Genetic enhancement of foxo3 in mesenchymal stem cells and their derivatives for treating ischemic stroke

Abstract

The application discloses application of FOXO3 genetically enhanced mesenchymal stem cells and derivatives thereof in treating ischemic stroke, and belongs to the field of biomedicine. It is found that FOXO3 genetically enhanced mesenchymal stem cells (F3-MSC) and derivatives thereof, exosomes (F3-Exo), have the effect of treating ischemic stroke, have stronger retention capacity, improve the motor sensory function of an ischemic stroke animal, and improve the cell ecology and microenvironment in the brain of the animal after stroke. It is shown that the FOXO3 genetically enhanced stem cells and derivatives thereof can be used as a treatment means for ischemic stroke. This further expands the current treatment strategy for ischemic stroke, provides an extremely potential candidate strategy for treating ischemic stroke, and will help to alleviate the increasingly serious social problem of cerebrovascular disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of FOXO3 genetically enhanced mesenchymal stem cells and their derivatives in the treatment of ischemic stroke. Background Technology

[0002] Due to global population growth and increasing life expectancy, the incidence of cerebrovascular diseases has been increasing year by year in recent decades. Stroke has become the second leading cause of death worldwide and the third leading cause of disability, imposing a huge economic burden and mental anguish on society and families. Ischemic stroke accounts for approximately 70-80% of all strokes, and ischemic stroke is caused by irreversible neuronal death due to prolonged hypoxia caused by obstruction of blood supply to the brain. After an ischemic stroke, promoting vascular recanalization and restoring blood flow perfusion to ischemic brain tissue as early as possible is crucial to preventing or mitigating neurological damage caused by ischemia. In recent years, stem cell transplantation has been widely studied in animal research and clinical trials for the treatment of ischemic stroke. Among them, mesenchymal stem cells (MSCs) are highly attractive candidates in regenerative medicine and cell immunotherapy due to their wide availability, strong potential to derive into various tissue cells, and ability to secrete cytokines to regulate the ecological microenvironment of organs and tissues. MSCs possess multiple functions, including regulating immune responses, anti-oxidative stress, anti-apoptosis, and promoting angiogenesis. Currently, clinical research using mesenchymal stem cells (MSCs) covers a wide range of diseases. However, due to the harsh microenvironment within the recipient's ischemic brain tissue, traditional wild-type MSC transplantation for ischemic stroke still faces numerous challenges, including low quality (including reduced differentiation capacity and increased senescence in late-stage passaged cells), low in vivo retention of transplanted MSCs, and low survival rates. Furthermore, the high carcinogenicity associated with transplanting xenogeneic cells hinders the wider clinical application of stem cell transplantation for ischemic stroke. Therefore, developing new strategies to improve the safety and efficacy of stem cell transplantation for ischemic stroke is imperative. "Genetically enhanced" cells, i.e., autologous or xenografted cells, can be targeted with gene editing technology to enhance cell viability, resilience, self-renewal capacity, directed differentiation, and anti-tumor transformation capabilities, thereby optimizing therapeutic efficacy. This can improve the survival rate and function of transplanted cells and reduce side effects.

[0003] FOXO3 is a longevity-related transcription factor and an aging-protective factor against vascular cell senescence. Genetically enhanced human mesenchymal stem cells (MSCs) were developed by recoding two specific nucleotides of FOXO3 to activate its activity. These genetically enhanced MSCs exhibit stronger regenerative capacity and a reduced risk of tumor development. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to treat ischemic stroke.

[0005] This invention provides any of the following applications of FOXO3 activity-enhancing proteins or FOXO3 proteins, or substances that increase the content and / or activity of FOXO3 proteins: X1. To prepare products for the treatment and / or prevention of ischemic stroke; X2. To prepare products that improve the motor function of people or animals with ischemic stroke; X3. To prepare products that improve the sensory abilities of the limbs in people or animals with ischemic stroke; X4. Prepare products that promote angiogenesis in the brain tissue of humans or animals with ischemic stroke. X5. Prepare products that reduce apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X6. Prepare products that improve the inflammatory level of brain tissue in humans or animals with ischemic stroke; X7. Prepare products that reduce glial scarring in the brain tissue of humans or animals with ischemic stroke; X8. Prepare products that improve the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X9. Prepare products that promote neuroregeneration in the brain tissue of humans or animals with ischemic stroke. X10. Products that improve the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; X11. Treatment and / or prevention of ischemic stroke; X12. Improves motor function in people or animals with ischemic stroke; X13. Improves limb sensory ability in people or animals with ischemic stroke; X14. Promotes angiogenesis in the brain tissue of humans or animals with ischemic stroke; X15. Reduces apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X16. Improves the inflammatory level of brain tissue in humans or animals with ischemic stroke; X17. Reduces glial scarring in the brain tissue of humans or animals with ischemic stroke; X18. Improves the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X19. Promotes neurogenesis in the brain tissue of humans or animals with ischemic stroke; X20 improves the level of mature neurons in the brain tissue of humans or animals with ischemic stroke.

[0006] The FOXO3 activity-enhancing protein is, as shown in A1), A2), or A3). A1) The amino acid sequence of the protein includes SEQ ID NO: 4; A2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, with one or more amino acid residues substituted and / or deleted and / or added; A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2); The FOXO3 protein is as follows: (C1), (C2), or (C3) C1) The amino acid sequence of the protein includes SEQ ID NO: 2; C2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, with one or more amino acid residues substituted and / or deleted and / or added; C3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of C1) or C2).

[0007] This invention provides the use of biomaterials related to FOXO3-enhancing proteins in the preparation of products having any of the following uses: X1. To prepare products for the treatment and / or prevention of ischemic stroke; X2. To prepare products that improve the motor function of people or animals with ischemic stroke; X3. To prepare products that improve the sensory abilities of the limbs in people or animals with ischemic stroke; X4. Prepare products that promote angiogenesis in the brain tissue of humans or animals with ischemic stroke. X5. Prepare products that reduce apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X6. Prepare products that improve the inflammatory level of brain tissue in humans or animals with ischemic stroke; X7. Prepare products that reduce glial scarring in the brain tissue of humans or animals with ischemic stroke; X8. Prepare products that improve the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X9. Prepare products that promote neuroregeneration in the brain tissue of humans or animals with ischemic stroke. X10. Products that improve the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; X11. Treatment and / or prevention of ischemic stroke; X12. Improves motor function in people or animals with ischemic stroke; X13. Improves limb sensory ability in people or animals with ischemic stroke; X14. Promotes angiogenesis in the brain tissue of humans or animals with ischemic stroke; X15. Reduces apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X16. Improves the inflammatory level of brain tissue in humans or animals with ischemic stroke; X17. Reduces glial scarring in the brain tissue of humans or animals with ischemic stroke; X18. Improves the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X19. Promotes neurogenesis in the brain tissue of humans or animals with ischemic stroke; X20 improves the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; The biomaterial is any one of B1) to B5) below: B1) Nucleic acid molecules encoding the FOXO3 activity-enhancing protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Cell lines containing the nucleic acid molecules described in B1), or cell lines containing the expression cassette described in B2); Derivatives of the cell lines described in B6 and B5).

