JP6468895B2 - Stem cell undifferentiated state maintenance agent and growth promoter - Google Patents

Description

  The present invention relates to an agent for maintaining an undifferentiated state of stem cells and an agent for promoting proliferation, or a wound healing agent, and a method for maintaining an undifferentiated state of stem cells and a method for promoting proliferation.

  When tissue of a vertebrate (especially a mammal) is damaged due to injury or disease, or aging, etc., the regeneration system works to try to recover the damage of the cell or organ. Stem cells (tissue stem cells, somatic stem cells) provided in the tissue play a major role in this action. Stem cells have the pluripotency to differentiate into all cells and organs, and this property is thought to lead to recovery by compensating for damaged parts of cells and organs. Expectations are gathered for regenerative medicine, which is the next generation of medicine using such stem cells.

  The most advanced tissue in stem cell research in mammals is the bone marrow. The bone marrow contains living hematopoietic stem cells, which have been shown to be the source of all blood cell regeneration. Furthermore, it has been reported that the bone marrow contains stem cells that can be differentiated into organs and tissues (for example, bone, cartilage, muscle, fat, etc.) in addition to hematopoietic stem cells (see Non-Patent Document 1).

  Furthermore, in recent years, it has been clarified that stem cells exist in all organs and tissues other than bone marrow, such as skin, liver, pancreas, fat, etc., and is responsible for the regeneration and homeostasis of each organ and tissue. It has been understood (see Non-Patent Documents 2 to 5). In addition, stem cells present in each organ or tissue are excellent in plasticity, and may be used for regeneration of organs and tissues that have been impossible to self-replicate until now.

  On the other hand, some of these stem cells are known to decrease with aging, and research on techniques to prevent the decrease of stem cells is actively conducted to maintain the homeostasis of each tissue ( Non-patent document 6). In recent years, stem cells have been isolated from living tissues in the field of cell transplantation and tissue engineering (regenerative medicine and regenerative beauty) in order to apply the ability (pluripotency) of stem cells to regeneration of organs and tissues. Development of techniques for propagation is in progress (Non-Patent Documents 7 and 8).

  In particular, when culturing stem cells in vitro, it is extremely important to proliferate while maintaining the pluripotency that is the ability of stem cells, that is, maintaining an undifferentiated state. If the undifferentiated state of the stem cells cannot be maintained during this culture and differentiation induction has progressed, the ability (pluripotency) of the finally prepared stem cells has been lost, and the desired effect ( Can not reproduce organs and tissues.

  From the above, when stem cells are used for cell transplantation treatment or tissue engineering (regenerative medicine or regenerative beauty) and regeneration of organs or tissues is desired, the stem cells must be able to be cultured while maintaining an undifferentiated state.

  To date, there have been several reports on techniques for proliferating stem cells while maintaining an undifferentiated state, but they are still under development. For example, embryonic stem cells (ES cells) and hematopoietic stem cells can be maintained undifferentiated by co-culture with supporting cells (stromal cells or feeder cells) (see Patent Literature 1 and Non-Patent Literatures 9 to 11). ). However, recently, there have been reports of cases of infection between different animals caused by feeder cells-derived endogenous viruses (see Non-Patent Document 12), and stem cell culture using feeder cells is a stem cell for medical purposes. It is not suitable for culturing.

  As another method, there is a method of maintaining an undifferentiated state of a stem cell by complexly combining cytokines. For example, mouse ES cells are maintained undifferentiated by adding LIF (Leukemia Inhibitory Factor) to the medium (see Patent Document 2 and Non-Patent Document 13). In addition, maintaining embryonic stem cells in the presence of early-acting cytokines thrombopoietin (TPO), interleukin 6 (IL-6), FLT-3 ligand, and stem cell factor (SCF) It has been reported for somatic stem cells (see Patent Literature 3 and Non-Patent Literature 14).

  However, cytokines are expensive and have problems such as collection raw materials and storage stability, and easy use is difficult. In addition, the effect of LIF is limited to a very specific cell lineage. In particular, in primate ES cells and somatic stem cells, it has been clarified that the addition of LIF alone cannot maintain an undifferentiated state. (See Non-Patent Document 10).

  As described above, all of the currently reported methods for maintaining the undifferentiated state of stem cells require complicated operations and have a low effect of maintaining the undifferentiated state. Therefore, in order to use stem cells for regenerative medicine, a technique for proliferating stem cells while maintaining an undifferentiated state has been demanded. That is, there has been a demand for a technique that can proliferate stem cells while maintaining an undifferentiated state in a safe, simple and efficient manner.

  In recent years, with the increase in diabetes and arteriosclerosis and aging, the number of patients with chronic wounds such as diabetic leg ulcers and pressure ulcers that are difficult to treat by skin grafting alone is increasing. In general, skin wounds wait for natural healing due to the healing power of the living body after applying emergency treatment such as cleaning of the wound. However, natural healing may take a long time depending on the degree of the wound. Especially, elderly people, diabetics and the like are slower in natural healing than young people and need to promote wound healing. As skin wound healing promoters, lysozyme chloride, solcothelin, and the like are known, but none of them has a sufficient wound healing promoting effect. In skin wound healing, tissue remodeling is performed, so that necessary cell migration and production of extracellular matrix such as collagen are promoted. Type 3 collagen, which is mainly produced in the early stage of wound healing and is important for tissue reconstruction, has a wound healing promoting effect and is used as a wound healing promoting agent (Non-patent Document 15 and Patent Document 4). Moreover, substance P (patent document 5) etc. are known as a substance which stimulates proliferation or differentiation of a stem cell and accelerates wound healing. However, since these substances are all proteins, they have problems in terms of cost and storage stability, and are not suitable when a large amount of skin regeneration treatment is required in a short period of time, such as therapeutic use. In addition, with increasing awareness of beauty and anti-aging, interest in skin rejuvenation beauty is increasing year by year for the purpose of improving wrinkles and sagging due to aging, ultraviolet rays, and pigmentation. Therefore, there is a demand for an easily obtainable, safe and inexpensive substance for skin regeneration medicine such as wound healing or skin regeneration beauty.

