Apelin expression enhancer, or a preventive and / or therapeutic agent for neurodegenerative diseases or cerebrovascular diseases.

Abstract

The present invention aims to provide a novel component involved in angiogenesis, and a preventive and / or therapeutic agent for neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases using the same. [Solution] The apelin expression promoter of the present invention is an apelin expression promoter in vascular endothelial cells comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. The preventive and / or therapeutic agent of the present invention is a preventive and / or therapeutic agent for neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells.

Description

[Technical Field]

[0001] The present invention relates to an apelin expression promoter, or an agent for the prevention and / or treatment of neurodegenerative diseases or cerebrovascular diseases. [Background technology]

[0002] Angiogenesis is the phenomenon in which new blood vessels branch and extend from existing blood vessels. Angiogenesis is deeply involved in various physiological functions and pathological conditions because it forms a new vascular network within tissues, enhancing the supply of oxygen and nutrients. Angiogenesis occurs when vascular endothelial cells proliferate and migrate, forming tubular structures. It is believed that promoting angiogenesis can lead to the treatment of ischemic diseases, accelerated wound healing and tissue regeneration, and the treatment of neurodegenerative diseases.

[0003] Among these, attention is being drawn to treatment approaches for Alzheimer's disease that involve promoting angiogenesis. For example, age-related changes in blood vessels occur at or before the onset of Alzheimer's disease, suggesting that vascular pathology may be a cause of age-related dementia, including Alzheimer's disease (Non-Patent Literature 1). In addition, other literature suggests that cerebrovascular damage may be involved in the onset and progression of Alzheimer's disease (Non-Patent Literature 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Marta Cortes Canteli et al., J Am Coll Cardiol, 2020 Mar 3;75(8):942-951 [Non-Patent Document 2] Philip B Gorelick et al., American Heart Association journal Stroke, 2011 Sep;42(9):2672-2713 [Overview of the project] [Problems that the invention aims to solve]

[0005] In light of the circumstances described above, the present invention aims to provide a novel component involved in angiogenesis, and an agent for the prevention and / or treatment of neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases using the same. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the inventors have for the first time discovered that mesenchymal stem cells, their culture supernatant, or cell secretions have an effect of promoting apelin expression in vascular endothelial cells. Here, apelin is a peptide released from vascular endothelial cells that is thought to be involved in angiogenesis and blood pressure reduction, and in recent years it has also been reported to be involved in suppressing brain aging. Furthermore, the inventors have revealed that mesenchymal stem cells, their culture supernatant, or cell secretions have excellent effects on promoting elongation, reticular structure formation, branching chain formation, proliferation, and migration in vascular endothelial cells. Based on these findings, the inventors conceived the idea that mesenchymal stem cells, their culture supernatant, or cell secretions can be used as an apelin expression promoter for vascular endothelial cells, thereby providing the above-mentioned various promoting effects to vascular endothelial cells, and that these promote angiogenesis and are effective in preventing and / or treating neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases, and have completed the present invention. In other words, the gist of the present invention is as follows.

[0007] [1] An apelin expression promoter in vascular endothelial cells, comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. [2] The apelin expression promoter of [1], wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or dental pulp-derived mesenchymal stem cells. [3] The vascular endothelial cells are brain vascular endothelial cells, and the apelin expression promoter of [1] or [2]. [4] The vascular endothelial cells are human umbilical vein endothelial cells (HUVECs) or human brain capillary endothelial cells (HBMECs), and are any of the apelin expression promoters from [1] to [3]. [5] The culture supernatant is substantially serum-free and is an apelin expression promoter of any of [1] to [4]. [6] The mesenchymal stem cells are mesenchymal stem cells cultured in serum-free medium, and any of the apelin expression promoters from [1] to [5]. [7] A vascular endothelial cell elongation promoter comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. [8] A vascular endothelial cell reticular formation promoter comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. [9] A vascular endothelial cell branching agent comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells.

[10] A vascular endothelial cell proliferation promoter comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells.

[11] A vascular endothelial cell migration promoter comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells.

[12] A vascular endothelial cell sugar coating promoter comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells.

[13] The vascular endothelial cells are brain vascular endothelial cells, or any of the agents from [7] to

[12] .

[14] The vascular endothelial cells are human umbilical vein endothelial cells (HUVECs) or human brain capillary endothelial cells (HBMECs), one of the agents from [7] to

[13] .

[15] A prophylactic and / or therapeutic agent for neurodegenerative diseases, demyelinating diseases or cerebrovascular diseases, comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatants of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells.

[16] The neurodegenerative disease is Alzheimer's disease,

[15] and / or a preventive and / or therapeutic agent for the neurodegenerative disease. [Effect of the Invention]

[0008] The apelin expression promoter of the present invention can efficiently promote the expression of apelin in vascular endothelial cells. Therefore, the present invention is expected to be effective in the prevention and / or treatment of neurodegenerative diseases, demyelinating diseases or cerebrovascular diseases. [Brief Description of the Drawings]

[0009] [Figure 1] Figure 1 is a microscopic photograph showing the effect of the culture supernatant of various mesenchymal stem cells on HUVEC performed in Example 1. [Figure 2] Figure 2 is a graph showing the effects (elongation, network structure formation, and branched chain formation) of the culture supernatant of various mesenchymal stem cells on HUVEC performed in Example 1. [Figure 3] Figure 3 is a microscopic photograph showing the effect of the culture supernatant of adipose-derived mesenchymal stem cells on HBMEC performed in Example 2. [Figure 4] Figure 4 is a graph showing the effects (elongation, network structure formation, and branched chain formation) of the culture supernatant of adipose-derived mesenchymal stem cells on HBMEC performed in Example 2. [Figure 5] Figure 5 is a graph showing the effect of the culture supernatant of adipose-derived mesenchymal stem cells on the cell proliferation (number of live cells) of HUVEC performed in Example 3. [Figure 6] Figure 6 is a graph showing the effect of the culture supernatant of adipose-derived mesenchymal stem cells on the cell proliferation (number of BrdU-positive cells) of HUVEC performed in Example 3. [Figure 7] Figure 7 is a microscopic photograph showing the effect of the culture supernatant of adipose-derived mesenchymal stem cells on the proliferation and migration of HUVEC performed in Example 4. [Figure 8] Figure 8 is a graph showing the effect of the culture supernatant of adipose-derived mesenchymal stem cells on the proliferation and migration of HUVEC performed in Example 4. [Figure 9] Figure 9 is the RNA-seq analysis result (heat map) performed in Example 5. [Figure 10]Figure 10 shows the results of the RNA-seq analysis (Volcano Plot) performed in Example 5. [Figure 11] Figure 11 is a graph showing the results of the RNA-seq analysis (GO term analysis) performed in Example 5. [Figure 12] Figure 12 is a photograph showing the results of the immunohistochemical staining performed in Example 6. [Figure 13] Figure 13 is a photograph showing the results of the immunohistochemical staining performed in Example 6. [Figure 14] Figure 14 is a photograph showing the results of the immunohistochemical staining performed in Example 6. [Figure 15] Figure 15 is a photograph showing the results of the fluorescence imaging performed in Example 6. [Figure 16] Figure 16 is a photograph showing the results of the fluorescence imaging performed in Example 6. [Figure 17] Figure 17 is a graph showing the results of the fluorescence imaging performed in Example 6 plotted as a graph. [Figure 18] Figure 18 is a photograph showing the results of the immunohistochemical staining performed in Example 7. [Figure 19] Figure 19 is a graph showing the results of the immunohistochemical staining performed in Example 7 plotted as a graph. [Figure 20] Figure 20 is a graph showing the results of the immunohistochemical staining performed in Example 7 plotted as a graph. [Figure 21] Figure 21 is a photograph showing the results of the immunohistochemical staining performed in Example 7. [Figure 22] Figure 22 is a graph showing the results of the immunohistochemical staining performed in Example 7 plotted as a graph. [Figure 23] Figure 23 is a graph showing the results of the immunohistochemical staining performed in Example 7 plotted as a graph. [Figure 24] Figure 24 is a photograph showing the results of the immunohistochemical staining performed in Example 7.

Mode for Carrying Out the Invention

[0010] The following describes in detail the apelin expression promoter, vascular endothelial cell elongation promoter, vascular endothelial cell reticular structure formation promoter, vascular endothelial cell branching chain formation promoter, vascular endothelial cell proliferation promoter, vascular endothelial cell migration promoter, neurodegenerative disease or cerebrovascular disease preventive and / or therapeutic agent, or gene expression promoter according to the present invention.

[0011] <Aperin expression promoter> The apelin expression promoter of the present invention is an apelin expression promoter in vascular endothelial cells comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. Using the apelin expression promoter of the present invention can promote apelin expression in vascular endothelial cells. The apelin expression promoter of the present invention may also contain other components, provided that they do not impair the effects of the present invention. The apelin expression promoter of the present invention will be described in detail below.

