WO2018034314A1 - Mesenchymal stem cell activation agent - Google Patents

Mesenchymal stem cell activation agent Download PDF

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WO2018034314A1
WO2018034314A1 PCT/JP2017/029483 JP2017029483W WO2018034314A1 WO 2018034314 A1 WO2018034314 A1 WO 2018034314A1 JP 2017029483 W JP2017029483 W JP 2017029483W WO 2018034314 A1 WO2018034314 A1 WO 2018034314A1
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mesenchymal stem
stem cell
cells
age
stem cells
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PCT/JP2017/029483
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French (fr)
Japanese (ja)
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本望　修
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北海道公立大学法人札幌医科大学
ニプロ株式会社
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Priority to JP2018534417A priority Critical patent/JP7076709B2/en
Publication of WO2018034314A1 publication Critical patent/WO2018034314A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention uses ⁇ -carotene as a mesenchymal stem cell activator and stem cell age-improving agent, and tissue repair by using the mesenchymal stem cell activator or stem cell age-improving agent and mesenchymal stem cells in combination -Regarding regenerative medicine.
  • MSC mesenchymal Stem Cell
  • neurotrophic / protective action by neurotrophic factor angiogenic action (recovery of cerebral blood flow), and nerve regeneration .
  • humoral factors such as BDNF (Brain Derived Neurotrophic Factor) and GDNF (Glial Derived Neurotrophic Factor) which are neurotrophic factors.
  • BDNF Brain Derived Neurotrophic Factor
  • GDNF Glial Derived Neurotrophic Factor
  • angiogenesis one is that MSC accumulated in the lesion site secretes angiogenic factors and induces angiogenesis, and the other is that the administered MSC itself is vascularized. It is to differentiate into endothelium to form new blood vessels.
  • MSC accumulated in the lesion promotes endogenous neurogenesis
  • the other is that the administered MSC itself becomes a nerve cell / glial cell. It is to differentiate.
  • all of the above-mentioned mechanisms of action are only speculations from the observed phenomenon, and a mechanism for treating cerebral infarction and spinal cord injury by intravenous administration of MSC has not been demonstrated.
  • ⁇ -carotene is a kind of carotenoid, has a strong antioxidant action, and is converted into vitamin A as needed in the body. Therefore, it is known that ⁇ -carotene has a function of maintaining normal mucosa, skin, immune function, and visual acuity, but its action on mesenchymal stem cells and its mechanism have not been elucidated.
  • the object of the present invention is to elucidate the action of physiologically active substances on the profile and therapeutic effects of mesenchymal stem cells (MSC), and based on the results, the development of cranial nerves, age-related changes, and damage healing are referred to as stem cell homeostasis. From a viewpoint, it is providing the new treatment method with respect to an intractable neurological disease.
  • MSC mesenchymal stem cells
  • mesenchymal stem cells in the bone marrow have a decreased number of cells and proliferative capacity as a result of aging.
  • administration of ⁇ -carotene activates mesenchymal stem cells, and It was confirmed that the number increased and showed high proliferation ability.
  • administration of ⁇ -carotene improved motor function and increased plasticity.
  • the present invention has been completed based on the above findings and relates to the following (1) to (11).
  • a mesenchymal stem cell activator comprising ⁇ -carotene as an active ingredient;
  • mesenchymal stem cell activator according to any one of (1) to (3) above, wherein the mesenchymal stem cells are CD24 negative; (5) An agent for improving stem cell age of mesenchymal stem cells, comprising ⁇ -carotene as an active ingredient; (6) The stem cell age improving agent according to (5), wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow or blood; (7) The agent for improving stem cell age according to (6) above, wherein the bone marrow or blood is bone marrow or blood of a subject receiving mesenchymal stem cells.
  • mesenchymal stem cells can be activated and the stem cell age can be rejuvenated by administration (intake) of ⁇ -carotene.
  • MSCs mesenchymal stem cells
  • ⁇ -carotene administration of ⁇ -carotene.
  • Figure 1 shows the bone marrow between the ginseng juice administration group (graph right) ingested ginseng juice from 24 to 62 weeks of age and the control group at 8 weeks of age (graph left) and 62 weeks of age (graph center). It is the graph which compared the proliferative ability of a leaf stem cell.
  • the vertical axis of the graph is the number of cells, and the horizontal axis is the number of passages (passage number (P), passage number (Day)). From the left of the graph, P0Day9 (9th day of primary culture), P1Day9 (1st passage, 3rd day), P2Day4 (2nd passage, 4th day), P3Day5 (3rd passage, day), P4Day6 (1st day) 4th passage, 6th day).
  • FIG. 2 compares the proliferative ability of bone marrow mesenchymal stem cells between the ⁇ -carotene administration group (graph right) in which ⁇ -carotene was intraperitoneally administered from 33 to 43 weeks of age and the same 43-week-old control group (graph left). It is a graph. The vertical axis of the graph is the number of cells, and the horizontal axis is the number of passages (passage number (P), passage number (Day)). From the left, P0Day9 (9th day of primary culture), P1Day9 (1st and 3rd day), P2Day4 (2nd and 4th day), P3Day5 (3rd and 5th day), P4Day6 (1st day) 4th passage, 6th day).
  • FIG. 3 compares the proliferative ability of bone marrow mesenchymal stem cells between the ginseng juice administration group (graph right) in which ginseng juice was orally ingested from 33 to 43 weeks of age and the same 43-week-old control group (graph left). It is a graph. The vertical axis of the graph is the number of cells, and the horizontal axis is the number of passages (passage number (P), passage number (Day)). From the left of the graph, P0Day9 (9th day of primary culture), P1Day9 (1st passage, 3rd day), P2Day4 (2nd passage, 4th day), P3Day5 (3rd passage, 5th day), P4Day6 ( 4th passage, 6th day).
  • FIG. 4 shows an outline of the experimental protocol of Example 2.
  • FIG. 5 is a graph comparing the improvement in motor function between the ⁇ -carotene administration group and the control group in cerebral infarction (subacute stage) model rats. The vertical axis of the graph is the maximum speed (m / min) that the rat can run, and the horizontal axis is the age of the rat.
  • FIG. 6 is a graph comparing the improvement in motor function between the MSC group and the DMEM group (control group) in cerebral infarction (chronic phase) model rats. The vertical axis of the graph is the maximum speed (m / min) that the rat can run, and the horizontal axis is the age of the rat.
  • the “mesenchymal stem cell activator” is a composition having an effect of reviving the number, proliferation ability and cell function of mesenchymal stem cells (MSC) which have decreased with aging or disease. Means.
  • the “mesenchymal stem cell activator” of the present invention has ⁇ carotene as an active ingredient, has the effect of activating MSC and rejuvenating its stem cell age.
  • the “mesenchymal stem cell activator” of the present invention can improve its tissue repair / regeneration ability and enhance the therapeutic effect of cerebral infarction, spinal cord injury, dementia and the like.
  • the “stem cell age-improving agent” is a composition for rejuvenating the stem cell age, and has a mesenchymal stem cell (MSC) cell number, proliferative ability and cell function that have decreased with aging and disease. Means a composition having a rejuvenating effect.
  • Stem cells are known to decrease in number, proliferation ability, differentiation ability, and stem cell functions associated with tissue repair / regeneration associated with aging. MSCs emerge from the bone marrow into peripheral blood, support systemic metabolism, and contribute to the maintenance of organs including blood vessels and epidermis. When the number and function of MSCs decline, aging progresses or they become ill (stem cell failure).
  • the “stem cell age” is an index indicating a cell age different from the actual age, in other words, a youth of a living body or a cell.
  • the “mesenchymal stem cell” is a stem cell having pluripotency and self-replicating ability that is present in a small amount in a stromal cell of a mesenchymal tissue, such as bone cell, chondrocyte, adipocyte, etc. In addition to differentiating into connective tissue cells, it is known to have the ability to differentiate into neurons and cardiomyocytes.
  • Mesenchymal stem cells exist throughout the body such as bone marrow, peripheral blood, umbilical cord blood, fetal embryo, placenta, fat, and brain.
  • mesenchymal stem cells derived from human bone marrow or blood (bone marrow mesenchymal stem cells).
  • human bone marrow mesenchymal stem cells are preferred.
  • Bone marrow mesenchymal stem cells 1) Expected to have significant effects, 2) Low risk of side effects, 3) Expected to supply sufficient donor cells, 4) Non-invasive treatment and autotransplantation possible 5) Low risk of infection, 6) No immune rejection, 7) No ethical problems, 8) Social acceptance, 9) Widespread establishment as general medical care There are advantages such as.
  • bone marrow transplantation is a treatment already used in clinical practice, and its safety has been confirmed.
  • bone marrow-derived stem cells are highly migratory and can reach the target damaged tissue not only by local transplantation but also by intravenous administration, and a therapeutic effect can be expected.
  • the cell may be a cell induced to differentiate from an ES cell or an induced pluripotent stem cell (iPS cell or the like), a cell established, or a cell isolated and proliferated from a living body.
  • the cells may be derived from other cells or autologous cells, but mesenchymal stem cells derived from autologous cells (derived from the patient's own cells) are preferred.
  • the mesenchymal stem cells are CD24 negative, which is a differentiation marker. It is preferable that the cells maintain an undifferentiated state.
  • CD24-negative mesenchymal stem cells are characterized by a high proliferation rate and a high survival rate after introduction into the living body.
  • ⁇ -carotene By using in combination with a mesenchymal stem cell activator or a stem cell age-improving agent ( ⁇ -carotene), It can be expected to establish a new therapeutic strategy and improve the therapeutic effect of refractory neurological diseases such as cerebral infarction, spinal cord injury, and dementia due to high MSC.
  • the inventors have also developed a method for obtaining such undifferentiated mesenchymal stem cells, the details of which are described in WO2009 / 002503.
  • the mesenchymal stem cells are positive for at least one selected from CD73, CD90, CD105, and CD200, and At least one selected from CD19, CD34, CD45, CD74, CD79 ⁇ , and HLA-DR is characterized by being negative.
  • the mesenchymal stem cells used in the present invention are positive for two or more of CD73, CD90, CD105, and CD200, and negative for four or more of CD19, CD34, CD45, CD74, CD79 ⁇ , and HLA-DR. It is characterized by being.
  • the mesenchymal stem cells used in the present invention are characterized by being positive for CD73, CD90, CD105, and CD200 and negative for CD19, CD34, CD45, CD74, CD79 ⁇ , and HLA-DR. It is done.
  • cells separated from bone marrow fluid or the like under conditions that do not substantially contact with an anticoagulant contain human serum (preferably autologous serum), and anticoagulation Growth is performed using a medium that does not contain an agent (such as heparin) or contains a very low concentration.
  • the density of the cells in the medium affects the nature of the cells and the direction of differentiation.
  • the properties of the cells change, so subculture at a maximum of 8,500 cells / cm 2 or less. More preferably, the subculture is performed when the number of cells reaches 5500 cells / cm 2 or more.
  • the medium is changed as few times as possible in consideration of the burden on the serum donor, for example, at least once a week, more preferably a week. Replace the medium once or twice.
  • the subculture is repeated until the total number of cells reaches 10 8 or more.
  • the number of cells required may vary depending on the purpose of use.
  • the number of mesenchymal stem cells required for transplantation for the treatment of cerebral infarction is considered to be 10 7 or more.