[0008] B1) The nucleic acid molecule described is as follows (b11) or (b12)): b11) The coding sequence is the DNA molecule of SEQ ID NO: 3 in the sequence listing; b12) has 75% or more identity with the nucleotide sequence defined in b11) and is a DNA molecule encoding the FOXO3 activity-enhancing protein; The derivative described in B6) is an exosome of the cell line described in B5).

[0009] B5) The cell line described is mesenchymal stem cells.

[0010] This invention provides the use of biomaterials related to FOXO3 protein in the preparation of products having any of the following uses: X1. To prepare products for the treatment and / or prevention of ischemic stroke; X2. To prepare products that improve the motor function of people or animals with ischemic stroke; X3. To prepare products that improve the sensory abilities of the limbs in people or animals with ischemic stroke; X4. Prepare products that promote angiogenesis in the brain tissue of humans or animals with ischemic stroke. X5. Prepare products that reduce apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X6. Prepare products that improve the inflammatory level of brain tissue in humans or animals with ischemic stroke; X7. Prepare products that reduce glial scarring in the brain tissue of humans or animals with ischemic stroke; X8. Prepare products that improve the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X9. Prepare products that promote neuroregeneration in the brain tissue of humans or animals with ischemic stroke. X10. Products that improve the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; X11. Treatment and / or prevention of ischemic stroke; X12. Improves motor function in people or animals with ischemic stroke; X13. Improves limb sensory ability in people or animals with ischemic stroke; X14. Promotes angiogenesis in the brain tissue of humans or animals with ischemic stroke; X15. Reduces apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X16. Improves the inflammatory level of brain tissue in humans or animals with ischemic stroke; X17. Reduces glial scarring in the brain tissue of humans or animals with ischemic stroke; X18. Improves the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X19. Promotes neurogenesis in the brain tissue of humans or animals with ischemic stroke; X20 improves the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; The biomaterial is any one of the following D1) to D5): D1) The nucleic acid molecule encoding the FOXO3 protein; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3); D5) Cell lines containing the nucleic acid molecules described in D1), or cell lines containing the expression cassette described in D2); Derivatives of the cell lines described in D6 and D5).

[0011] Wherein, the nucleic acid molecule described in D1) is as follows: d11) or d12): d11) The coding sequence is the DNA molecule of SEQ ID NO: 1 in the sequence listing; The nucleotide sequence defined by d12) has 75% or more identity with the nucleotide sequence defined by d11) and is a DNA molecule encoding the FOXO3 protein; The derivative described in D6) is an exosome of the cell line described in D5).

[0012] The cell line described in D5 is a mesenchymal stem cell.

[0013] This invention provides a product having any of the following functions, containing the FOXO3 activity-enhancing protein, or the FOXO3 activity-enhancing protein, or the substance that increases the content and / or activity of FOXO3 protein, or the biomaterial: Y1. Treatment and / or prevention of ischemic stroke; Y2. Improves motor function in people or animals with ischemic stroke; Y3. Improves limb sensory ability in people or animals with ischemic stroke; Y4. Promotes angiogenesis in the brain tissue of humans or animals with ischemic stroke; Y5. Reduces apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; Y6. Improves the inflammatory level of brain tissue in humans or animals with ischemic stroke; Y7. Reduces glial scarring in the brain tissue of humans or animals with ischemic stroke; Y8. Improves the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; Y9. Promotes neurogenesis in the brain tissue of humans or animals with ischemic stroke; Y10 improves the level of mature neurons in the brain tissue of humans or animals with ischemic stroke.

[0014] This invention also provides the application of FOXO3 genetically enhanced mesenchymal stem cells in the preparation of products having any of the following functions: X1. Enhance the survival ability of transplanted stem cells in patients with ischemic stroke. X2. Improve motor and sensory function in ischemic stroke patients; X3. Improves apoptosis in brain tissue after ischemic stroke; X4. Improves the elevated inflammatory response caused by ischemic stroke. X5. Improves glial scar formation caused by ischemic stroke; X6. Promotes angiogenesis after ischemic stroke; X7. Promotes neurogenesis after ischemic stroke. In the above applications, the cell line is FOXO3-enhanced mesenchymal stem cell (FOXO3).2SA / 2SA hMSCs, FOXO3 2SA / 2SA hMSCs are derived from FOXO3 2SA / 2SA hESCs, FOXO3 2SA / 2SA hESCs were obtained from the human embryonic stem cell line H9 ESC via recombinant adenovirus gene-targeted editing, FOXO3 2SA / 2SA hESCs were obtained by mutating the T at positions 757 and 943 of the FOXO3 gene (the wild-type FOXO3 gene) in the human embryonic stem cell H9 ESC cell line to G. The mutated FOXO3 gene is a FOXO3 activity-enhancing gene. Compared with the FOXO3 protein, the activity of the FOXO3 activity-enhancing protein is enhanced.

[0015] This invention also provides a derivative of FOXO3 genetically enhanced mesenchymal stem cells, which has the following applications in products: X1. Improves motor and sensory function in ischemic stroke patients; X2. Improves apoptosis in brain tissue after ischemic stroke; X3. Improves the elevated inflammatory response caused by ischemic stroke. X4. Improves glial scar formation caused by ischemic stroke; X5. Promotes angiogenesis after ischemic stroke; X6. Promotes neurogenesis after ischemic stroke. In the above applications, the derivative is an exosome secreted by mesenchymal stem cells.

[0016] In the above applications, the enhanced survival ability of transplanted stem cells in animals with ischemic stroke can be reflected in the prolonged expression time of luciferase in the brains of mice with ischemic stroke.

[0017] In the above applications, the improvement of motor and sensory function in animals with ischemic stroke is reflected in promoting the recovery of limb motor function and superficial sensory function after ischemic stroke.

[0018] In the above applications, the apoptosis of brain tissue refers to neuronal apoptosis caused by ischemic stroke.

[0019] In the above applications, the elevated inflammatory response refers to the increase in microglia, activated inflammatory cells, and inflammatory mediators caused by ischemic stroke.

[0020] In the above applications, the glial scar formation refers to the proliferation of astrocytes caused by ischemic stroke.

[0021] In the above applications, the angiogenesis refers to increased cerebral blood flow and increased expression of vascular endothelial cells on the injured side after a stroke. In the above applications, the neurogenesis refers to the increased expression levels of immature neurons and mature neurons that occur after ischemic stroke.

[0022] In the above applications, the cell line is FOXO3-enhanced mesenchymal stem cell (FOXO3). 2SA / 2SA hMSCs, FOXO3 2SA / 2SA hMSCs are derived from FOXO3 2SA / 2SA hESCs, FOXO3 2SA / 2SA hESCs were obtained from the human embryonic stem cell line H9 ESC via recombinant adenovirus gene-targeted editing, FOXO3 2SA / 2SA hESCs were obtained by mutating the T at positions 757 and 943 of the FOXO3 gene (the wild-type FOXO3 gene) in the human embryonic stem cell H9 ESC cell line to G. The mutated FOXO3 gene is a FOXO3 activity-enhancing gene. Compared with the FOXO3 protein, the activity of the FOXO3 activity-enhancing protein is enhanced.