[0012] The subjects treated with the apelin expression promoter of the present invention are not particularly limited, but mammals are preferred. Examples of mammals include humans, non-human primates (monkeys, gorillas, chimpanzees, etc.), carnivores (dogs, cats, etc.), rodents (mice, rats, guinea pigs, etc.), even-toed ungulates (pigs, cattle, sheep, llamas, alpacas, etc.), and odd-toed ungulates (horses, donkeys, etc.), with humans being preferred.

[0013] [Aperine] Apelin is an endogenous peptide ligand that exerts diverse physiological activities by binding to the APJ receptor (apelin receptor coupled to a G protein). Apelin is widely expressed in the body and has been elucidated to have many physiological functions, including vasodilation, enhancement of myocardial contractility, fluid regulation, metabolic regulation, cardiovascular development, skeletal muscle regeneration, and neuroprotection. Apelin is released mainly from vascular endothelial cells as a polypeptide consisting of 77 amino acids. The released apelin polypeptide is cleaved into peptides of, for example, 13, 17, or 36 amino acids, and is called apelin-13, apelin-17, and apelin-36, respectively. Of these, apelin-13, which has the highest activity, has a sequence that is conserved from mouse to human, and in blood vessels, it enlarges the lumen of capillaries and controls vascular permeability.

[0014] The APJ receptor has been shown to function as an endogenous antagonistic system that inhibits the blood pressure-raising effect of angiotensin II by forming a heterodimer with the angiotensin II receptor AT1. In addition, the apelin-APJ receptor system has been shown to exhibit a potent proliferative effect on vascular endothelial cells and to be involved in various physiological angiogenesis and angiogenesis in pathological conditions.

[0015] In this specification, apelin expression enhancement refers to the enhancement of the expression of at least one apelin polypeptide and its fragments (e.g., apelin-13, apelin-17, apelin-36, etc.), as well as the expression of the gene encoding apelin. These enhancements can be confirmed by methods such as qPCR, RNA sequencing, Northern blotting, Western blotting, ELISA, and immunostaining.

[0016] [Vascular endothelial cells] Blood vessels, excluding capillaries, have a three-layered structure: the intima, media, and adventitia. Endothelial cells, along with the basement membrane, constitute the intima and are located on the innermost layer of the blood vessel. Capillaries, on the other hand, are composed only of the basement membrane and endothelial cells. Endothelial cells not only serve as components of blood vessels, but also act as a site for the exchange of substances such as oxygen and nutrients between blood and tissues, and further produce various physiologically active substances to maintain the function of tissues and organs. In this specification, endothelial cells may sometimes be simply referred to as endothelial cells.

[0017] Endothelial cells are present in blood vessels throughout almost the entire body, but their function and properties differ depending on the tissue. Examples of endothelial cells include cerebral vascular endothelial cells, coronary artery endothelial cells, saphenous vein endothelial cells, pulmonary artery endothelial cells, aortic endothelial cells, cutaneous vascular endothelial cells, cutaneous microvascular endothelial cells, uterine microvascular endothelial cells, pulmonary microvascular endothelial cells, cardiac microvascular endothelial cells, umbilical vein endothelial cells, and umbilical artery endothelial cells.

[0018] In recent years, in developed countries, the number of patients with neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases has been increasing due to aging populations and changes in lifestyle. Neurodegenerative diseases, demyelinating diseases, and cerebrovascular diseases pose a significant risk to patients' life expectancy and quality of life, while effective treatment methods are limited. The apelin expression promoter of the present invention shows excellent effects on cerebral vascular endothelial cells, and is therefore thought to be effective in preventing and / or treating neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases by promoting cerebral angiogenesis and providing neuroprotective effects. Furthermore, apelin has also been reported to have a repair effect on the aged brain (Masumi Ito et al., Nature Aging, 2021, 1, 284-294). For these reasons, cerebral vascular endothelial cells are preferred as the target vascular endothelial cells for the apelin expression promoter of the present invention.

[0019] Specific neurodegenerative diseases include, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease, white matter lesions, amyotrophic lateral sclerosis, spinocerebellar degeneration, multiple system atrophy, frontotemporal dementia, progressive supranuclear palsy, and Creutzfeldt-Jakob disease. Demyelinating diseases include, for example, multiple sclerosis and neuromyelitis optica. Cerebrovascular diseases include, for example, ischemic cerebrovascular disease (cerebral infarction), cerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, epidural hemorrhage, cerebral aneurysm, arteriovenous malformation, cerebral small vessel disease, cavernous hemangioma, traumatic brain injury, moyamoya disease, hypertensive encephalopathy, cerebrovascular vasculitis, and vascular dementia.

[0020] The diseases to which the apelin expression promoter of the present invention can be applied include Alzheimer's disease, Parkinson's disease, ischemic cerebrovascular disease (cerebral infarction), cerebral hemorrhage (especially during the recovery phase), subarachnoid hemorrhage (especially during the recovery and regeneration phase), white matter lesions, Huntington's disease, multiple sclerosis, cerebral aneurysm (especially during the recovery phase), cerebral small vessel disease, cerebral cavernous hemangioma, traumatic brain injury, or Moyamoya disease, with Alzheimer's disease being more preferred. As mentioned in Non-Patent Documents 1 and 2 above, Alzheimer's disease is closely related to abnormalities in cerebral blood vessels, and the promotion of angiogenesis through apelin expression enhancement of the present invention may be an effective treatment method for Alzheimer's disease.

[0021] Generally, mesenchymal stem cells are thought to be unable to cross the blood-brain barrier in healthy organisms. On the other hand, as shown in the examples, when cerebral blood vessels are damaged due to diseases such as neurodegenerative diseases (especially Alzheimer's disease), it is thought that the blood-brain barrier loosens, allowing mesenchymal stem cells to pass through. Therefore, the apelin expression promoter of the present invention can exert an apelin expression promoting effect targeting cerebral blood vessels, even when it contains mesenchymal stem cells.

[0022] Furthermore, because aperin possesses diverse physiological activities, the aperin expression promoter of the present invention is also effective for diseases in areas other than the brain. For example, the aperin expression promoter of the present invention is considered effective in preventing and / or treating various diseases in which improvement is expected through angiogenesis, such as ischemic heart disease, hypertension, metabolic disorders such as diabetes, and kidney disease, as well as in treating fibrosis (fibrous diseases) and wounds.

[0023] The apelin expression promoter of the present invention also has an effect of promoting apelin expression in cultured endothelial cells. By using the apelin expression promoter of the present invention on cultured endothelial cells, it becomes possible to analyze these cultured endothelial cells and perform various tests. Examples of cultured endothelial cells include human umbilical vein endothelial cells (HUVEC), human brain capillary endothelial cells (HBMEC), human umbilical artery endothelial cells (HUAEC), human coronary artery endothelial cells (HCAEC), human saphenous vein endothelial cells (HSaVEC), human pulmonary artery endothelial cells (HPAEC), human aortic endothelial cells (HAoEC), human cutaneous microvascular endothelial cells (HDMEC), human cutaneous vascular endothelial cells (HDBEC), human cutaneous lymphatic endothelial cells (HDLEC), human pulmonary microvascular endothelial cells (HPMEC), human cardiac microvascular endothelial cells (HCMEC), and human uterine microvascular endothelial cells (HUtMEC), among others (primary or subcultured cells). When using cultured endothelial cells as vascular endothelial cells, cultured human umbilical vein endothelial cells (HUVECs) or human brain capillary endothelial cells (HBMECs) are preferred as the vascular endothelial cells targeted by the apelin expression promoter of the present invention.

[0024] [Mesenchymal stem cells] Mesenchymal stem cells are pluripotent progenitor cells first isolated from bone marrow by Friedenstein in 1982. They have been shown to exist in various tissues, including bone marrow, umbilical cord, and adipose tissue, and are expected to be a new therapeutic method for various intractable diseases. Recently, it has been discovered that cells with equivalent function exist in stromal cells of adipose tissue, placenta, umbilical cord, and amniotic membranes. Therefore, mesenchymal stem cells are sometimes referred to as stromal cells.

[0025] Mesenchymal stem cells are thought to be therapeutically useful not only for the cells themselves, but also for their culture supernatant or cellular secretions. The culture supernatant or cellular secretions of mesenchymal stem cells are rich in various cytokines, growth factors, chemokines, and exosomes secreted by the stem cells, and numerous studies have shown that these physiologically active substances have effects such as tissue repair and anti-inflammatory properties. Mesenchymal stem cell culture supernatant is thought to be effective, for example, for osteoarthritis and rheumatoid arthritis.

[0026] In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more cells belonging to the mesenchyme (such as osteocytes, cardiomyocytes, chondrocytes, tendon cells, and adipocytes) and that can proliferate while maintaining that ability. The term "mesenchymal stem cells" as used in the present invention refers to the same cells as stromal cells, and there is no particular distinction between the two. They may also be simply referred to as mesenchymal cells. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ.

[0027] Examples of mesenchymal stem cells used in the present invention include those derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. Preferred mesenchymal stem cells in the present invention are adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or dental pulp-derived mesenchymal stem cells, with adipose-derived mesenchymal stem cells being more preferred.