  • 10 7 mesenchymal stem cells can be obtained in about 12 days.
  • the proliferated mesenchymal stem cells may be stored by a technique such as cryopreservation (for example, in a deep freezer at ⁇ 152 ° C.) until use, if necessary.
  • a medium containing serum preferably human serum, more preferably autologous serum
  • dextran DMSO (medium for mammalian cells such as RPMI)
  • DMSO medium for mammalian cells
  • cells can be suspended and stored frozen at -150 ° C. in a cryopreservation solution containing 20.5 mL of normal filter-sterilized RPMI, 20.5 mL of autologous serum collected from patients, 5 mL of dextran, and 5 mL of DMSO.
  • Cryoserve and Dextran manufactured by Nipro Corporation can use low molecular dextran L injection manufactured by Otsuka Pharmaceutical, but are not limited thereto.
  • a “medicine repair / regeneration drug containing mesenchymal stem cells” is a cell preparation containing the above-mentioned “mesenchymal stem cells (MSC)”, and is a damaged site or an aging site due to aging (particularly, Nervous system tissues such as the brain and spinal cord are repaired and regenerated (WO 2009/034708). Furthermore, the inventors have also found that administration of MSC improves synapse formation and plasticity (Japanese Patent Application Nos. 2016-091286 and 2016-091300).
  • Synapse formation means that axons extending from nerve cells properly extend to the vicinity of the target cells and axons that establish neural connections, reach the target, and between the axon end and the target cells and axons. It is a process of forming synapses, and an important process of forming correct neural circuits. When the MSC according to the present invention reaches the affected area, it has the effect of reconstructing the neural circuit by differentiating into nerve cells and axons and forming synapses.
  • Plastic plasticity refers to a phenomenon in which nerve cells and brain circuits create an optimal treatment system according to the environment and needs.
  • the MSC according to the present invention also has a function of promoting “brain plasticity” that functions beyond the normal range so that an uninjured site compensates for the function of the damaged site.
  • MSC meenchymal stem cells
  • the number of mesenchymal stem cells contained in the “medicament for tissue repair / regeneration containing mesenchymal stem cells” is 10 7 or more, preferably 5 ⁇ 10 7 or more, more preferably 10 8 or more, and further preferably 5 ⁇ 10 8 or more.
  • tissue repair / regeneration drug containing mesenchymal stem cells is preferably a parenteral preparation, more preferably a parenteral systemic preparation, particularly an intravenous preparation.
  • parenteral preparation preferably a parenteral systemic preparation, particularly an intravenous preparation.
  • dosage form suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections, injections prepared at the time of use, and grafts.
  • a preparation for parenteral administration is in the form of an aqueous or non-aqueous isotonic sterile solution or suspension, for example, a pharmacologically acceptable carrier or vehicle, specifically, sterile water or saline, Medium (especially medium used for culturing mammalian cells such as RPMI), physiological buffer solution such as PBS, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, excipient, vehicle, preservative, binding
  • a pharmacologically acceptable carrier or vehicle specifically, sterile water or saline, Medium (especially medium used for culturing mammalian cells such as RPMI), physiological buffer solution such as PBS, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, excipient, vehicle, preservative, binding
  • physiological buffer solution such as PBS, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, excipient, vehicle, preservative, binding
  • Aqueous solutions for injection include, for example, physiological saline, culture media, physiological buffers such as PBS, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. And may be used in combination with an appropriate solubilizing agent such as alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol or a nonionic surfactant such as polysorbate 80, HCO-50, or the like.
  • physiological saline such as PBS
  • isotonic solutions containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc.
  • an appropriate solubilizing agent such as alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol or a nonionic surfactant such as polysorbate 80, HCO-50, or the like.
  • the “mesenchymal stem cell activator” or “stem cell age-improving agent” ( ⁇ -carotene) of the present invention can enhance the therapeutic effect of MSC by activating MSC and rejuvenating stem cell age.
  • the mesenchymal stem cell activator or stem cell age-improving agent of the present invention is used in combination with "a tissue repair / regeneration drug containing mesenchymal stem cells" to improve the therapeutic effect of MSC administration. Can be expected to do. Below, the effect is described concretely.
  • a cerebral infarction is a pathological condition in which cerebral ischemia is caused by occlusion or stenosis of a cerebral artery, and brain tissue becomes necrotic or close to this condition.
  • Mesenchymal stem cells have a protective effect on the brain (parenchyma and blood vessels), and in acute and subacute cerebral infarction, administration of MSC reduces infarct volume and improves behavioral function.
  • Administration of a mesenchymal stem cell activator or a stem cell age-improving agent can be expected to enhance the effect.
  • Necrotic cells and damaged nerve fibers do not return to their original state in the chronic phase. Therefore, in chronic cerebral infarction, in addition to preventing recurrence, recovery of non-dead cells and cells that have stopped functioning around necrotic cells and reducing the disease state are the main treatment. Has been considered. However, administration of MSC promotes reconstruction of the neural circuit and compensation by normal tissue, thereby making it possible to restore motor function and brain function even in chronic cerebral infarction. Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to enhance the effect.
  • MSCs of the present invention are amyotrophic lateral sclerosis (ALS), Parkinson's disease, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), black It is also useful for neurodegenerative diseases such as striatal degeneration (SND), Shy-Drager syndrome, olive bridge cerebellar atrophy (OPCA), spinocerebellar degeneration (SCD) and the like.
  • Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to further enhance its therapeutic effect.
  • vascular dementia can be treated with mesenchymal stem cells .
  • Alzheimer-type dementia and vascular dementia have similar pathological conditions, and in Alzheimer-type dementia, improvement of cognitive function by MSC can be expected.
  • Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to further enhance the therapeutic effect of MSC on dementia.
  • MSC of the present invention is used for schizophrenia, manic depression, personality disorder, mood disorder, psychological development disorder, stress-related disorder, autism, learning disorder, It is also useful for mental disorders such as emotional disorders, mental retardation, sleep disorders, eating disorders, identity disorders, dissociative disorders, adaptation disorders, alcoholic disorders, addictions.
  • Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to further enhance its therapeutic effect.
  • the MSC of the present invention improves higher-order functions such as attention disorder, memory disorder, aphasia, forgetfulness, apraxia, executive function, and emotional disorder.
  • administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to enhance the effect.
  • Life extension MSC of the present invention has effects of rejuvenation, physical strength enhancement and life extension in healthy individuals, and in patients with intractable neurological diseases such as dementia and cerebral infarction and spinal cord injury, Combined with improvement of motor function, it has a life extension effect after treatment.
  • Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to enhance the effect.
  • the dose of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention is not particularly limited, and is appropriately determined according to the administration (intake) method, the age of the subject, and the health condition.
  • the administration route of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention is not particularly limited, but oral administration (intake) is simple and preferable.
  • the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention contains ⁇ -carotene, its form / shape is not particularly limited, and may be a food or drink such as carrot juice or a supplement.
  • the mesenchymal stem cell activator of the present invention may be formulated into a liquid, tablet, capsule or the like together with a pharmacologically acceptable carrier.
  • Example 1 Effect on ⁇ -carotene mesenchymal stem cells
  • ginseng juice oral intake
  • SD rats female
  • the carrot juice administration group consists of 100% carrot juice from Chiba Prefecture (made by Ihashi Sangyo Co., Ltd. (Ikiiki Carrot Museum Sukoyaka)), about 100 ml / animal / day, and MF food (made by Oriental Yeast Co., Ltd.) as a free feed for 24 weeks.
  • DMEM Dulbecco's modified Eagle's medium
  • the control group was reared with free feeding (food: MF) and free water, and bone marrow (two femurs and two tibias) was collected at 8 and 62 weeks of age.
  • the collected bone marrow cells were subcultured 4 times in the same manner as in (1), and the number of cells was counted at the time of subculture. Evaluation was carried out by comparing the number of cultured cells (average number of 5 or 8 cells) per passage of the collected bone marrow up to P4.
  • ⁇ -carotene has a significant MSC activation effect and stem cell age rejuvenation effect. That is, by ingesting ⁇ -carotene, it can be expected that the tissue maintenance / repair / regeneration effect of MSC is improved, and the therapeutic effect of rejuvenation and illness (cerebral infarction, spinal cord injury, dementia, etc.) is improved.
  • Example 2 Effect of ⁇ -carotene administration in cerebral infarction (subacute stage) model rats Materials and Methods (1) Cerebral Infarction (Subacute Phase) Model Rat A rat middle cerebral artery occlusion (MCAO) model was used as a cerebral infarction model. As previously reported, 9-week-old female SD rats (200-250 g) were anesthetized with ketamine (75 mg / kg) and xylazine (10 mg / kg), and 20.0-22.0 mm embolic thread (MONOSOF) was externally necked. Inserted from the artery, permanent middle cerebral artery occlusion was performed (Honma T. et al., Exp. Neurol. 2006; 199: 56-66.
  • MCAO Cerebral Infarction
  • MCAO Middle cerebral artery occlusion
  • FIG. 4 shows the protocol of this embodiment.
  • Reference Example 1 Effects of MSC administration in cerebral infarction (chronic phase) model rats Materials / Methods (1) Preparation of rat bone marrow-derived mesenchymal stem cells Experiments were performed in accordance with animal experiment management regulations of Sapporo Medical University. According to previous reports, bone marrow obtained from femurs of adult SD rats was diluted to 25 ml with Dulbecco's modified Eagle's medium (DMEM) and heat-inactivated 10% FBS, 2 mM l-glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin was added and incubated for 3 days at 37 ° C. in 5% CO 2 atmosphere (Kim S. et al., Brain Res.
  • DMEM Dulbecco's modified Eagle's medium
  • MSC group MS rats of SD rats 8 weeks after MCAO, DMEM containing 1.0 ⁇ 10 6 P2 in 1 ml were administered from the femoral vein.
  • DMEM group 1 ml of DMEM was administered from the femoral vein.
  • the present invention is useful for establishing a new therapeutic strategy and improving the therapeutic effect of intractable neurological diseases such as cerebral infarction, spinal cord injury, and dementia caused by MSC.

Abstract

The present invention pertains to the use of β carotene as a mesenchymal stem cell activation agent and as a stem cell age improving agent, and tissue repair and regenerative medicine through combination use of the mesenchymal stem cell activation agent or the stem cell age improving agent and mesenchymal stem cells.

Description

間葉系幹細胞活性化剤Mesenchymal stem cell activator
［関連出願］
　本明細書は、本願の優先権の基礎である特願２０１６－１６０２６８号（２０１６年８月１８日出願）の明細書に記載された内容を包含する。
［技術分野］
　本発明は、間葉系幹細胞活性化剤及び幹細胞年齢改善剤としてのβカロテンの利用、ならびに、前記間葉系幹細胞活性化剤又は幹細胞年齢改善剤と間葉系幹細胞を併用することによる組織修復・再生医療に関する。 [Related applications]
This specification includes the contents described in the specification of Japanese Patent Application No. 2016-160268 (filed on August 18, 2016), which is the basis of the priority of the present application.
[Technical field]
The present invention uses β-carotene as a mesenchymal stem cell activator and stem cell age-improving agent, and tissue repair by using the mesenchymal stem cell activator or stem cell age-improving agent and mesenchymal stem cells in combination -Regarding regenerative medicine.
