A method for pretreating mesenchymal stem cells and products and application thereof in drugs for cerebral stroke

Abstract

This invention, entitled "A Method and Product for Pretreatment of Mesenchymal Stem Cells and Its Application in Stroke Drugs," belongs to the field of biotechnology. The technical problem to be solved is to improve the function and therapeutic effect of mesenchymal stem cells. The key technical solution is a method for pretreatment of mesenchymal stem cells, including: culturing cells using an activation medium, culturing cells using a directional medium, and culturing cells under hypoxic conditions using an adaptation medium; the activation medium contains complete medium, IFN-γ, TNF-α, and IL-1β; the directional medium contains complete medium, sodium valproate, 5-azacytidine, and all-trans retinoic acid; the adaptation medium is a complete medium containing basal medium and free of animal serum. The pretreatment method of this invention enhances the anti-inflammatory, neuroprotective, and angiogenic capabilities of mesenchymal stem cells, showing significant therapeutic effects on stroke, and since it does not use animal serum, it meets clinical requirements.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to mesenchymal stem cells, specifically to a method for pretreating mesenchymal stem cells and the application of the product in stroke drugs. Background Technology

[0002] Stroke, also known as cerebrovascular accident, is characterized by high incidence, high disability rate, high mortality rate, and high recurrence rate. Acute ischemic stroke (AIS) has a short treatment window, and its pathological process exhibits a complex temporal and spatial cascade, involving multiple damaging pathways such as inflammatory eruption, neuronal apoptosis, and blood-brain barrier disruption. Current treatment options, including thrombolysis and thrombectomy, and supplemental neuroprotective agents, all have limitations that are difficult to overcome.

[0003] Multiple studies have shown that mesenchymal stem cells (MSCs) possess the ability to self-renew and differentiate into various cell types and tissues. They exhibit low immunogenicity, secrete a variety of cytokines, regulate the immune system, and promote the repair of damaged tissues and organs, making them suitable for treating various diseases. MSCs can exert their immunomodulatory and neurorepair functions through a "multi-pathway synergistic repair" therapeutic mechanism, involving cell replacement and repair, secretion of neurotrophic factors, promotion of angiogenesis, and inhibition of inflammation and apoptosis, simultaneously acting on key nodes of the ischemic cascade in acute myeloid inflammatory syndrome (AIS). Although MSC transplantation therapy matches the pathogenesis of AIS and has a good safety profile, there is still room for improvement in its efficacy.

[0004] Relevant patent documents retrieved: This document, published in China (CN115433710A) on December 6, 2022, discloses a method, product, and application of curcumin-pretreated olfactory mesenchymal stem cells. Using olfactory mesenchymal stem cells as a carrier, curcumin is added to their culture medium for co-culture to prepare curcumin-pretreated olfactory mesenchymal stem cells. The main application is in the preparation of drugs for treating cerebral hemorrhage. Specific applications include that curcumin-pretreated olfactory mesenchymal stem cells can improve the survival rate of olfactory mesenchymal stem cells after transplantation in patients with cerebral hemorrhage, reduce oxidative stress, inhibit neuronal death, improve cerebral edema and blood-brain barrier disruption, thereby promoting brain injury repair and ultimately effectively improving neurological deficits caused by cerebral hemorrhage.

[0005] Relevant non-patent literature retrieved: The journal or book title is *Chinese Journal of Cell Biology*, and the article title is "Resveratrol-Pretreated Adipose-Mesenchymal Stem Cells Protect CCl4-Induced Hepatocyte Injury by Regulating Autophagy," Volume 47, Publication Date: May 2025. This article discloses the therapeutic effect of resveratrol (Res) pretreatment of adipose-derived mesenchymal stem cells on carbon tetrachloride (CCl4)-induced hepatocyte injury. The results show that resveratrol-pretreated adipose-derived mesenchymal stem cells can repair liver injury by promoting autophagy, and the increase in autophagy may be related to the inhibition of the PI3K / AKT / mTOR pathway.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Literature CN115433710A discloses the application of pretreated mesenchymal stem cells in the treatment of cerebral hemorrhage, while the literature "Resveratrol-Pretreated Adipose-Derived Mesenchymal Stem Cells Protect CCl4-Induced Hepatocyte Injury by Regulating Autophagy" discusses their therapeutic effects on liver injury. Neither of these studies addresses a treatment option for stroke. There is an urgent need to provide a pretreatment method for mesenchymal stem cells that can treat stroke. Summary of the Invention

[0007] The purpose of this invention is to provide: The application of a method and product for pretreatment of mesenchymal stem cells in stroke drugs, and related technologies, to solve technical problems such as providing a method for pretreatment of mesenchymal stem cells, or combinations thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] Definitions of standard terminology can be found in the reference book *Stem Cell Biology* (Publisher: Science Press; Publication Date: January 2003; Edition: 1st edition).

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as cell culture, cell passage, inoculation, centrifugation, and intravenous injection, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] As used in this article, "stem cell" refers to undifferentiated cells capable of self-renewal and differentiation into multiple cell types. Stem cells are a type of cell with unlimited or immortal self-renewal capacity, capable of producing at least one type of highly differentiated daughter cell.

[0014] The term "mesenchymal stem cells (MSCs)" as used in this article refers to a type of adult stem cell with self-renewal capacity, multi-lineage differentiation potential, and immunomodulatory functions; they are also known as "mesenchymal stromal cells" or "mesenchymal cells." MSCs are widely derived and can be isolated from various tissues, including bone marrow, adipose tissue, Wharton's jelly of the umbilical cord, placenta, and dental pulp. MSC surface markers include specific proteins such as CD73, CD90, and CD105, but they generally do not express hematopoietic stem cell markers (such as CD34 and CD45). MSCs possess self-renewal capacity and retain stem cell characteristics after division; they can also differentiate into multiple lineages, such as bone, cartilage, adipose tissue, and nerve cells; the paracrine function of MSCs regulates the microenvironment through the secretion of cytokines or exosomes, inhibiting inflammatory responses and promoting tissue regeneration and repair.

[0015] The term "primary cells" used in this article refers to cells that are cultured immediately after being removed from body tissues, generally denoted by P0. Primary cells mainly originate from human fetal cells, adult cells, and cells from other animals. After being removed, these cells undergo certain processing and culture to form primary cell lines. Primary cells largely retain their original biological characteristics from the body, including morphology, structure, and function. Primary cells have a limited number of passages and generally cannot be passaged indefinitely like passaged cells.

[0016] The term "cell passage" used in this article refers to a cell population capable of continuous passage and proliferation in an in vitro environment, primarily expanded by splitting the culture and transferring it to a new container. The culture process involves three typical phases: the free phase, the logarithmic growth phase, and the stationary phase. The logarithmic growth phase exhibits the best cell viability and is suitable for experimental manipulation. After successful primary cell culture, as the culture time increases and cells continue to divide, contact inhibition occurs between cells, slowing or even halting growth. Insufficient nutrients and accumulation of metabolites can also hinder growth. Once the cells have reached confluence in the culture flask, they need to be diluted and divided into multiple flasks to allow for continued cell growth. Passaging provides a large number of cells for experimental needs.

[0017] The term "cell passage" as used in this article refers to the number of times cells have been artificially passaged since their original source (such as tissue isolation or cryopreservation and thawing). Cell passage reflects the "age accumulation" of a cell population in vitro; the higher the passage, the more proliferative stress and environmental exposure the cells have experienced.

[0018] The term "serum-free culture medium" as used in this article refers to a synthetic culture medium that can maintain cell proliferation in vitro for a relatively long period of time without the addition of serum. The basic components of serum-free culture medium generally include two parts: a basal medium and additives. These are mixed in a specific ratio to form a complete culture medium used for the in vitro culture of cells in biopharmaceutical and vaccine production.