[0028] In the present invention, adipose tissue refers to tissue containing adipocytes and stromal cells including microvascular cells, and is, for example, tissue obtained by surgically excising or aspirating subcutaneous fat of a mammal.

[0029] In this invention, the umbilical cord is a white, tubular tissue that connects the fetus and the placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's jelly), the umbilical cord matrix itself, etc., and contains a large amount of mesenchymal stem cells.

[0030] In this invention, bone marrow refers to the soft tissue that fills the cavity of bone and is a hematopoietic organ. Bone marrow fluid is present in the bone marrow, and the cells present in it are called bone marrow cells. Bone marrow cells include red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes, as well as mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells, etc. Bone marrow cells can be collected, for example, from human ilium, long bones, or other bones.

[0031] In the present invention, the species of mesenchymal stem cells is preferably a mammal similar to those exemplified above as the target species for the apelin expression promoter of the present invention, and more preferably a human.

[0032] Mesenchymal stem cells may be cells provided by companies such as PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, and Corning, or cells prepared by methods well known to those skilled in the art. Furthermore, mesenchymal stem cells may be primary cells isolated from donor tissue or established cell lines.

[0033] In the present invention, the mesenchymal stem cells are preferably mesenchymal stem cells cultured in serum-free medium. That is, the apelin expression promoter of the present invention preferably contains one or more selected from the group consisting of mesenchymal stem cells cultured in serum-free medium, the culture supernatant of mesenchymal stem cells cultured in serum-free medium, and cellular secretions of mesenchymal stem cells cultured in serum-free medium.

[0034] [Supernatant of mesenchymal stem cell culture] The culture supernatant of mesenchymal stem cells used in this invention is the supernatant of the culture medium recovered from the culture vessel of mesenchymal stem cells. The culture supernatant of mesenchymal stem cells is obtained by the metabolism of mesenchymal stem cells, which consumes some components such as nutrients from the original culture medium (hereinafter sometimes referred to as the raw material medium), and at the same time releases various components from the mesenchymal stem cells. The culture supernatant of mesenchymal stem cells contains various physiologically active substances such as various cytokines, growth factors, chemokines, and exosomes. From the viewpoint of infection risk, it is preferable that the culture supernatant used in this invention does not contain biological raw materials as much as possible. In particular, it is preferable that the culture supernatant used in this invention is substantially serum-free, and more preferably completely serum-free.

[0035] As the raw material culture medium for the culture supernatant, any medium known to those skilled in the art can be used, as long as the mesenchymal stem cells are viable. Specifically, examples include minimal essential mediums (MEM) such as Eagle medium, Dulbecco's modified Eagle medium (DMEM), minimal essential medium α (MEM-α), mesenchymal cell basal medium (MSCBM), Ham's F-12 and F-10 medium, DMEM / F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove modified Dulbecco's medium (IMDM), McCoy modified medium, etc., as well as mixed media of these. From the viewpoint of infection risk, it is preferable that the raw material culture medium does not contain biological raw materials as much as possible. In particular, it is preferable that the raw material culture medium be serum-free.

[0036] The raw material culture medium is preferably a medium suitable for culturing vascular endothelial cells. By using the culture supernatant derived from a vascular endothelial cell medium, the apelin expression promoter of the present invention can effectively act on the target vascular endothelial cells. Examples of such raw material culture media include "EGM medium (manufactured by Lonza Corporation)", "EGM-2 medium (manufactured by Lonza Corporation)", "MCDB 131 medium", "VascuLife® medium (manufactured by Lifeline Cell Technology)", and "Endothelial Cell Medium (manufactured by ScienceCell Research Laboratories)". However, the raw material culture medium may also be a medium for mesenchymal stem cells or a medium for other cells.

[0037] Furthermore, the culture medium may contain other components as needed, in addition to the culture medium exemplified above. Examples of other components include amino acids such as glutamine, sugars such as glucose, metal salts such as sodium chloride and magnesium sulfate, trace metals such as selenium, lipids (excluding lysophosphatidic acid and its salts), vitamins such as pantothenic acid, albumin, insulin, transferrin, insulin, growth factors (e.g., epidermal growth factor, basic fibroblast growth factor), growth factors, cytokines and other proteins, polysaccharides, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts.

[0038] The culture conditions for obtaining the culture supernatant of mesenchymal stem cells can be any known culture conditions. Specifically, the culture temperature is preferably 30°C to 40°C, more preferably 30°C to 37°C, and even more preferably 37°C. The CO2 concentration during culture is preferably 1% to 10%, more preferably 2% to 7%, and even more preferably 5%. The O2 concentration during culture is preferably 0% to 95%, more preferably 1% to 25%, even more preferably 5% to 22%, and particularly preferably 17% to 21%. Furthermore, during culture, subculturing of mesenchymal stem cells and change of culture medium may be performed as needed, and the timing and method are not particularly limited as long as they are appropriate for each type of mesenchymal stem cell, and can be carried out in the same manner as conventionally while observing the morphology of the mesenchymal stem cells.

[0039] The passage number of mesenchymal stem cells used is preferably 1 to 10 (P1 to P10), and more preferably 3 to 6 (P3 to P6). The culture period for obtaining the culture supernatant of mesenchymal stem cells (the period of culture in the raw material medium) is preferably 10 hours to 200 hours, more preferably 20 hours to 120 hours, and even more preferably 50 hours to 100 hours. During the culture of mesenchymal stem cells, the medium may be changed or passaged if necessary. In this case, the mixed supernatant of the medium collected at different times may be used as the culture supernatant for the apelin expression promoter of the present invention.

[0040] As for the culture method of mesenchymal stem cells, general planar adherent culture in which cells are attached to culture vessels such as flasks, dishes, and plates for cell culture may be used; suspension culture in which mesenchymal stem cells are attached to microbeads, microcarriers, etc., and the microbeads, microcarriers, etc. are suspended in the culture medium; culture may be performed by suspension and agitation; culture may be performed by attaching the cells to a fibrous scaffold such as a microfiber; or spheroid culture may be used.

[0041] The culture vessel is not particularly limited as long as it is capable of culturing mesenchymal stem cells, but examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, etc.

[0042] The culture medium recovered from the mesenchymal stem cell culture may be used as is, but it is preferable to remove cell debris by centrifugation and / or filter filtration. The culture supernatant may also be diluted with water, physiological saline, buffer, etc., concentrated by ultrafiltration, or treated by dialysis, as needed. Furthermore, optional components (for example, the other components mentioned above as examples of components added to the raw material culture medium) may be added to the culture supernatant as needed. In addition, any pharmaceutical components may be added to the culture supernatant.

[0043] The culture supernatant is preferably sterilized by aseptic treatment. The endotoxin content in the culture supernatant is preferably 2.5 EU / mL or less.

[0044] The mesenchymal stem cells used to obtain the culture supernatant can be any known cells. Furthermore, when obtaining the culture supernatant, it is preferable to pre-culture the mesenchymal stem cells in a mesenchymal stem cell medium for a certain period of time before culturing them in the raw material medium, and then culture the mesenchymal stem cells in the desired raw material medium. As mentioned above, a medium optimized for culturing cells other than mesenchymal stem cells (a medium not optimized for culturing mesenchymal stem cells) can be used as the raw material medium. Therefore, by ensuring good growth conditions of the mesenchymal stem cells through pre-culturing, it is thought that the survival, metabolism, and proliferation of the mesenchymal stem cells will be promoted when they are cultured in the raw material medium, and a high-quality culture supernatant can be obtained.

[0045] For pre-culture of mesenchymal stem cells, known media may be used, for example: Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use), PromoCell; Mesenchymal Stem Cell Growth Medium XF (Ready-to-use), PromoCell; MSCGM BulletKittm; MSCGMtm Mesenchymal Stem Cell Growth Medium BulletKittm (Lonza); Xeno-free medium for human mesenchymal stem cells (MSC NutriStem® XF, Biological Industries); MesenCult-ACF Plus (Veritas); StemXVivotm Serum-Free Human MSC Expansion Media (R&D Systems, Corning); Serum-free medium for adipose-derived stem cells (KBM ADSC-4, Kojin Bio); Serum-free medium for mesenchymal stem cells (R:STEM Medium for Examples include hMSC High Growth (manufactured by Rohto Pharmaceutical Co., Ltd.).

[0046] The pH of the culture medium for mesenchymal stem cells used in pre-culture is not particularly limited as long as it is within a range that is pharmaceutically, pharmacologically (pharmaceutically), or physiologically acceptable. The pH of the culture medium used in pre-culture according to this embodiment may be, for example, 4.0 to 9.5, preferably 5.0 to 9.0, more preferably 6.0 to 8.5, and even more preferably 6.5 to 8.4.

[0047] The osmotic pressure ratio of the culture medium for mesenchymal stem cells used in pre-culture can be adjusted to a range acceptable to the body as needed. An appropriate osmotic pressure ratio can be set appropriately depending on the use and method of use of the culture medium, but for example, it can be 0.5 to 2, preferably 0.75 to 1.5, and more preferably 0.95 to 1.19. The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to 286 mOsm (osmotic pressure of 0.9 w / v% sodium chloride aqueous solution) based on the 18th edition of the Japanese Pharmacopoeia, and the osmotic pressure is measured by referring to the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. The standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) can be prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500-650°C for 40-50 minutes, then allowing it to cool in a desiccator (silica gel), accurately weighing 0.900 g of it, dissolving it in purified water to make exactly 100 mL, or by using a commercially available standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution).