  So far, oleanolic acid and / or ursolic acid has an inhibitory effect on keratinocyte differentiation synergistically with retinol (Patent Document 6), and pentacyclic triterpenes such as ursolic acid and betulinic acid have been found in adipose tissue. Although it has been reported that the precursor adipocyte has a differentiation-suppressing effect or a proliferation-suppressing effect (Patent Documents 7 to 8), the effect of these triterpenes on the stem cell differentiation (maintaining undifferentiated state) and the promotion of stem cell proliferation are reported. The effect is not known.

JP 2004-24089 A JP-T-2002-525042 Japanese Patent No. 3573354 Japanese Patent Laid-Open No. 7-17844 JP 2006-124389 A JP 2000-503030 Gazette International Publication No. 2003/039270 International Publication No. 2003/011267

Pittenger M.M. F. Et al., Science, 1999, Vol. 284, pp. 143-147 Goodell M.M. A. Et al., Nat. Med. , 1997, Vol. 3, pp. 1337-1345 Zulewski H. Et al., Diabetes, 2001, Vol. 50, pp. 521-533 Suzuki A. Et al., Hepatology, 2000, Vol. 32, pp. 1230-1239 Zuk P.M. A. Et al., Tissue Engineering, 2001, Vol. 7, pp. 211-228 Koji Hasegawa, Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 Hiroshi Mabuchi et al., Journal of Japanese Society for Regenerative Medicine, 2007, Vol. 6, pp. 263-268 All Kitagawa et al., Journal of Japanese Society for Regenerative Medicine, 2008, Vol. 7, pp. 14-18 Thomson J.M. A. Et al., Proc. Natl. Acad. Sci. USA, 1995, Vol. 92, pp. 7844-7848 Thomson J.M. A. Et al., Science, 1998, Vol. 282, pp. 1145-1147 Reubinoff B.E. E. Et al., Nature Biotech. , 2000, Vol. 18, pp. 399-404 van der Laan L.L. J. et al. Et al., Nature, 2000, Vol. 407, pp. 90-94 Smith A. G. Et al., Dev. Biol. , 1987, Vol. 121, pp. 1-9 Madlambayan G.M. J. et al. J. et al. Hematother. Stem Cell Res. , 2001, Vol. 10, pp. 481-492 R. H. CHAMPION et al, Textbook of Dermatology, vol. 1: 342-343, 1998.

  In view of the above-described circumstances, the present invention finds a new substance capable of efficiently proliferating stem cells while maintaining an undifferentiated state, and provides the stem cell as an undifferentiated state maintaining agent or a growth promoter for stem cells. Is an issue. Another object of the present invention is to provide a wound healing agent that has a skin wound healing action, can be easily used in the field of skin regenerative medicine or cosmetics, has high safety, and is inexpensive.

  As a result of intensive studies to solve the above problems, the present inventors have found that triterpenic acid, which is contained in many plants and exhibits various physiological activities, is an excellent undifferentiated state for stem cells that have not been known so far. It has been found that it has a maintenance effect, a growth promoting effect, and a wound healing effect, and has completed the present invention.

That is, the present invention includes the following.
(1) A stem cell undifferentiated state maintaining agent containing triterpenic acid as an active ingredient.
(2) A stem cell proliferation promoter containing triterpenic acid as an active ingredient.
(3) A wound healing agent containing triterpenic acid as an active ingredient.
(4) The agent according to any one of (1) to (3), wherein the triterpenic acid is oleanolic acid or ursolic acid.
(5) A pharmaceutical or quasi drug containing the agent according to any one of (1) to (4).
(6) A method for producing stem cells, comprising a step of culturing stem cells in a medium containing triterpenic acid.
(7) A method for maintaining an undifferentiated state of stem cells, comprising a step of culturing stem cells in a medium containing triterpenic acid.
(8) A method for promoting the proliferation of stem cells, comprising a step of culturing stem cells in a medium containing triterpenic acid.

  According to the present invention, stem cells can be efficiently proliferated while maintaining an undifferentiated state. According to the present invention, a wound can be effectively healed. Therefore, the present invention can greatly contribute to the fields of regenerative medicine and regenerative beauty.

Hereinafter, the present invention will be described in detail.
The stem cell undifferentiated state maintenance agent or growth promoter or wound healing agent according to the present invention contains triterpenic acid as an active ingredient.

  Triterpenic acid is a 30-carbon pentacyclic triterpene compound in which 6 isoprene units having 5 carbon atoms are bonded and a carboxy group is located at the 28-position. Examples of the triterpenic acid that can be used in the present invention include oleanolic acid, ursolic acid, betulinic acid, maslinic acid, corosolic acid, tormic acid, etc. Among them, oleanolic acid or ursolic acid is preferable.

  The triterpenic acid used in the present invention may be one of the above-mentioned triterpenic acids, but may be a mixture of two or more.

  The triterpenic acid used in the present invention can be obtained by chemical synthesis, but it can also be obtained by extracting from a plant material known to contain the triterpenic acid by a known method and purifying it. Moreover, a commercial item can also be utilized.