[0019] The term "cryopreservative" as used in this article refers to a substance (usually a solution) that can protect cells from freezing damage. Adding a cryopreservative to a cell suspension protects cells from both solution damage and ice crystal damage. Cryopreservatives bind to water molecules in the solution, causing hydration, weakening the water crystallization process, increasing the viscosity of the solution, and thus reducing ice crystal formation. Simultaneously, cryopreservatives can maintain a certain molar concentration inside and outside the cell, reducing the concentration of electrolytes in the unfrozen solution inside and outside the cell, thereby protecting the cell from solute damage. Cryopreservatives can be divided into permeable and non-permeable categories based on whether they penetrate the cell membrane.

[0020] The term "cell resuscitation" as used in this article refers to the process of thawing cells frozen in liquid nitrogen or at -80°C and then reculturing them to resume growth. When cells are brought back to room temperature, their morphology and structure remain normal, and biochemical reactions resume. Unlike cell cryopreservation, the cell resuscitation process requires rapid warming to prevent water from entering the cells during thawing, forming ice crystals, and affecting cell survival.

[0021] In a first aspect, the present invention provides: a method for pretreating mesenchymal stem cells, comprising: culturing cells using an activation medium, culturing cells using a directional medium, and culturing cells under hypoxic conditions using an adaptation medium; The activation medium contains complete culture medium, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The directional culture medium contains complete culture medium, sodium valproate, 5-azacytidine, and all-trans retinoic acid; The adaptation culture medium is a complete culture medium containing basal medium and animal serum-free medium.

[0022] These include technical characteristics: culture time, culture medium, hypoxia conditions, and cells.

[0023] Specifically, the incubation time using activation medium, directional medium, and adaptation medium is 6-72 hours (h).

[0024] Preferably, the culture time using the activation medium is 12-48 hours; more preferably, the culture time using the activation medium is 24 hours.

[0025] Preferably, the culture time using the directional culture medium is 12-48 hours; more preferably, the culture time using the directional culture medium is 24 hours.

[0026] Preferably, the culture time using the adaptation medium is 12-72 hours; more preferably, the culture time using the adaptation medium is 24 hours.

[0027] The activation medium contained complete culture medium, 1-100 ng / mL IFN-γ, 1-100 ng / mL TNF-α, and 1-100 ng / mL IL-1β; According to some embodiments of the present invention, the concentration of IFN-γ in the activation culture medium is selected from 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, and any intermediate value range.

[0028] According to some embodiments of the present invention, the activation culture medium contains IFN-γ at concentrations of 1-10 ng / mL, 1-20 ng / mL, 1-30 ng / mL, 1-40 ng / mL, 1-50 ng / mL, 1-60 ng / mL, 1-70 ng / mL, 1-80 ng / mL, 1-90 ng / mL, 1-100 ng / mL, 10-20 ng / mL, 10-30 ng / mL, 10-40 ng / mL, etc. g / mL, 10-50ng / mL, 10-60ng / mL, 10-70ng / mL, 10-80ng / mL, 10-90ng / mL, 10-100ng / mL, 20-30ng / m L, 20-40ng / mL, 20-50ng / mL, 20-60ng / mL, 20-70ng / mL, 20-80ng / mL, 20-90ng / mL, 20-100ng / mL, 30 -40ng / mL, 30-50ng / mL, 30-60ng / mL, 30-70ng / mL, 30-80ng / mL, 30-90ng / mL, 30-100ng / mL, 40-50 ng / mL, 40-60ng / mL, 40-70ng / mL, 40-80ng / mL, 40-90ng / mL, 40-100ng / mL, 50-60ng / mL, 50-70ng / The following values ​​can be selected: mL, 50-80 ng / mL, 50-90 ng / mL, 50-100 ng / mL, 60-70 ng / mL, 60-80 ng / mL, 60-90 ng / mL, 60-100 ng / mL, 70-80 ng / mL, 70-90 ng / mL, 70-100 ng / mL, 80-90 ng / mL, 80-100 ng / mL, 90-100 ng / mL, and any range in between.

[0029] According to some embodiments of the present invention, the activation culture medium includes TNF-α concentrations of 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, and any intermediate value range may be selected.

[0030] According to some embodiments of the present invention, the activation culture medium includes TNF-α at concentrations of 1-10 ng / mL, 1-20 ng / mL, 1-30 ng / mL, 1-40 ng / mL, 1-50 ng / mL, 1-60 ng / mL, 1-70 ng / mL, 1-80 ng / mL, 1-90 ng / mL, 1-100 ng / mL, 10-20 ng / mL, 10-30 ng / mL, and 10-40 ng / mL. g / mL, 10-50ng / mL, 10-60ng / mL, 10-70ng / mL, 10-80ng / mL, 10-90ng / mL, 10-100ng / mL, 20-30ng / m L, 20-40ng / mL, 20-50ng / mL, 20-60ng / mL, 20-70ng / mL, 20-80ng / mL, 20-90ng / mL, 20-100ng / mL, 30 -40ng / mL, 30-50ng / mL, 30-60ng / mL, 30-70ng / mL, 30-80ng / mL, 30-90ng / mL, 30-100ng / mL, 40-50 ng / mL, 40-60ng / mL, 40-70ng / mL, 40-80ng / mL, 40-90ng / mL, 40-100ng / mL, 50-60ng / mL, 50-70ng / The following values ​​can be selected: mL, 50-80 ng / mL, 50-90 ng / mL, 50-100 ng / mL, 60-70 ng / mL, 60-80 ng / mL, 60-90 ng / mL, 60-100 ng / mL, 70-80 ng / mL, 70-90 ng / mL, 70-100 ng / mL, 80-90 ng / mL, 80-100 ng / mL, 90-100 ng / mL, and any range in between.

[0031] According to some embodiments of the present invention, the concentration of IL-1β in the activation culture medium is selected from 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, and any intermediate value range.

[0032] According to some embodiments of the present invention, the activation culture medium contains IL-1β at concentrations of 1-10 ng / mL, 1-20 ng / mL, 1-30 ng / mL, 1-40 ng / mL, 1-50 ng / mL, 1-60 ng / mL, 1-70 ng / mL, 1-80 ng / mL, 1-90 ng / mL, 1-100 ng / mL, 10-20 ng / mL, 10-30 ng / mL, 10-40 ng / mL, etc. g / mL, 10-50ng / mL, 10-60ng / mL, 10-70ng / mL, 10-80ng / mL, 10-90ng / mL, 10-100ng / mL, 20-30ng / m L, 20-40ng / mL, 20-50ng / mL, 20-60ng / mL, 20-70ng / mL, 20-80ng / mL, 20-90ng / mL, 20-100ng / mL, 30 -40ng / mL, 30-50ng / mL, 30-60ng / mL, 30-70ng / mL, 30-80ng / mL, 30-90ng / mL, 30-100ng / mL, 40-50 ng / mL, 40-60ng / mL, 40-70ng / mL, 40-80ng / mL, 40-90ng / mL, 40-100ng / mL, 50-60ng / mL, 50-70ng / The following values ​​can be selected: mL, 50-80 ng / mL, 50-90 ng / mL, 50-100 ng / mL, 60-70 ng / mL, 60-80 ng / mL, 60-90 ng / mL, 60-100 ng / mL, 70-80 ng / mL, 70-90 ng / mL, 70-100 ng / mL, 80-90 ng / mL, 80-100 ng / mL, 90-100 ng / mL, and any range in between.

[0033] Preferably, the activation medium contains complete culture medium, 60 ng / mL IFN-γ, 60 ng / mL TNF-α, and 20 ng / mL IL-1β.

[0034] The directional culture medium contains complete culture medium, 10 nM-10 mM sodium valproate, 0.5-50 μM 5-azacytidine, and 1-100 μM all-trans retinoic acid; According to some embodiments of the present invention, the directional culture medium includes sodium valproate at concentrations of 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, and any intermediate value may be selected.

[0035] According to some embodiments of the present invention, the concentration range of sodium valproate in the directional culture medium is 10nM-100nM, 10nM-1μM, 10nM-10μM, 10nM-100μM, 10nM-1mM, 10nM-10mM, 100nM-1μM, 100nM-10μM, 100nM-100μM, 100nM-1mM, 100nM-10mM, 1μM-10μM, 1μM-10μM, 1μM-100μM, 1μM-1mM, 1μM-10mM, 10μM-100μM, 10μM-10mM, 100μM-1mM, 100μM-10mM, 1mM-10mM, and any intermediate value may be selected.