[0048] Other components may be added to the culture medium for mesenchymal stem cells as needed. Examples of other components include the other components mentioned above as examples of components to be added to the raw material culture medium.

[0049] The culture conditions for mesenchymal stem cells in pre-culture can be the same as those exemplified above as the culture conditions for mesenchymal stem cells used to obtain the culture supernatant.

[0050] [Mesenchymal stem cell secretions] In this specification, "cellular secretions" refers to the totality of biomolecules secreted extracellularly by mesenchymal stem cells. Cellular secretions may include soluble factors (proteins, peptides, nucleic acids, lipids, etc.) and extracellular vesicles. Cellular secretions can be obtained by performing column chromatography, affinity chromatography, ultracentrifugation, etc., on the culture supernatant of mesenchymal stem cells to concentrate fine particles such as exosomes, and / or remove other components.

[0051] Cellular secretions may be diluted with water, saline solution, buffer, etc., as needed. Furthermore, optional components (e.g., pharmaceutical components) may be added to the cellular secretions as needed.

[0052] (Method of administration) The method of administering the apelin expression promoter of the present invention is not particularly limited, but intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, lumbar puncture, or nasal administration are preferred, with intravascular administration or lumbar puncture being more preferred. In addition to pharmaceuticals, the apelin expression promoter of the present invention can also be used as quasi-drugs, cosmetics, research reagents, and foods (supplements, etc.).

[0053] <An agent that promotes the elongation of vascular endothelial cells> The vascular endothelial cell elongation promoter of the present invention comprises one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. It is determined that using the vascular endothelial cell elongation promoter of the present invention can promote angiogenesis through the promotion of vascular endothelial cell elongation. The vascular endothelial cell elongation promoter of the present invention may also contain other components, to the extent that they do not impair the effects of the present invention.

[0054] In the vascular endothelial cell elongation promoter of the present invention, the details of each cell, the details of mesenchymal stem cells, the culture supernatant of mesenchymal stem cells, and the cellular secretions of mesenchymal stem cells, as well as the method of use, are the same as those of the apelin expression promoter of the present invention, so redundant explanations will be omitted.

[0055] In this specification, promoting vascular endothelial cell elongation refers to increasing the cell length of vascular endothelial cells. The cell length of vascular endothelial cells is one indicator of the proliferative activity of vascular endothelial cells.

[0056] The promotion of vascular endothelial cell elongation can be confirmed, for example, by the method described in the examples. Specifically, to confirm the promotion of vascular endothelial cell elongation, vascular endothelial cells (e.g., HUVEC, HBMEC, etc.) are cultured in a plate for a predetermined time (e.g., 4 to 8 hours, preferably 6 hours) using mesenchymal stem cells, mesenchymal stem cell supernatant, mesenchymal stem cell secretions, or a control. Then, five images are taken from each well of the plate and analyzed using software (e.g., "ImageJ") to measure the cell length. More specifically, the sum A of the cell lengths of all cells is measured for each image, and the average value of the sum A of the five images is calculated. The obtained average value is taken as the cell length B of that well, and the cell length B is measured for multiple wells, and the mean and variance are calculated. If the cell length is significantly increased compared to the control (preferably an increase of 5% or more, more preferably an increase of 10% or more), it is recognized that mesenchymal stem cells, mesenchymal stem cell supernatant, or mesenchymal stem cell secretions promoted the elongation of vascular endothelial cells.

[0057] <An agent that promotes the formation of the vascular endothelial cell network structure> The present invention provides a vascular endothelial cell reticular structure formation promoter that comprises one or more selected from the group consisting of mesenchymal stem cells, culture supernatants of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. It is determined that using the vascular endothelial cell reticular structure formation promoter of the present invention can promote angiogenesis by promoting the formation of a vascular endothelial cell reticular structure. The vascular endothelial cell reticular structure formation promoter of the present invention may also contain other components, to the extent that they do not impair the effects of the present invention.

[0058] In the present invention's vascular endothelial cell reticular structure formation promoter, the details of each cell, the details of the mesenchymal stem cell culture supernatant, and the method of use are the same as those of the apelin expression promoter of the present invention, so redundant explanations will be omitted.

[0059] In this specification, promoting the formation of a vascular endothelial cell reticular structure refers to increasing the number of meshes (networks) formed by the bonding of elongated vascular endothelial cells. The vascular endothelial cell reticular structure is one indicator of the strength of cell adhesion between vascular endothelial cells.

[0060] The promotion of vascular endothelial cell reticular structure formation can be confirmed, for example, by the method described in the examples. Specifically, to confirm the promotion of vascular endothelial cell reticular structure formation, vascular endothelial cells (e.g., HUVEC, HBMEC, etc.) are cultured in a plate for a predetermined time (e.g., 4 to 8 hours, preferably 6 hours) using mesenchymal stem cells, mesenchymal stem cell supernatant, mesenchymal stem cell secretions, or a control. Then, five images from each well of the plate are analyzed using software (e.g., "ImageJ") and the number of meshes (#Mesh) is measured. If the number of meshes is significantly increased compared to the control (preferably an increase of 5% or more, more preferably an increase of 10% or more), it is recognized that mesenchymal stem cells, mesenchymal stem cell supernatant, or mesenchymal stem cell secretions promoted the formation of vascular endothelial cell reticular structure.

[0061] <An agent that promotes branching of vascular endothelial cells> The branching chain formation promoter for vascular endothelial cells of the present invention comprises one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. It is determined that using the branching chain formation promoter for vascular endothelial cells of the present invention can promote angiogenesis by promoting branching chain formation of vascular endothelial cells. The branching chain formation promoter for vascular endothelial cells of the present invention may also contain other components, to the extent that they do not impair the effects of the present invention.

[0062] In the branching chain formation promoter of vascular endothelial cells of the present invention, the details of each cell, the details of the culture supernatant of mesenchymal stem cells, and the method of use are the same as those of the apelin expression promoter of the present invention, so redundant explanations will be omitted.

[0063] In this specification, promoting branching of vascular endothelial cells refers to increasing the number of junctions formed by the connection of elongated vascular endothelial cells. The branching of vascular endothelial cells is one of the important indicators of angiogenesis.

[0064] The promotion of branching chain formation in vascular endothelial cells can be confirmed, for example, by the method described in the examples. Specifically, to confirm the promotion of branching chain formation in vascular endothelial cells, for example, vascular endothelial cells (e.g., HUVEC, HBMEC, etc.) are cultured in a plate for a predetermined time (e.g., 4 to 8 hours, preferably 6 hours) using mesenchymal stem cells, mesenchymal stem cell supernatant, mesenchymal stem cell secretions, or a control. Then, five images from each well of the plate are analyzed using software (e.g., "ImageJ") and the number of junctions (#Junction) is measured. If the number of junctions is significantly increased compared to the control (preferably an increase of 10% or more, more preferably an increase of 20% or more), it is recognized that mesenchymal stem cells, mesenchymal stem cell supernatant, or mesenchymal stem cell secretions promoted branching chain formation in vascular endothelial cells.

[0065] <An agent that promotes the proliferation of vascular endothelial cells> The vascular endothelial cell proliferation promoter of the present invention comprises one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. It is determined that using the vascular endothelial cell proliferation promoter of the present invention can promote angiogenesis through the promotion of vascular endothelial cell proliferation. The vascular endothelial cell proliferation promoter of the present invention may also contain other components, to the extent that they do not impair the effects of the present invention.

[0066] In the vascular endothelial cell proliferation promoter of the present invention, the details of each cell, the details of mesenchymal stem cells, the culture supernatant of mesenchymal stem cells, and the cellular secretions of mesenchymal stem cells, as well as the method of use, are the same as those of the apelin expression promoter of the present invention, so redundant explanations will be omitted.

[0067] In this specification, promoting the proliferation of vascular endothelial cells means, for example, increasing the proliferation rate of vascular endothelial cells or increasing the rate of DNA replication as measured by BrdU uptake, etc.

[0068] The promotion of vascular endothelial cell proliferation can be confirmed, for example, by the method described in the examples. Specifically, to confirm the promotion of vascular endothelial cell proliferation, for example, vascular endothelial cells (e.g., HUVEC, HBMEC, etc.) are cultured on a plate for a predetermined time (e.g., 36-60 hours, preferably 48 hours) using mesenchymal stem cells, mesenchymal stem cell supernatant, mesenchymal stem cell secretions, or a control. Then, the number of viable cells on the plate is measured, or DAPI staining and BrdU staining are performed after BrdU incorporation. The number of viable cells and / or the percentage of BrdU-positive cells relative to the total number of DAPI-positive cells (BrdU) are then compared with the control. + / DAPI + If the amount is significantly increased (preferably by 100% or more), it is recognized that the mesenchymal stem cells, the culture supernatant of mesenchymal stem cells, or the cellular secretions of mesenchymal stem cells promoted the proliferation of vascular endothelial cells.