[0023] In the above applications, the glial scar formation refers to the proliferation of astrocytes caused by ischemic stroke.

[0024] In the above applications, the angiogenesis refers to increased cerebral blood flow and increased expression of vascular endothelial cells on the injured side after a stroke.

[0025] In the above applications, the neurogenesis refers to the increased expression levels of immature neurons and mature neurons that occur after ischemic stroke.

[0026] This invention discovers that FOXO3-enhanced mesenchymal stem cells (F3-MSCs) and their derivative, exosomes (F3-Exo), have therapeutic effects on ischemic stroke. They exhibit enhanced survival rates, improve motor and sensory functions in animals with ischemic stroke, and enhance the cellular ecology and microenvironment in the brain after stroke. This demonstrates that FOXO3-enhanced stem cells and their derivatives can serve as a treatment for ischemic stroke. This further expands current treatment strategies for ischemic stroke, providing a highly promising candidate strategy that may help alleviate the increasingly serious social problem of cerebrovascular disease.

[0027] This invention discovers that FOXO3-enhanced mesenchymal stem cells (F3-MSCs) and their derivative, exosomes (F3-Exo), have therapeutic effects on ischemic stroke. They exhibit enhanced survival rates, improve motor and sensory functions in animals with ischemic stroke, and enhance the cellular ecology and microenvironment in the brain after stroke. This demonstrates that FOXO3-enhanced stem cells and their derivatives can serve as a treatment for ischemic stroke. This further expands current treatment strategies for ischemic stroke, providing a highly promising candidate strategy that may help alleviate the increasingly serious social problem of cerebrovascular disease.

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0029] Figure 1 The results show the tracing and motor-sensory-behavioral outcomes of transplanted mesenchymal stem cells. Specifically, A presents representative images and statistical results of transplanted wild-type mesenchymal stem cells and FOXO3 genetically enhanced stem cells; B presents statistical results of the cylinder experiment in behavioral experiments; C presents statistical results of the adhesive tape removal experiment in behavioral experiments; and D presents the results of the cerebral blood flow ratio test. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns indicates no significant difference.

[0030] Figure 2 Immunofluorescence staining results show that transplanted mesenchymal stem cells can improve the brain tissue cellular microenvironment damage caused by ischemic stroke. A shows the fluorescence staining results of apoptotic cells; B shows the fluorescence staining results of microglia; C shows the fluorescence staining results of activated immune cells; D shows the fluorescence staining results of the inflammatory mediator TNF-α; E shows the fluorescence staining results of IL-1β; and F shows a representative fluorescence staining image of astrocytes and statistical results of glial scar length. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns indicates no significant difference. Scale bar 100 μm.

[0031] Figure 3Immunofluorescence staining results and laser speckle pattern detection results of cerebral blood flow were used to demonstrate that transplanted mesenchymal stem cells can promote the recovery of the brain tissue cellular microenvironment induced by ischemic stroke. In the figures, A represents the fluorescence staining results of vascular endothelium; B represents the laser speckle pattern detection results of cerebral blood flow; C represents the fluorescence staining results of immature neurons; and D represents the immunofluorescence staining results of mature neurons. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns indicates no significant difference.

[0032] Figure 4 This data represents the motor and sensory behavioral results of transplanted mesenchymal stem cell derivative exosomes and the immunofluorescence staining results demonstrating the improvement of brain tissue cellular microenvironment damage caused by ischemic stroke. In the figures, A represents the statistical results of the cylinder experiment in the behavioral experiments; B represents the statistical results of the adhesive tape removal experiment in the behavioral experiments; and C represents the cerebral blood flow detection results. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns indicates no significant difference.

[0033] Figure 5 Immunofluorescence staining results demonstrate that transplantation of mesenchymal stem cell derivative exosomes can improve the brain tissue cellular microenvironment damage caused by ischemic stroke. A shows the fluorescence staining results of apoptotic cells; B shows the fluorescence staining results of microglia; C shows the fluorescence staining results of activated immune cells; D shows the fluorescence staining results of the inflammatory mediator IL-1β; E shows the fluorescence staining results of TNF-αβ; and F shows a representative fluorescence staining image of astrocytes and statistical results of glial scar length. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns indicates no significant difference. Scale bar 100 μm.

[0034] Figure 6 Immunofluorescence staining results demonstrate that transplanted mesenchymal stem cell derivative exosomes promote the recovery of the brain tissue cellular microenvironment induced by ischemic stroke. In the figures, A represents the fluorescence staining results of vascular endothelium; B represents the fluorescence staining results of immature neurons; and C represents the immunofluorescence staining results of mature neurons. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns indicates no significant difference. Detailed Implementation

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0036] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results are expressed as mean ± standard deviation and were tested using one-way ANOVA.

[0037] The FOXO3-enhanced mesenchymal stem cells (F3-MSCs) in the following examples are the FOXO3 described in the literature "FOXO3-Engineered Human ESC-Derived Vascular Cells Promote Vascular Protection and Regeneration. Cell Stem Cell. 2019; 24(3):447-461". 2SA / 2SA hMSCs (i.e., FOXO3-enhanced mesenchymal stem cells), FOXO3 2SA / 2SA hMSCs were derived from FOXO3 in this literature. 2SA / 2SA hESCs. FOXO3 2SA / 2SA hESCs were obtained from the human embryonic stem cell line H9 ESC via recombinant adenovirus gene-targeted editing, FOXO3 2SA / 2SA hESCs were obtained by mutating the T at positions 757 and 943 of the FOXO3 gene (denoted as the FOXO3 wild-type gene) in the human embryonic stem cell H9 ESC cell line to G, respectively. The mutated FOXO3 gene was designated as the FOXO3 enhancer gene. The sequences of the FOXO3 wild-type gene and the FOXO3 enhancer gene are SEQ ID NO: 1 and 3 in the sequence listing, respectively, encoding the FOXO3 wild-type protein shown in SEQ ID NO: 2 and the FOXO3 enhancer protein shown in SEQ ID NO: 4. The FOXO3 wild-type protein has serine at positions 253 and 315, while the FOXO3 enhancer protein has alanine at positions 253 and 315. Compared with the FOXO3 wild-type protein, the FOXO3 enhancer protein exhibits enhanced activity.

[0038] Among them, the human embryonic stem cell H9 ESC cell line is abbreviated as H9 cell, a product of WiCell, catalog number WA09(H9)-DL-7.

[0039] FOXO3 gene obtained above was used to enhance embryonic stem cells FOXO3 2SA / 2SA hESCs yielded FOXO3-enhanced mesenchymal stem cells. 2SA / 2SAThe specific steps for hMSCs are as follows: (1) FOXO3 2SA / 2SA hESCs undergo embryoid differentiation to obtain embryoid bodies; (2) The embryoid obtained in step (1) is cultured until fibrous cells appear; (3) After completing step (2), collect cells, passage and culture them; (4) After completing step (3), select cells that are positive for CD73, CD90, and CD105; these are FOXO3 enhanced mesenchymal stem cells. 2SA / 2SA hMSCs.