[0036] According to some embodiments of the present invention, the directional culture medium includes 5-azacytidine at concentrations of 0.5 μM, 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and any intermediate value may be selected.

[0037] According to some embodiments of the present invention, the concentration range of 5-azacytidine in the directional culture medium is 0.5-1 μM, 0.5-10 μM, 0.5-20 μM, 0.5-30 μM, 0.5-40 μM, 0.5-50 μM, 1-10 μM, 1-20 μM, 1-30 μM, 1-40 μM, 1-50 μM, 10-20 μM, 10-30 μM, 10-40 μM, 10-50 μM, 20-30 μM, 20-40 μM, 20-50 μM, 30-40 μM, 30-50 μM, 40-50 μM, and any intermediate range may be selected.

[0038] According to some embodiments of the present invention, the directional culture medium includes all-trans retinoic acid at concentrations of 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, and any intermediate value may be selected.

[0039] According to some embodiments of the present invention, the concentration range of all-trans retinoic acid in the directional culture medium is 1-10 μM, 1-20 μM, 1-30 μM, 1-40 μM, 1-50 μM, 1-60 μM, 1-70 μM, 1-80 μM, 1-90 μM, 1-100 μM, 10-20 μM, 10-30 μM, 10-40 μM, 10-50 μM, 10-60 μM, 10-70 μM, 10-80 μM, 10-90 μM, 10-100 μM, 20-30 μM, 20-40 μM, 20-50 μM, 20-60 μM, 20-70 μM, 20-80 μM, 20-90 μM, 20-100 μM. The following values ​​are available: 30-40μM, 30-50μM, 30-60μM, 30-70μM, 30-80μM, 30-90μM, 30-100μM, 40-50μM, 40-60μM, 40-70μM, 40-80μM, 40-90μM, 40-100μM, 50-60μM, 50-70μM, 50-80μM, 50-90μM, 50-100μM, 60-70μM, 60-80μM, 60-90μM, 60-100μM, 70-80μM, 70-90μM, 70-100μM, 80-90μM, 80-100μM, 90-100μM, and any value in between.

[0040] Preferably, the directional culture medium contains complete culture medium, 1 mM sodium valproate, 2 μM 5-azacytidine and 10 μM all-trans retinoic acid.

[0041] Specifically, the low-oxygen conditions are 1-10% oxygen (O2) and 3-8% carbon dioxide (CO2); preferably, the low-oxygen conditions are 2% O2 and 5% CO2.

[0042] Specifically, the concentration of each component in the culture medium is the final concentration.

[0043] The cells mentioned are selected from P1-P10 generation mesenchymal stem cells; According to some embodiments of the present invention, the cells may be selected from P1 generation, P2 generation, P3 generation, P4 generation, P5 generation, P6 generation, P7 generation, P8 generation, P9 generation, and P10 generation. Preferably, the cells are P4-P6 generation cells; More preferably, the cells are P4 generation cells.

[0044] Specifically, the mesenchymal stem cells are selected from any one or more of the following: umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, peripheral blood mesenchymal stem cells, uterine blood mesenchymal stem cells, hair follicle mesenchymal stem cells, and urine mesenchymal stem cells. Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0045] According to some embodiments of the present invention, the order of cell culture using the culture medium is not limited.

[0046] Preferably, the pretreatment time using three different culture media is 72 hours in total, including at least four different treatment methods: (1) First, use the activation medium to culture the cells under normal oxygen conditions for 24 hours, then use the orientation medium to culture the cells under normal oxygen conditions for 24 hours, and finally use the adaptation medium to culture the cells under low oxygen conditions for 24 hours. (2) First, use the activating medium to culture the cells under normal oxygen conditions for 48 hours, and then use the directional medium to culture the cells under low oxygen conditions for 24 hours; (3) First, use the activating medium to culture under low oxygen conditions for 24 hours, and then use the directional medium to culture under low oxygen conditions for 48 hours; (4) First, use the adaptation medium to culture the cells under hypoxic conditions for 24 hours, then use the activation medium to culture the cells under normoxic conditions for 24 hours, and finally use the orientation medium to culture the cells under normoxic conditions for 24 hours.

[0047] More preferably, the pretreatment sequence is as follows: first, cells are cultured in activating medium under normoxic conditions for 24 hours; then, cells are cultured in directional medium under normoxic conditions for 24 hours; and finally, cells are cultured in acclimatization medium under hypoxic conditions for 24 hours.

[0048] Secondly, the present invention provides mesenchymal stem cells pretreated by the above-described method.

[0049] Thirdly, the present invention provides the application of the above-mentioned mesenchymal stem cells in the preparation of drugs for treating nervous system diseases, inflammatory diseases, autoimmune diseases, cardiovascular diseases and metabolic diseases.

[0050] Specifically, the neurological diseases mentioned include, but are not limited to, stroke, Alzheimer's disease, Parkinson's disease, epilepsy, depression, amyotrophic lateral sclerosis, Huntington's disease, and multiple sclerosis.

[0051] Specifically, the inflammatory diseases mentioned include, but are not limited to, osteoarthritis, periodontitis, liver failure, fatty liver, cirrhosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, and diabetic foot ulcers.

[0052] Specifically, the autoimmune diseases mentioned include, but are not limited to, graft-versus-host disease (GvHD), autoimmune uveitis, systemic lupus erythematosus, lupus nephritis, systemic sclerosis, rheumatoid arthritis, and autoimmune hepatitis.

[0053] Specifically, the cardiovascular diseases mentioned include, but are not limited to, myocardial infarction, lower limb ischemia, angina pectoris, premature beats, heart failure, and cardiomyopathy.

[0054] Specifically, the metabolic diseases mentioned include, but are not limited to, obesity, type 2 diabetes, type 1 diabetes, diabetic ketoacidosis, hypercholesterolemia, hyperuricemia, and geriatric syndrome.

[0055] Specifically, the drug may also include a pharmaceutically acceptable carrier.

[0056] Furthermore, the pharmaceutically acceptable carrier is selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0057] Specifically, the dosage form of the drug is an injection.

[0058] Fourthly, the present invention provides a medicament for treating nervous system diseases, inflammatory diseases, autoimmune diseases, cardiovascular diseases, and metabolic diseases, wherein the medicament comprises the pretreated mesenchymal stem cells described above.

[0059] Specifically, the drug may also include a pharmaceutically acceptable carrier.

[0060] The core principle and mechanism of action of this invention are as follows: Stimulation by pro-inflammatory factors can mimic the inflammatory environment of the initial stage of injury in vivo. When MSCs arrive at the site of inflammation, they first encounter a cytokine storm, which can rapidly pre-activate the immunomodulatory function of MSCs, enhancing their ability to inhibit T cells and macrophages, as well as their immunomodulatory, anti-inflammatory, and trophic factor secretion capabilities. Among them, IFN-γ acts on the JAK-STAT1 pathway, upregulating indoleamine 2,3-dioxygenase (IDO), consuming tryptophan and producing kynurenine, strongly inhibiting the proliferation of T cells and NK cells, and inducing regulatory T cells; TNF-α, through the NF-κB pathway, upregulates COX-2, TSG-6, and IL-6, promotes the secretion of prostaglandin E2 (PGE2), inhibits the polarization of macrophages towards the pro-inflammatory M1 type, and promotes their transformation towards the anti-inflammatory M2 type; IL-1β upregulates chemokines and adhesion molecules such as IL-8, MCP-1, and ICAM-1, enhancing the chemotactic ability of MSCs towards the site of inflammation and their adhesion to immune cells, making their "localization" more precise.

