 37°C in the dark for 30 minutes. The absorbance of each well was measured using a microplate reader to obtain the standard curve and calculate the sample concentration. Loading buffer and lysis buffer were used to prepare the loading system. After mixing, the plate was placed in a metal bath at 100°C for 9 minutes to denature the proteins. The plates were then stored at -20°C for short-term storage and -80°C for long-term storage.

[0141] Western Blot:

[0142] (1) Glue preparation: Clean a 1.00mm thick glass plate, assemble the glue preparation device, add deionized water and test for leaks for 10 minutes. If the leak is not obvious, proceed to the next step. Prepare and add the separating glue, press it flat with deionized water, let it solidify for 30 minutes, pour off the upper layer of deionized water, add the prepared concentrated glue, insert the comb, and let it stand for 15 minutes;

[0143] (2) Electrophoresis: Assemble the electrophoresis system, add electrophoresis buffer to the electrophoresis tank, remove the comb, and add the marker and sample. Run the stacking gel at a constant voltage of 90V for 15 minutes; after the samples are compacted, run the separating gel at a constant voltage of 120V for 60 minutes.

[0144] (3) Transfer: Cut the gel according to the molecular weight of the target protein, cut the PVDF membrane, put it in methanol to activate, assemble the transfer sandwich, keep the current at 300mA, and adjust the time according to the molecular weight of the target protein.

[0145] (4) Sealing: Place the strip in 5% skim milk powder and seal it in a shaker at room temperature for 2 hours;

[0146] (5) Incubation of primary antibody: Prepare primary antibody using antibody dilution buffer according to the instructions and incubate overnight at 4°C;

[0147] (6) Washing the membrane: Wash the incubated strips twice with TBST and once with TBS, each time for 10 minutes;

[0148] (7) Incubation of secondary antibody: Prepare secondary antibody using TBST according to the instructions and incubate on a shaker at room temperature for 1 hour;

[0149] (8) Washing the membrane: TBST twice, TBS once, 10 minutes each time;

[0150] (9) Color development: Add Western Blue or ECL color development solution to develop color. After stopping the color development, scan the bands for analysis.

[0151] The results of TTC staining for cerebral ischemia volume and neurological function impairment scores after ischemic stroke showed that, compared with intravenous administration and intraperitoneal injection (IV and IP), nasal administration (in) has unique advantages such as convenience, non-invasiveness, and high absorption while achieving the same therapeutic effect (see...). Figure 6 ).

[0152] The effects of 1 mg / mL hMSC-L and 1 mg / mL hMSC-M treatment on motor function recovery in stroke-affected mice were evaluated using the angle-turning test and body-lifting test. The angle-turning test primarily assesses asymmetric sensorimotor impairment in mice and can detect unilateral abnormalities in sensory and motor function in stroke-affected mice. Statistical results showed that administration of hMSC-L via intravenous (in), intravenous (iv), and intraperitoneal (ip) routes significantly improved sensory and motor function abnormalities in mice (see [link to study]. Figure 7 (A and B in the text), after treatment with hMSC-M, also showed the same effect as hMSC-L (see A and B in the text). Figure 7 (C and D in the text).

[0153] The body elevation test was used to assess the asymmetry of motor function in mice. Healthy mice exhibited comparable frequency and intensity of body elevation bilaterally, but MCAO mice with neurological deficits on the ischemic side showed a relatively lower proportion of rotations towards the affected side. Statistical results showed that administration of hMSC-L via all three routes significantly improved the proportion of mice rotating to the right (see [link to results]). Figure 7 (B and D in the middle)

[0154] After establishing the MACO model, HE staining revealed disordered cell arrangement, vacuolation, and nuclear atrophy in the MCAO group. These phenomena were alleviated after treatment with hMSC-M at concentrations of 1 mg / mL and 2 mg / mL. Statistical results showed that 1 mg / mL and 2 mg / mL hMSC-M significantly improved the number of damaged cells in mouse brain tissue, with the 2 mg / mL group showing a more significant reduction in the number of damaged cells in mouse brain tissue (see...). Figure 8(A and C in the text).