[0040] Wild-type mesenchymal stem cells (WT-MSCs) were prepared using the human embryonic stem cell H9 ESC cell line according to the above method.

[0041] In the following examples, exosomes were collected using ultracentrifugation, following the method described in the literature "Exosomes from antler stem cells alleviate mesenchymal stem cell senescence and osteoarthritis. Protein Cell 13, 220-226". The specific steps are as follows: (1) First, in order to prevent exosome contamination caused by donkey serum, the donkey serum was subjected to an exosome depletion process by ultracentrifugation at 100,000 g for 16 hours. (2) Add the donkey serum obtained in step (1) to the mesenchymal stem cell culture medium and use it for the 7th generation of hMSCs. Collect the culture medium replaced each time the medium is changed for exosome isolation. (3) After completing step (2), centrifuge the conditioned medium at 500 g for 5 minutes and filter it through a 0.22 μm filter membrane to remove cells and cell debris. After ultracentrifuging at 100,000 g for 2 hours at 4°C, remove the supernatant and wash the exosomes with 1 mL of physiological saline. Then centrifuge again at 100,000 g for 2 hours at 4°C; finally, collect the exosomes.

[0042] (4) After completing step (3), exosomes were quantified using negative staining transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).

[0043] The ischemic stroke mouse model in the following examples was established using electrocoagulation to block the middle cerebral artery, following the method described in the literature "Modeling stroke in mice: permanent coagulation of the distal middlecerebral artery. J Vis Exp, e51729". The specific steps are as follows: Male C57 / B6J mice (Violent Living Laboratory Animal Technology Co., Ltd.) aged 8-12 weeks were anesthetized via intraperitoneal injection of tribromoethanol. After anesthesia, the mice were placed supine on the operating table, their limbs were secured with tape, and the hair between the left ear and eye was removed with depilatory cream to expose the surgical site. After disinfecting the surgical area with povidone-iodine, an incision approximately 1 cm long was made along the long axis of the skin at that location using ophthalmic scissors. After blunt dissection of the temporalis muscle with a scalpel, the skull was removed using a cranial drill to expose the brain tissue and the surface middle cerebral artery. Electrocoagulation was performed on the proximal and distal ends of the exposed distal bifurcation of the middle cerebral artery using a coagulation pen. Then, cerebral blood flow perfusion images were recorded and analyzed using a laser speckle imaging system (RWD Life Sciences, Beijing, CN) to assess changes in the blood flow ratio in the ischemic hemisphere on the surgical side and to evaluate the ischemic area. Subsequently, 6 μL of normal saline (carrier) or saline containing WT-MSCs or F3-MSCs was transcranially injected into the three perilesional sites of the ischemic area (1 × 10⁻⁶). 5 (1 cell / 1 μL, 2 μL per injection site). For the exosome injection procedure, prepare an exosome solution with a concentration of 100 μg / mL and administer it in 6 μL of physiological saline. Return the mice to their home environment after they recover.

[0044] The sham surgery group only used a cranial drill to dissect the skull, without electrocoagulating the middle cerebral artery or injecting cells or exosomes; the remaining steps were the same as the surgical group.

[0045] In the following examples, 5-10 mice were used in each group for model establishment and treatment in animal experiments, and 6-8 mice were used in each group for different detection methods. Unless otherwise specified, the cell culture environment was 37°C and 5% CO2.

[0046] The lentivirus expressing luciferase Luciferas is the "luciferase(Control)-expressed vector" in the literature "SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2. Pan et al. Cellresearch. (2016) 26: 190-205".

[0047] Example 1: Detection of the survival ability of FOXO3-enhanced mesenchymal stem cells after transplantation into the brain tissue of ischemic stroke patients. Fluorescent tracing of transplanted cells (1) Wild-type mesenchymal stem cells and FOXO3-enhanced mesenchymal stem cells of the same passage were infected with lentiviruses expressing luciferase. The culture medium used was mesenchymal stem cell culture medium (mesenchymal stem cell culture medium: composed of MEM medium (Gibco, 12571-063), fetal bovine serum (Gibco, 10270-106), penicillin / streptomycin (Gibco, 15140-122) and recombinant human fibroblast growth factor (JPC, bFGF); the culture medium was prepared by mixing MEM medium and fetal bovine serum at a volume ratio of 90:10 and then adding penicillin / streptomycin and recombinant human fibroblast growth factor; the concentration of penicillin / streptomycin in the culture medium was 1 g / 100 ml, and the concentration of recombinant human fibroblast growth factor was 10 ng / ml).

[0048] (2) 3-5 days after infection, when the cell confluence reaches 100%, use TrypLE digestion to collect and count the cells.

[0049] (3) After step (2) is completed, 100 μL contains 1×10 6 The cell density was determined by resuspending the cells obtained in step (2) in MEM-α medium to obtain two cell suspensions. 100 μL of each of the two cell suspensions was mixed with Luciferase substrate (D-Luciferin Firefly, potassium salt, GOLDBIO) to obtain the respective cell and substrate mixtures. Then, the relationship between fluorescence intensity and cell number was determined using an ELISA reader.

[0050] (4) After step (3) is completed, the two cell mixtures obtained in step (3) are diluted separately using MEM-α medium to obtain a concentration of 1×10⁻⁶. 5 Two cell suspensions were administered at 1 μL per mouse. These two cell suspensions were injected at three points along the edge of the ischemic brain tissue during the establishment of a mouse model of ischemic stroke. Each mouse received a total of 6 μL of these suspensions, and the day of injection was designated as day 0. Mice injected with cell suspensions derived from wild-type mesenchymal stem cells and FOXO3-enhanced mesenchymal stem cells were designated as the wild-type mesenchymal stem cell treatment group (ischemic stroke + WT-MSC group) and the FOXO3-enhanced mesenchymal stem cell treatment group (ischemic stroke + F3-MSC group), respectively.

[0051] (5) After step (4) is completed, the level of luc fluorescence in the myocardium and the ROI value are monitored by in vivo imaging system on days 1, 2, 3, 4, 5, 6, 7 and 8 after injection, so as to reflect the level of transplanted cells surviving in brain tissue.

[0052] The results are as follows Figure 1 The results showed that FOXO3-enhanced mesenchymal stem cells had a greater survival rate than wild-type mesenchymal stem cells, with significant differences observed on days 4-7 post-injection.

[0053] Example 2: Detection of the effect of FOXO3-enhanced mesenchymal stem cells on improving neurological function after ischemic stroke. I. Treatment of Ischemic Stroke in Mice (1) Wild-type mesenchymal stem cells and FOXO3-enhanced mesenchymal stem cells of the same generation were cultured in mesenchymal stem cell culture medium. When the cell confluence reached 100%, cells were collected and counted using TrypLE digestion.