　間葉系幹細胞（Ｍｅｓｅｎｃｈｙｍａｌ　Ｓｔｅｍ　Ｃｅｌｌ：ＭＳＣ）には脳（実質及び血管）の保護作用があることが知られている。脳梗塞後のＭＳＣ投与は、梗塞体積を減らし、行動機能を改善することが、実験的梗塞モデルを用いて確認されている（非特許文献１～３、特許文献１）。また、ＭＳＣの静脈投与による脳梗塞患者の治療も多数実施され、運動機能や損傷部位の改善が報告されている（非特許文献４、特許文献２）。 It is known that a mesenchymal stem cell (Mesenchymal Stem Cell: MSC) has a protective effect on the brain (parenchyma and blood vessels). It has been confirmed using experimental infarction models that MSC administration after cerebral infarction reduces infarct volume and improves behavioral function (Non-patent Documents 1 to 3, Patent Document 1). In addition, many treatments for cerebral infarction patients by intravenous administration of MSC have been carried out, and improvements in motor function and damaged sites have been reported (Non-patent Document 4, Patent Document 2).
　脊髄損傷患者についても、ＭＳＣの静脈投与により、機能回復、及び軸索再生の促進、損傷部位の低減が認められている。ＭＳＣの効果は、これまで急性期の脊髄損傷患者においては多数報告されているが、慢性期の患者に対する研究は限られており、その効果は十分確認されていない。 Spinal cord injury patients have also been found to recover function, promote axonal regeneration, and reduce the damaged area by intravenous administration of MSC. Although the effect of MSC has been reported so far in patients with spinal cord injury in the acute phase, studies on patients in the chronic phase are limited, and the effect has not been fully confirmed.
　ＭＳＣの治療メカニズムについては、多数の作用機序が推測されており、これらは神経栄養因子による神経栄養・保護作用、血管新生作用（脳血流の回復）、神経再生の３つに分類される。神経栄養・保護作用は、神経栄養因子であるＢＤＮＦ（Brain Derived Neurotrophic Factor）やＧＤＮＦ（Glial Derived Neurotrophic Factor）等の液性因子を介して発揮されることが予測される。血管新生作用には、２つのメカニズムが考えられ、一つは病巣部に集積したＭＳＣが血管新生因子等を分泌し血管新生を誘導することであり、もう一つは投与されたＭＳＣ自身が血管内皮に分化して新たな血管を形成することである。神経再生作用も、２つのメカニズムが考えられ、一つは病巣部に集積したＭＳＣが内因性の神経形成を促進することであり、もう一つは投与されたＭＳＣ自身が神経細胞・グリア細胞へと分化することである。しかしながら、上記の作用機序はいずれも観察された現象からの推測にすぎず、ＭＳＣの静脈投与によって脳梗塞や脊髄損傷が治療されるメカニズムは実証されていない。 Numerous mechanisms of action have been speculated for the treatment mechanism of MSC, and these are classified into three categories: neurotrophic / protective action by neurotrophic factor, angiogenic action (recovery of cerebral blood flow), and nerve regeneration . It is predicted that the neurotrophic / protective action is exerted through humoral factors such as BDNF (Brain Derived Neurotrophic Factor) and GDNF (Glial Derived Neurotrophic Factor) which are neurotrophic factors. There are two possible mechanisms for angiogenesis: one is that MSC accumulated in the lesion site secretes angiogenic factors and induces angiogenesis, and the other is that the administered MSC itself is vascularized. It is to differentiate into endothelium to form new blood vessels. There are two possible mechanisms of nerve regeneration: one is that MSC accumulated in the lesion promotes endogenous neurogenesis, and the other is that the administered MSC itself becomes a nerve cell / glial cell. It is to differentiate. However, all of the above-mentioned mechanisms of action are only speculations from the observed phenomenon, and a mechanism for treating cerebral infarction and spinal cord injury by intravenous administration of MSC has not been demonstrated.
　間葉系幹細胞のプロファイルは加齢とともに変化する。脂肪由来の幹細胞について、セレンやβカロテンなどの抗酸化剤及びサイトカインを含む培地で培養することによりテロメラーゼ活性が向上することが報告されている（特許文献３）。βカロテンはカロテノイドの一種で、強い抗酸化作用を有し、体内では必要に応じてビタミンＡに変換される。そのため、βカロテンには、粘膜や皮膚、免疫機能、視力を正常に維持する機能があることが知られているが、間葉系幹細胞に対する作用やそのメカニズムについては解明されていない。 The profile of mesenchymal stem cells changes with age. It has been reported that stem cells derived from fat are improved in telomerase activity by culturing in a medium containing an antioxidant and cytokines such as selenium and β-carotene (Patent Document 3). β-carotene is a kind of carotenoid, has a strong antioxidant action, and is converted into vitamin A as needed in the body. Therefore, it is known that β-carotene has a function of maintaining normal mucosa, skin, immune function, and visual acuity, but its action on mesenchymal stem cells and its mechanism have not been elucidated.
ＷＯ２００２／０００８４９号WO2002 / 000849 ＷＯ２００９／０３４７０８号WO2009 / 034708 特表２０１５－５０００１８号Special table 2015-500018
　本発明の課題は、間葉系幹細胞（ＭＳＣ）のプロファイル及び治療効果に対する生理活性物質の作用を解明し、その結果に基づき、脳神経の発達・加齢変化・損傷治癒について、幹細胞の恒常性という観点から、難治性神経疾患に対する新たな治療方法を提供することにある。 The object of the present invention is to elucidate the action of physiologically active substances on the profile and therapeutic effects of mesenchymal stem cells (MSC), and based on the results, the development of cranial nerves, age-related changes, and damage healing are referred to as stem cell homeostasis. From a viewpoint, it is providing the new treatment method with respect to an intractable neurological disease.
　発明者らは、骨髄内の間葉系幹細胞は、加齢により細胞数が減少し増殖能も低下するが、βカロテン投与により間葉系幹細胞が活性化され、骨髄内の間葉系幹細胞の数が増え、高い増殖能を示すようになることを確認した。また、亜急性期の脳梗塞モデルにおいて、βカロテンを投与することで、運動機能の改善や可塑性の亢進がみられることを確認した。 The inventors of the present invention have found that mesenchymal stem cells in the bone marrow have a decreased number of cells and proliferative capacity as a result of aging. However, administration of β-carotene activates mesenchymal stem cells, and It was confirmed that the number increased and showed high proliferation ability. In addition, in a subacute cerebral infarction model, it was confirmed that administration of β-carotene improved motor function and increased plasticity.
　本発明は上記の知見に基づいて完成されたものであり、以下の（１）～（１１）に関する。
（１）βカロテンを有効成分とする、間葉系幹細胞活性化剤；
（２）間葉系幹細胞が骨髄又は血液に由来する間葉系幹細胞である、上記（１）に記載の間葉系幹細胞活性化剤；
（３）骨髄又は血液が、間葉系幹細胞の投与を受ける対象の骨髄又は血液である、上記（２）に記載の間葉系幹細胞活性化剤；
（４）間葉系幹細胞がＣＤ２４陰性である、上記（１）～（３）のいずれかに記載の間葉系幹細胞活性化剤；
（５）βカロテンを有効成分とする、間葉系幹細胞の幹細胞年齢改善剤；
（６）間葉系幹細胞が骨髄又は血液に由来する間葉系幹細胞である、上記（５）に記載の幹細胞年齢改善剤；
（７）骨髄又は血液が、間葉系幹細胞の投与を受ける対象の骨髄又は血液である、上記（６）に記載の幹細胞年齢改善剤；
（８）間葉系幹細胞がＣＤ２４陰性である、上記（５）～（７）のいずれかに記載の間葉系幹細胞年齢改善剤；
（９）ＣＤ２４陰性の間葉系幹細胞を含む組織修復・再生用医薬と併用することを特徴とする、上記（１）～（４）のいずれかに記載の間葉系幹細胞活性化剤又は上記（５）～（８）のいずれかに記載の幹細胞年齢改善剤；
（１０）上記（１）～（４）のいずれかに記載の間葉系幹細胞活性化剤又は上記（５）～（８）のいずれかに記載の幹細胞年齢改善剤と併用される、ＣＤ２４陰性の間葉系幹細胞を含む組織修復・再生用医薬；
（１１）上記（１）～（４）のいずれかに記載の間葉系幹細胞活性化剤又は上記（５）～（８）のいずれかに記載の幹細胞年齢改善剤と、ＣＤ２４陰性の間葉系幹細胞を含む細胞製剤とを組み合わせてなる、組織修復・再生用医薬。なお、上記「組織修復・再生用医薬」としては、脳梗塞、脊髄損傷、神経変性疾患、又は認知症の治療、高次機能の改善、寿命延長のための医薬が挙げられる。 The present invention has been completed based on the above findings and relates to the following (1) to (11).
(1) a mesenchymal stem cell activator comprising β-carotene as an active ingredient;
(2) The mesenchymal stem cell activator according to (1) above, wherein the mesenchymal stem cell is a mesenchymal stem cell derived from bone marrow or blood;
(3) The mesenchymal stem cell activator according to (2) above, wherein the bone marrow or blood is bone marrow or blood of a subject receiving mesenchymal stem cells.
(4) The mesenchymal stem cell activator according to any one of (1) to (3) above, wherein the mesenchymal stem cells are CD24 negative;
(5) An agent for improving stem cell age of mesenchymal stem cells, comprising β-carotene as an active ingredient;
(6) The stem cell age improving agent according to (5), wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow or blood;
(7) The agent for improving stem cell age according to (6) above, wherein the bone marrow or blood is bone marrow or blood of a subject receiving mesenchymal stem cells.
(8) The mesenchymal stem cell age-improving agent according to any one of (5) to (7) above, wherein the mesenchymal stem cells are CD24 negative;
(9) The mesenchymal stem cell activator according to any one of (1) to (4) above, which is used in combination with a tissue repair / regeneration medicament containing CD24-negative mesenchymal stem cells (5) to the stem cell age-improving agent according to any one of (8);
(10) CD24 negative in combination with the mesenchymal stem cell activator according to any one of (1) to (4) above or the stem cell age-improving agent according to any one of (5) to (8) above Tissue repair / regenerative medicine containing mesenchymal stem cells;
(11) The mesenchymal stem cell activator according to any one of (1) to (4) above or the stem cell age improving agent according to any one of (5) to (8) above, and a CD24-negative mesenchyme A drug for tissue repair / regeneration, which is combined with a cell preparation containing stem cells. Examples of the “medicine for tissue repair / regeneration” include drugs for treatment of cerebral infarction, spinal cord injury, neurodegenerative disease, or dementia, improvement of higher-order functions, and extension of life span.
　本発明により、βカロテンの投与（摂取）により間葉系幹細胞（ＭＳＣ）を活性化させ、幹細胞年齢を若返らせることができることが確認された。これにより、ＭＳＣによる脳梗塞、脊髄損傷、認知症などの難治性神経疾患の新たな治療戦略の確立や、若返り可能となる。 According to the present invention, it was confirmed that mesenchymal stem cells (MSCs) can be activated and the stem cell age can be rejuvenated by administration (intake) of β-carotene. This makes it possible to establish and rejuvenate new treatment strategies for intractable neurological diseases such as cerebral infarction, spinal cord injury, and dementia caused by MSC.