[0061] Epigenetic regulation induces the secretion of neurotrophic factors: On the basis of pre-activation of cells, the combined use of epigenetic drugs such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase inhibitors can more effectively activate genes related to neuroprotection and repair, achieving functional superposition. Sodium valproate is an HDAC inhibitor that increases histone acetylation levels by inhibiting HDAC activity, making chromatin structure more open and thereby activating the transcription of a series of neurotrophic factors and anti-inflammatory genes, such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF). 5-Azacytidine (5AZA) is a DNA methyltransferase inhibitor that inhibits DNA methyltransferases, leading to a decrease in overall genomic DNA methylation levels. This relieves transcriptional inhibition of specific genes, such as those related to neuroprotection and repair, and has a synergistic effect with the HDAC inhibitor, enhancing the neurotrophic factor secretion capacity of MSCs to a level that cannot be achieved by a single component. All-trans retinoic acid (RA) is a nuclear receptor agonist that, after entering the cell, binds to retinoic acid receptors / vitamin A AX receptors, forming a complex that acts as a transcription factor, directly binding to the promoter regions of specific genes and initiating the expression of target genes involved in neural differentiation and cell cycle regulation. These three components work together to promote the secretion of neurotrophic factors by MSCs, resulting in a stronger neuroprotective and repairing effect on stroke lesions after transplantation.

[0062] Hypoxia treatment can simulate the final "adaptation" of MSCs to the pathological microenvironment after they migrate to the ischemic core of the tissue. Hypoxia can further enhance the proliferation, migration, anti-apoptosis and secretion of MSCs by stabilizing hypoxia-inducible factor 1α (HIF-1α), upregulating vascular endothelial growth factor (VEGF), promoting the expression of angiogenesis and anti-inflammatory factors, and enhancing the migration and homing ability and tissue repair ability of MSCs.

[0063] By combining the above three mechanisms, the anti-inflammatory, angiogenesis, and neuroprotective effects of MSCs can be promoted simultaneously, significantly improving the treatment efficacy of AIS.

[0064] The present invention has at least the following beneficial effects: This invention employs a three-step induction method to pretreat MSCs. Pretreatment did not affect cell viability, and the levels of IDO, PGE2, VEGF, BDNF, GDNF, hepatocyte growth factor (HGF), fibroblast growth factor-2 (FGF-2), and angiopoietin-1 (ANG-1) secreted by pretreated MSCs significantly increased. When pretreated MSCs were co-cultured with PBMCs, the number of PBMCs decreased significantly, while the concentrations of IL-17, TNF-α, and IFN-γ decreased, and the concentration of interleukin-10 (IL-10) increased, demonstrating significant immunomodulatory effects. This pretreatment method enhances the anti-inflammatory, neuroprotective, homing, and angiogenesis-promoting capabilities of MSCs, improving the efficacy of stem cell therapy for AIS.

[0065] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for treating neurological disorders, inflammatory diseases, autoimmune diseases, cardiovascular diseases, and metabolic diseases, the method comprising administering the aforementioned mesenchymal stem cells to a subject.

[0066] Specifically, the pretreatment method for mesenchymal stem cells includes: culturing cells using an activation medium, culturing cells using a directional medium, and culturing cells under hypoxic conditions using an adaptation medium. The activation medium contains complete culture medium, IFN-γ, TNF-α, and IL-1β; The directional culture medium contains complete culture medium, sodium valproate, 5-azacytidine, and all-trans retinoic acid; The adaptation culture medium is a complete culture medium containing basal medium and animal serum-free medium.

[0067] The incubation time using activation medium, directional medium, and adaptation medium is 6-72 hours.

[0068] Furthermore, the activation medium contains complete medium, 1-100 ng / mL IFN-γ, 1-100 ng / mL TNF-α, and 1-100 ng / mL IL-1β.

[0069] Furthermore, the directional culture medium contains complete culture medium, 10 nM-10 mM sodium valproate, 0.5-50 μM 5-azacytidine, and 1-100 μM all-trans retinoic acid.

[0070] Furthermore, the pretreatment time using three different culture media totaled 72 hours, including at least four different treatment methods: (1) First, use the activation medium to culture the cells for 24 hours, then use the orientation medium to culture the cells for 24 hours, and finally use the adaptation medium to culture the cells under low oxygen conditions for 24 hours. (2) First, use the activation medium to culture the cells for 48 hours, and then use the directional medium to culture the cells under low oxygen conditions for 24 hours; (3) First, use the activating medium to culture under low oxygen conditions for 24 hours, and then use the directional medium to culture under low oxygen conditions for 48 hours; (4) First, use the adaptation medium to culture the cells under hypoxic conditions for 24 hours, then use the activation medium to culture the cells under normoxic conditions for 24 hours, and finally use the orientation medium to culture the cells under normoxic conditions for 24 hours. Attached Figure Description

[0071] Figure 1 This is a schematic diagram of a mesenchymal stem cell pretreatment method.

[0072] Figure 2 This represents the cell viability results from in vitro cell experiments.

[0073] Figure 3 The results show the concentrations of indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), and vascular endothelial growth factor (VEGF) in in vitro cell experiments.

[0074] Figure 4 The results show the concentrations of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF) in in vitro cell experiments.

[0075] Figure 5 The results show the concentrations of fibroblast growth factor 2 (FGF-2) and angiopoietin 1 (ANG-1) in in vitro cell experiments.

[0076] Figure 6 The results show the levels of interleukin-17 (IL-17) and interleukin-10 (IL-10) in in vitro cell experiments.

[0077] Figure 7 The results show the levels of tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) in in vitro cell experiments.

[0078] Figure 8 The results represent the immune cell suppression rate, the proportion of Th1 cells in the CD4+ subset, and the proportion of Treg cells in the CD4+ subset in in vitro cell experiments.

[0079] Figure 9 The graph shows the infarct area, cerebral infarction area, and neurological function score results.

[0080] Figure 10 Image showing the results of TUNEL staining.

[0081] Figure 11 This is a graph showing the number of microglia in Example 3.

[0082] Figure 12 The image shows the expression of insulin-like growth factor 1 (IGF-1) and the number of CD31-positive blood vessels in Example 3.

[0083] Figure 13 The image shows the NIHSS score and mRS score results of clinical case 1 in Example 4.

[0084] Figure 14 The graph shows the NIHSS score, mRS score, and BI score results of clinical case 2 in Example 5.

[0085] Figure 15 The graph shows the NIHSS and mRS scores of the placebo group in comparative clinical case 3. Detailed Implementation

[0086] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0087] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0088] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0089] A schematic diagram of the mesenchymal stem cell pretreatment method of the present invention is shown below. Figure 1 As shown.

[0090] Example 1: Culture of human umbilical cord mesenchymal stem cells (hUC-MSCs), cell viability and secretion factor detection experiments. 1. Umbilical cord MSCs culture: Obtain a fresh neonatal umbilical cord sample, clean it with physiological saline, disinfect it with medical alcohol, clean it again, cut the umbilical cord into small segments, cut the amnion, remove the blood vessels, peel off 2g of Wharton's jelly tissue, and cut it into 1-3mm pieces with sterile scissors. 3Small pieces of umbilical cord Wharton's jelly tissue were transferred to a T75 cell culture flask. 10 mL of serum-free complete culture medium (serum-free DMEM / F12 basal medium + 5% platelet lysate (the platelet lysate content can be adjusted to 2%-10% depending on experimental needs)) was added, pH 7.2-7.4. The flask was then incubated at 37°C with 5% CO2. After the cells detached, the complete culture medium was replaced. Once the cell confluence reached 80%, all culture medium was aspirated and discarded. The cells were washed with sodium chloride injection solution and then digested with 2 mL of 0.05% recombinant trypsin for 2-5 minutes. After cell detachment, 10 mL of PBS buffer was added to stop the digestion. The cell suspension was transferred to a centrifuge tube, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium to obtain P0 generation cells. 5 × 10^5 P0 generation cells were transferred to a new T75 cell culture flask and cultured using the same method to obtain P1 generation cells. Thus, the cells were passaged to generation P4, and seeded into 6-well plates at a rate of 1×10^5 cells per well. 2 mL of complete culture medium was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator.