[0054] (2) After step (1) is completed, the two cell mixtures are diluted separately using MEM-α medium to obtain a concentration of 1×10⁻⁶. 5 Two cell suspensions were administered at 1 μL per mouse. These two cell suspensions were injected at three points along the edge of the ischemic brain tissue during the establishment of a mouse model of ischemic stroke. Each mouse received a total of 6 μL of these suspensions, and the day of injection was designated as day 0. Mice injected with cell suspensions derived from wild-type mesenchymal stem cells and FOXO3-enhanced mesenchymal stem cells were designated as the wild-type mesenchymal stem cell treatment group (ischemic stroke + WT-MSC group) and the FOXO3-enhanced mesenchymal stem cell treatment group (ischemic stroke + F3-MSC group), respectively.

[0055] In addition, a control group (ischemic stroke + Vehicle group, i.e. ischemic stroke group) was injected with 6 μL of normal saline simultaneously, and a control group (Sham group) was set up for simultaneous treatment.

[0056] II. Detection of the effect of FOXO3-enhanced mesenchymal stem cells on improving neurological function and cerebral blood flow after ischemic stroke. 1) Cylinder test On day 7 after injection, mice in each group were placed in a vertical transparent glass cylinder (diameter: 8.8 cm; height: 17.5 cm) for acclimatization training. Once acclimatized, the formal experiment began. A mirror was placed behind the mice to observe their behavior. The number of paw contacts after the mice had fully retted their hind limbs was recorded, including the left forepaw, right forepaw, and both forepaws. At least 20 forelimb contacts were recorded for subsequent analysis. After each test, the gas cylinder was rinsed with water and a 70% ethanol solution to remove any odor. The left / right ratio of independent forepaw use was calculated for statistical purposes. This experiment was used to assess the limb coordination function of mice; the left / right ratio decreased significantly with impaired neurological function.

[0057] The test results are as follows Figure 1 As shown in Figure B, compared with the Sham group, the forelimb usage rate in the ischemic stroke + Vehicle group was significantly lower, indicating a significant decline in motor coordination function in this group of mice. The forelimb usage rates in the ischemic stroke + WT-MSC group and the ischemic stroke + F3-MSC group were improved compared with the ischemic stroke + Vehicle group. FOXO3-enhanced mesenchymal stem cell transplantation improved the motor function of ischemic stroke mice, indicating that it can improve the motor function of ischemic stroke mice.

[0058] 2) Adhesive tape removal test On day 7 after injection, mice in each group were placed in a vertical transparent glass cylinder (diameter: 8.8 cm; height: 17.5 cm) for acclimatization training. Once acclimatized, the formal experiment began. A small piece of tape (3 mm × 3 mm) was placed on the forepaw on the side opposite the stroke hemisphere. The time required for contact and removal of the tape (maximum 120 seconds) was recorded for subsequent analysis. This experiment was used to assess limb sensory function in mice; in cases of decreased neurological function, both contact and removal times were significantly prolonged.

[0059] The test results are as follows Figure 1 As shown in Figure C, compared with the Sham group, the adhesive tape contact time and adhesive tape removal time were significantly increased in the ischemic stroke + Vehicle group, indicating a significant decline in limb sensory function in this group of mice. The limb sensory abilities of the ischemic stroke + WT-MSC group and the ischemic stroke + F3-MSC group were improved compared with the ischemic stroke + Vehicle group. FOXO3-enhanced mesenchymal stem cell transplantation enhanced limb sensory ability in ischemic stroke mice, indicating that it can improve limb sensory ability in ischemic stroke mice.

[0060] 3) Cerebral blood flow was detected using a laser speckle imaging system (RWD Life Sciences, Beijing, CN). On day 7 after injection, mice in each group were anesthetized by intraperitoneal injection of tribromoethanol and fixed in a stereotactic position. The heads were shaved and the skin was prepared. Cerebral blood flow perfusion images were recorded and analyzed using a laser speckle imaging system (RWD Life Sciences, Beijing, CN). The ratio of cerebral blood flow on the surgical side to that on the healthy side was calculated to understand the change in the blood flow ratio of the ischemic hemisphere on the surgical side.

[0061] The test results are as follows Figure 1 As shown in Figure D, compared with the Sham group, cerebral blood flow on the surgical side of the ischemic stroke + Vehicle group was significantly reduced. The cerebral blood flow ratios of the ischemic stroke + WT-MSC group and the ischemic stroke + F3-MSC group were improved compared with the ischemic stroke + Vehicle group, and the cerebral blood flow ratio of the ischemic stroke + F3-MSC group was significantly higher than that of the ischemic stroke + WT-MSC group. FOXO3-enhanced mesenchymal stem cell transplantation restored the cerebral blood flow reduction caused by ischemic stroke, indicating that it can promote angiogenesis in the brain tissue of mice with ischemic stroke.

[0062] III. Detection of the regulatory effect of FOXO3 on the brain tissue cellular microenvironment after ischemic stroke by mesenchymal stem cells 1) TUNEL staining for apoptotic cell markers in brain tissue sections After step one, brain tissue was collected from four groups of mice. The collected brain tissue was fixed with 4% PFA, dehydrated with 30% sucrose solution, and then frozen sectioned to a thickness of 15 micrometers. Immunofluorescence was used to detect the presence of transplantation in the mouse brain tissue sections. The specific steps are as follows: (1) Wash brain tissue sections twice with PBS.

[0063] (2) The sections were permeabilized with 0.4% Triton-100 for 30 minutes.

[0064] (3) Block the slices treated in step (2) with 5% BSA at room temperature for 30 minutes.

[0065] (4) Wash the slices after BSA incubation in step (3) with PBS three times, for 5 minutes each time.

[0066] (5) Staining was performed using the one-step TUNEL cell apoptosis detection kit (C1088, Beyotime) provided by Beyotime Biotechnology Co., Ltd.

[0067] (6) Wash the slices after incubation in step (5) three times with PBS for 5 minutes each time.

[0068] (7) Mount the tissue sections from step (6) with coverslips and observe and photograph them using a laser confocal microscope.

[0069] Staining results as follows Figure 2 As shown in Figure A, the number of TUNEL-positive cells in brain tissue sections from each group of mice was counted. The results showed that the proportion of TUNEL-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of TUNEL-positive cells in the brain tissue of the ischemic stroke + F3-MSC group was significantly lower than that of the ischemic stroke + WT-MSC group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced mesenchymal stem cell transplantation significantly improved the apoptosis level of nerve cells in brain tissue.

[0070] 2) IBA1 staining, a marker for microglia, in brain tissue sections. (1) Wash brain tissue sections twice with PBS.

[0071] (2) The sections were permeabilized with 0.4% Triton-100 for 30 minutes.

[0072] (3) Block the slices treated in step (2) with 5% BSA at room temperature for 30 minutes.

[0073] (4) After processing the slides in step (3), add antibody diluted in the antibody diluent according to the ratio and incubate at 4 degrees overnight. The antibody diluent contains 5% BSA and primary antibody, which is microglial cell marker IBA1 antibody (Woke, no. 019-19741, 1:200 dilution).

[0074] (5) Wash the slices after antibody incubation in step (4) with PBS three times, for 5 minutes each time.

[0075] (6) After rinsing with PBS, the sections were incubated at room temperature for 45 minutes with the corresponding fluorescent secondary antibody and nuclear dye Hoechst 33342 (Lifetechnology, H3570, 1:1000 dilution). The fluorescent secondary antibody used was rabbit anti-sheep fluorescent secondary antibody (Invitrogen, A32723).