For example, oleanolic acid (molecular formula: C ₃₀ H ₄₈ O ₃ , molecular weight: 456.7, CAS registration number: 508-02-1) represented by the following structural formula (I) can be obtained by chemical synthesis, but olives It is known to be contained in plants such as grapes and perillas. From these plant materials, lower alcohols (ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.) are used as extraction solvents. Alternatively, extraction is performed using one or more organic solvents such as ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.), and after solvent extraction, the solvent phase is concentrated, diluted, filtered, dried, etc. It can be obtained by performing purification by chromatography or the like. A commercially available product of oleanolic acid (for example, a commercially available product manufactured by Wako Pure Chemical Industries, Ltd.) can also be used.

Ursolic acid (molecular formula: C ₃₀ H ₄₈ O ₃ , molecular weight: 456.7, CAS registration number: 77-52-1) is represented by the following structural formula (II) and can be obtained by chemical synthesis. It can be obtained from a plant material such as rosemary, apple, kiwi, sage, basil, peppermint, lavender and the like by solvent extraction using the same extraction solvent as above. A commercial product of ursolic acid (for example, a commercial product manufactured by Sigma) can also be used.

  The oleanolic acid and ursolic acid may be pharmacologically acceptable salts. Examples of pharmacologically acceptable salts include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt and aluminum salt, and salts with organic bases such as triethylamine, triethanolamine and piperazine. However, it is not limited to these.

  Since the above-mentioned triterpenic acid has an action of efficiently proliferating stem cells while maintaining an undifferentiated state at a biological level or a culture level, it can be used as an undifferentiated state maintaining agent or a growth promoting agent for stem cells. Furthermore, the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention can be used as an additive for cell culture or a research reagent for efficiently proliferating stem cells while maintaining the undifferentiated state. Can do. The above-mentioned triterpenic acid can also be used as a wound healing agent because it has an excellent action for promoting wound healing.

  The above-mentioned action of maintaining the undifferentiated state of stem cells by triterpenic acid, the action of promoting the proliferation of stem cells, or the action of wound healing is enhanced by the combined use of fatty acid glycerides. Examples of fatty acid glycerides include acyl groups derived from unsaturated fatty acids having 16 to 22 carbon atoms, such as acyl groups derived from docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, α-linolenic acid, γ-linolenic acid, linoleic acid, and oleic acid. Fatty acid glycerides having The fatty acid glyceride may be any of monoglyceride, diglyceride and triglyceride, but is preferably diglyceride, more preferably 1,3-diglyceride in which the above unsaturated fatty acid is bonded to the 1st and 3rd positions of glycerin. More preferred is 1,3-diglyceride to which linoleic acid is bound. In the case of diglyceride and triglyceride, the unsaturated fatty acid bonded to the 1-position, 2-position or 3-position of glycerin may be the same or different. These fatty acid glycerides can be obtained by a method of squeezing / extracting / extracting from plant raw materials or animal raw materials, an enzymatic method, or an organic synthetic method. Moreover, the commercial item generally used in the manufacture field | areas, such as a foodstuff, cosmetics, and a pharmaceutical, can also be utilized.

  In the present invention, when triterpenic acid and fatty acid glyceride are used in combination, the ratio is not particularly limited, and can be appropriately changed to each type of triterpenic acid and fatty acid glyceride, and examples thereof include 10:90 to 90:10.

  By applying the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention to stem cells of mammals including humans, the undifferentiated state of stem cells can be maintained and the proliferation of stem cells can be promoted. . Stem cells to which the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention is applied are not particularly limited as long as they meet the object of the present invention. For example, embryonic stem cells (ES cells); bone marrow, blood , Somatic stem cells present in the skin (epidermis, dermis, subcutaneous tissue), fat, hair follicle, brain, nerve, liver, pancreas, kidney, muscle and other tissues; stem cells artificially prepared by gene transfer etc. (Artificial pluripotent stem cells: iPS cells). Preferably, the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is more effective for stem cells derived from bone marrow, blood, skin, or adipose tissue. Examples of ES cells include ES cells established by culturing early embryos before implantation, ES cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei, And ES cells obtained by modifying genes on the chromosomes of those ES cells using genetic engineering techniques. Such ES cells can be prepared, for example, by a method known per se, but can be obtained from a predetermined institution, and further commercially available products can be purchased. In addition, these stem cells may be primary cultured cells, subcultured cells, or frozen cells.

  Furthermore, the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention can be applied to stem cells derived from all mammals as long as it has equivalent characteristics with respect to the direction of differentiation of the stem cells and the process of differentiation. Is possible. For example, the stem cell undifferentiated state maintaining agent or the growth promoting agent according to the present invention is a stem cell of a mammal such as a human, monkey, mouse, rat, guinea pig, rabbit, cat, dog, horse, cow, sheep, goat or pig. Can be effective.

  The application of the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention to the stem cell may be in vitro or in vivo, and in any case, the action can be exerted. Therefore, the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is obtained by culturing stem cells in a culture medium for stem cell culture to which an effective amount thereof has been added, or by administering to a mammal including a human. , Can maintain the undifferentiated state of stem cells and promote proliferation.

  Stem cell undifferentiated state maintenance agent or growth promoter according to the present invention, because the active ingredient triterpenic acid has an excellent stem cell undifferentiated state maintenance action and proliferation promoting action, skin, osteoblast, cartilage, muscle, It is effective for treating, ameliorating, and preventing damage or damage to tissues or organs by acting on stem cells of tissues or organs in vivo such as nerves, fats, and livers. In addition, since stem cells decrease or decrease in function with aging and the like, the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention reduces the stem cells or functions of organs or organs in the living body. It is effective in treating, ameliorating and preventing related diseases. Here, as diseases related to tissue or organ damage or damage, stem cell decrease or functional decline, for example, in the case of skin, wrinkles, tarmi, spots, dullness, rough skin, thickened skin, open pores, acne scars , Wounds, scars, keloids and the like, and also includes scalp and hair damage such as thinning and hair loss. For bone-related, osteoporosis, fractures (vertebral compression fracture, femoral neck fracture, etc.), for cartilage diseases, osteoarthritis, rheumatoid arthritis, disc herniation, etc. For nerve-related, spinal cord injury, facial nerve palsy , Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, memory decline with age, blood related, aplastic anemia, leukemia, cardiovascular related myocardial infarction, obstructive arteriosclerosis, etc. Related to periodontal disease, alveolar bone damage due to alveolar pyorrhea, ophthalmology related, retinitis pigmentosa, age-related macular degeneration, glaucoma, etc. liver / pancreas related include hepatitis, cirrhosis, diabetes, etc. It is not limited to.