[0091] 2. Three-step induction pretreatment: Step 1: When the P4 generation cells are in the logarithmic growth phase, have good morphology, and have a confluence of about 70%-80% (about 24 hours after passage), add three pro-inflammatory factors to the culture medium: IFN-γ (final concentration 60 ng / mL), TNF-α (final concentration 60 ng / mL), and IL-1β (final concentration 20 ng / mL), and continue culturing for 24 hours.

[0092] Step 2: Discard all the culture medium containing pro-inflammatory factors in the well plate and replace it with a complete culture medium containing three epigenetic regulatory molecules: sodium valproate (final concentration 1mM), 5-azacytidine (final concentration 2μM), and all-trans retinoic acid (final concentration 10μM), and continue culturing for 24 hours.

[0093] Step 3: Discard all the culture medium containing epigenetic regulatory molecules in the well plate and replace it with the complete culture medium without animal serum used for conventional culture. Place the cells in a hypoxic incubator at 37°C containing 2% O2 and 5% CO2 for 24 hours.

[0094] 3. Cell viability detection: In the third step, after 24 hours of treatment, all culture supernatant was collected, washed with physiological saline, and then the cells were digested with trypsin. P4 generation cells were collected by centrifugation, resuspended, and 10 μL of the cell suspension was mixed thoroughly with acridine orange / propidium iodide (AO / PI). Cell viability was detected using an automated fluorescence cell counter. There was no significant difference between the example and the comparative example. Figure 2 ).

[0095] 4. Cryopreservation of MSCs: Add MSCs to a cryopreservation solution containing DMSO and a serum substitute. The cryopreservation solution mainly contains three components: DMSO with a final concentration ranging from 2.5% to 15%, preferably 7.5% to 10%, with a preferred value of 7.5%; serum substitute with a final concentration ranging from 2.5% to 20%, preferably 5% to 10%, with a preferred value of 5%; and the remaining volume consisting of one of the following three components: PBS, 0.9% sodium chloride injection, or compound electrolyte injection. The preferred cell cryopreservation density is 5×10^6 / mL to 5×10^7 / mL, with a preferred value of 2×10^7 / mL. Cool the MSCs to -80°C using a programmed temperature control device, and then transfer them to a gas-phase liquid nitrogen tank for storage.

[0096] 5. Cytokine level detection: The levels of cytokines in the culture supernatant were detected using ELISA (R&D, DaYou, ELISA). Compared with the untreated MSCs control group, the levels of IDO, PGE2, VEGF, BDNF, GDNF, HGF, FGF-2, and ANG-1 secreted by MSCs pretreated with a three-step induction method were significantly increased. Figures 3-5 ).

[0097] Example 2: Immunocyte proliferation inhibition experiment 1. Cultivation of PBMCs: Frozen human peripheral blood mononuclear cells (PBMCs) were revived, washed and resuspended, and cultured in T175 culture flasks with 20 mL of RPMI 1640 complete medium (serum-free RPMI 1640 basal medium + 10% platelet lysate) in an incubator at 37°C and 5% CO2 for 24 h.

[0098] 2. Wrap: Six-well plates were coated with PBS buffer containing 5 μg / mL anti-human CD3 monoclonal antibody (Tonglihaiyuan GMP-TL101) and 5 μg / mL anti-human CD28 monoclonal antibody (Tonglihaiyuan GMP-TL102) to activate PBMCs. The plates were incubated at 4°C for 12 hours and then washed with PBS. (The concentration of the two antibodies can be selected from 1 to 10 μg / mL.)

[0099] 3. Co-culture of MSCs: Resuscitated frozen MSCs were seeded into 6-well plates at a 1:5 ratio with PBMCs, containing 4 × 10^5 MSCs and 2 × 10^6 PBMCs per well. The plates were cultured in RPMI 1640 complete medium with 100 IU / mL IL-2 added to promote PBMC activation and proliferation (range: 10 IU / mL-1000 IU / mL). A negative control consisted of PBMCs cultured alone without MSCs or IL-2, while a positive control consisted of PBMCs cultured alone with IL-2 added but without MSCs.

[0100] 4. Chemokine concentration detection: After 5 days of culture, all culture supernatant was collected, cells were removed by centrifugation, and the chemokine content was detected using ELISA (R&D, DaYou, ELISA). Compared with PBMCs cultured alone, when pretreated MSCs were co-cultured with PBMCs, the number of PBMCs decreased significantly, while the concentrations of IL-17, TNF-α, and IFN-γ decreased, and the concentration of IL-10 increased. Figures 6-7 ).

[0101] 5. Detection of immune cell count: All adherent cells were digested and mixed with suspended cells in the supernatant. The mixture was co-stained with 1 μL each of CD90 and CD45 antibodies, and the number of CD45+CD90- PBMCs was detected by flow cytometry. The number of PBMCs co-cultured with pretreated MSCs was significantly reduced compared to PBMCs co-cultured with untreated MSCs. Inhibition rate (%) = [1 - (number of PBMCs in experimental group / number of PBMCs in positive control group)] × 100%. Pretreated MSCs had a negative immunomodulatory effect on human PBMCs in vitro. Figure 8 ).

[0102] 6. Detection of the ratio of Th1 cells to Treg cells: After incubation with CD4 and IFN-γ, CD4 and CD25 antibodies (Biolegend), the cells were detected by flow cytometry.

[0103] Experimental methods: 1. Th1 cell surface staining: Prepare 1×10^6 PBMCs, wash with PBS, and add CD4-FITC and CD25-PE antibodies (Treg tubes) or CD4-FITC antibody only (Th1 tubes). Vortex to mix and incubate at 4°C in the dark for 30 minutes. Wash: Add 2 mL of flow cytometry staining buffer, centrifuge at 300-400g for 5 minutes, and discard the supernatant.

[0104] 2. Th1 cell fixation, membrane rupture, and intracellular staining 1) Fixation and permeabilization: Add 1 mL of freshly prepared fixation / permeabilization working solution to the Th1 detection tube and gently vortex to resuspend the cells. Incubate at room temperature in the dark for 30-60 minutes.

[0105] 2) Permeabilization washing: Add 2 mL of 1× membrane rupture washing buffer, centrifuge at 300-400g for 5 minutes at room temperature, and discard the supernatant. Repeat this washing once.

[0106] 3) Intracellular factor staining: Resuspend the cells in approximately 100 μL of residual liquid. Add IFN-γ-APC antibody and incubate at room temperature in the dark for 30 minutes.

[0107] 4) Final wash: Add 2 mL of 1× membrane permeabilization wash buffer and centrifuge once. Finally, resuspend the cells with an appropriate amount of flow cytometry staining buffer, ready for flow cytometry analysis. Treg detection tubes are resuspended directly after surface staining.

[0108] 3. Data Acquisition and Analysis 1) Use flow cytometry for detection. First, use FSC / SSC to gate and exclude cell debris, then circle the lymphocyte population.

[0109] 2) In the lymphocyte phylum, circle the CD4+ T cell population. Th1 cell detection: In the CD4+ cell phylum, analyze the expression of IFN-γ; IFN-γ+ cells are Th1 cells. Treg cell detection: In the CD4+ cell phylum, analyze the expression of CD25; CD25+ cells are Treg cells.

[0110] 3) Record the percentage of Th1 cells and Treg cells among CD4+ T cells.

[0111] The proportion of CD4+IFN-γ+ Th1 cells decreased, while the proportion of CD4+CD25+ Treg cells increased. Figure 8 This indicates that pro-inflammatory subsets in PBMCs were significantly suppressed, while regulatory subsets were induced to expand, demonstrating that pretreated MSCs have significant immunomodulatory effects.

[0112] Example 3: Drug Efficacy Experiment in an Animal Stroke Model 1. Preparation of MSCs formulations: P4 generation MSCs (or P3-P10 generation MSCs, or directly revived cryopreserved cells) that have undergone a three-step induction pretreatment are revived. Compound electrolyte injection solution (which can be replaced with 9% sodium chloride injection) is added to adjust the MSC density to 1×10^7 / mL (the density range can be 2×10^6 / mL-2×10^7 / mL). The mixture is thoroughly mixed to prepare a mesenchymal stem cell injection solution, and then examined for appearance, sterility, mycoplasma, and endotoxins. The finished formulation is stored and transported at 2-8℃ for use in animal experiments involving cell transplantation.