[0076] (7) Wash the slices after incubation in step (6) three times with PBS for 5 minutes each time.

[0077] (8) Mount the tissue sections from step (7) with coverslips and observe and photograph them using a laser confocal microscope.

[0078] Staining results as follows Figure 2As shown in Figure B, the number of IBA1-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of IBA1-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of IBA1-positive cells in the brain tissue of the ischemic stroke + F3-MSC group was significantly lower than that of the ischemic stroke + WT-MSC group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced mesenchymal stem cell transplantation significantly improved the level of microglia.

[0079] 3) CD68 staining, a marker of activated immune cells, in brain tissue sections. The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with CD68 (Abcam, no. ab125212, 1:200 dilution).

[0080] Staining results as follows Figure 2 As shown in Figure C, the number of CD68-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of CD68-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of CD68-positive cells in the brain tissue of the ischemic stroke + F3-MSC group was significantly lower than that of the ischemic stroke + WT-MSC group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced mesenchymal stem cell transplantation significantly improved the level of activated inflammatory cells.

[0081] 4) Staining of brain tissue sections with the inflammatory mediator TNF-α The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with TNF-α (Abcam, no. ab1793, 1:100 dilution).

[0082] Staining results as follows Figure 2 As shown in Figure D, the number of TNF-α-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of TNF-α-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of TNF-α-positive cells in the brain tissue of the ischemic stroke + F3-MSC group was significantly lower than that of the ischemic stroke + WT-MSC group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced mesenchymal stem cell transplantation significantly improved the expression level of the inflammatory mediator TNF-α.

[0083] 5) Staining of brain tissue sections with the inflammatory mediator IL-1β The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with IL-1β (Santa Cruz, no. sc-52012, 1:100 dilution).

[0084] Staining results as follows Figure 2 As shown in Figure E, the number of IL-1β-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of IL-1β-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of CD68-positive cells in the brain tissue of the ischemic stroke + F3-MSC group was significantly lower than that of the ischemic stroke + WT-MSC group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced mesenchymal stem cell transplantation significantly improved the expression level of the inflammatory mediator IL-1β.

[0085] 6) GFAP staining of brain tissue sections (a marker for astrocytes) The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with GFAP (Abcam, no. ab4674, 1:400 dilution).

[0086] Staining results as follows Figure 2 As shown in Figure F, the length of glial scars in the brain tissue sections of mice in each group was statistically analyzed. The results showed that glial scars formed by GFAP-positive cells appeared in the brain tissue of the ischemic stroke group. The length of glial scars in the brain tissue of the ischemic stroke + F3-MSC group was significantly lower than that in the ischemic stroke + WT-MSC group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced mesenchymal stem cell transplantation significantly reduced the length of glial scars formed by astrocytes.

[0087] 7) CD31 staining, a marker for vascular endothelial cells, in brain tissue sections. The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with CD31 (Abcam, no. ab28364, 1:100 dilution).

[0088] Staining results as follows Figure 3 As shown in Figure A, the number of CD31-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of CD31-positive cells in the brain tissue of the ischemic stroke group decreased, while the proportion of CD31-positive cells in the brain tissue of the ischemic stroke + F3-MSC group was significantly higher than that of the ischemic stroke + WT-MSC group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced mesenchymal stem cell transplantation significantly improved the level of vascular endothelial cells.

[0089] 8) DCX staining of immature neuron markers in brain tissue sections The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with DCX (Cell signaling Technology, no. 4604S, 1:100 dilution).

[0090] Staining results as follows Figure 3 As shown in Figure B, the number of DCX-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of DCX-positive cells in the brain tissue of the ischemic stroke group increased, and FOXO3-enhanced mesenchymal stem cell transplantation significantly improved the level of immature neurons.

[0091] 9) NeuN staining of mature neurons in brain tissue sections The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with NeuN (Abcam, no. ab104224, 1:200 dilution).

[0092] Staining results as follows Figure 3 As shown in Figure C, the number of NeuN-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of NeuN-positive cells in the brain tissue of the ischemic stroke group was decreased, and the proportion of NeuN-positive cells in the brain tissue of the ischemic stroke + F3-MSC group was significantly higher than that of the ischemic stroke + WT-MSC group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced mesenchymal stem cell transplantation significantly improved the level of mature neurons.

[0093] Example 3: Detection of the effect of FOXO3-enhanced mesenchymal stem cell-derived exosomes on improving neurological function after ischemic stroke. I. Treatment of Ischemic Stroke in Mice (1) Seventh generation wild-type mesenchymal stem cells and FOXO3-enhanced mesenchymal stem cells were collected in conditioned medium and then exosomes were collected by ultracentrifugation.

[0094] (2) After step (1) was completed, the two types of exosomes were diluted with physiological saline to obtain two exosome suspensions with a concentration of 100 μg / mL. The two exosome suspensions were injected at three points at the edge of the ischemic brain tissue during the establishment of the ischemic stroke mouse model. Each mouse was injected with a total of 6 μL, and the day of injection was recorded as day 0 of injection. Among them, the mice injected with exosome suspensions obtained from wild-type mesenchymal stem cells and FOXO3-enhanced mesenchymal stem cells were respectively recorded as the wild-type exosome treatment group (ischemic stroke + WT-Exo group) and the FOXO3-enhanced exosome treatment group (ischemic stroke + F3-Exo group).

[0095] In addition, a control group (ischemic stroke + Vehicle group, i.e. ischemic stroke group) was injected with 6 μL of normal saline simultaneously, and a control group (Sham group) was set up for simultaneous treatment.

[0096] II. Detection of the effect of FOXO3-enhanced mesenchymal stem cell-derived exosomes on improving neurological function and cerebral blood flow after ischemic stroke. 1) Cylinder test The testing procedure is the same as the cylinder test in Example 2.

[0097] The test results are as follows Figure 4 As shown in Figure A, compared with the Sham group, the forelimb usage rate in the ischemic stroke + Vehicle group was significantly lower, indicating a significant decline in motor coordination function in this group of mice. The forelimb usage rates in the ischemic stroke + WT-Exo group and the ischemic stroke + F3-Exo group were improved compared with the ischemic stroke + Vehicle group. Injection of FOXO3-enhanced mesenchymal stem cell-derived exosomes improved the motor function of ischemic stroke mice, indicating that it can improve the motor function of ischemic stroke mice.

[0098] 2) Adhesive tape removal test The testing procedure is the same as the tape removal test in Example 2.

[0099] The test results are as follows Figure 4 As shown in Figure B, compared with the Sham group, the adhesive tape contact time and adhesive tape removal time were significantly increased in the ischemic stroke + Vehicle group, indicating a significant decline in limb sensory function in this group of mice. The limb sensory abilities of the ischemic stroke + WT-Exo group and the ischemic stroke + F3-Exo group were improved compared with the ischemic stroke + Vehicle group. FOXO3-enhanced mesenchymal stem cell transplantation enhanced limb sensory ability in ischemic stroke mice, indicating that it can improve limb sensory ability in ischemic stroke mice.