  The wound-healing agent according to the present invention is effective for healing a skin wound that is an injury of the epidermis or dermis of the skin because triterpenic acid as an active ingredient has an excellent action for promoting wound healing. Here, as a skin wound, a serious injury degree and a depth are not specifically limited. For example, in addition to general wounds such as cuts, tears, split wounds, abrasions, crush wounds, wounds, stab wounds, bites, etc., pressure ulcers, burns, burns, diabetic ulcers, lower limb ulcers, lower limb aneurysms, etc. .

  The content of triterpenic acid in the undifferentiated state maintenance agent or growth promoter for stem cells according to the present invention, or the wound healing agent is not particularly limited. For example, the total amount of the drug is 0.00001 in terms of dry matter. It is preferable that it is 10 to 10 weight%, and it is more preferable to set it as 0.0001 to 1 weight%. If it is less than 0.00001% by weight, the effect may not be sufficiently exhibited.

  When the stem cell undifferentiated state maintaining agent or growth promoter or wound healing agent according to the present invention is administered in vivo, it can be administered as it is, but it is suitable within the range not impairing the effects of the present invention. It can be provided by being added to various compositions such as cosmetics, quasi-drugs, pharmaceuticals, and foods and drinks together with additives. The pharmaceutical of the present invention includes drugs used for animals, that is, veterinary medicine.

  When the stem cell undifferentiated state maintenance agent or growth promoter or wound healing agent according to the present invention is blended in cosmetics or quasi drugs, the dosage form is an aqueous solution system, a solubilization system, an emulsification system, a powder system. Any of a powder dispersion system, an oil liquid system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, or a water-oil-powder three-layer system may be used. In addition, the cosmetics and quasi-drugs include various components, additives, bases, etc. that are usually used in compositions for external use with skin, together with an agent for maintaining an undifferentiated state of stem cells, a growth promoter, or a wound healing agent. It selects according to a kind, mix | blends suitably, and can manufacture according to a well-known method in this field | area. The form may be liquid, emulsion, cream, gel, paste, spray or the like. Examples of the ingredients for the external composition for skin include oils and fats (olive oil, palm oil, evening primrose oil, jojoba oil, castor oil, hardened castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons ( Liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.) , Esters (isopropyl myristate, isopropyl palmitate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid) Pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and salts thereof, vitamins, plant / animal extracts, various Surfactants, humectants, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, fragrances and the like can be mentioned.

  As types of cosmetics and quasi-drugs, for example, lotion, milky lotion, gel, beauty essence, general cream, sunscreen cream, pack, mask, face wash, cosmetic soap, foundation, funny, bath preparation, body lotion, Examples include body shampoos, hair shampoos, hair conditioners, hair restorers and the like.

  When the agent for maintaining the undifferentiated state of stem cells or the growth promoting agent or wound healing agent according to the present invention is added to a pharmaceutical product, it is mixed with a pharmacologically and pharmaceutically acceptable additive and applied to the affected part. Can be formulated into various preparations suitable for the preparation. The pharmacologically and pharmaceutically acceptable additives include pharmaceutical bases and carriers, excipients, diluents, binders, lubricants, and coatings that are appropriately selected according to the dosage form and application. Agents, disintegrants or disintegration aids, stabilizers, preservatives, preservatives, extenders, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, propellants A coloring agent, a sweetening agent, a corrigent, a fragrance, and the like may be added as appropriate, and various preparations that can be administered systemically or locally orally or parenterally by various known methods may be used. When the pharmaceutical product of the present invention is provided in each of the above-described forms, it can be produced by a method usually used by those skilled in the art, for example, the method shown in the General Formulation [2] Formulation of the Japanese Pharmacopoeia.

  The form of the pharmaceutical product of the present invention is not particularly limited. For example, tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspensions, elixirs Oral preparations such as pills, injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, suppositories, ointments, lotions, eye drops, sprays, transdermal Examples include skin absorption agents, transmucosal absorption agents, and parenteral preparations such as patches. Moreover, it may be a dry product which is redissolved when used, and in the case of an injectable preparation, it is provided in the state of a unit dose ampule or a multi-dose container.

  The form suitable for using the stem cell undifferentiated state maintaining agent or growth promoting agent or wound healing agent according to the present invention as a pharmaceutical for treating, ameliorating and preventing the skin-related damage or disease is an external preparation. Examples thereof include ointments, creams, gels, liquids, patches (paps, plasters), foams, sprays, sprays, and the like. The ointment refers to a homogeneous semi-solid external preparation, and includes an oily ointment, an emulsion ointment, and a water-soluble ointment. The gel is an external preparation in which a water-insoluble component hydrate compound is suspended in an aqueous liquid. The liquid preparation refers to a liquid external preparation and includes lotions, suspensions, emulsions, liniments and the like. In particular, when used as a skin wound healing agent, ointments, creams, liquids and sprays are more preferred because of the skin wound healing effect and ease of use.