図１は、人参ジュースを２４～６２週齢にかけて経口摂取させた人参ジュース投与群（グラフ右）と、８週齢時（グラフ左）及び６２週齢時（グラフ中央）の対照群の骨髄間葉系幹細胞の増殖能を比較したグラフである。グラフ縦軸は細胞数、横軸は継代日数（継代数（Ｐ）、継代日数（Ｄａｙ））。グラフ左からＰ０Ｄａｙ９（初代培養９日目）、Ｐ１Ｄａｙ９（第１継代、３日目）、Ｐ２Ｄａｙ４（第２継代、４日目）、Ｐ３Ｄａｙ５（第３継代、日目）、Ｐ４Ｄａｙ６（第４継代、６日目）。Figure 1 shows the bone marrow between the ginseng juice administration group (graph right) ingested ginseng juice from 24 to 62 weeks of age and the control group at 8 weeks of age (graph left) and 62 weeks of age (graph center). It is the graph which compared the proliferative ability of a leaf stem cell. The vertical axis of the graph is the number of cells, and the horizontal axis is the number of passages (passage number (P), passage number (Day)). From the left of the graph, P0Day9 (9th day of primary culture), P1Day9 (1st passage, 3rd day), P2Day4 (2nd passage, 4th day), P3Day5 (3rd passage, day), P4Day6 (1st day) 4th passage, 6th day). 図２は、βカロテンを３３～４３週齢にかけて腹腔内投与したβカロテン投与群（グラフ右）と、同じ４３週齢の対照群（グラフ左）の骨髄間葉系幹細胞の増殖能を比較したグラフである。グラフ縦軸は細胞数、横軸は継代日数（継代数（Ｐ）、継代日数（Ｄａｙ））。左からＰ０Ｄａｙ９（初代培養９日目）、Ｐ１Ｄａｙ９（第１継代、３日目）、Ｐ２Ｄａｙ４（第２継代、４日目）、Ｐ３Ｄａｙ５（第３継代、５日目）、Ｐ４Ｄａｙ６（第４継代、６日目）。FIG. 2 compares the proliferative ability of bone marrow mesenchymal stem cells between the β-carotene administration group (graph right) in which β-carotene was intraperitoneally administered from 33 to 43 weeks of age and the same 43-week-old control group (graph left). It is a graph. The vertical axis of the graph is the number of cells, and the horizontal axis is the number of passages (passage number (P), passage number (Day)). From the left, P0Day9 (9th day of primary culture), P1Day9 (1st and 3rd day), P2Day4 (2nd and 4th day), P3Day5 (3rd and 5th day), P4Day6 (1st day) 4th passage, 6th day). 図３は、人参ジュースを３３～４３週齢にかけて経口摂取させた人参ジュース投与群（グラフ右）と、同じ４３週齢の対照群（グラフ左）の骨髄間葉系幹細胞の増殖能を比較したグラフである。グラフ縦軸は細胞数、横軸は継代日数（継代数（Ｐ）、継代日数（Ｄａｙ））。グラフ左からＰ０Ｄａｙ９（初代培養９日目）、Ｐ１Ｄａｙ９（第１継代、３日目）、Ｐ２Ｄａｙ４（第２継代、４日目）、Ｐ３Ｄａｙ５（第３継代、５日目）、Ｐ４Ｄａｙ６（第４継代、６日目）。FIG. 3 compares the proliferative ability of bone marrow mesenchymal stem cells between the ginseng juice administration group (graph right) in which ginseng juice was orally ingested from 33 to 43 weeks of age and the same 43-week-old control group (graph left). It is a graph. The vertical axis of the graph is the number of cells, and the horizontal axis is the number of passages (passage number (P), passage number (Day)). From the left of the graph, P0Day9 (9th day of primary culture), P1Day9 (1st passage, 3rd day), P2Day4 (2nd passage, 4th day), P3Day5 (3rd passage, 5th day), P4Day6 ( 4th passage, 6th day). 図４は、実施例２の実験プロトコルの概要を示す。FIG. 4 shows an outline of the experimental protocol of Example 2. 図５は、脳梗塞（亜急性期）モデルラットにおける、βカロテン投与群と対照群の運動機能の改善を比較したグラフである。グラフ縦軸はラットが走行できる最大速度（ｍ/分）、横軸はラットの週齢。FIG. 5 is a graph comparing the improvement in motor function between the β-carotene administration group and the control group in cerebral infarction (subacute stage) model rats. The vertical axis of the graph is the maximum speed (m / min) that the rat can run, and the horizontal axis is the age of the rat. 図６は、脳梗塞（慢性期）モデルラットにおける、ＭＳＣ群とＤＭＥＭ群（対照群）の運動機能の改善を比較したグラフである。グラフ縦軸はラットが走行できる最大速度（ｍ/分）、横軸はラットの週齢。FIG. 6 is a graph comparing the improvement in motor function between the MSC group and the DMEM group (control group) in cerebral infarction (chronic phase) model rats. The vertical axis of the graph is the maximum speed (m / min) that the rat can run, and the horizontal axis is the age of the rat.
［間葉系幹細胞活性化剤］
　本発明にかかる「間葉系幹細胞活性化剤」とは、加齢や病気に伴って低下した間葉系幹細胞（ＭＳＣ）の細胞数や増殖能や細胞機能を、復活させる効果を有する組成物を意味する。 [Mesenchymal stem cell activator]
The “mesenchymal stem cell activator” according to the present invention is a composition having an effect of reviving the number, proliferation ability and cell function of mesenchymal stem cells (MSC) which have decreased with aging or disease. Means.
　本発明の「間葉系幹細胞活性化剤」は、βカロテンを有効成分とし、ＭＳＣを活性化し、その幹細胞年齢を若返らせる効果を有する。本発明の「間葉系幹細胞活性化剤」は、ＭＳＣと併用することで、その組織修復・再生能を向上させ、脳梗塞、脊髄損傷、認知症等の治療効果を高めることができる。 The “mesenchymal stem cell activator” of the present invention has β carotene as an active ingredient, has the effect of activating MSC and rejuvenating its stem cell age. When used together with MSC, the “mesenchymal stem cell activator” of the present invention can improve its tissue repair / regeneration ability and enhance the therapeutic effect of cerebral infarction, spinal cord injury, dementia and the like.
［幹細胞年齢改善剤］
　本発明にかかる「幹細胞年齢改善剤」とは、幹細胞年齢を若返らせる組成物であって、加齢や病気に伴って低下した間葉系幹細胞（ＭＳＣ）の細胞数や増殖能や細胞機能を、復活させる効果を有する組成物を意味する。
　幹細胞は、加齢とともにその数や増殖能や分化能、及びそれに伴う組織修復・再生等の幹細胞機能の低下が知られている。ＭＳＣは骨髄から末梢血に出て、全身の新陳代謝を支持し、血管や表皮を含む各臓器の維持に貢献する。ＭＳＣの数や機能が衰えると、加齢が進んだり、病気になったりする（ｓｔｅｍ ｃｅｌｌ　ｆａｉｌｕｒｅ）。同じ年齢でも、運動機能や健康状態は異なり、寿命も各人で異なる。このような加齢の個人差は、ＭＳＣの活性化度に依存していると考えられ、個々の生体の加齢（年齢）の本体はＭＳＣ活性の低下にあると考えられる。本発明にかかる「幹細胞年齢」とは、このような実年齢とは異なる細胞年齢、換言すれば、生体や細胞の若さを示す指標である。 [Stem cell age improver]
The “stem cell age-improving agent” according to the present invention is a composition for rejuvenating the stem cell age, and has a mesenchymal stem cell (MSC) cell number, proliferative ability and cell function that have decreased with aging and disease. Means a composition having a rejuvenating effect.
Stem cells are known to decrease in number, proliferation ability, differentiation ability, and stem cell functions associated with tissue repair / regeneration associated with aging. MSCs emerge from the bone marrow into peripheral blood, support systemic metabolism, and contribute to the maintenance of organs including blood vessels and epidermis. When the number and function of MSCs decline, aging progresses or they become ill (stem cell failure). Even at the same age, motor function and health status are different, and life expectancy varies from person to person. Such individual differences in aging are considered to depend on the degree of activation of MSC, and the body of aging (age) of each living organism is considered to be in a decrease in MSC activity. The “stem cell age” according to the present invention is an index indicating a cell age different from the actual age, in other words, a youth of a living body or a cell.
［間葉系幹細胞］
　本発明にかかる「間葉系幹細胞」とは、間葉系組織の間質細胞の中に微量に存在する多分化能および自己複製能を有する幹細胞であり、骨細胞、軟骨細胞、脂肪細胞などの結合組織細胞に分化するだけでなく、神経細胞や心筋細胞への分化能を有することが知られている。 [Mesenchymal stem cells]
The “mesenchymal stem cell” according to the present invention is a stem cell having pluripotency and self-replicating ability that is present in a small amount in a stromal cell of a mesenchymal tissue, such as bone cell, chondrocyte, adipocyte, etc. In addition to differentiating into connective tissue cells, it is known to have the ability to differentiate into neurons and cardiomyocytes.
　間葉系幹細胞は、骨髄、末梢血、臍帯血、胎児胚、胎盤、脂肪、脳など全身に存在するが、本発明においてはヒト骨髄又は血液由来の間葉系幹細胞（骨髄間葉系幹細胞）、とくにヒト骨髄間葉系幹細胞が好ましい。骨髄間葉系幹細胞は、１）顕著な効果が期待できる、２）副作用の危険性が低い、３）充分なドナー細胞の供給が期待できる、４）非侵襲的な治療であり自家移植が可能である、５）感染症のリスクが低い、６）免疫拒絶反応の心配がない、７）倫理的問題がない、８）社会的に受け入れられやすい、９）一般的な医療として広く定着しやすいなどの利点がある。さらに、骨髄移植療法は、既に臨床の現場で用いられている治療であり、安全性も確認されている。また、骨髄由来の幹細胞は遊走性が高く、局所への移植ばかりか、静脈内投与によっても目的の損傷組織へ到達し、治療効果が期待できる。 Mesenchymal stem cells exist throughout the body such as bone marrow, peripheral blood, umbilical cord blood, fetal embryo, placenta, fat, and brain. In the present invention, mesenchymal stem cells derived from human bone marrow or blood (bone marrow mesenchymal stem cells). In particular, human bone marrow mesenchymal stem cells are preferred. Bone marrow mesenchymal stem cells: 1) Expected to have significant effects, 2) Low risk of side effects, 3) Expected to supply sufficient donor cells, 4) Non-invasive treatment and autotransplantation possible 5) Low risk of infection, 6) No immune rejection, 7) No ethical problems, 8) Social acceptance, 9) Widespread establishment as general medical care There are advantages such as. Furthermore, bone marrow transplantation is a treatment already used in clinical practice, and its safety has been confirmed. In addition, bone marrow-derived stem cells are highly migratory and can reach the target damaged tissue not only by local transplantation but also by intravenous administration, and a therapeutic effect can be expected.
　細胞は、ＥＳ細胞や誘導多能性幹細胞（ｉＰＳ細胞等）から分化誘導した細胞であっても、株化された細胞であっても、生体から単離・増殖させた細胞であってもよい。細胞は、他家細胞由来でも自家細胞由来であってもよいが、自家細胞由来（患者自身の細胞に由来する）間葉系幹細胞が好ましい。 The cell may be a cell induced to differentiate from an ES cell or an induced pluripotent stem cell (iPS cell or the like), a cell established, or a cell isolated and proliferated from a living body. . The cells may be derived from other cells or autologous cells, but mesenchymal stem cells derived from autologous cells (derived from the patient's own cells) are preferred.