[0069] In angiogenesis, vascular endothelial cells need to actively divide and increase their number in order to create new vascular structures. Therefore, the proliferation of vascular endothelial cells is one of the important indicators of angiogenesis.

[0070] <An agent that promotes the migration of vascular endothelial cells> The vascular endothelial cell migration promoter of the present invention comprises one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. It is determined that using the vascular endothelial cell migration promoter of the present invention can promote angiogenesis through the promotion of vascular endothelial cell migration. The vascular endothelial cell migration promoter of the present invention may also contain other components, to the extent that they do not impair the effects of the present invention.

[0071] In the vascular endothelial cell migration promoter of the present invention, the details of each cell, mesenchymal stem cells, the culture supernatant of mesenchymal stem cells, and the cellular secretions of mesenchymal stem cells, as well as the method of use, are the same as those of the apelin expression promoter of the present invention, so redundant explanations will be omitted.

[0072] In this specification, promoting the migration of vascular endothelial cells means, for example, accelerating the rate at which proliferating vascular endothelial cells invade adjacent areas where vascular endothelial cells are not present.

[0073] The promotion of vascular endothelial cell migration can be confirmed, for example, by the method described in the examples. For specific confirmation of the promotion of vascular endothelial cell migration, for example, vascular endothelial cells (e.g., HUVEC, HBMEC, etc.) are cultured on a plate to form a monolayer (for example, a cell density of 5 × 10⁶ in a 24-well culture plate). 5 (To make a cell / well). Next, a wound is made in the monolayer using a micropipette tip or the like. Then, the cells are cultured in a plate for a predetermined time (e.g., 36 to 60 hours, preferably 48 hours) using mesenchymal stem cells, mesenchymal stem cell supernatant, mesenchymal stem cell secretions, or a control. Then, using a fluorescence microscope (e.g., Keyence Corporation's "BZ-X800"), the wound closure rate of each cell is calculated using the following formula based on the wound area at 0 hours (initial wound area) and the wound area at 48 hours (final wound area). If the wound closure rate is significantly improved compared to the control (preferably an improvement of 5% or more), it is considered that the mesenchymal stem cells, mesenchymal stem cell supernatant, or mesenchymal stem cell secretions promoted the migration of vascular endothelial cells. Wound closure rate [%] = 100 × (Initial wound area - Final wound area) / Initial wound area

[0074] In angiogenesis, new blood vessels are formed when endothelial cells migrate and extend from existing blood vessels. Therefore, the migration of endothelial cells is one of the important indicators of angiogenesis.

[0075] <An agent that promotes the sugar coating of vascular endothelial cells> The present invention provides a sugar coating-promoting agent for vascular endothelial cells, comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. It is determined that using the sugar coating-promoting agent for vascular endothelial cells of the present invention can promote sugar coating formation of vascular endothelial cells and the restoration (re-thickening) of thinned sugar coatings. The sugar coating-promoting agent for vascular endothelial cells of the present invention may also contain other components, as long as they do not impair the effects of the present invention.

[0076] The sugar coating is a layered structure that covers the outside of the cell membrane of vascular endothelial cells, and its thickness is, for example, several hundred nanometers. This sugar coating is mainly composed of proteoglycans, polysaccharides, etc. Furthermore, the sugar coating is known to reflect the health status of vascular endothelial cells; it is relatively thick in a normal state, but can thin with age or damage associated with various pathological conditions. Thinning of the sugar coating may be involved in a decline in endothelial function (maintenance of blood flow, endothelial protective function, antithrombotic effect, etc.). In addition, it has been reported that thinning of the sugar coating layer with age is involved in the breakdown of the blood-brain barrier (Shi et al., Nature 2025). Therefore, the sugar coating formation promoter for vascular endothelial cells of the present invention may normalize brain function, for example, by restoring the function of a broken blood-brain barrier.

[0077] In the present invention's sugar coating-promoting agent for vascular endothelial cells, the details of each cell, mesenchymal stem cells, the culture supernatant of mesenchymal stem cells, and the cellular secretions of mesenchymal stem cells, as well as the method of use, are the same as those for the apelin expression-promoting agent of the present invention, so redundant explanations are omitted.

[0078] In this specification, promotion of sugar coating formation in vascular endothelial cells refers, for example, to the thickening of thinned sugar coatings in vascular endothelial cells or the restoration of lost sugar coatings.

[0079] The formation of a sugar coating on vascular endothelial cells can be confirmed, for example, by the method described in the examples. Specifically, to confirm the formation of a sugar coating on vascular endothelial cells, mesenchymal stem cells, mesenchymal stem cell culture supernatant, mesenchymal stem cell secretions, or a control are administered (e.g., via tail vein administration) to mice exhibiting thinning of the sugar coating (e.g., aged mice or Alzheimer's model mice) and reared for a predetermined period (e.g., 1 to 4 weeks). Administration may be performed two or more times as needed. Then, the sugar coating markers (e.g., lectins) of vascular endothelial cells (cerebral vascular endothelial cells are preferred) are observed by immunofluorescence staining. If the thickness of the sugar coating and the expression level of the markers are significantly improved compared to the control, it can be concluded that mesenchymal stem cells, mesenchymal stem cell culture supernatant, or mesenchymal stem cell secretions promoted the formation of a sugar coating on vascular endothelial cells.

[0080] <Agents for the prevention and / or treatment of neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases> The preventive and / or therapeutic agent for neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases of the present invention (hereinafter sometimes simply referred to as "preventive and / or therapeutic agent") comprises one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. By using the preventive and / or therapeutic agent of the present invention, neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases can be effectively prevented and / or treated through effects such as promoting the elongation of vascular endothelial cells, promoting the formation of reticular structures, promoting branching chain formation, promoting proliferation, promoting migration, inhibiting microglia, suppressing inflammation, attracting asterocytes to the periphery of amyloid-beta plaques, or promoting sugar coating formation. The preventive and / or therapeutic agent of the present invention may also contain other components, to the extent that they do not impair the effects of the present invention.

[0081] In the preventive and / or therapeutic agent of the present invention, the details of each cell, mesenchymal stem cells, the culture supernatant of mesenchymal stem cells, and the cellular secretions of mesenchymal stem cells are the same as those of the apelin expression promoter of the present invention, so redundant explanations are omitted.

[0082] As described above, the promotion of cerebral angiogenesis, the inhibitory effect on microglia, the anti-inflammatory effect, the attraction effect of amyloid-beta to asterocytes around plaques, or the promotion of sugar coating formation of vascular endothelial cells are thought to be effective in preventing and / or treating neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases. Specific neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases are similar to those exemplified in the apelin expression promoter of the present invention. As mentioned in Non-Patent Documents 1 and 2 above, Alzheimer's disease is closely related to abnormalities in cerebral blood vessels, and Alzheimer's disease is preferred as the neurodegenerative disease.

[0083] <Gene expression promoter> The gene expression promoter of the present invention is an expression promoter of at least one gene selected from the group consisting of apelin, angiocline factors such as PGF, DEPP1, KRT18, UACA, PTX3, RGCC, RGS3, PTP4A3, CD34, TCF4, PRSS23, PALD1, PODXL, CCND1, Protocadherin12, TM4SF18, UBE2J1, and NUDT4, comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells. The gene whose expression is promoted by the gene expression promoter of the present invention is preferably at least one gene selected from the group consisting of apelin, angiocline factors such as PGF, CD34, and Protocadherin12. The case in which the present invention is an apelin expression promoter that promotes the expression of apelin has been described above. If the present invention is an angiocrine expression promoter such as PGF, the present invention can promote angiogenesis of vascular endothelial cells by promoting the expression of angiocrine factors such as PGF. If the present invention is a CD34 expression promoter, the present invention can promote angiogenesis by promoting the undifferentiated state of vascular endothelial cells by promoting the expression of CD34. If the present invention is a protocadherin 12 expression promoter, the present invention can promote angiogenesis by increasing the intercellular adhesion of vascular endothelial cells by promoting the expression of protocadherin 12. [Examples]

[0084] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[0085] [Example 1: Effects of culture supernatants of various mesenchymal stem cells on HUVEC] (Preparation of culture supernatant for mesenchymal stem cells) The following were prepared as mesenchymal stem cells, mesenchymal stem cell culture medium, and culture dishes. Each mesenchymal stem cell was passaged to stage 4 (P4). The mesenchymal stem cells were cultured at 37°C under 5% CO2 conditions. Adipose-derived mesenchymal stem cells (ADMSC): "#PT-5006" manufactured by Lonza Corporation Umbilical cord-derived mesenchymal stem cells (UCMSC): Thermo Fisher Scientific "Life technologies #KW-4009" Bone marrow-derived mesenchymal stem cells (BMMSC): Thermo Fisher Scientific "Life technologies #KW-4309" Dental pulp-derived mesenchymal stem cells (DPMSC): "#PT-5025" manufactured by Lonza Corporation Culture medium for mesenchymal stem cells: Proprietary preparation (substantially the same composition as "R:STEM Medium for hMSC High Growth" manufactured by Rohto Pharmaceutical Co., Ltd.) Culture dish: Corning "100-mm cell culture dish (CellBIND®, Corning #3296)"

[0086] Each mesenchymal stem cell (ADMSC, UCMSC, BMMSC, or DPMSC) was cultured for 24 hours until 50-60% confluence using the above-mentioned culture dish (Corning #3296) and mesenchymal stem cell medium. Then, the medium for each mesenchymal stem cell was replaced with 10 mL of large vascular endothelial cell medium "EGM-2 medium" (manufactured by Lonza Co., Ltd.) (equivalent to the raw material medium). Each mesenchymal stem cell was then cultured for a further 72 hours (hereinafter, the period of culture in the raw material medium may be referred to as "MSC culture time"). The medium (condition medium CM) for each mesenchymal stem cell was then collected. Each condition medium CM was centrifuged at 3,000 rpm for 10 minutes to remove cell debris. After cell debris removal, each condition medium CM was filtered through a 22 μm filter (Merck Millipore), and the filtered condition medium CM was used as the culture supernatant for each mesenchymal stem cell. Separately, 10 mL of the aforementioned "EGM-2 medium" was incubated in the above-mentioned culture dish (Corning #3296) without cells for the same time as the MSC culture time (72 hours), and the culture medium after incubation was used as a control.