  The pharmaceutical agent of the present invention functions as a prophylactic agent that suppresses the onset of the above diseases and / or a therapeutic agent that improves the normal state. Since the active ingredient of the pharmaceutical of the present invention is derived from natural products, it is very safe and has no side effects. Therefore, when used as a medicament for the treatment, amelioration, and prevention of the aforementioned diseases, humans, mice, rats, rabbits It can be administered orally or parenterally in a wide range of doses to mammals such as dogs and cats.

  The content of the stem cell undifferentiated state maintaining agent or growth promoter, or wound healing agent in the cosmetics, pharmaceuticals, and quasi drugs of the present invention is not particularly limited, but triterpenic acid relative to the total weight of the preparation (composition). In terms of the dry solid content, 0.001 to 30% by weight is preferable, and 0.01 to 10% by weight is more preferable. If the amount is less than 0.001% by weight, the effect is low, and if the amount exceeds 30% by weight, the effect is hardly increased. The above amount is merely an example, and may be set and adjusted as appropriate in consideration of the type and form of the composition, the general amount used, the efficacy and the effect. Moreover, about the addition method of the active ingredient in formulation, it may add previously, may be added in the middle of manufacture, and should just select suitably in consideration of workability | operativity.

  In addition, the stem cell undifferentiated state maintenance agent or growth promoter or wound healing agent according to the present invention can also be added to food and drink. In addition, in the present invention, the food and drink is a general food and drink, a food that can be ingested for the purpose of maintaining and promoting health other than pharmaceuticals, for example, health food, functional food, health functional food, or special use Used to include food. The health food includes foods provided under the names of nutritional supplements, health supplements, supplements, and the like. Functional health foods are defined by the Food Sanitation Law or Health Promotion Law, and include specific health foods and functional nutrition foods that can display the effects of specific health, the function of nutritional components, the reduction of disease risk, and the like.

  The form of the food or drink may be any form suitable for edible use, for example, solid, liquid, granular, granular, powder, capsule, cream, or paste. In particular, the shape in the case of the above health foods, for example, tablets, rounds, capsules, powders, granules, fine granules, troches, liquids (including syrups, milks, suspensions) Etc. are preferred.

  The types of food and drink include bread, noodles, confectionery, dairy products, processed fishery and livestock products, processed oils and fats, processed foods, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks) , Milk beverages, etc.) and concentrated beverages and powders for adjustment of the beverages, but are not limited thereto.

  The food / beverage products of the present invention may be appropriately blended with additives usually used depending on the type. Any additive that is acceptable under the Food Sanitation Law can be used as the additive. For example, sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, stevia; citric acid, malic acid Acidulants such as tartaric acid; excipients such as dextrin and starch; binders, diluents, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspensions Examples include turbidizers and preservatives.

When the food / beverage products of this invention are general food / beverage products, it can manufacture by including the process of adding triterpenic acid in the normal manufacturing process of the food / beverage products. In the case of health foods, it may be according to the above-mentioned pharmaceutical production method. For example, in tablet supplements, additives such as excipients are added to and mixed with triterpenic acid, and pressure is applied with a tableting machine or the like. Can be produced by molding. Moreover, other materials (for example, vitamins such as vitamin C, vitamin B ₂ , vitamin B ₆ , minerals such as calcium, dietary fiber, etc.) can be added as necessary.

  The blending amount of triterpenic acid in the food and drink of the present invention may be any amount that can exhibit the undifferentiated state maintaining effect and proliferation promoting effect of stem cells and the wound healing effect, but the general intake of the target food and drink, food and drink What is necessary is just to set suitably in consideration of the form of goods, an effect and an effect, taste property, palatability, cost, etc.

  The present invention also relates to a method for promoting stem cell proliferation while culturing stem cells in a medium containing triterpenic acid while maintaining the undifferentiated state of the stem cells. In other words, the method according to the present invention can be referred to as a method for producing a stem cell, a method for maintaining an undifferentiated state of a stem cell, or a method for promoting the proliferation of a stem cell, comprising a step of culturing the stem cell in a medium containing triterpenic acid. .

  In the method according to the present invention, a culture medium generally used for maintaining and proliferating the undifferentiated state of stem cells may be used for culturing stem cells. For example, a basic medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids) necessary for stem cell survival and proliferation, specifically, Dulbecco's Modified Eagle Medium (D-MEM) ), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium (Nentient Mixture F-12 (D-MEM / F-M / G). Hanks' solution (Hank's balanced salt solution) etc. are mentioned. In addition, basic fibroblast growth factor (bFGF) and / or leukocyte migration inhibitory factor (LIF) may be added to the medium as growth factors. Further, if necessary, the medium may be epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27. -It may contain supplements, N2-supplements, ITS-supplements, antibiotics and the like.

  In addition to the above components, it is preferable that serum is contained in the medium at a content of 1 to 20%. However, since serum has different components depending on the lot and there are variations in its effects, it is preferably used after lot check.

  Commercially available media include Mesenchymal stem cell basal medium manufactured by Invitrogen, Mesenchymal stem cell basal medium manufactured by Sanko Junyaku Co., Ltd., MF medium manufactured by TOYOBO, Hanks' solution manufactured by Sigma (Hank's balanced salt solution) ) Etc. can be used.

  The incubator used for stem cell culture is not particularly limited as long as stem cells can be cultured. Examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. .

  The incubator may be non-cell-adhesive or adhesive, and is appropriately selected according to the purpose. As the cell-adhesive incubator, for the purpose of improving the adhesion with cells, a cell-treated culture vessel treated with a cell support substrate using an extracellular matrix or the like may be used. Examples of the cell support substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

  The concentration of triterpenic acid added to the medium used for stem cell culture can be appropriately determined according to the content of triterpenic acid in the above-described stem cell undifferentiated state maintenance agent or growth promoter according to the present invention. The concentration is 0.1 to 1000 μg / mL, preferably 1 to 100 μg / mL. In addition, triterpenic acid may be periodically added to the medium during the stem cell culture period.