　後述するように、間葉系幹細胞が医薬として、本発明に係る間葉系幹細胞活性化剤又は幹細胞年齢改善剤（βカロテン）と併用される場合、間葉系幹細胞は分化マーカーであるＣＤ２４陰性であり、未分化状態を維持した細胞であることが好ましい。ＣＤ２４陰性の間葉系幹細胞は、増殖率および生体内導入後の生存率が高いという特徴を有し、間葉系幹細胞活性化剤又は幹細胞年齢改善剤（βカロテン）と併用することによって、より高いＭＳＣによる脳梗塞、脊髄損傷、認知症などの難治性神経疾患の新たな治療戦略の確立及び治療効果の改善が期待できる。発明者らは、こうした未分化な間葉系幹細胞の取得方法も開発しており、その詳細はＷＯ２００９／００２５０３号に記載されている。 As described later, when mesenchymal stem cells are used in combination with the mesenchymal stem cell activator or stem cell age-improving agent (β-carotene) according to the present invention as a medicine, the mesenchymal stem cells are CD24 negative, which is a differentiation marker. It is preferable that the cells maintain an undifferentiated state. CD24-negative mesenchymal stem cells are characterized by a high proliferation rate and a high survival rate after introduction into the living body. By using in combination with a mesenchymal stem cell activator or a stem cell age-improving agent (β-carotene), It can be expected to establish a new therapeutic strategy and improve the therapeutic effect of refractory neurological diseases such as cerebral infarction, spinal cord injury, and dementia due to high MSC. The inventors have also developed a method for obtaining such undifferentiated mesenchymal stem cells, the details of which are described in WO2009 / 002503.
　本発明に係る間葉系幹細胞活性化剤あるいは幹細胞年齢改善剤と併用される場合、ＣＤ２４のほか、間葉系幹細胞は、ＣＤ７３、ＣＤ９０、ＣＤ１０５、及びＣＤ２００から選ばれる少なくとも１以上が陽性、及び／又はＣＤ１９、ＣＤ３４、ＣＤ４５、ＣＤ７４、ＣＤ７９α、及びＨＬＡ－ＤＲから選ばれる少なくとも１以上が陰性であることで特徴づけられる。好ましくは、本発明で使用される間葉系幹細胞は、ＣＤ７３、ＣＤ９０、ＣＤ１０５、及びＣＤ２００の２以上が陽性であり、ＣＤ１９、ＣＤ３４、ＣＤ４５、ＣＤ７４、ＣＤ７９α、及びＨＬＡ－ＤＲの４以上が陰性であることで特徴づけられる。より好ましくは本発明で使用される間葉系幹細胞は、ＣＤ７３、ＣＤ９０、ＣＤ１０５、及びＣＤ２００が陽性であり、ＣＤ１９、ＣＤ３４、ＣＤ４５、ＣＤ７４、ＣＤ７９α、及びＨＬＡ－ＤＲが陰性であることで特徴づけられる。 When used in combination with the mesenchymal stem cell activator or stem cell age improver according to the present invention, in addition to CD24, the mesenchymal stem cells are positive for at least one selected from CD73, CD90, CD105, and CD200, and At least one selected from CD19, CD34, CD45, CD74, CD79α, and HLA-DR is characterized by being negative. Preferably, the mesenchymal stem cells used in the present invention are positive for two or more of CD73, CD90, CD105, and CD200, and negative for four or more of CD19, CD34, CD45, CD74, CD79α, and HLA-DR. It is characterized by being. More preferably, the mesenchymal stem cells used in the present invention are characterized by being positive for CD73, CD90, CD105, and CD200 and negative for CD19, CD34, CD45, CD74, CD79α, and HLA-DR. It is done.
　発明者らが開発した前記方法では、骨髄液等から抗凝固剤（ヘパリン等）と実質的に接触しない条件で分離した細胞を、ヒト血清（好ましくは、自家血清）を含み、かつ、抗凝固剤（ヘパリン等）を含まないかあるいは極めて低濃度で含む培地を用いて増殖させる。 In the method developed by the inventors, cells separated from bone marrow fluid or the like under conditions that do not substantially contact with an anticoagulant (such as heparin) contain human serum (preferably autologous serum), and anticoagulation Growth is performed using a medium that does not contain an agent (such as heparin) or contains a very low concentration.
　培地における細胞の密度は、細胞の性質および分化の方向性に影響を与える。間葉系幹細胞の場合、培地中の細胞密度が８，５００個／ｃｍ2を超えると、細胞の性質が変化してしまうため、最大でも８，５００個／ｃｍ2以下で継代培養させることが好ましく、より好ましくは、５，５００個／ｃｍ2以上になった時点で継代培養させる。 The density of the cells in the medium affects the nature of the cells and the direction of differentiation. In the case of mesenchymal stem cells, if the cell density in the medium exceeds 8,500 cells / cm 2 , the properties of the cells change, so subculture at a maximum of 8,500 cells / cm 2 or less. More preferably, the subculture is performed when the number of cells reaches 5500 cells / cm 2 or more.
　発明者らが開発した前記方法ではヒト血清含有培地を使用するため、血清ドナーの負担を考慮して、培地交換はなるべく少ない回数であることが望ましく、例えば、少なくとも週１回、より好ましくは週１～２回の培地交換を行う。 In the method developed by the inventors, since a human serum-containing medium is used, it is desirable that the medium is changed as few times as possible in consideration of the burden on the serum donor, for example, at least once a week, more preferably a week. Replace the medium once or twice.
　培養は、細胞の総数が１０8個以上になるまで継代培養を繰り返し行う。必要とされる細胞数は、使用目的に応じて変化し得るが、例えば、脳梗塞の治療のための移植に必要とされる間葉系幹細胞の数は、１０7個以上と考えられている。発明者らが開発した方法によれば、１２日間程度で１０7個の間葉系幹細胞を得ることができる。 The subculture is repeated until the total number of cells reaches 10 8 or more. The number of cells required may vary depending on the purpose of use. For example, the number of mesenchymal stem cells required for transplantation for the treatment of cerebral infarction is considered to be 10 7 or more. . According to the method developed by the inventors, 10 7 mesenchymal stem cells can be obtained in about 12 days.
　増殖した間葉系幹細胞は、必要に応じて、使用されるまで凍結保存などの手法で（例えば、－１５２℃のディープフリーザーにて）保存してもよい。凍結保存には、血清（好ましくはヒト血清、より好ましくは自家血清）、デキストラン、ＤＭＳＯを含む培地（ＲＰＭＩ等の哺乳動物細胞用の培地）を凍結保存液として使用する。例えば、通常の濾過滅菌したＲＰＭＩ２０．５ｍＬと、患者から採取した自己血清２０．５ｍＬ、デキストラン５ｍＬ、ＤＭＳＯ ５ｍＬを含む凍結保存液に細胞を懸濁して－１５０℃で凍結保存することができる。例えば、ＤＭＳＯとしては、ニプロ株式会社製のクライオザーブ、デキストランは大塚製薬製の低分子デキストランＬ注を使用できるが、これらに限定されない。 The proliferated mesenchymal stem cells may be stored by a technique such as cryopreservation (for example, in a deep freezer at −152 ° C.) until use, if necessary. For cryopreservation, a medium containing serum (preferably human serum, more preferably autologous serum), dextran, DMSO (medium for mammalian cells such as RPMI) is used as a cryopreservation solution. For example, cells can be suspended and stored frozen at -150 ° C. in a cryopreservation solution containing 20.5 mL of normal filter-sterilized RPMI, 20.5 mL of autologous serum collected from patients, 5 mL of dextran, and 5 mL of DMSO. For example, as DMSO, Cryoserve and Dextran manufactured by Nipro Corporation can use low molecular dextran L injection manufactured by Otsuka Pharmaceutical, but are not limited thereto.
［間葉系幹細胞を含む組織修復・再生用医薬］
　本発明にかかる「間葉系幹細胞を含む組織修復・再生用医薬」は、前述した「間葉系幹細胞（ＭＳＣ）」を含む細胞製剤であって、損傷部位もしくは加齢による老化した部位（とくに脳や脊髄等の神経系組織）を修復・再生させる（ＷＯ２００９／０３４７０８号）。さらに、発明者らは、ＭＳＣの投与は、シナプス形成や可塑性を向上させることも見出している（特願２０１６－０９１２８６、特願２０１６－０９１３００）。 [Medicine for tissue repair / regeneration including mesenchymal stem cells]
A “medicine repair / regeneration drug containing mesenchymal stem cells” according to the present invention is a cell preparation containing the above-mentioned “mesenchymal stem cells (MSC)”, and is a damaged site or an aging site due to aging (particularly, Nervous system tissues such as the brain and spinal cord are repaired and regenerated (WO 2009/034708). Furthermore, the inventors have also found that administration of MSC improves synapse formation and plasticity (Japanese Patent Application Nos. 2016-091286 and 2016-091300).
　シナプス形成とは、神経細胞から伸びた軸索が、神経結合を成立させる標的細胞や軸策付近まで適切に伸長し、標的に到達して、軸索末端と標的細胞や軸策との間にシナプスを形成させる過程であり、正しい神経回路形成の重要なプロセスである。本発明に係るＭＳＣは、患部に到達すると、神経細胞や軸策に分化し、シナプスを形成することで、神経回路を再建する効果を有する。 Synapse formation means that axons extending from nerve cells properly extend to the vicinity of the target cells and axons that establish neural connections, reach the target, and between the axon end and the target cells and axons. It is a process of forming synapses, and an important process of forming correct neural circuits. When the MSC according to the present invention reaches the affected area, it has the effect of reconstructing the neural circuit by differentiating into nerve cells and axons and forming synapses.
　脳の可塑性とは、神経細胞や脳回路が環境や必要に応じて最適の処理システムを作り上げる現象を言う。本発明に係るＭＳＣは、損傷を受けていない部位が、損傷部位の機能を代償するように、通常範囲を超えて機能する「脳の可塑性」を促進する機能も有する。 “Plastic plasticity” refers to a phenomenon in which nerve cells and brain circuits create an optimal treatment system according to the environment and needs. The MSC according to the present invention also has a function of promoting “brain plasticity” that functions beyond the normal range so that an uninjured site compensates for the function of the damaged site.
　さらに、発明者らは、「間葉系幹細胞（ＭＳＣ）」の投与は、投与された対象の寿命を延長させる効果を有することも見出している。 Furthermore, the inventors have also found that administration of “mesenchymal stem cells (MSC)” has an effect of extending the life of the administered subject.
　本発明にかかる「間葉系幹細胞を含む組織修復・再生用医薬」に含まれる間葉系幹細胞の細胞数は多い程好ましいが、対象への投与時期や、培養に要する時間を勘案すると、効果を示す最小量であることが実用的である。したがって、好ましい態様において、間葉系幹細胞の細胞数は、１０7個以上、好ましくは５×１０7個以上、より好ましくは１０8個以上、さらに好ましくは５×１０8個以上含まれる。 The larger the number of mesenchymal stem cells contained in the “medicament for tissue repair / regeneration containing mesenchymal stem cells” according to the present invention, the more preferable, but the effect is considered in consideration of the administration time to the subject and the time required for culture. It is practical that the minimum amount of Therefore, in a preferred embodiment, the number of mesenchymal stem cells is 10 7 or more, preferably 5 × 10 7 or more, more preferably 10 8 or more, and further preferably 5 × 10 8 or more.