[0100] 3) Cerebral blood flow was detected using a laser speckle imaging system (RWD Life Sciences, Beijing, CN). The detection steps are the same as those for cerebral blood flow detection in Example 2.

[0101] The test results are as follows Figure 4 As shown in Figure C, compared with the Sham group, cerebral blood flow on the surgical side of the ischemic stroke + Vehicle group was significantly reduced. The cerebral blood flow ratios of the ischemic stroke + WT-Exo group and the ischemic stroke + F3-Exo group were improved compared with the ischemic stroke + Vehicle group, and the cerebral blood flow ratio of the ischemic stroke + F3-Exo group was significantly higher than that of the ischemic stroke + WT-Exo group. FOXO3-enhanced mesenchymal stem cell-derived exosome transplantation could also restore the decreased cerebral blood flow caused by ischemic stroke, indicating that it can promote angiogenesis in the brain tissue of ischemic stroke mice.

[0102] III. Detection of the regulatory effect of FOXO3-enhanced mesenchymal stem cell-derived exosomes on the cellular microenvironment of brain tissue after ischemic stroke. 1) TUNEL staining for apoptotic cell markers in brain tissue sections The detection steps are the same as the TUNEL staining in Example 2.

[0103] Staining results as follows Figure 5 As shown in Figure A, the number of TUNEL-positive cells in brain tissue sections of mice in each group was counted. The results showed that the proportion of TUNEL-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of TUNEL-positive cells in the brain tissue of the ischemic stroke + F3-Exo group was significantly lower than that of the ischemic stroke + WT-Exo group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced treatment with exosomes derived from mesenchymal stem cells significantly improved the apoptosis level of nerve cells in brain tissue.

[0104] 2) IBA1 staining, a marker for microglia, in brain tissue sections. The immunofluorescence staining procedure for brain tissue sections is the same as the immunofluorescence staining procedure for IBA-1 in Example 2.

[0105] Staining results as follows Figure 5 As shown in Figure B, the number of IBA1-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of IBA1-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of IBA1-positive cells in the brain tissue of the ischemic stroke + F3-Exo group was significantly lower than that of the ischemic stroke + WT-Exo group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced treatment with exosomes derived from mesenchymal stem cells significantly improved the level of microglia.

[0106] 3) CD68 staining, a marker of activated immune cells, in brain tissue sections. The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with CD68 (Abcam, no. ab125212, 1:200 dilution).

[0107] Staining results as follows Figure 5 As shown in Figure C, the number of CD68-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of CD68-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of CD68-positive cells in the brain tissue of the ischemic stroke + F3-Exo group was significantly lower than that of the ischemic stroke + WT-Exo group and the ischemic stroke + Vehicle group. This indicates that FOXO3 enhances the level of activation of inflammatory cells by mesenchymal stem cell-derived exosome therapy.

[0108] 4) Staining of brain tissue sections with the inflammatory mediator TNF-α The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with TNF-α (Abcam, no. ab1793, 1:100 dilution).

[0109] Staining results as follows Figure 5 As shown in Figure E, the number of TNF-α-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of TNF-α-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of TNF-α-positive cells in the brain tissue of the ischemic stroke + F3-Exo group was significantly lower than that of the ischemic stroke + WT-Exo group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced treatment with exosomes derived from mesenchymal stem cells significantly improved the expression level of the inflammatory mediator TNF-α.

[0110] 5) Staining of brain tissue sections with the inflammatory mediator IL-1β The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with IL-1β (Santa Cruz, no. sc-52012, 1:100 dilution).

[0111] Staining results as follows Figure 5 As shown in Figure D, the number of IL-1β-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of IL-1β-positive cells in the brain tissue of the ischemic stroke group was increased, and the proportion of IL-1β-positive cells in the brain tissue of the ischemic stroke + F3-Exo group was significantly lower than that of the ischemic stroke + WT-Exo group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced treatment with exosomes derived from mesenchymal stem cells significantly improved the expression level of the inflammatory mediator IL-1β.

[0112] 6) GFAP staining of brain tissue sections (a marker for astrocytes) The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with GFAP (Abcam, no. ab4674, 1:400 dilution).

[0113] Staining results as follows Figure 5As shown in Figure F, the length of glial scars in the brain tissue sections of mice in each group was statistically analyzed. The results showed that glial scars formed by GFAP-positive cells appeared in the brain tissue of the ischemic stroke group. The length of glial scars in the brain tissue of the ischemic stroke + F3-Exo group was significantly lower than that of the ischemic stroke + WT-Exo group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced treatment with exosomes derived from mesenchymal stem cells significantly reduced the length of glial scars formed by astrocytes.

[0114] 7) CD31 staining, a marker for vascular endothelial cells, in brain tissue sections. The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with CD31 (Abcam, no. ab28364, 1:100 dilution).

[0115] Staining results as follows Figure 6 As shown in Figure A, the number of CD31-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of CD31-positive cells in the brain tissue of the ischemic stroke group decreased, while the proportion of CD31-positive cells in the brain tissue of the ischemic stroke + F3-Exo group was significantly higher than that of the ischemic stroke + WT-Exo group and the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced treatment with exosomes derived from mesenchymal stem cells significantly improved the expression level of CD31 in vascular endothelial cells.

[0116] 8) DCX staining of immature neuron markers in brain tissue sections The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with DCX (Cell signaling Technology, no. 4604S, 1:100 dilution).

[0117] Staining results as follows Figure 6 As shown in Figure B, the number of DCX-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of DCX-positive cells in the brain tissue of the ischemic stroke group increased, and FOXO3-enhanced treatment with exosomes derived from mesenchymal stem cells significantly improved the level of immature neurons.

[0118] 9) NeuN staining of mature neurons in brain tissue sections The immunofluorescence staining procedure for brain tissue sections was the same as that for IBA1 in Example 2, except that the primary antibody was replaced with NeuN (Abcam, no. ab104224, 1:200 dilution).

[0119] Staining results as follows Figure 6As shown in Figure C, the number of NeuN-positive cells in the brain tissue sections of mice in each group was counted. The results showed that the proportion of NeuN-positive cells in the brain tissue of the ischemic stroke group was decreased, while the proportion of NeuN-positive cells in the brain tissue of the ischemic stroke + F3-Exo group was significantly higher than that of the ischemic stroke + Vehicle group. This indicates that FOXO3-enhanced treatment with exosomes derived from mesenchymal stem cells significantly improved the level of mature neurons.