[0087] (Preparation of vascular endothelial cells) As vascular endothelial cells, "Human umbilical vein endothelial cell (HUVEC)" (passage 4 (P4)) manufactured by Lonza Corporation was cultured at 37°C and 5% CO2 in the aforementioned "EGM-2 medium" and an additive factor kit ("#CC-3162" manufactured by Lonza Corporation).

[0088] (Conditioning culture) 48-well plates were coated with Corning Matrigel and incubated at 37°C and 5% CO2 for 30 minutes. Next, the HUVEC cells described above were seeded onto the gel and cultured for 6 hours using the culture supernatant of each mesenchymal stem cell or a control. During the culture, at 2, 4, and 6 hours, microscopic images of the tubular structures formed by vascular endothelial cells were acquired using a phase-contrast microscope (Figure 1). Subsequently, five images from each well were analyzed using ImageJ software, and the cell length, mesh count, and junction count of HUVEC cells were graphed (Figure 2). The graph in Figure 2 shows the relative values ​​of each parameter when the measured values ​​of each parameter of HUVEC cultured with the control are set to 100% (the same applies to the graphs in Figures 4 and 5 described later).

[0089] The cell length, reticular count, and junction count of HUVECs indicate vascular endothelial cell elongation, reticular structure formation, and branching, respectively, and are all indicators involved in angiogenesis. As is clear from Figures 1 and 2, the culture supernatant of each mesenchymal stem cell promoted HUVEC elongation, reticular structure formation, and branching. From these results, it was confirmed that the culture supernatant of mesenchymal stem cells has an effect on vascular endothelial cells that is involved in angiogenesis.

[0090] [Example 2: Effects of culture supernatant of adipose-derived mesenchymal stem cells on HBMEC] (Preparation of culture supernatant for mesenchymal stem cells) The culture supernatant of adipose-derived mesenchymal stem cells (ADMSCs) was prepared using the same procedure as in Example 1, except that "Endothelial Cell Medium (Catalog: No. 1001)" from ScienCell Research Laboratories was used as the raw material culture medium.

[0091] (Preparation of vascular endothelial cells) As vascular endothelial cells, "Human Brain Microvascular Endothelial Cells (HBMEC)" (passage 1) from ScienCell Research Laboratories, which are brain vascular endothelial cells, were cultured in "Endothelial Cell Medium (Catalog: No. 1001)" from ScienCell Research Laboratories at 37°C and 5% CO2.

[0092] (Conditioning culture) Except for using the above-mentioned HBMEC as vascular endothelial cells and using the above-mentioned adipose-derived mesenchymal stem cell (ADMSC) culture supernatant or control as the culture supernatant, the procedure was carried out in the same manner as in Example 1, and the effect of the mesenchymal stem cell culture supernatant on HBMEC was measured. The results are shown in Figures 3 and 4.

[0093] As is clear from Figures 3 and 4, the culture supernatant of mesenchymal stem cells promoted elongation, reticular formation, and branching of HBMECs, similar to HUVECs. Therefore, it was confirmed that the culture supernatant of mesenchymal stem cells also has an effect on cerebral vascular endothelial cells that contributes to angiogenesis.

[0094] [Example 3: Effect of culture supernatant of adipose-derived mesenchymal stem cells on HUVEC cell proliferation] (Preparation of culture supernatant for mesenchymal stem cells) The culture supernatant and control were prepared using the same procedure as in Example 1, except that the MSC culture time was changed to 96 hours.

[0095] The above-mentioned (HUVEC) (passage 4 (P4)) was seeded in a 96-well plate and cultured for 24 hours at 37°C and 5% CO2 in the above-mentioned "EGM-2 medium" and additive factor kit. Subsequently, the medium was replaced with the culture supernatant or control of the above-mentioned adipose-derived mesenchymal stem cells (ADMSC), and cultured for a further 48 hours.

[0096] (Evaluation of cell count) The proliferation rate of each cell was measured using a viable cell counting kit (Dojin Chemical Research Institute Co., Ltd. "Cell Counting Kit-8 (#343-07623)"). Specifically, the absorbance (optical density) at 450 nm was measured using a microplate reader (Thermo Fisher Scientific "Multiskan FC"). In the measurement, the background control value was subtracted from each measurement value to obtain the average absorbance for each of the three duplicate sets. The measurement results are shown in Figure 5.

[0097] (Evaluation of BrdU-positive cells) Each cell was evaluated by uptake of bromodeoxyuridine (BrdU) from Sigma-Aldrich and by immunofluorescence staining. Specifically, BrdU was added to each cell to a final concentration of 50 μg / mL, incubated for 2 hours, and then fixed. Next, for immunofluorescence staining, each cell was incubated in a solution of phosphate-buffered saline (PBS) with 0.1% Triton X-100 added. Then, each cell was stained with mouse monoclonal anti-BrdU antibody (Roche) (dilution ratio 1:1000). After washing each cell with PBS, it was incubated again with Alexa Fluor 488-labeled anti-mouse IgG secondary antibody (Molecular Probes) (dilution ratio 1:2500). After washing each cell with PBS, pair staining was performed with DAPI. The obtained specimens were observed using a confocal laser microscope (Olympus Corporation (FV3000)) and image processing was performed using Adobe Photoshop. This results in the proportion of BrdU-positive cells to the total number of DAPI-positive cells (BrdU + / DAPI + The following percentage was calculated. This percentage represents the proportion of cells in a proliferative state among living cells. The measurement results are shown in Figure 6.

[0098] As is clear from Figures 5 and 6, the culture supernatant of mesenchymal stem cells was found to increase both the cell number and the proportion of cells in a proliferating state in HUVECs, thus promoting cell proliferation.

[0099] [Example 4: Effects of culture supernatant of adipose-derived mesenchymal stem cells on cell proliferation and migration of HUVECs] The above HUVEC has a cell density of 5 × 10 5 Cells were seeded in a 24-well culture plate to reach a cell / well level and cultured at 37°C and 5% CO2 using the EGM-2 medium described above. The following day, a monolayer of HUVECs was formed in the wells. Next, the HUVEC monolayer was wound using a micropipette tip. Then, the culture medium in the wells was replaced with the culture supernatant or control of adipose-derived mesenchymal stem cells (ADMSCs) and maintained for 48 hours. During maintenance, time-lapse images of the monolayer were taken using an all-in-one fluorescence microscope (KEYENCE Corporation "BZ-X800") (at 0 hours, 6 hours, or 8 hours, Figure 7). The wound closure rate of each cell was then calculated using the following formula based on the wound area at 0 hours (initial wound area) and the wound area at 48 hours (final wound area). The results are shown in Figure 8. The wound area refers to the area in the monolayer on the well where cells have been removed by manipulation with a micropipette tip. Wound closure rate [%] = 100 × (Initial wound area - Final wound area) / Initial wound area

[0100] As is clear from Figures 7 and 8, the culture supernatant of mesenchymal stem cells accelerated the closure of wounds in the HUVEC monolayer compared to the control. This wound closure occurs as HUVECs migrate to the wound area while proliferating. Therefore, it was confirmed that the culture supernatant of mesenchymal stem cells promotes the proliferation and migration of HUVECs.

[0101] [Example 5: Changes in gene expression in HUVECs due to culture supernatant of mesenchymal stem cells] Each mesenchymal stem cell (ADMSC) was cultured for 24 hours until it reached 50-60% confluence using the above-mentioned culture dish (Corning #3296) and mesenchymal stem cell medium. Then, the culture medium for each mesenchymal stem cell was replaced with 10 mL of large vascular endothelial cell medium "EGM-2 medium" (manufactured by Lonza Co., Ltd.) (equivalent to the raw material medium). Each mesenchymal stem cell was then cultured for a further 48 hours (hereinafter, the period of culture in the raw material medium may be referred to as "MSC culture time"). The culture medium (condition medium CM) for each mesenchymal stem cell was then collected. Each condition medium CM was centrifuged at 3,000 rpm for 10 minutes to remove cell debris. After cell debris removal, each condition medium CM was filtered through a 22 μm filter (Merck Millipore), and the filtered condition medium CM was used as the culture supernatant for each mesenchymal stem cell. Separately, 10 mL of the aforementioned "EGM-2 medium" was incubated in the above-mentioned culture dish (Corning #3296) without cells for the same time as the MSC culture time (48 hours), and the culture medium after incubation was used as a control.