Stem cell culture conditions may be in accordance with normal conditions used for stem cell culture, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably 36 to 37 ° C. The CO ₂ gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. In addition, it is preferable to perform culture | cultivation replacement | exchange once every 2-3 days, and it is more preferable to carry out every day. The culture conditions can also be set by varying as appropriate within the range in which the stem cells can survive and proliferate.

  Stem cell undifferentiated state maintenance is performed, for example, in the presence of the stem cell undifferentiated state maintaining agent according to the present invention as compared to the stem cell cultured in the absence of the stem cell undifferentiated state maintaining agent according to the present invention. The stem cell undifferentiated marker gene expression level in the stem cells can be evaluated by whether or not it is maintained at the mRNA level or the protein level at the same level as the expression level at the start of culture. Examples of the stem cell undifferentiation marker gene include Nanog gene (Cell Res. 2007 Jan; 17 (1): 42-9. Review. Nanog and transitional networks in Jemantic in Pamp. .

  Examples of the method for measuring the level of expression of stem cell undifferentiated marker gene include, for example, RT-PCR using primers and probes specific to stem cell undifferentiated marker gene, quantitative PCR, Northern blotting and the like at the mRNA level. On the protein level, for example, immunological methods such as ELISA using an antibody specific for a protein encoded by a stem cell undifferentiation marker gene, flow cytometry, Western blotting and the like can be mentioned.

  As a result of measuring the expression level, the expression level of the stem cell undifferentiation marker gene in the stem cell at the start of culture (100% undifferentiated state) and the stem cell after culturing for a predetermined time in the presence of the stem cell undifferentiated state maintaining agent of the present invention When the relative ratio of the expression level of the stem cell undifferentiation marker gene is greater than the same relative ratio (control) when cultured in the absence of the stem cell undifferentiated state maintenance agent of the present invention, the stem cell undifferentiated state It can be determined that it was maintained.

  In addition, the proliferation promotion of the stem cell is achieved by, for example, the stem cell cultured in the presence of the stem cell proliferation promoter according to the present invention, as compared with the stem cell cultured in the absence of the stem cell proliferation promoter according to the present invention. It can be evaluated by whether or not the number of cells is significantly increased. The number of cells can be measured using a commercially available cell number measurement kit by, for example, the MTT method or the WST method. As a result of the measurement, the relative ratio between the number of stem cells at the start of culture and the number of stem cells cultured for a predetermined time in the presence of the stem cell proliferation promoter of the present invention is the non-existence of the stem cell proliferation promoter of the present invention. It can be determined that the proliferation of stem cells could be promoted when the relative ratio (control) was greater when cultured in the presence.

  Stem cells prepared by the above-described method according to the present invention can be used as a transplant material (cell transplant agent), and can be performed by the same method as conventional bone marrow transplantation or umbilical cord blood transplantation.

  According to the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention or the method according to the present invention, triterpenic acid alone, separately from the medium or mixed with the medium, and the undifferentiated state of the stem cell It can also be provided as a reagent kit for maintenance or growth promotion. The kit can include an instruction manual or the like as necessary. Alternatively, triterpenic acid can be mixed with a medium and provided as a medium for maintaining the undifferentiated state of stem cells or promoting growth.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

Table 1 shows the triterpenic acid and fatty acid glycerides used in the evaluation tests of the examples and their structures. The following commercial products were used for triterpenic acid and fatty acid glycerides.
Oleanolic acid: Wako Pure Chemical Industries, Ltd. Ursolic acid: Sigma
1-palmitin-3-linolein: India Fine Chemical Company
1,3-Dilinolein: NU-CHEK-PREP

[Example 1] Evaluation of undifferentiated state maintaining effect and growth promoting effect of triterpenic acid on stem cells Hereinafter, undifferentiated state on stem cells using triterpenic acid, fatty acid glyceride, or a mixture of triterpenic acid and fatty acid glyceride as a test substance Experimental examples and results of the maintenance effect and the growth promoting effect are shown. When a mixture of triterpenic acid and fatty acid glyceride was used, the ratio was 1: 1 (weight ratio).

(Experimental example 1) Evaluation of an undifferentiated state maintenance effect with respect to a stem cell Dulbecco's Modified Eagle Medium culture solution (product made from Gibco), fetal bovine serum (FBS, 15%, product made from Sigma), nucleoside solution (100 times dilution, Dainippon Pharmaceutical Co., Ltd.), non-essential amino acid solution (100-fold dilution, Dainippon Pharmaceutical Co., Ltd.), β2-mercaptoethanol solution (100-fold dilution, Dainippon Pharmaceutical Co., Ltd.), L-glutamine solution (100-fold dilution, large Mouse embryonic stem cells (mouse ES cells: manufactured by Cosmo Bio) using a medium prepared by adding Nippon Pharmaceutical Co., Ltd., penicillin (100 units / mL, manufactured by Sigma) and streptomycin (100 μg / mL, manufactured by Sigma). ) were seeded 5x10 ⁵ cells in 6cm dish coated with gelatin (Sigma Co., Ltd.), the test The substance was added to the medium to a final concentration of 0.0005% and the culture was continued for 3 days.

  After 3 days of culture, the cells were collected, washed twice with PBS (−), and RNA was extracted from the cells with Trizol Reagent (Invitrogen). 2-STEP real-time PCR kit (Applied Biosystems) was used to reverse-transcribe the extracted RNA into cDNA, and then ABI7300 (Applied Biosystems) was used for real-time PCR (95 ° C: 15 seconds, 60 ° C) using the following primer set: : 30 seconds, 40 cycles), and Nanog (undifferentiated marker: Cell Res. 2007 Jan; 17 (1): 42-9. Revie. Nanog and transcribable networks in emp. The gene expression of was confirmed. Other operations were carried out in accordance with established methods.