　本発明にかかる「間葉系幹細胞を含む組織修復・再生用医薬」は、好ましくは非経口投与製剤、より好ましくは非経口全身投与製剤、特に静脈内投与製剤である。非経口投与に適した剤形としては、溶液性注射剤、懸濁性注射剤、乳濁性注射剤、用時調製型注射剤等の注射剤や移植片などが挙げられる。非経口投与用製剤は、水性または非水性の等張性無菌溶液または懸濁液の形態であり、例えば、薬理学上許容される担体もしくは媒体、具体的には、滅菌水や生理食塩水、培地（とくに、ＲＰＭＩ等の哺乳動物細胞の培養に用いられる培地）、ＰＢＳなどの生理緩衝液、植物油、乳化剤、懸濁剤、界面活性剤、安定剤、賦形剤、ビヒクル、防腐剤、結合剤等を適宜組み合わせて、適切な単位投与形態に製剤化される。 The “tissue repair / regeneration drug containing mesenchymal stem cells” according to the present invention is preferably a parenteral preparation, more preferably a parenteral systemic preparation, particularly an intravenous preparation. Examples of the dosage form suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections, injections prepared at the time of use, and grafts. A preparation for parenteral administration is in the form of an aqueous or non-aqueous isotonic sterile solution or suspension, for example, a pharmacologically acceptable carrier or vehicle, specifically, sterile water or saline, Medium (especially medium used for culturing mammalian cells such as RPMI), physiological buffer solution such as PBS, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, excipient, vehicle, preservative, binding An appropriate unit dosage form is formulated by appropriately combining agents and the like.
　注射用の水溶液としては、例えば、生理食塩水、培地、ＰＢＳなどの生理緩衝液、ブドウ糖やその他の補助剤を含む等張液、例えばＤ－ソルビトール、Ｄ－マンノース、Ｄ－マンニトール、塩化ナトリウム等が挙げられ、適当な溶解補助剤、例えばアルコール、具体的にはエタノール、ポリアルコール、プロピレングリコール、ポリエチレングリコールや非イオン性界面活性剤、例えばポリソルベート８０、ＨＣＯ－５０等と併用してもよい。 Aqueous solutions for injection include, for example, physiological saline, culture media, physiological buffers such as PBS, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. And may be used in combination with an appropriate solubilizing agent such as alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol or a nonionic surfactant such as polysorbate 80, HCO-50, or the like.
［間葉系幹細胞活性化剤又は幹細胞年齢改善剤と間葉系幹細胞を含む組織修復・再生用医薬との併用］
　本発明の「間葉系幹細胞活性化剤」又は「幹細胞年齢改善剤」（βカロテン）は、ＭＳＣを活性化させ、幹細胞年齢を若返らせることで、ＭＳＣの治療効果を高めることができる。具体的に言えば、本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤を、「間葉系幹細胞を含む組織修復・再生用医薬」と併用することで、ＭＳＣ投与による治療効果を改善することが期待できる。以下に、その効果を具体的に記載する。 [Combination of a mesenchymal stem cell activator or stem cell age-improving agent with a tissue repair / regeneration drug containing mesenchymal stem cells]
The “mesenchymal stem cell activator” or “stem cell age-improving agent” (β-carotene) of the present invention can enhance the therapeutic effect of MSC by activating MSC and rejuvenating stem cell age. Specifically, the mesenchymal stem cell activator or stem cell age-improving agent of the present invention is used in combination with "a tissue repair / regeneration drug containing mesenchymal stem cells" to improve the therapeutic effect of MSC administration. Can be expected to do. Below, the effect is described concretely.
(1)脳梗塞の治療
　脳梗塞は、脳動脈の閉塞または狭窄のために脳虚血を来たし、脳組織が壊死またはこれに近い状態になる病態を言う。間葉系幹細胞には脳（実質及び血管）の保護作用があり、急性期や亜急性期の脳梗塞においては、ＭＳＣの投与は、梗塞体積を減らし、行動機能を改善するが、本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与は、その効果をより高めることが期待できる。 (1) Treatment of cerebral infarction A cerebral infarction is a pathological condition in which cerebral ischemia is caused by occlusion or stenosis of a cerebral artery, and brain tissue becomes necrotic or close to this condition. Mesenchymal stem cells have a protective effect on the brain (parenchyma and blood vessels), and in acute and subacute cerebral infarction, administration of MSC reduces infarct volume and improves behavioral function. Administration of a mesenchymal stem cell activator or a stem cell age-improving agent can be expected to enhance the effect.
　壊死した細胞や損傷を受けた神経線維は慢性期になると元には戻らない。そのため、慢性期の脳梗塞においては、再発の防止とともに、壊死した細胞の周辺に存在する、死滅していない細胞や、機能停止している細胞を回復させ、病状を軽減することが治療の中心と考えられてきた。しかし、ＭＳＣの投与は、神経回路の再建と正常組織による代償を促進させることで、慢性期の脳梗塞においても、運動機能や脳機能の回復が可能になる。本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与は、その効果をより高めることが期待できる。 Necrotic cells and damaged nerve fibers do not return to their original state in the chronic phase. Therefore, in chronic cerebral infarction, in addition to preventing recurrence, recovery of non-dead cells and cells that have stopped functioning around necrotic cells and reducing the disease state are the main treatment. Has been considered. However, administration of MSC promotes reconstruction of the neural circuit and compensation by normal tissue, thereby making it possible to restore motor function and brain function even in chronic cerebral infarction. Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to enhance the effect.
(2)脊髄損傷の治療
　脊髄を含む中枢神経系は末梢神経と異なり、一度損傷すると修復・再生されることはない。とくに、瘢痕化の進んだ慢性期脊髄損傷に対する治療は難しく、ＥＳ細胞を用いた臨床試験も試みられたが成功に至っていない。しかし、間葉系幹細胞の投与は、神経回路の再建と正常組織による代償を促進させることで、亜急性期のみならず、慢性期の脳梗塞においても、運動機能や神経機能の回復が可能になる。本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与は、その効果をより高めることが期待できる。 (2) Treatment of spinal cord injury The central nervous system including the spinal cord is different from the peripheral nerve, and once it is damaged, it cannot be repaired or regenerated. In particular, it is difficult to treat chronic spinal cord injury with advanced scarring, and clinical trials using ES cells have been attempted but have not been successful. However, administration of mesenchymal stem cells promotes reconstruction of neural circuits and compensation by normal tissues, enabling recovery of motor and neural functions not only in the subacute phase but also in chronic strokes Become. Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to enhance the effect.
(3)神経変性疾患の治療
　本発明のＭＳＣは、筋萎縮性側索硬化症（ＡＬＳ）、パーキンソン病、進行性核上性麻痺（ＰＳＰ）、ハンチントン病、多系統萎縮症（ＭＳＡ）、黒質線状体変性症（ＳＮＤ）、シャイ・ドレーガー症候群、オリーブ橋小脳萎縮症（ＯＰＣＡ）、脊髄小脳変性症（ＳＣＤ）等の神経変性疾患にも有用である。本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与は、その治療効果をより高めることが期待できる。 (3) Treatment of neurodegenerative diseases MSCs of the present invention are amyotrophic lateral sclerosis (ALS), Parkinson's disease, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), black It is also useful for neurodegenerative diseases such as striatal degeneration (SND), Shy-Drager syndrome, olive bridge cerebellar atrophy (OPCA), spinocerebellar degeneration (SCD) and the like. Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to further enhance its therapeutic effect.
(4)認知症の治療
　発明者らは、脳卒中易発性高血圧自然発症ラットにおいて、ＭＳＣの静脈投与により認知機能が改善され、血管性認知症が間葉系幹細胞により治療できることを実証している。アルツハイマー型認知症と血管性認知症は病態が似ており、アルツハイマー型認知症においても、ＭＳＣによる認知機能の改善が期待できる。本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与は、認知症に対するＭＳＣの治療効果をより高めることが期待できる。 (4) Treatment of dementia The inventors have demonstrated that cognitive function is improved by intravenous administration of MSC in cerebral stroke-prone spontaneously hypertensive rats, and vascular dementia can be treated with mesenchymal stem cells . Alzheimer-type dementia and vascular dementia have similar pathological conditions, and in Alzheimer-type dementia, improvement of cognitive function by MSC can be expected. Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to further enhance the therapeutic effect of MSC on dementia.
(5)精神疾患の治療
　上記した疾患のほか、本発明のＭＳＣは、統合失調症、躁うつ病、人格障害、気分障害、心理発達障害、ストレス関連障害、自閉症、学習障害、行動・情緒障害、精神遅滞、睡眠障害、摂食障害、同一性障害、解離性障害、適応障害、アルコール性障害、依存症、等の精神疾患にも有用である。本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与は、その治療効果をより高めることが期待できる。 (5) Treatment of mental illness In addition to the above-mentioned diseases, MSC of the present invention is used for schizophrenia, manic depression, personality disorder, mood disorder, psychological development disorder, stress-related disorder, autism, learning disorder, It is also useful for mental disorders such as emotional disorders, mental retardation, sleep disorders, eating disorders, identity disorders, dissociative disorders, adaptation disorders, alcoholic disorders, addictions. Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to further enhance its therapeutic effect.
(6)高次機能
　本発明のＭＳＣは、運動機能や単純な認知機能の改善に加えて、注意障害、記憶障害、失語症、失念、失行、遂行機能、情緒障害等の高次機能を改善することもできるが、本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与は、その効果をより高めることが期待できる。 (6) Higher-order function In addition to improving motor function and simple cognitive function, the MSC of the present invention improves higher-order functions such as attention disorder, memory disorder, aphasia, forgetfulness, apraxia, executive function, and emotional disorder. However, administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to enhance the effect.
(7)寿命延長
　本発明のＭＳＣは、健常人においては、若返り、体力増強、寿命延長の効果を有し、認知症ならびに脳梗塞及び脊髄損傷などの難治性神経疾患患者においては、神経機能や運動機能の改善と合わせて、治療後の寿命延長効果を有する。本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与は、その効果をより高めることが期待できる。 (7) Life extension MSC of the present invention has effects of rejuvenation, physical strength enhancement and life extension in healthy individuals, and in patients with intractable neurological diseases such as dementia and cerebral infarction and spinal cord injury, Combined with improvement of motor function, it has a life extension effect after treatment. Administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention can be expected to enhance the effect.
　本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与量は、特に限定されず、投与（摂取）方法、対象の年齢、健康状態に応じて適宜決定される。 The dose of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention is not particularly limited, and is appropriately determined according to the administration (intake) method, the age of the subject, and the health condition.
　本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与経路は特に限定されないが、経口投与（摂取）が簡便で好ましい。 The administration route of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention is not particularly limited, but oral administration (intake) is simple and preferable.
　本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤はβカロテンを含む限り、その形態・形状は特に限定されず、人参ジュースのような飲食品であっても、サプリメントであってもよい。また、本発明の間葉系幹細胞活性化剤は、薬理学的に許容し得る担体とともに、液剤、錠剤、カプセル剤などの形態に製剤化されていてもよい。 As long as the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention contains β-carotene, its form / shape is not particularly limited, and may be a food or drink such as carrot juice or a supplement. . In addition, the mesenchymal stem cell activator of the present invention may be formulated into a liquid, tablet, capsule or the like together with a pharmacologically acceptable carrier.