[0113] 2. Modeling of acute ischemic stroke due to ischemia-reperfusion: 2.1 Selection of Experimental Animals: The middle cerebral artery (MCA) is a common site of ischemic brain injury in clinical practice. Therefore, the middle cerebral artery occlusion (MCAO) model is widely used in the study of ischemic stroke. The suture occlusion method for constructing the MCAO model involves inserting a specially made suture through one side of the internal carotid artery into the bifurcation of the middle cerebral artery and the anterior cerebral artery to block blood flow and cause cerebral infarction. It has advantages such as no craniotomy and uniform ischemic localization, and is currently the most widely used method for constructing cerebral ischemia models both domestically and internationally. The vascular structure of SD rats is similar to that of humans, and experimental conditions are easy to standardize. Therefore, the suture occlusion method MCAO model of SD rats is an ideal model for studying cerebral ischemia. In addition, according to reports by JW Simpkins and RL Roof et al., estrogen and progesterone have neuroprotective effects on ischemic cerebral infarction in adult rats. To eliminate the influence of estrogen and progesterone on the experimental results, male SD rats were selected as experimental animals in this experiment.

[0114] 2.2 Grouping: The animals were divided into 3 groups: sham surgery group (negative control group), model control group (positive control group), and cell therapy group, with 10 animals in each group.

[0115] 2.3 Modeling process: a) Anesthesia induction: Rats were placed in an anesthesia induction box filled with 3.0% isoflurane for anesthesia induction.

[0116] b) Fixation: After anesthesia induction, the animals are transferred to the operating table and anesthesia is maintained using a small animal gas anesthesia machine with 2.0%-2.5% isoflurane at 200 mL / min. The eyelid reflex and pain response of the rats are observed. Surgery can only begin after the eyelid reflex and pain response in the limbs and tail have disappeared.

[0117] c) Ischemia-reperfusion surgery: A. Dissection and Exposure of Blood Vessels: After preparing the surgical area, the rat is placed under a surgical microscope. The rat's skin is incised along the midline with ophthalmic scissors, approximately 2 cm in length. Using a right-sided paracervical approach, the muscles of the right neck are bluntly dissected and retracted with microforceps to expose the right common carotid artery (CCA). Then, the external carotid artery (ECA) and internal carotid artery (ICA) are further dissected upwards along the common carotid artery. B. Ligate the ECA and temporarily clamp the ICA; thread a suture through the proximal and distal ends of the CCA, tie the distal end tightly, and tie a loose knot at the proximal end for later use; cut a small opening between the two sutures. C. Insertion of the suture plug: Insert the 4-0 suture plug through the incision in the CCA, and then slowly and gently push it into the internal carotid artery. Pause at the arterial clip in the ICA and further tighten the pre-ligated suture (to avoid excessive bleeding when pushing the suture plug). Then remove the arterial clip that is blocking blood flow to the ICA, and immediately push the suture plug into the ICA until it enters the intracranial cavity. At this time, be careful not to insert the suture into the pterygopalatine artery, another branch of the internal carotid artery (the pterygopalatine artery is an extracranial branch of the ICA; when the suture plug enters to a depth of about 10mm in this artery, it should not be inserted further. At this point, slightly withdraw the suture plug, adjust the direction, and repeat the operation). D. Fixing the suture plug and suturing the incision: When the suture plug is inserted to a depth of approximately 18 mm from the bifurcation of the common carotid artery, if a slight resistance is felt, it indicates that the tip of the suture plug has entered the anterior cerebral artery (CAA), and the lateral wall of the suture plug has blocked the opening of the middle cerebral artery. At this point, stop insertion and record the time. Remove the arterial clip on the CCA, and close the incision after observing no active bleeding. E. Cerebral reperfusion: Ischemic rats were placed at room temperature and induced anesthesia after 90 minutes. While maintaining anesthesia, the suture thrombus was slowly and gently pulled to return the head end to the external carotid artery, thus achieving reperfusion of the middle cerebral artery. F. Disinfect the incision with iodine solution.

[0118] 2.4 Drug administration: 24 hours after ischemia-reperfusion, pretreated MSCs were administered via tail vein injection at a dose of 1×10^7 cells / cell. The corresponding model control group was given an equal volume of cell solvent.

[0119] 2.5 Observation and Sampling: Observe behavior and various symptoms daily, and measure weight; 2.6 Rat neurological function scoring: Before modeling (D-1) and 4 days (D5) and 6 days (D7) after modeling, the rat neurological function scoring scale (mNSS) was used to score the animals' behavior, including motor function test, sensory test, balance test and reflex and abnormal movement test; Table 1. Rats' Neurological Function Scoring Scale (mNSS)

[0120] 2.7 Eight days after modeling, the animals in each group were dissected and brain tissue samples were collected. 2.8 Pathological Evaluation (TTC Staining) Method: The brain of euthanized animals was rapidly removed and frozen at -20°C until hardened. The rat brain was then cut into 2mm thick sections, starting from the olfactory bulb and moving posteriorly, totaling 6-8 sections. Each section was placed in a six-well plate containing 0.5% TTC solution (Sigma-Aldrich, BCCC3620, prepared in PBS) and incubated at 37°C in the dark for 20 minutes. After incubation, the sections were stored in 10% formaldehyde solution in the dark. Normal tissue stained rose-red, while infarcted tissue appeared white. Each brain plane was placed sequentially on filter paper, photographed with a digital camera, and the white tissue was carefully removed and weighed. The percentage of infarcted tissue weight relative to the total brain weight was used as the infarct extent (%), or the infarct extent was calculated using image processing. The inhibition rate (%) of each drug treatment group was calculated based on the infarction range. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (Infarction range of model group - Infarction range of treatment group) / Infarction range of model group × 100%.

[0121] 3. Treatment results: Compared to the model control group, the MSCs treatment group showed a significant reduction in cerebral infarction area, an infarction inhibition rate exceeding 30%, and a significant decrease in the mNSS score. Figure 9 The results showed that intervention with pretreated MSCs significantly inhibited cerebral infarction in AIS rats and significantly improved neurological function.

[0122] 4. Mechanism of action study: Fourteen days after modeling, three animals were taken from each of the sham surgery group, model control group, and cell therapy group. The injured and uninjured sides of the brain were fixed in 10% neutral buffered formalin and embedded in paraffin. Pathological sections of the infarcted area tissue were stained with TUNEL and immunofluorescence stained with Iba1 (Abcam), CD68, IGF-1, and CD31 antibodies (all antibodies from Abcam).

[0123] Immunofluorescence staining method: 1) Antigen retrieval: Heat in citrate buffer for 30 minutes to remove cross-links. 2) Blocking: Incubate the sample with blocking buffer to prevent nonspecific binding. 3) Primary antibody incubation: Incubate the sample with a specific primary antibody to ensure that the antibody binds to the target antigen. 4) Secondary antibody incubation: Incubate the sample with fluorescently labeled secondary antibody to ensure that the secondary antibody can bind to the primary antibody. 5) Cleaning and sealing: Clean to remove excess antibodies, and seal the sample on a glass slide with a sealing agent. 6) Observation: Use a fluorescence microscope to observe the antigen-antibody complex.

[0124] The results show that ( Figures 10-12 Compared with the model-only group, the number of apoptotic cells in the brain tissue of the MSCs treatment group was significantly reduced, the number of Iba1+CD68+ microglia was reduced, IGF-1 expression was upregulated, and the density of CD31-positive blood vessels was increased. This demonstrates that pretreated MSCs treatment can inhibit brain tissue cell apoptosis caused by AIS, inhibit microglia activation and excessive astrocyte proliferation, and promote angiogenesis.