Primer set for Nanog (undifferentiation marker):
ATGCCTGCAGGTTTTTCATCC (SEQ ID NO: 1)
GAGGCAGGTCTTCAGAGGAA (SEQ ID NO: 2)
Primer set for Gapdh (internal standard):
TGCACCACCAACTGCCTTAGC (SEQ ID NO: 3)
TCTTCTGGGTGGCAGTGATG (SEQ ID NO: 4)

The effect of maintaining the undifferentiated state is that the expression level of Nanog mRNA in mouse ES cells at the start of culture was calculated as a ratio relative to the expression level of Gapdh mRNA as an internal standard (Nanog gene expression level / Gapdh gene expression). The amount of the relative expression level of Nanog in ES cells after 3 days of culture was calculated and evaluated.
The test results are shown in Table 2 below.

  As shown in Table 2, both oleanolic acid and ursolic acid, which are triterpenic acids, have a remarkable effect of maintaining the undifferentiated state of stem cells, and 1-palmitin-3-linolein, which is a fatty acid glyceride, The effect was enhanced by the combined use of 1,3-dilinolein. From the above, it was clarified that triterpenic acid has an excellent effect of maintaining the undifferentiated state of stem cells. In addition to the stem cells used in this experimental example, a similar test was performed on somatic stem cells. As a result, a remarkable effect of maintaining the undifferentiated state of stem cells was observed.

(Experimental example 2) Evaluation of proliferation promoting effect on stem cells Human somatic stem cells (manufactured by DS PharmaBiomedical) cultured using a human stem cell culture medium (manufactured by TOYOBO) were seeded at 3 × 10 ^{5 in} a 6 cm dish and tested. Triterpenic acid or fatty acid glyceride was added as a substance to a final concentration of 0.0005%, and the culture was continued for 3 days. When a mixture of triterpenic acid and fatty acid glyceride was used as the test substance, the ratio was 1: 1 (weight ratio).

  After culturing for 3 days, the cells were washed three times with PBS (−) and then collected by a rubber policeman, and the number of each cell was counted.

When the total number of cells when the test substance was not added was used as a control and the control was 100 (%), the increase / decrease (%) of the number of cells when the test substance was added was calculated, and the stem cell proliferation promoting effect was evaluated.
These test results are shown in Table 3 below.

  As shown in Table 3, a remarkable effect of promoting the proliferation of stem cells was observed for oleanolic acid and ursolic acid, which are triterpenic acids. In addition, the effect was enhanced by using these in combination with 1-palmitin-3-linolein and 1,3-dilinolein, which are fatty acid glycerides. When the control value described above was 100%, the number of human somatic stem cells at the start of culture was 25%. From the above, it was clarified that triterpenic acid has an excellent effect of promoting the proliferation of stem cells. In addition to the stem cells used in this experimental example, a similar test was performed on embryonic stem cells (ES cells), and a remarkable effect of promoting stem cell proliferation was observed.

[Example 2] In vitro wound healing test (scratch assay)
As a method for evaluating the effectiveness of promoting wound healing, a commonly performed scratch assay (Japanese Patent Laid-Open No. 2013-18756) was performed. Specifically, 5 × 10 ⁵ human somatic stem cells are seeded in a 3.5 cm dish and cultured until confluent using a human stem cell culture solution, and then the bottom surface of the dish is scratched with the tip of a sterilized chip to form a cell sheet. A gap was created. Thereafter, the test substance was replaced with a medium added to a final concentration of 0.0005%, and the recovery rate of the cell gap was evaluated 48 hours later. Note that a mark that can be used as a reference point is provided in the vicinity of the scratch so that the same area can be obtained under a phase-contrast microscope when images before and after recovery are acquired.

  The scratch region after 48 hours was calculated by using commercially available image analysis software as [scratch region after 48 hours] = 100 × [scratch region area at 48 hours] / [scratch region area at 0 hours]. The test results are shown in Table 4 below.

  As shown in Table 4, oleanolic acid and ursolic acid, which are triterpene acids, showed a remarkable in vitro wound healing effect. In addition, the effect was enhanced by using these in combination with 1-palmitin-3-linolein and 1,3-dilinolein, which are fatty acid glycerides.

  As shown in each of the above experimental examples, by applying triterpenic acid to stem cells, it is possible to promote the proliferation of stem cells while maintaining an undifferentiated state easily and efficiently compared to conventional techniques. Became. Moreover, the triterpenic acid of this invention showed the very outstanding wound healing effect.

[Example 3] Example of product formulation An example of a product containing oleanolic acid or ursolic acid as triterpenic acid and 1-palmitin-3-linolein or 1,3-dilinolein as a fatty acid glyceride used in combination with triterpenic acid is shown below. .

(Formulation Example 1) Lotion Formulation Blending amount (% by weight)
1. Oleanolic acid 0.02
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4). Xanthan gum 0.02
5. Citric acid 0.01
6). Sodium citrate 0.1
7). Ethanol 5.0
8). Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
10. Fragrance 0.1
11. Purified water remaining amount After components 2-6 and 11 and components 1 and 7-10 are uniformly dissolved, both are mixed and filtered to prepare a lotion.

(Formulation example 2) Cream 1
Formulation amount (% by weight)
1. Ursolic acid 0.1
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12 Ethyl paraoxybenzoate 0.05
13.1,3-Butylene glycol 8.5
14 Purified water remaining amount Components 1 to 9 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 11-14 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. Next, an aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. Then, component 10 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product (cream 1).