[リハビリテーション]
　本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤の投与とＭＳＣの併用による治療は、さらにリハビリテーションと併用することにより、格段にその効果が向上することが期待できる。脳梗塞や脊髄損傷患者において、リハビリテーションが可塑性を向上させることは公知であるが、本発明の間葉系幹細胞活性化剤又は幹細胞年齢改善剤（βカロテン）投与とＭＳＣ投与に、リハビリテーションを併用することにより、可塑性促進機能はさらに相乗的に向上する。実施方法は特に限定されず、ロボットによるリハビリテーションや、ＢＭＩ(brain machine interface)による訓練でもよい。 [Rehabilitation]
It is expected that the effects of the administration of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention and the combined use of MSC will be remarkably improved by further using rehabilitation. Although it is known that rehabilitation improves plasticity in patients with cerebral infarction or spinal cord injury, rehabilitation is used in combination with MSC administration and mesenchymal stem cell activator or stem cell age improver (β-carotene) administration of the present invention. As a result, the plasticity promoting function is further synergistically improved. The implementation method is not particularly limited, and may be rehabilitation by a robot or training by BMI (brain machine interface).
　以下、実施例により本発明について具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.
実施例１．βカロテンの間葉系幹細胞に対する効果
１．材料・方法
（１）人参ジュース（経口摂取）の効果
　ＳＤラット（雌）を無作為に人参ジュース投与群（ｎ＝５）と対照群（８週及び６２週各々　ｎ＝５）に分けた。人参ジュース投与群は、千葉県産１００％人参ジュース（伊橋産業株式会社（イキイキにんじん館すこやか）製）を約１００ｍｌ／匹／日、ＭＦ餌（オリエンタル酵母株式会社製）は自由給餌として、２４週から６２週まで投与し、６２週齢時に骨髄（大腿骨２本，脛骨２本分）を採取した。対照群は、自由給餌（餌：ＭＦ）・自由給水で飼育し、８週齢及び６２週齢時に骨髄（大腿骨２本，脛骨２本分）を採取した。 Example 1. Effect on β-carotene mesenchymal stem cells Materials / Methods (1) Effects of ginseng juice (oral intake) SD rats (female) were randomly divided into a ginseng juice administration group (n = 5) and a control group (8 weeks and 62 weeks each n = 5). The carrot juice administration group consists of 100% carrot juice from Chiba Prefecture (made by Ihashi Sangyo Co., Ltd. (Ikiiki Carrot Museum Sukoyaka)), about 100 ml / animal / day, and MF food (made by Oriental Yeast Co., Ltd.) as a free feed for 24 weeks. To 62 weeks, and bone marrow (2 femurs, 2 tibias) was collected at the age of 62 weeks. The control group was reared with free feeding (food: MF) and free water, and bone marrow (two femurs and two tibias) was collected at 8 and 62 weeks of age.
　採取した骨髄は、既報にしたがい、ダルベッコの改変イーグル培地（ＤＭＥＭ）で２５ｍｌに希釈し、加熱不活化した１０％　ＦＢＳ、２ｍＭ　ｌ－グルタミン、１００Ｕ／ｍｌ　ペニシリン、０．１ｍｇ／ｍｌ　ストレプトマイシンを添加し、５％ＣＯ2雰囲気下３７℃で３日間インキュベートした（Kim S. et al., Brain Res. 2006;1123:27-33. Ukai R. et al.,J. Neurotrauma. 2007;24:508-520.）。上記方法で細胞を４回継代して培養し、各継代時に細胞数をカウントした。 Collected bone marrow was diluted to 25 ml with Dulbecco's modified Eagle's medium (DMEM) and added with heat-inactivated 10% FBS, 2 mM l-glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin according to a previous report. Incubated for 3 days at 37 ° C. in a 5% CO 2 atmosphere (Kim S. et al., Brain Res. 2006; 1123: 27-33. Ukai R. et al., J. Neurotrauma. 2007; 24: 508- 520.). Cells were subcultured 4 times by the above method, and the number of cells was counted at each passage.
　同様の実験を３３週から４３週までの人参ジュース投与についても行い、投与の有無を比較した（人参ジュース投与群（ｎ＝５）と対照群（ｎ＝８））。
　評価は、採取した骨髄の継代毎培養細胞数（５匹の平均細胞数）を第４継代（Ｐａｓｓａｇｅ＝Ｐ４）まで追って比較することで実施した。 A similar experiment was conducted for ginseng juice administration from week 33 to 43, and the presence or absence of administration was compared (ginseng juice administration group (n = 5) and control group (n = 8)).
Evaluation was carried out by comparing the number of cultured cells (average number of 5 mice) per passage of the collected bone marrow until the fourth passage (Passage = P4).
（２）βカロテン（腹腔内投与）の効果
　ＳＤラット（雌）を、無作為にβカロテン投与群（ｎ＝５）と対照群（ｎ＝８）に分けた。βカロテン投与群は、３３週から４３週まで水溶性βカロテン（協和発酵バイオ株式会社製）を約０．１μｇ／０．５ｍｌ／日腹腔内投与し、ＡＩＮ７６Ａ餌（Ｈｏｋｕｄｏ社製）は約３０ｇ／日で摂餌、自由給水で飼育し、骨髄（大腿骨２本，脛骨２本分）を採取した。対照群は、自由給餌（餌：ＭＦ）・自由給水で飼育し、８週齢及び６２週齢時に骨髄（大腿骨２本，脛骨２本分）を採取した。採取した骨髄細胞を（１）と同様の方法で４回継代して培養し、継代時に細胞数をカウントした。
　評価は、採取した骨髄の継代毎培養細胞数（５匹又は８匹の平均細胞数）をＰ４まで追って比較することで実施した。 (2) Effect of β-carotene (intraperitoneal administration) SD rats (female) were randomly divided into a β-carotene administration group (n = 5) and a control group (n = 8). In the β-carotene administration group, water-soluble β-carotene (produced by Kyowa Hakko Bio Co., Ltd.) was administered intraperitoneally from about 33 to 43 weeks, and AIN76A diet (produced by Hokudo) was about 30 g. Per day, fed with free water and bone marrow (2 femurs, 2 tibias) was collected. The control group was reared with free feeding (food: MF) and free water, and bone marrow (two femurs and two tibias) was collected at 8 and 62 weeks of age. The collected bone marrow cells were subcultured 4 times in the same manner as in (1), and the number of cells was counted at the time of subculture.
Evaluation was carried out by comparing the number of cultured cells (average number of 5 or 8 cells) per passage of the collected bone marrow up to P4.
２．結果
（１）人参ジュース（２４－６２週　経口投与：図１）
　対照群のデータからわかるよう、８週齢時（グラフ左）に比べて６２週齢時（グラフ中央）では、老齢化により細胞数の減少が認められるが、２４週齢から６２週齢にかけて人参ジュースを経口摂取させた群（グラフ右）では、６２週齢時の対照群（グラフ中央）に比べて著しい細胞数の増加が観察され、若年のラット（８週齢の対照群）に近い増殖能を有することが確認された。
（２）βカロテン（３３－４３週　腹腔内投与：図２）
　図２に示されるとおり、３３週齢から４３週齢にかけてβカロテンを腹腔内投与した群では、同じ４３週齢の対照群に比較して、著しい細胞数の増加（増殖能）が観察された。このことは、人参ジュースの効果がβカロテンによるものであったことを示す。
（３）人参ジュース（３３－４３週　経口投与：図３）
　図３に示されるとおり、３３週齢から４３週齢にかけて人参ジュースを経口摂取させた群では、同じ４３週齢の対照群に比較して、著しい細胞数の増加（増殖能）が観察された。 2. Results (1) Carrot juice (24-62 weeks oral administration: Fig. 1)
As can be seen from the control group data, the number of cells decreased due to aging at 62 weeks of age (graph center) compared to 8 weeks of age (graph left), but ginseng from 24 weeks to 62 weeks of age. In the group in which the juice was orally ingested (right graph), a marked increase in the number of cells was observed compared to the control group at 62 weeks of age (middle of the graph), and the growth was close to that of young rats (8 weeks old control group). It was confirmed to have the ability.
(2) β-carotene (33-43 weeks intraperitoneal administration: Fig. 2)
As shown in FIG. 2, in the group in which β-carotene was intraperitoneally administered from 33 to 43 weeks of age, a marked increase in cell number (proliferative ability) was observed compared to the same 43-week-old control group. . This indicates that the effect of ginseng juice was due to β-carotene.
(3) Carrot juice (33-43 weeks oral administration: Fig. 3)
As shown in FIG. 3, in the group in which ginseng juice was orally ingested from 33 to 43 weeks of age, a marked increase in cell number (proliferative ability) was observed compared to the same 43-week-old control group. .
３．考察
　本実施例の結果から、βカロテンには、有意なＭＳＣ活性化効果、幹細胞年齢の若返り効果があることが確認された。すなわち、βカロテンを摂取することにより、ＭＳＣの組織維持・修復・再生効果が向上し、若返りや、病気（脳梗塞、脊髄損傷、認知症など）の治療効果を向上させることが期待できる。 3. Discussion From the results of this Example, it was confirmed that β-carotene has a significant MSC activation effect and stem cell age rejuvenation effect. That is, by ingesting β-carotene, it can be expected that the tissue maintenance / repair / regeneration effect of MSC is improved, and the therapeutic effect of rejuvenation and illness (cerebral infarction, spinal cord injury, dementia, etc.) is improved.
実施例２．脳梗塞（亜急性期）モデルラットにおけるβカロテン投与の効果
１．材料・方法
（１）脳梗塞（亜急性期）モデルラット
　脳梗塞モデルとして、ラット中大脳動脈閉塞（ＭＣＡＯ）モデルを使用した。既報にしたがい、９週齢の雌性ＳＤラット（２００－２５０ｇ）をケタミン（７５ｍｇ／ｋｇ）及びキシラジン（１０ｍｇ／ｋｇ）で麻酔し、２０．０－２２．０ｍｍの塞栓糸（ＭＯＮＯＳＯＦ）を外頸動脈から挿入して、中大脳動脈永久閉塞を行った（Honma T. et al., Exp. Neurol. 2006;199:56-66. Sasaki M. et al., Methods Mol. Biol. 2009;549:187-195.）。１１週齢時にＤＷＩ－ＭＲＩを撮影して初期梗塞体積を評価した。初期梗塞体積が基準（２００ｍｍ3）に満たない動物は実験から除外し、ラットを無作為にβカロテン投与群（ｎ＝２）と対照群（ｎ＝２）に分けた。 Example 2 Effect of β-carotene administration in cerebral infarction (subacute stage) model rats Materials and Methods (1) Cerebral Infarction (Subacute Phase) Model Rat A rat middle cerebral artery occlusion (MCAO) model was used as a cerebral infarction model. As previously reported, 9-week-old female SD rats (200-250 g) were anesthetized with ketamine (75 mg / kg) and xylazine (10 mg / kg), and 20.0-22.0 mm embolic thread (MONOSOF) was externally necked. Inserted from the artery, permanent middle cerebral artery occlusion was performed (Honma T. et al., Exp. Neurol. 2006; 199: 56-66. Sasaki M. et al., Methods Mol. Biol. 2009; 549: 187-195.). DWI-MRI was taken at the age of 11 weeks to evaluate the initial infarct volume. Animals with an initial infarct volume below the standard (200 mm 3 ) were excluded from the experiment and rats were randomly divided into β-carotene treated groups (n = 2) and control groups (n = 2).