[0102] (RNA sequencing analysis) RNA sequencing analysis was performed on HUVECs cultured in the culture supernatant or control of adipose-derived mesenchymal stem cells (ADMSCs) using the following methods.

[0103] To extract total RNA from the sample, the RNA extraction kit "Direct-Zol RNA miniprep kit (Zymo Research)" was used. The quality of the extracted total RNA was confirmed using the nucleic acid quality testing system "Agilent 2200 TapeStation (Agilent Technologies, Inc.)". Next, a sequencing library was prepared from the obtained RNA using the cDNA preparation kit "SMART-Seq v4 Ultra Low Input Kit for Sequencing (Takara Bio Inc.)".

[0104] Next, the prepared sequencing library was sequenced using the "Illumina Novaseq 6000" sequencing system (manufactured by Illumina Corporation). This generated 150 bp paired-end reads. The obtained reads were then mapped to the human reference genome (GENCODE / GRCh38[hg38]) using the data analysis software "DRAGEN" (manufactured by Illumina Corporation).

[0105] Next, a heatmap was created showing differences in gene expression levels (differentially expressed genes) when cultured with the culture supernatant versus when cultured with the control (Figure 9). As shown in Figure 9, when cultured with the culture supernatant ("CD-ADMSC treated HUVEC" in the figure), the expression levels of the following genes increased compared to the control ("Control HUVEC" in the figure) ("upDEGs" in the figure). DEPP1 KRT18 APLN (Apelin) UACA PTX3 RGCC RGS3 PGF (Placenta growth factor) PTP4A3 CD34 TCF4 PRSS23 PALD1 PODXL CCND1 PCDH12 (Protocadherin12) TM4SF18 UBE2J1 NUDT4

[0106] Next, a Volcano Plot of differentially expressed genes was created (Figure 10). In the Volcano Plot, the vertical axis (unit: -log(FDR)) represents significance (higher on the upper side indicates greater significance), and the horizontal axis (unit: log FC) shows the degree of difference in expression levels between the two conditions (cultured in culture supernatant or cultured in control) (the center (log FC=0) indicates no change, the further to the left of the center the expression level decreases, and the further to the right the expression level increases).

[0107] Furthermore, Gene Ontology (GO) terminology analysis was performed using differentially expressed genes. In this analysis, Expression Miner 2.0 was used to provide functional annotations for each gene. The GO terminology analysis results are shown in Figure 11. In Figure 11, the vertical axis of the graph (unit: -log) 10 The (p-adj) axis indicates significance (higher on the right side indicates greater significance), and the horizontal axis shows the category (GO:MF, GO:CC, GO:BP, RE:AC). As shown in Figure 11, 20 genes with high significance were found among the differentially expressed genes. Table 1 shows the details of the function of these 20 genes (g:Profiler (https: / / biit.cs.ut.ee / gprofiler / gost)).

[0108] [Table 1]

[0109] As shown in Figure 9, HUVECs cultured in the culture supernatant of mesenchymal stem cells showed increased apelin expression. Therefore, it was confirmed that the culture supernatant of mesenchymal stem cells functions as an excellent apelin expression promoter. Furthermore, as mentioned above, apelin is a gene that plays an important role in angiogenesis. Therefore, it is judged that the promotion of angiogenesis of vascular endothelial cells by the culture supernatant of mesenchymal stem cells (particularly promotion of elongation, promotion of reticular structure formation, promotion of branching chain formation, promotion of proliferation or migration) is carried out via apelin. In addition to its angiogenesis-promoting effect, apelin also has neuroprotective effects as mentioned above and is thought to be closely related to Alzheimer's disease. Therefore, it is judged that the culture supernatant of mesenchymal stem cells is also effective as a preventive and / or therapeutic agent for neurodegenerative diseases (particularly Alzheimer's disease), demyelinating diseases, or cerebrovascular diseases.

[0110] Furthermore, as shown in Figure 9, HUVECs cultured in the culture supernatant of mesenchymal stem cells also showed increased expression of placental growth factor (PGF), one of the angiocline factors. PGF is also known as a major factor involved in angiogenesis. Therefore, it is thought that the culture supernatant of mesenchymal stem cells functions as an excellent angiocline factor expression enhancer or PGF expression enhancer, and promotes angiogenesis of vascular endothelial cells through the enhancement of angiocline factor or PGF expression.

[0111] As shown in Figure 9, HUVECs cultured in the culture supernatant of mesenchymal stem cells also showed increased CD34 expression. Some reports indicate that CD34 is expressed in undifferentiated endothelial cells that are close to stem cells or progenitor cells. Therefore, it is thought that the culture supernatant of mesenchymal stem cells also functions as an excellent CD34 expression promoter and contributes to promoting the undifferentiated state of vascular endothelial cells through the promotion of CD34 expression.

[0112] In addition, as shown in Figure 9, HUVECs cultured in the culture supernatant of mesenchymal stem cells also showed increased expression of Protocadherin 12. Protocadherin 12 is a cell adhesion molecule and is known to be expressed in angiogenic endothelium. Therefore, it is possible that the culture supernatant of mesenchymal stem cells also functions as an excellent Protocadherin 12 expression promoter, potentially promoting angiogenesis by enhancing intercellular adhesion of vascular endothelial cells.

[0113] In addition, as shown in Figure 11 and Table 1, many of the genes that showed differential expression in HUVECs cultured in the culture supernatant of mesenchymal stem cells were genes related to the regulation of the cell cycle (especially mitosis). Therefore, it should be considered that the culture supernatant of mesenchymal stem cells may promote angiogenesis by promoting the proliferation of vascular endothelial cells.

[0114] [Example 6: In vivo dynamics of mesenchymal stem cells in an Alzheimer's disease model animal] (Preparation of Alzheimer's model animals) APP mutant mice (APPSWE) were air-shipped from Taconic, Inc. in the United States, underwent microbiological cleaning (SPF conversion) at Shimizu Materials Co., Ltd., and then bred in an SPF room (APP tg mice). As mentioned above, the APP gene codes for amyloid precursor protein and is known as one of the causative genes for familial Alzheimer's disease. Amyloid precursor protein is cleaved on the membrane by enzymes called secretases, and among these, cleavage by β-secretase and γ-secretase produces amyloid-beta. When a mutation occurs in the APP gene, an increase in amyloid-beta production, an increase in the Aβ42 / Aβ40 ratio, and increased amyloid-beta aggregation often occur.

[0115] Immunohistochemical staining of the brains of 2-month-old wild-type mice (WT) and Alzheimer's disease model mice (APP tg) was performed. Antibodies against CD31, a marker for vascular endothelial cells, and Iba1, a marker for microglia, were used for staining. The presence of microglia indicates the occurrence of inflammation. The results are shown in Figures 12 and 13. As is clear from Figures 12 and 13, at 2 months of age, Alzheimer's disease model mice (APP tg) showed almost no change in vascular endothelial cells or microglia compared to wild-type mice (WT). On the other hand, in Alzheimer's disease model mice, amyloid-beta (Abeta) accumulation was already confirmed to have begun at 2 months of age (not shown).

[0116] Immunohistochemical staining of the brains of 7.5-month-old wild-type mice (WT) and Alzheimer's disease model mice (APPtg) was performed. Antibodies against amyloid-beta (Abeta) and Iba1, a microglia marker, were used for staining. Nuclear staining with DAPI (4',6-diamino-2-phenylindole) was also performed simultaneously. The results are shown in Figure 14. As is clear from Figure 14, APPtg mice showed increased microglia (indicating inflammation) and accumulation of amyloid-beta.

[0117] (Measurement of the dynamics of mesenchymal stem cells in the body) Corning's "Cell BIND® dish (Catalog No.: Corning3296)" was used as the culture vessel, and serum-free medium for mesenchymal stem cells (R: STEM Medium for hMSC High Growth, manufactured by Rohto Pharmaceutical Co., Ltd.) was used as the culture medium. Adipose-derived mesenchymal stem cells (AD MSCs, manufactured by Lonza, passage number 5, human origin) were cultured in a three-dimensional manner for three days. Subsequently, the adipose-derived mesenchymal stem cells were harvested, and fluorescent dye (Levity Japan's "IVISense 680. Fluorescent Cell Labeling Dye") was introduced into the cells for fluorescent labeling.