(Formulation example 3) Cream 2
In Formulation Example 2, cream 2 was prepared by replacing 0.1% by weight of ursolic acid with 0.05% by weight of ursolic acid and 0.05% by weight of 1-palmitin-3-linolein.

(Formulation Example 4) Cream 3
In Formulation Example 2, Cream 3 was prepared by replacing 0.1% by weight of ursolic acid with 0.05% by weight of oleanolic acid and 0.05% by weight of 1,3-dilinolein.

(Formulation Example 5) Emulsion
Formulation amount (% by weight)
1. Oleanolic acid 0.1
2. Squalane 5.0
3. Olive oil 5.0
4). Jojoba oil 5.0
5. Cetanol 1.5
6). Glycerol monostearate 2.0
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Polyoxyethylene sorbitan monooleate (20E.O.) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12 Methyl paraoxybenzoate 0.2
13. Purified water remaining amount Components 1 to 8 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 10 to 13 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring.

(Formulation example 6) Gel formulation Formulation amount (% by weight)
1. Ursolic acid 0.1
2. Ethanol 5.0
3. Methyl paraoxybenzoate 0.1
4). Polyoxyethylene hydrogenated castor oil (20 EO) 0.1
5. Perfume proper amount 6.1,3-butylene glycol 5.0
7). Glycerin 5.0
8). Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. Purified water remaining amount Ingredients 1 to 5 and ingredients 6 to 11 are uniformly dissolved and mixed to obtain a product.

(Prescription Example 7) Ointment 1
Formulation amount (% by weight)
1. Oleanolic acid 2.0
2. Polyoxyethylene cetyl ether (30E.O.) 2.0
3. Glycerol monostearate 10.0
4). Liquid paraffin 5.0
5. Cetanol 6.0
6). Methyl paraoxybenzoate 0.1
7). Propylene glycol 10.0
8). Purified water remaining amount Components 1 to 5 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 6 to 8 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled to 30 ° C. while stirring to obtain a product (ointment 1).

(Prescription Example 8) Ointment 2
In Formulation Example 7, ointment 2 was prepared by replacing 2.0% by weight of oleanolic acid with 1.0% by weight of oleanolic acid and 1.0% by weight of 1-palmitin-3-linolein.

(Prescription Example 9) Ointment 3
In Formulation Example 7, ointment 3 was obtained by replacing 2.0% by weight of oleanolic acid with 1.0% by weight of ursolic acid and 1.0% by weight of 1,3-dilinolein.

(Prescription Example 10) Pack Formulation Blending amount (% by weight)
1. Ursolic acid 0.1
2. Polyvinyl alcohol 12.0
3. Ethanol 5.0
4.1,3-Butylene glycol 8.0
5. Methyl paraoxybenzoate 0.2
6). Polyoxyethylene hydrogenated castor oil (20 EO) 0.5
7). Citric acid 0.1
8). Sodium citrate 0.3
9. Perfume appropriate amount10. Purified water remaining amount Ingredients 1 to 10 are uniformly dissolved to obtain a product.

(Formulation Example 11) Tablet Formulation Formulation amount (% by weight)
1. Oleanolic acid 1.0
2. Dried corn starch 25.0
3. Carboxymethylcellulose calcium 20.0
4). Microcrystalline cellulose 40.0
5. Polyvinylpyrrolidone 8.0
6). Talc 6.0
Components 1-5 are mixed, then 10% water is added as a binder and dried after extrusion granulation. Ingredient 6 is added to the molded granules, mixed and compressed into tablets. One tablet is 0.52 g.

(Prescription Example 12) Beverage Formulation Blending amount (% by weight)
1. Ursolic acid 0.01
2. Stevia 0.05
3. Malic acid 5.0
4). Fragrance 0.1
5. Purified water remaining amount Ingredients 1 to 4 are dissolved in a part of the purified water of component 5 with stirring. The remaining purified water of Component 5 is then added and mixed, heated to 90 ° C. and filled into a 50 mL glass bottle.

  As an application example of the present invention, application to regenerative medicine and regenerative beauty is expected. For example, by utilizing the present invention, it becomes possible to easily and efficiently prepare undifferentiated stem cells used for regenerative medicine and regenerative beauty. Further, the triterpenic acid according to the present invention is directly injected or applied to the stem cells after transplantation of the stem cells or in the tissue by oral administration, application, pasting, etc., so that the stem cells are undifferentiated. It is possible to grow while maintaining the state. The triterpenic acid according to the present invention can be used as an excellent wound healing agent.

  That is, the present invention can be used as a method for preparing stem cells and / or an agent for maintaining an undifferentiated state of a stem cell, a growth promoting agent, or a wound healing agent in regenerative medicine and regenerative beauty.

Claims (8)

An agent for maintaining an undifferentiated state of stem cells in a medium, comprising triterpenic acid as an active ingredient. An agent for promoting proliferation of stem cells in a medium, comprising triterpenic acid as an active ingredient. A wound healing agent comprising a mixture of triterpenic acid and fatty acid diglyceride as an active ingredient.   The agent according to any one of claims 1 to 3, wherein the triterpenic acid is oleanolic acid or ursolic acid. A medicinal product or quasi-drug for maintaining stem cell undifferentiated state or promoting stem cell proliferation, comprising a mixture of triterpenic acid and fatty acid diglyceride as an active ingredient .   A method for producing stem cells, comprising a step of culturing stem cells in a medium containing triterpenic acid.   A method for maintaining an undifferentiated state of stem cells, comprising a step of culturing stem cells in a medium containing triterpenic acid.   A method for promoting the proliferation of stem cells, comprising a step of culturing stem cells in a medium containing triterpenic acid.