（２）リハビリテーション
　βカロテン投与群は、生理食塩水０．５ｍｌとβカロテン（協和発酵バイオ株式会社製）０．１ｍｇを連日腹腔内投与した。対照群は、生理食塩水０．５ｍｌを連日腹腔内投与した。すべてのラットに毎日シクロスポリンＡ（１０ｍｇ／ｋｇ）を腹腔内投与した。リハビリテーションとして、１２週から毎日トレッドミル上を毎日２０分間走らせた（トレッドミル　角度０度、速度８～１２ｍ／分）。 (2) Rehabilitation In the β-carotene administration group, 0.5 ml of physiological saline and 0.1 mg of β-carotene (manufactured by Kyowa Hakko Bio Co., Ltd.) were intraperitoneally administered daily. In the control group, 0.5 ml of physiological saline was intraperitoneally administered every day. All rats received cyclosporin A (10 mg / kg) intraperitoneally daily. As a rehabilitation, a treadmill was run for 20 minutes every day from 12 weeks (treadmill angle 0 degree, speed 8-12 m / min).
（３）運動機能
　運動負荷試験により運動評価を行った。毎週トレッドミル（角度２０度）にて強制走行をさせ、最大走行速度（m/分）を測定した。図４に本実施例のプロトコルを示す。 (3) Motor function The motor was evaluated by an exercise load test. The vehicle was forced to run on a treadmill (angle 20 degrees) every week, and the maximum running speed (m / min) was measured. FIG. 4 shows the protocol of this embodiment.
２．結果
　図５に示すとおり、βカロテン投与群では運動機能の改善を認めたが、対照群では運動機能に変化は認められなかった。これはβカロテン投与により、損傷組織の再生と可塑性が亢進したためと考えられる。 2. Results As shown in FIG. 5, improvement in motor function was observed in the β-carotene administration group, but no change in motor function was observed in the control group. This is thought to be due to the increased regeneration and plasticity of the damaged tissue by β-carotene administration.
参考例１．脳梗塞（慢性期）モデルラットにおけるＭＳＣ投与の効果
１．材料・方法
（１）ラット骨髄由来間葉系幹細胞の調製
　実験は札幌医科大学の動物実験管理規定にしたがって実施した。既報に従い、成熟ＳＤラットの大腿骨から得た骨髄をダルベッコの改変イーグル培地（ＤＭＥＭ）で２５ｍｌに希釈し、加熱不活化した１０％　ＦＢＳ、２ｍＭ　ｌ－グルタミン、１００Ｕ／ｍｌ　ペニシリン、０．１ｍｇ／ｍｌ　ストレプトマイシンを添加し、５％ＣＯ2雰囲気下３７℃で３日間インキュベートした（Kim S. et al., Brain Res. 2006;1123:27-33. Ukai R. et al.,J. Neurotrauma. 2007;24:508-520.）。コンフルエントになるまで培養し、接着細胞をトリプシン－ＥＤＴＡで剥離し、１×１０4ｃｅｌｌｓ／ｍｌの密度で３回継代培養して間葉系幹細胞（ＭＳＣ）を得た。 Reference Example 1 1. Effects of MSC administration in cerebral infarction (chronic phase) model rats Materials / Methods (1) Preparation of rat bone marrow-derived mesenchymal stem cells Experiments were performed in accordance with animal experiment management regulations of Sapporo Medical University. According to previous reports, bone marrow obtained from femurs of adult SD rats was diluted to 25 ml with Dulbecco's modified Eagle's medium (DMEM) and heat-inactivated 10% FBS, 2 mM l-glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin was added and incubated for 3 days at 37 ° C. in 5% CO 2 atmosphere (Kim S. et al., Brain Res. 2006; 1123: 27-33. Ukai R. et al., J. Neurotrauma. 2007) ; 24: 508-520.). The cells were cultured until confluent, adherent cells were detached with trypsin-EDTA, and subcultured 3 times at a density of 1 × 10 4 cells / ml to obtain mesenchymal stem cells (MSC).
（２）脳梗塞（慢性期）モデルラット
　９週齢の雌性ＳＤラット（２００－２５０ｇ）をケタミン（７５ｍｇ／ｋｇ）及びキシラジン（１０ｍｇ／ｋｇ）で麻酔し、２０．０－２２．０ｍｍの塞栓糸（ＭＯＮＯＳＯＦ）を外頸動脈から挿入して、中大脳動脈永久閉塞（ＭＣＡＯ)を行った。初期梗塞体積が基準（２００ｍｍ3）に満たない動物は実験から除外し、以下のとおりラットを無作為にＭＳＣ群（ｎ＝８）とＤＭＥＭ群（ｎ＝８）に分け、ＭＣＡＯ後８週が経過した慢性期に移植を行った。
　ＭＳＣ群：ＭＣＡＯの８週後のＳＤラットのＭＳＣ、Ｐ２　１．０×１０6個を１ｍｌ中に含むＤＭＥＭを大腿静脈より投与した。
　ＤＭＥＭ群：１ｍｌのＤＭＥＭを大腿静脈より投与した。 (2) Cerebral infarction (chronic phase) model rat A 9-week-old female SD rat (200-250 g) was anesthetized with ketamine (75 mg / kg) and xylazine (10 mg / kg), and an embolus of 20.0-22.0 mm was obtained. A thread (MONOSOF) was inserted from the external carotid artery and permanent middle cerebral artery occlusion (MCAO) was performed. Animals whose initial infarct volume did not meet the criteria (200 mm 3 ) were excluded from the experiment, and rats were randomly divided into MSC group (n = 8) and DMEM group (n = 8) as follows, and 8 weeks after MCAO Transplantation was performed during the chronic phase.
MSC group: MS rats of SD rats 8 weeks after MCAO, DMEM containing 1.0 × 10 6 P2 in 1 ml were administered from the femoral vein.
DMEM group: 1 ml of DMEM was administered from the femoral vein.
（３）リハビリテーション
　投与翌日より、リハビリテーション(トレッドミル　角度０度、速度８～１２ｍ／分、２０分)を行い、シクロスポリン投与(１０ｍｇ/ｋｇ)を移植１週間は連日、その後は隔日投与を行った。全例において、移植翌日から毎日リハビリテーション(トレッドミル　角度０度、速度８～１２ｍ／分、２０分)を行った。 (3) Rehabilitation From the next day of administration, rehabilitation (treadmill angle 0 degree, speed 8-12 m / min, 20 minutes) was performed, and cyclosporine (10 mg / kg) was administered every day for one week after transplantation, and thereafter every other day. . In all cases, daily rehabilitation (treadmill angle 0 degree, speed 8-12 m / min, 20 minutes) was performed from the day after transplantation.
（４）運動機能
　運動負荷試験により運動評価を行った。毎週トレッドミル（角度２０度）にて強制走行をさせ、最大走行速度（ｍ/分）を測定した。 (4) Motor function Motor evaluation was performed by an exercise load test. The vehicle was forced to run on a treadmill (angle 20 degrees) every week, and the maximum running speed (m / min) was measured.
２．結果
　図６に示すとおり、ＭＳＣ群では運動機能の改善を認めたが、ＤＭＥＭ群では変化は認められなかった。このことから、脳梗塞慢性期にＭＳＣを投与すると運動機能の改善が見られることが確認された。これはＭＳＣ投与により、損傷組織の再生と可塑性が亢進したためと考えられる。 2. Results As shown in FIG. 6, improvement in motor function was observed in the MSC group, but no change was observed in the DMEM group. From this, it was confirmed that when MSC was administered in the chronic stage of cerebral infarction, the motor function was improved. This is presumably because the regeneration and plasticity of damaged tissues were enhanced by administration of MSC.
３．考察
　本参考例の結果と実施例１及び２の結果から、βカロテン投与（摂取）することで、損傷組織の再生と可塑性の亢進が向上することが示唆される。また、ＭＳＣ投与にβカロテン投与（摂取）を併用することで、ＭＳＣによる損傷組織の再生と可塑性の亢進がより向上することが示唆される。 3. Discussion The results of the present reference example and the results of Examples 1 and 2 suggest that administration of β-carotene (ingestion) improves the regeneration of damaged tissue and the enhancement of plasticity. Moreover, it is suggested that the combined use of β-carotene (ingestion) with MSC administration improves the regeneration of damaged tissue and the enhancement of plasticity by MSC.
　本発明は、ＭＳＣによる脳梗塞、脊髄損傷、認知症などの難治性神経疾患の新たな治療戦略の確立及び治療効果の改善に有用である。 The present invention is useful for establishing a new therapeutic strategy and improving the therapeutic effect of intractable neurological diseases such as cerebral infarction, spinal cord injury, and dementia caused by MSC.
　本明細書中で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書中にとり入れるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated into this specification as they are.

Claims (9)

  1. 　βカロテンを有効成分とする、間葉系幹細胞活性化剤。 A mesenchymal stem cell activator containing β-carotene as an active ingredient.
  2. 　間葉系幹細胞が骨髄又は血液に由来する間葉系幹細胞である、請求項１に記載の間葉系幹細胞活性化剤。 The mesenchymal stem cell activator according to claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow or blood.
  3. 　骨髄又は血液が、間葉系幹細胞の投与を受ける対象の骨髄又は血液である、請求項２に記載の間葉系幹細胞活性化剤。 3. The mesenchymal stem cell activator according to claim 2, wherein the bone marrow or blood is bone marrow or blood of a subject to receive mesenchymal stem cells.
  4. 　間葉系幹細胞がＣＤ２４陰性である、請求項１～３のいずれか１項に記載の間葉系幹細胞活性化剤。 The mesenchymal stem cell activator according to any one of claims 1 to 3, wherein the mesenchymal stem cells are CD24 negative.
  5. 　βカロテンを有効成分とする、間葉系幹細胞の幹細胞年齢改善剤。 An agent for improving stem cell age of mesenchymal stem cells, containing β-carotene as an active ingredient.
  6. 　間葉系幹細胞が骨髄又は血液に由来する間葉系幹細胞である、請求項５に記載の幹細胞年齢改善剤。 The stem cell age-improving agent according to claim 5, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow or blood.
  7. 　骨髄又は血液が、間葉系幹細胞の投与を受ける対象の骨髄又は血液である、請求項６に記載の幹細胞年齢改善剤。 7. The stem cell age-improving agent according to claim 6, wherein the bone marrow or blood is bone marrow or blood of a subject receiving mesenchymal stem cells.
  8. 　間葉系幹細胞がＣＤ２４陰性である、請求項５～７のいずれか１項に記載の幹細胞年齢改善剤。 The stem cell age improving agent according to any one of claims 5 to 7, wherein the mesenchymal stem cells are CD24-negative.
  9. 　ＣＤ２４陰性の間葉系幹細胞を含む組織修復・再生用医薬と併用することを特徴とする、請求項１～４のいずれか１項に記載の間葉系幹細胞活性化剤又は請求項５～８のいずれか１項に記載の幹細胞年齢改善剤。 The mesenchymal stem cell activator according to any one of claims 1 to 4, or the medicinal stem cell activator according to any one of claims 1 to 4, wherein the mesenchymal stem cell activator according to any one of claims 1 to 4 is used in combination. The stem cell age-improving agent according to any one of the above.
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