[0120] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Any of the following applications of FOXO3-enhancing proteins or FOXO3 proteins, or substances that increase the content and / or activity of FOXO3 proteins: X1. To prepare products for the treatment and / or prevention of ischemic stroke; X2. To prepare products that improve the motor function of people or animals with ischemic stroke; X3. To prepare products that improve the sensory abilities of the limbs in people or animals with ischemic stroke; X4. Prepare products that promote angiogenesis in the brain tissue of humans or animals with ischemic stroke. X5. Prepare products that reduce apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X6. Prepare products that improve the inflammatory level of brain tissue in humans or animals with ischemic stroke; X7. Prepare products that reduce glial scarring in the brain tissue of humans or animals with ischemic stroke; X8. Prepare products that improve the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X9. Prepare products that promote neuroregeneration in the brain tissue of humans or animals with ischemic stroke. X10. Products that improve the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; X11. Treatment and / or prevention of ischemic stroke; X12. Improves motor function in people or animals with ischemic stroke; X13. Improves limb sensory ability in people or animals with ischemic stroke; X14. Promotes angiogenesis in the brain tissue of humans or animals with ischemic stroke; X15. Reduces apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X16. Improves the inflammatory level of brain tissue in humans or animals with ischemic stroke; X17. Reduces glial scarring in the brain tissue of humans or animals with ischemic stroke; X18. Improves the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X19. Promotes neurogenesis in the brain tissue of humans or animals with ischemic stroke; X20 improves the level of mature neurons in the brain tissue of humans or animals with ischemic stroke.

2. The application according to claim 1, characterized in that: The FOXO3 activity-enhancing protein is as follows: A1), A2), or A3). A1) The amino acid sequence of the protein includes SEQ ID NO: 4; A2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, with one or more amino acid residues substituted and / or deleted and / or added; A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2); The FOXO3 protein is as follows: (C1), (C2), or (C3) C1) The amino acid sequence of the protein includes SEQ ID NO: 2; C2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, with one or more amino acid residues substituted and / or deleted and / or added; C3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of C1) or C2).

3. Application of biomaterials related to FOXO3-enhancing proteins in the preparation of products with any of the following uses: X1. To prepare products for the treatment and / or prevention of ischemic stroke; X2. To prepare products that improve the motor function of people or animals with ischemic stroke; X3. To prepare products that improve the sensory abilities of the limbs in people or animals with ischemic stroke; X4. Prepare products that promote angiogenesis in the brain tissue of humans or animals with ischemic stroke. X5. Prepare products that reduce apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X6. Prepare products that improve the inflammatory level of brain tissue in humans or animals with ischemic stroke; X7. Prepare products that reduce glial scarring in the brain tissue of humans or animals with ischemic stroke; X8. Prepare products that improve the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X9. Prepare products that promote neuroregeneration in the brain tissue of humans or animals with ischemic stroke. X10. Products that improve the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; X11. Treatment and / or prevention of ischemic stroke; X12. Improves motor function in people or animals with ischemic stroke; X13. Improves limb sensory ability in people or animals with ischemic stroke; X14. Promotes angiogenesis in the brain tissue of humans or animals with ischemic stroke; X15. Reduces apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X16. Improves the inflammatory level of brain tissue in humans or animals with ischemic stroke; X17. Reduces glial scarring in the brain tissue of humans or animals with ischemic stroke; X18. Improves the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X19. Promotes neurogenesis in the brain tissue of humans or animals with ischemic stroke; X20 improves the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; The biomaterial is any one of B1) to B5) below: B1) A nucleic acid molecule encoding the FOXO3 activity-enhancing protein as described in claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Cell lines containing the nucleic acid molecules described in B1), or cell lines containing the expression cassette described in B2); Derivatives of the cell lines described in B6 and B5).

4. The application according to claim 3, characterized in that: B1) The nucleic acid molecule described is as follows (b11) or (b12)): b11) The coding sequence is the DNA molecule of SEQ ID NO: 3 in the sequence listing; b12) has 75% or more identity with the nucleotide sequence defined in b11) and is a DNA molecule encoding the FOXO3 activity-enhancing protein; The derivative described in B6) is an exosome of the cell line described in B5).

5. The application according to claim 3 or 4, characterized in that: B5) The cell line described is mesenchymal stem cells.

6. Application of biomaterials related to FOXO3 protein in the preparation of products with any of the following uses: X1. To prepare products for the treatment and / or prevention of ischemic stroke; X2. To prepare products that improve the motor function of people or animals with ischemic stroke; X3. To prepare products that improve the sensory abilities of the limbs in people or animals with ischemic stroke; X4. Prepare products that promote angiogenesis in the brain tissue of humans or animals with ischemic stroke. X5. Prepare products that reduce apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X6. Prepare products that improve the inflammatory level of brain tissue in humans or animals with ischemic stroke; X7. Prepare products that reduce glial scarring in the brain tissue of humans or animals with ischemic stroke; X8. Prepare products that improve the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X9. Prepare products that promote neuroregeneration in the brain tissue of humans or animals with ischemic stroke. X10. Products that improve the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; X11. Treatment and / or prevention of ischemic stroke; X12. Improves motor function in people or animals with ischemic stroke; X13. Improves limb sensory ability in people or animals with ischemic stroke; X14. Promotes angiogenesis in the brain tissue of humans or animals with ischemic stroke; X15. Reduces apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; X16. Improves the inflammatory level of brain tissue in humans or animals with ischemic stroke; X17. Reduces glial scarring in the brain tissue of humans or animals with ischemic stroke; X18. Improves the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; X19. Promotes neurogenesis in the brain tissue of humans or animals with ischemic stroke; X20 improves the level of mature neurons in the brain tissue of humans or animals with ischemic stroke; The biomaterial is any one of the following D1) to D5): D1) A nucleic acid molecule encoding the FOXO3 protein as described in claim 1 or 2; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3); D5) Cell lines containing the nucleic acid molecules described in D1), or cell lines containing the expression cassette described in D2); Derivatives of the cell lines described in D6 and D5).

7. The application according to claim 6, characterized in that: D1) The nucleic acid molecule described is as follows (d11) or (d12): d11) The coding sequence is the DNA molecule of SEQ ID NO: 1 in the sequence listing; The nucleotide sequence defined by d12) has 75% or more identity with the nucleotide sequence defined by d11) and is a DNA molecule encoding the FOXO3 protein; The derivative described in D6) is an exosome of the cell line described in D5).

8. The application according to claim 6 or 7, characterized in that: The cell line described in D5 is a mesenchymal stem cell.

9. A product having any of the following functions, comprising the FOXO3 activity-enhancing protein as described in claim 1 or 2, or the FOXO3 activity-enhancing protein as described in claim 1 or 2, or the substance for increasing the content and / or activity of FOXO3 protein as described in claim 1, or any of the biomaterials described in claims 3-8: Y1. Treatment and / or prevention of ischemic stroke; Y2. Improves motor function in people or animals with ischemic stroke; Y3. Improves limb sensory ability in people or animals with ischemic stroke; Y4. Promotes angiogenesis in the brain tissue of humans or animals with ischemic stroke; Y5. Reduces apoptosis of cells in the brain tissue of humans or animals with ischemic stroke; Y6. Improves the inflammatory level of brain tissue in humans or animals with ischemic stroke; Y7. Reduces glial scarring in the brain tissue of humans or animals with ischemic stroke; Y8. Improves the level of vascular endothelial cells in the brain tissue of humans or animals with ischemic stroke; Y9. Promotes neurogenesis in the brain tissue of humans or animals with ischemic stroke; Y10 improves the level of mature neurons in the brain tissue of humans or animals with ischemic stroke.

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