[0125] 5. Conclusion: In a rat model of ischemia-reperfusion injury (AIS), compared with the control group, the MSCs treatment group (using MSCs pretreated with a three-step induction process) showed a significant reduction in cerebral infarction area and a significant decrease in the mNSS score, with good safety. In a permanent rat model of AIS, MSCs treatment improved prognosis by reducing cerebral infarction area, increasing infarction inhibition rate, reducing neurological deficits, and decreasing apoptotic cells. The mechanism of action includes inhibiting microglia activation and astrocyte overgrowth, upregulating IGF-1 expression, promoting neural repair, and promoting angiogenesis by increasing CD31-positive vessel density, ultimately achieving a synergistic effect of inflammation regulation, apoptosis inhibition, and neurovascular regeneration.

[0126] Examples 4-5: Clinical Trials 1. Preparation of MSCs formulations: Resuscitate P4 generation MSCs (or P1-P10 generation MSCs) that have undergone a three-step induction pretreatment, add compound electrolyte injection (which can be replaced with 0.9% sodium chloride injection) and a final concentration of 5% human serum albumin (HSA, selectable concentration range 2%-10%), adjust the MSC cell density to 2×10^6 / mL (MSC density selectable range 5×10^5 / mL-5×10^6 / mL), mix thoroughly, and prepare a mesenchymal stem cell injection solution. Perform tests on biological characteristics, biological safety, microbiological safety, and exogenous and endogenous viral factors. The finished formulation is stored and transported at 2-8℃ for cell transplantation therapy.

[0127] 2. Patient treatment: This double-blind, randomized, controlled clinical trial was conducted at Beijing Tiantan Hospital, Yan'an University Xianyang Hospital, and Linyi People's Hospital, and passed ethical and academic reviews. Patients were diagnosed, met the inclusion criteria, and did not meet the exclusion criteria. They were fully informed and signed informed consent forms.

[0128] Within 72 hours of symptom onset, patients received a single intravenous infusion of mesenchymal stem cell injection (selectable dose range: 5.0 × 10^7 / infusion to 2.0 × 10^8 / infusion, 1-3 infusions, at intervals of 7 ± 2 days; treatment time window: acute phase 0-7 days after onset, early subacute phase 7 days-3 months, late subacute phase 3-6 months, chronic phase more than 6 months; Example 4: single intravenous infusion, dose of 5.0 × 10^7; Example 5: single intravenous infusion, dose of 2.0 × 10^8 MSCs) or placebo (cell-free, solvent-only, for comparative purposes). Adverse reactions were closely monitored after treatment, with follow-up for 90-360 days.

[0129] 3. Rating: Before and after treatment, NIHSS scores (Tables 2-3), mRS scores, and / or BI scores were assessed to evaluate the improvement in patients' neurological and motor functions.

[0130] The National Institutes of Health Stroke Scale (NIHSS) in the United States: The NIHSS score covers important neurological function assessments and can systematically quantify the assessment results. The score can assess the degree of neurological deficits in stroke patients; baseline assessment can assess the severity of stroke, and treatment effectiveness can be regularly evaluated after treatment.

[0131] Table 2 NIHSS Scoring Table

[0132] Table 3 NIHSS Scoring Table

[0133] Modified Rankin Scale (mRS): The mRS is used to measure the recovery of neurological function in patients after stroke. It mainly assesses the patient's ability to live independently and walk, and is divided into 7 levels.

[0134] Table 4 Modified Rankin Scale (mRS)

[0135] The Barthel Index (BI) is a clinical tool used to assess an individual's basic activities of daily living, reflecting the patient's level of functional independence in life.

[0136] Table 5. Barthel Index Rating Scale (BI)

[0137] 4. Results: The results are as follows Figures 13-15 As shown.

[0138] Clinical Case 1 (Example 4 Experimental Group): The subject was diagnosed with AIS (first stroke) upon admission, with an NIHSS score of 8 and an mRS score of 4; CT results showed: multiple infarcts in the left centrum semiovale, falx cerebri calcification (CS), new cerebral infarction, multiple stenosis of varying degrees in the intracranial segment of the left internal carotid artery, including severe stenosis in the communicating segment; occlusion of the left middle cerebral artery with surrounding collateral formation; and thin V4 segment of the left vertebral artery, which did not merge into the basilar artery at its terminal point.

[0139] Concomitant medications: Aspirin enteric-coated tablets (100mg, qd), metformin hydrochloride tablets (100mg, tid), dapagliflozin tablets (10mg, qd), and atorvastatin calcium capsules (20mg, qd) are all taken continuously.

[0140] The subjects received a single intravenous infusion of 5.0 × 10^7 cells. Their vital signs remained stable before and after the infusion. The 360-day visit was completed on November 18, 2024. The mRS score was 1 from day 90 to day 360, and the BI score was 100 on day 360.

[0141] No adverse events (AEs) related to the investigational drug occurred during the entire trial period, and no drug-induced leukemia (DLT) events occurred.

[0142] Clinical Case 2 (Example 5 Experimental Group): The subject was diagnosed with AIS (first stroke, cardiogenic) upon admission. The mRS score before the onset was 0, the NIHSS score for this stroke was 18, the mRS score for this stroke was 4, and the BI score was 20. CT results showed: 1. Cerebral infarction in the left frontoparietal temporal lobe, left basal ganglia, and left lateral ventricle; 2. Stenosis of the M1 segment of the left middle cerebral artery (CS left middle cerebral artery stenosis).

[0143] Combined medications: folic acid tablets (5mg, qd), mecobalamin tablets (0.5mg, tid), idebenone tablets (30mg, tid), warfarin tablets (1.875mg, qd), and atorvastatin calcium tablets (20mg, qd), all of which are taken continuously.

[0144] The experimental drug was administered once via intravenous infusion of 2.0 × 10^8 cells. The vital signs of the subjects remained stable before and after the infusion, and no adverse events occurred.

[0145] No adverse events (AEs) related to the investigational drug occurred during the entire trial period, and no drug-induced leukemia (DLT) events occurred.

[0146] Clinical Case 3 (Comparative Example 6, Placebo Group): The subject was diagnosed with AIS (first stroke) upon admission, with a pre-stroke mRS score of 0, an NIHSS score of 18, and an mRS score of 4 during this stroke.

[0147] A head CT scan revealed multiple diffusion-restricted lesions in the left temporoparietal insula and left basal ganglia. A CTA scan showed occlusion of the origin of the left internal carotid artery and the left middle cerebral artery.

[0148] Concomitant medications: Aspirin enteric-coated tablets (100mg, qn) and atorvastatin calcium tablets (20mg, qn) were both taken continuously. During hospitalization, the patient received 100mg of phosphatidylcholine phosphate for injection for 14 days and 0.3g of cypermethrin for injection for 7 days.

[0149] A single intravenous infusion of 30 mL of cell-free solvent resulted in stable vital signs in the subjects before and after the infusion, with no adverse events occurring.

[0150] Regarding efficacy, although the subject's NIHSS score improved within 14 days, the primary efficacy endpoint, mRS, remained unchanged from baseline to the end of the visit. The BI score on days 180 and 360 was 35, showing no change.

[0151] 4.1 Safety: No adverse events (AEs) related to the investigational drug occurred during the entire trial, and no drug-induced leukemia (DLT) events occurred. The safety and tolerability of the MSCs were good.

[0152] 4.2 Validity: In the placebo group, the NIHSS score decreased from 18 to 13 within 7 days, showing some improvement. However, the primary efficacy endpoint, mRS, remained unchanged at 4 from baseline until the end of the 360-day visit.

[0153] After treatment, the subjects in the experimental group showed an improvement of ≥6 points in their NIHSS scores (Example 4: baseline 8 points, improved to 2 points after 30 days; Example 5: baseline 18 points, improved to 5 points after 90 days). The improvement in motor coordination function was more significant and could be maintained for a long time. Functional independence rate (mRS) and activities of daily living (BI) also showed varying degrees of improvement (Example 4: mRS baseline 4 points, improved to 1 point after 90 days; Example 5: mRS baseline 4 points, improved to 1 point after 180 days; BI baseline 20 points, improved to 100 points after 180 days), indicating good recovery of neurological function.