[0155] Nissl staining results showed a decrease in the number and disordered arrangement of Nissl bodies in the brains of MCAO group mice. These phenomena were alleviated after treatment with hMSC-M at concentrations of 1 mg / mL and 2 mg / mL. Statistical results showed that 1 mg / mL and 2 mg / mL significantly increased the number of Nissl bodies in mouse brain tissue, with the 2 mg / mL group showing a significantly greater increase in the number of Nissl bodies (see [link to data]). Figure 8 (B and D in the text). The above results indicate that treatment with hMSC secretory factors and cellular secretory components significantly improved the pathological damage of brain tissue in ischemic mice, and the therapeutic effect was more significant with high concentrations of secretory factors and cellular secretory components.

[0156] By examining the expression levels of major pro-inflammatory cytokine genes in the brains of mice following ischemic stroke, it was found that the transcriptional levels of IFN-γ, TNF-α, and IL-1β in the brain significantly decreased after treatment with hMSC-L and hMSC-M. In particular, the decreases in IFN-γ and TNF-α in the brains of mice in the hMSC-M group were more significant than those in the hMSC-L group (see...). Figure 9 ).

[0157] Furthermore, hMSC-L and hMSC-M treatments can significantly downregulate blood-brain barrier leakage volume, and the degree of angiogenesis may be more significant after hMSC-M treatment (see...). Figure 11 and Figure 12 ).

[0158] Following treatment with hMSCs, PPAR-γ function was further activated, nuclear translocation was significantly enhanced, and phosphorylation levels decreased. Inhibition of PPAR-γ using T0070907 significantly suppressed neutrophil activation induced by hMSC-L and hMSC-M, manifested as downregulation of nuclear PPAR-γ expression and upregulation of p-PPAR-γ expression. After stimulation with hMSC-L and hMSC-M, the proportion of N2 neutrophils was significantly upregulated, while the proportion of N1 neutrophils was significantly decreased. Furthermore, inhibition of PPAR-γ significantly shifted the polarization of neutrophils in both groups towards the N1 type (see...). Figure 10 , Figure 13 and Figure 14 ).

[0159] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for preparing a mesenchymal stem cell secretory component with high IL-10 expression, characterized in that, Includes the following steps: S1. Expansion culture: A sufficient amount of human mesenchymal stem cells are obtained through expansion culture as the first mesenchymal stem cells; S2. Induction Culture: The first mesenchymal stem cells obtained in S1 were placed in an induction culture medium and cultured at 2-5% O2, 5% CO2, and 37℃ for 12-24 h to obtain the second mesenchymal stem cells. The induction culture medium was prepared by adding cytokines to the basal culture medium. The cytokines were human IFN-γ mimic peptide and TNF-α mimic peptide. The amino acid sequence of the human IFN-γ mimic peptide is shown in SEQ ID NO.1, and the amino acid sequence of the TNF-α mimic peptide is NKHNRKI. S3. Harvesting Culture: Discard the culture medium obtained in S2, wash the cells twice with physiological saline, harvest the cells by trypsin digestion, and then... 6 Inoculate the inoculum at a rate of 1 / mL into an ultra-low adsorption 96U substrate, centrifuge, add α-MEM medium containing 5-10% human AB serum, and incubate at 5% O2, 5% CO2, and 37℃ for 12 h. S4. Repeated freeze-thaw cycles: Freeze the 96-well plate at -80°C for 8 hours, thaw at room temperature, repeat twice, and collect the cell lysate, which is the secretory component of mesenchymal stem cells with high IL-10 expression.

2. The preparation method according to claim 1, characterized in that, The expansion culture described in S1 involves reviving human mesenchymal stem cells in a 37°C water bath, then seeding them in basal culture medium and culturing them at 5% O2, 5% CO2, and 37°C until the cell confluence reaches 70%–90%.

3. The preparation method according to claim 1, characterized in that, The amount of human IFN-γ mimic peptide added in S2 is 200-1000 U / mL, and the amount of TNF-α mimic peptide added is 5-10 ng / mL.

4. The preparation method according to claim 3, characterized in that, The optimal addition amount of the human IFN-γ mimic peptide S2 is 600 U / mL, and the optimal addition amount of the TNF-α mimic peptide is 10 ng / mL.

5. The preparation method according to claim 1, characterized in that, The basal culture medium described in S2 is α-MEM medium containing double antibiotics and 10-20% FBS or double antibiotics and 10-20% human AB serum.

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