[0118] Wild-type (WT) and APP tg mice, both 12 months old, were prepared and fed a low-fluorescence diet. Fluorescently labeled adipose-derived mesenchymal stem cells were extracted from the tail vein of each mouse in 5 × 10⁻¹⁴⁻¹ 5 Individual injections were administered. A separate control group was prepared and administered only physiological saline (NS). After each mouse was reared for 24 hours, fluorescence imaging was performed using an optical imaging device (IVIS Imaging Lumina XR, manufactured by Levity Japan). Fluorescence imaging was performed to observe fluorescence in the lungs, liver, heart, and brain to confirm how much of the administered adipose-derived stem cells migrated to each site. The results of the fluorescence imaging are shown in Figures 15 and 16, and the fluorescence intensity is graphed in Figure 17.

[0119] For fluorescence imaging, a total of five mice were measured: wild-type (WT) mice administered with saline (NS), wild-type (WT) mice administered with adipose-derived mesenchymal stem cells (AD MSC), APP tg mice administered with saline (NS), and APP tg mice administered with adipose-derived mesenchymal stem cells (AD MSC) (2 individuals). In Figure 15, the results of the lungs, and in Figure 16, the results of the heart and brain, the results of the aforementioned five individuals are shown from left to right. In Figure 15, the results of the liver, the results of the aforementioned five individuals are shown in the order of top left, top right, bottom left, bottom center, and bottom right.

[0120] As shown in Figures 15-17, in wild-type mice (WT), administered adipose-derived mesenchymal stem cells (AD MSCs) migrated to the lungs, liver, and heart, but not to the brain. On the other hand, in APP tg mice, administered adipose-derived mesenchymal stem cells (AD MSCs) migrated to the lungs, liver, heart, and brain. This is thought to be due to the weakening of the blood-brain barrier in APP tg mice, which allowed adipose-derived mesenchymal stem cells to pass through, and the fact that inflammation occurring in the brain attracted adipose-derived mesenchymal stem cells.

[0121] From the above experiments, it was confirmed that in patients with neurodegenerative diseases represented by Alzheimer's disease, mesenchymal stem cells can be transferred into the brain and act by intravenous administration of mesenchymal stem cells.

[0122] (Example 7: Administration of Mesenchymal Stem Cells to 10-Month-Old Alzheimer's Model Mice) For the aforementioned APP tg mice (10 months old) (n = 8), adipose-derived mesenchymal stem cells were injected into the tail vein at a dose of 1×10 6 cells. After that, the mice were bred for 2 weeks, and then 1×10 6 more adipose-derived mesenchymal stem cells were injected into the tail vein. After that, the mice were bred for 2 weeks and then euthanized, and dissection and immunohistochemical staining (microscopic photography) of the brain were performed. Separately, as a control group, APP tg mice (n = 8) were prepared in the same manner except that normal saline was administered twice instead of adipose-derived mesenchymal stem cells. In the staining, an antibody against amyloid β and an antibody against Iba1, a marker of microglia involved in inflammation, were used. At the same time, nuclear staining with DAPI was performed. The results of immunohistochemical staining are shown in Fig. 18. In Fig. 18, representative ones among the stained objects are indicated by solid arrows or dotted arrows. Also, the upper graph in Fig. 19 is a graph showing the area ratio (%) of amyloid β in the immunohistochemical staining image. The lower graph in Fig. 19 is a graph showing the area (pixels) of aggregated amyloid β in the immunohistochemical staining image. The graph in Fig. 20 is a graph showing the ratio (%) of Iba1-positive cells among all cells contained in the visual field in the immunohistochemical staining image.

[0123] As shown in Figs. 18 to 20, the amyloid β plaques (present in a spotted pattern in Fig. 18) were fewer in the adipose-derived mesenchymal stem cell administration group. Also, the expression of Iba1 (present in a spotted pattern in Fig. 18), which indicates the occurrence of inflammation, was fewer in the adipose-derived mesenchymal stem cell administration group. From the above results, in the administration to 10-month-old mice, the administration of adipose-derived mesenchymal stem cells to Alzheimer's model mice had an effect of suppressing the aggregation of amyloid β in the brain.

[0124] Immunohistochemical staining (microscopic imaging) was further performed on the brains of the aforementioned 10-month-old mice. For staining, antibodies against amyloid-beta and antibodies against GFAP, a marker for asterocytes, were used. Asterocytes are known to accumulate around amyloid-beta plaques. Nuclear staining with DAPI was also performed simultaneously. The results of the immunohistochemical staining are shown in Figure 21. In Figure 21, representative staining targets are indicated by solid or dotted arrows. The graph in Figure 22 shows the area percentage of amyloid-beta (%) or GFAP (%) in the immunohistochemical staining images. The graph in Figure 23 is a scatter plot showing the area percentage of amyloid-beta (%) on the vertical axis and the area percentage of GFAP (%) on the horizontal axis for the control group (upper) and the adipose-derived mesenchymal stem cell administration group (lower), respectively, in the immunohistochemical staining images, with the regression line and coefficient of determination (R) calculated. 2 ) is also shown.

[0125] As shown in Figures 21-23, asterocytes were observed to be concentrated around amyloid-beta plaques. The correlation between amyloid-beta and asterocytes was slightly greater in the group administered adipose-derived mesenchymal stem cells. Asterocytes have the function of accumulating around amyloid-beta plaques and degrading amyloid-beta, and it was judged that adipose-derived mesenchymal stem cells tended to promote this aggregation of asterocytes. However, the effect of adipose-derived mesenchymal stem cells on asterocytes was not that clear, and it is highly likely that they exert preventive or therapeutic effects through microglia, angiogenesis, and glycosylation, which will be discussed later.

[0126] Immunohistochemical staining (microscopic imaging) was performed on the brains of the aforementioned 10-month-old mice. For staining, a fluorescently labeled lectin that can bind to the sugar coating of vascular endothelium was used. Nuclear staining with DAPI was also performed simultaneously. The results of the immunohistochemical staining are shown in Figure 24. In Figure 24, representative staining targets are indicated by solid arrows.

[0127] The sugar coating is known to reflect the health of vascular endothelial cells and may be involved in endothelial function (maintaining blood flow, endothelial protective function, antithrombotic effect, etc.). As shown in Figure 24, lectins (linear) were more strongly expressed in the group administered adipose-derived mesenchymal stem cells. This suggests that the preventive or therapeutic effect of adipose-derived mesenchymal stem cells is likely mediated through the normalization (re-thickening) of the sugar coating.

[0128] The antibodies and other substances used in immunohistochemistry are listed below.

[0129] (Primary antibody) Anti-CD31 antibody: rat CD31 (BD 553370) (manufactured by BD Biosciences) Anti-Iba1 antibody: goat Iba-1 (Fujifilm 011-27991) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Anti-amyloid β antibodies: mouse Aβ (IBL 10323) (manufactured by Immuno-Biological Laboratories) and rabbit Aβ (CST 8243) (manufactured by Cell Signaling Technology) Anti-GFAP antibody: rabbit GFAP (Sigma-Aldrich "G9269") Fluorescently labeled lectin: Lycopersicon Esculentum (Tomato) Lectin, DyLight 649 (Thermo Fisher Scientific "L32472")

[0130] (Secondary antibody) Anti-mouse secondary antibody: mouse 488 (Invitrogen A21202) (manufactured by Thermo Fisher Scientific) Anti-rabbit secondary antibody: rabbit 488 (Invitrogen A21206) (manufactured by Thermo Fisher Scientific) Anti-mouse secondary antibody: mouse 594 (Invitrogen A21203) (manufactured by Thermo Fisher Scientific) Anti-rat secondary antibody: rat 647 (Abcam ab150155) (manufactured by Abcam) Anti-goat secondary antibody: goat 647 (Invitrogen A21447) (manufactured by Thermo Fisher Scientific) [Industrial applicability]

[0131] The apelin expression promoter of the present invention can efficiently promote apelin expression in vascular endothelial cells. Furthermore, the vascular endothelial cell elongation promoter, vascular endothelial cell reticular structure formation promoter, vascular endothelial cell branching chain formation promoter, vascular endothelial cell proliferation promoter, or vascular endothelial cell migration promoter of the present invention can impart desired promoting effects to vascular endothelial cells. Furthermore, the preventive and / or therapeutic agent for neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases of the present invention can provide novel therapeutic methods for neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases.

Claims

1. An apelin expression promoter in vascular endothelial cells, comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells.

2. The apelin expression promoter according to claim 1, wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or dental pulp-derived mesenchymal stem cells.

3. The apelin expression promoter according to claim 1 or 2, wherein the vascular endothelial cells are cerebral vascular endothelial cells.

4. The apelin expression promoter according to claim 1 or 2, wherein the culture supernatant substantially does not contain serum.

5. The apelin expression promoter according to claim 1 or 2, wherein the mesenchymal stem cells are mesenchymal stem cells cultured in serum-free medium.

6. A preventive and / or therapeutic agent for neurodegenerative diseases, demyelinating diseases, or cerebrovascular diseases, comprising one or more selected from the group consisting of mesenchymal stem cells, culture supernatant of mesenchymal stem cells, and cellular secretions of mesenchymal stem cells.

7. The preventive and / or therapeutic agent according to claim 6, wherein the neurodegenerative disease is Alzheimer's disease.

Images