[0154] Example 6 1. Umbilical cord MSCs culture is the same as in Example 1.

[0155] 2. Preprocessing Step 1: When the P4 generation cells are in the logarithmic growth phase, have good morphology, and have a confluence of about 70%-80% (about 24 hours after passage), add three pro-inflammatory factors to the culture medium: IFN-γ (final concentration 1 ng / mL), TNF-α (final concentration 1 ng / mL), and IL-1β (final concentration 1 ng / mL), and continue culturing for 6 hours.

[0156] Step 2: Discard all the culture medium containing pro-inflammatory factors in the well plate and replace it with a complete culture medium containing three epigenetic regulatory molecules: sodium valproate (final concentration 10 nM), 5-azacytidine (final concentration 0.5 μM), and all-trans retinoic acid (final concentration 1 μM), and continue culturing for 6 hours.

[0157] Step 3: Discard all the culture medium containing epigenetic regulatory molecules in the well plate and replace it with the complete culture medium without animal serum used for conventional culture. Place the cells in a hypoxic incubator at 37°C containing 1% O2 and 5% CO2 for 6 hours.

[0158] Example 7 1. Umbilical cord MSCs culture is the same as in Example 1.

[0159] 2. Preprocessing Step 1: When the P4 generation cells are in the logarithmic growth phase, have good morphology, and have a confluence of about 70%-80% (about 24 hours after passage), add three pro-inflammatory factors to the culture medium: IFN-γ (final concentration 100ng / mL), TNF-α (final concentration 100ng / mL), and IL-1β (final concentration 100ng / mL), and continue culturing for 72 hours.

[0160] Step 2: Discard all the culture medium containing pro-inflammatory factors in the well plate and replace it with a complete culture medium containing three epigenetic regulatory molecules: sodium valproate (final concentration 10mM), 5-azacytidine (final concentration 50μM), and all-trans retinoic acid (final concentration 100μM), and continue culturing for 72 hours.

[0161] Step 3: Discard all the culture medium containing epigenetic regulatory molecules in the well plate and replace it with the complete culture medium without animal serum used for conventional culture. Place the cells in a hypoxic incubator at 37°C containing 10% O2 and 5% CO2 for 72 hours.

[0162] Example 8 The only difference from the pretreatment in Example 1 is the order. The culture order in Example 8 is as follows: after activating the culture medium under normal oxygen conditions of 21% O2 and 5% CO2 for 48 hours, it is switched to directional culture medium and cultured under low oxygen conditions of 2% O2 and 5% CO2 for 24 hours, for a total of 72 hours.

[0163] Example 9 The only difference from the pretreatment in Example 1 is the order. The culture order in Example 9 is as follows: cultured under low oxygen conditions of 2% O2 and 5% CO2 throughout the process, first treated with activation medium for 24 hours, then treated with directional medium for 48 hours, for a total of 72 hours.

[0164] Example 10 The only difference from the pretreatment in Example 10 is the order. The culture order in Example 10 is as follows: first, culture in an adaptation medium under hypoxic conditions of 2% O2 and 5% CO2 for 24 hours; then, culture in an activation medium under normoxic conditions of 21% O2 and 5% CO2 for 24 hours; and finally, culture in a directional medium under normoxic conditions for 24 hours, for a total of 72 hours.

[0165] Comparative Example 1 The only difference between the pretreatment in Example 1 and that in Comparative Example 1 is the composition of the activation medium in the first step. The activation medium in Comparative Example 1 is a complete medium that does not contain pro-inflammatory factors but contains basic fibroblast growth factor (bFGF, final concentration 5 ng / mL). All other pretreatment steps are the same.

[0166] Comparative Example 2 The only difference between the pretreatment in Example 1 and the pretreatment in Example 1 is that the concentration of the components in the activation medium in the first step is different. The activation medium contains IFN-γ (final concentration 1 μg / mL), TNF-α (final concentration 1 μg / mL), and IL-1β (final concentration 1 μg / mL). All other pretreatment steps are the same.

[0167] Comparative Example 3 The only difference from the pretreatment in Example 1 is that the directional culture medium in the second step does not contain epigenetic regulatory molecules and only uses a complete culture medium without animal serum; the other pretreatment steps are the same.

[0168] Comparative Example 4 The only difference between the pretreatment in Example 1 and that in the second step is the concentration of the components in the directional culture medium. The directional culture medium is a complete culture medium containing sodium valproate (final concentration 1 nM), 5-azacytidine (final concentration 10 nM), and all-trans retinoic acid (final concentration 10 nM). All other pretreatment steps are the same.

[0169] Comparative Example 5 The only difference from the pretreatment in Example 1 is that the culture conditions in the third step are different. In Comparative Example 5, the culture conditions are normal oxygen content, not hypoxia. The cells are placed in an incubator at 37°C containing 21% O2 and 5% CO2 for 24 hours.

[0170] Comparative Example 6 The difference from the pretreatment in Example 1 is that the culture medium components and oxygen content conditions are different. In Comparative Example 6, a complete culture medium without animal serum was used throughout the process, and the culture conditions were normal oxygen content, not hypoxic conditions. The cells were placed in an incubator at 37°C containing 21% O2 and 5% CO2 for 72 hours.

[0171] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for pretreating mesenchymal stem cells, characterized in that, include: Cells were cultured using activating medium, directional medium, and adaptation medium under hypoxic conditions. The activation medium contains complete culture medium, IFN-γ, TNF-α, and IL-1β; The directional culture medium contains complete culture medium, sodium valproate, 5-azacytidine, and all-trans retinoic acid; The adaptation culture medium is a complete culture medium containing basal medium and animal serum-free medium.

2. The method according to claim 1, characterized in that, The incubation time using activating medium, directional medium, and adaptation medium is 6 to 72 hours.

3. The method according to claim 1, characterized in that, The activation medium contains complete medium, 1-100 ng / mL IFN-γ, 1-100 ng / mL TNF-α and 1-100 ng / mL IL-1β.

4. The method according to claim 1, characterized in that, The directional culture medium contains complete culture medium, 10 nM-10 mM sodium valproate, 0.5-50 μM 5-azacytidine and 1-100 μM all-trans retinoic acid.

5. The method according to claim 4, characterized in that, The directional culture medium contains complete culture medium, 1 mM sodium valproate, 2 μM 5-azacytidine and 10 μM all-trans retinoic acid.

6. The method according to claim 1, characterized in that, The mesenchymal stem cells mentioned above are selected from any one or more of the following tissue sources: umbilical cord, placenta, fat, bone marrow, dental pulp, umbilical cord blood, peripheral blood, uterine blood, hair follicles, and urine.

7. The method according to any one of claims 1-6, characterized in that, The pretreatment time using three different culture media totaled 72 hours, including at least four different treatment methods: (1) First, use the activation medium to culture the cells under normal oxygen conditions for 24 hours, then use the orientation medium to culture the cells under normal oxygen conditions for 24 hours, and finally use the adaptation medium to culture the cells under low oxygen conditions for 24 hours. (2) First, use the activating medium to culture the cells under normal oxygen conditions for 48 hours, and then use the directional medium to culture the cells under low oxygen conditions for 24 hours; (3) First, culture the cells in activating medium under low oxygen conditions for 24 hours, and then culture the cells in directional medium under low oxygen conditions for 48 hours. (4) First, use the adaptation medium to culture the cells under low oxygen conditions for 24 hours, then use the activation medium to culture the cells under normal oxygen conditions for 24 hours, and finally use the orientation medium to culture the cells under normal oxygen conditions for 24 hours.

8. Mesenchymal stem cells pretreated by the method according to any one of claims 1-7.

9. The use of the mesenchymal stem cells according to claim 8 in the preparation of medicaments for treating nervous system diseases, inflammatory diseases, autoimmune diseases, cardiovascular diseases and metabolic diseases.

10. A medicament for treating nervous system diseases, inflammatory diseases, autoimmune system diseases, cardiovascular system diseases, and metabolic diseases, characterized in that, The drug comprises the mesenchymal stem cells as described in claim 8.

Images