JP2020094003A - Agent for maintaining the undifferentiated state of stem cell and agent for promoting the proliferation of stem cell - Google Patents

Abstract

To discover a new material that can proliferate efficiently a stem cell while maintaining its undifferentiated state, and provide it as an agent for maintaining the undifferentiated state of a stem cell or an agent for promoting the proliferation of it.SOLUTION: An agent for maintaining the undifferentiated state of a stem cell and an agent for promoting the proliferation of a stem cell contains Laminaria japonica extract as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to an agent for maintaining an undifferentiated state of stem cells and a growth promoter, and a method for maintaining an undifferentiated state of stem cells and a method for promoting growth.

In vertebrate tissues (especially mammals), when cells or organs are damaged due to injury or disease, or aging, a regeneration system works to try to recover the damage to the cells or organs. Stem cells (tissue stem cells, somatic stem cells, adult stem cells) included in the tissue play a major role in this action. Stem cells have pluripotency to differentiate into all cells and organs, and it is believed that this property leads to recovery by compensating for damaged parts of cells and organs. Expectations are gathering for regenerative medicine, which is the next-generation medical treatment that applies such stem cells.

The most advanced tissue for stem cell research in mammals is the bone marrow. It was revealed that living hematopoietic stem cells are present in the bone marrow and are the source of all blood cell regeneration. Further, it has been reported that bone marrow contains, in addition to hematopoietic stem cells, stem cells capable of differentiating into organs or tissues (eg, bone, cartilage, muscle, fat, etc.) (see Non-Patent Document 1).

Furthermore, in recent years, in addition to bone marrow, it has been clarified that stem cells are present in all organs and tissues such as skin, liver, pancreas, and fat, and it is responsible for the regeneration and homeostasis of each organ and tissue. It has become clear (see Non-Patent Documents 2 to 5). In addition, stem cells present in each organ or tissue have excellent plasticity, and may be used for regeneration of organs or tissues, which until now could not self-renew.

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 carried out in order to maintain homeostasis of each tissue ( Non-Patent Document 6). In addition, in recent years, in order to apply the ability (pluripotency) of stem cells to the regeneration of organs and tissues, in the fields of cell transplantation therapy and tissue engineering (regenerative medicine and regenerative beauty), stem cells are separated from living tissues and then cultured. Development of a technique for breeding 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, which is the ability of the stem cells, that is, the undifferentiated state. If the undifferentiated state of stem cells cannot be maintained during this culture and the induction of differentiation has progressed, the finally prepared stem cells will have lost their ability (pluripotency), and the desired effect ( Regeneration of organs and tissues) cannot be exerted.

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

To date, there have been some reports on techniques for growing 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-culturing with supporting cells (stromal cells or feeder cells) (see Patent Document 1 and Non-Patent Documents 9 to 11). ). However, recently, an example of infection between heterologous animals due to an endogenous virus derived from a feeder cell has been reported (see Non-Patent Document 12), and culture of stem cells using feeder cells is intended for medical purposes. Is not suitable for culturing.

Another method is to maintain the undifferentiated state of stem cells 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, in the presence of the early acting cytokine thrombopoietin (TPO), interleukin 6 (IL-6), FLT-3 ligand, and stem cell factor (SCF), it is possible to maintain the undifferentiated state of embryonic stem cells, It has been reported in somatic stem cells and the like (see Patent Document 3 and Non-Patent Document 14).

However, cytokines are expensive and have problems such as raw materials to be collected and storability, so that easy use is difficult. In addition, the effect of LIF was extremely limited to a specific cell lineage, and it was revealed that addition of LIF alone cannot maintain an undifferentiated state in ES cells and somatic stem cells of primates. (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 utilize the stem cells for regenerative medicine, there has been a demand for a technique for proliferating the stem cells while maintaining an undifferentiated state. That is, there has been a demand for a technique capable of proliferating stem cells safely, simply and efficiently while maintaining an undifferentiated state.

Mackonbu (true kelp, scientific name: Saccharina japonica) is an alga that belongs to the kelp family, Kombu family, Kurofutsumu genus (Kombus genus), and has been eaten in Japan for a long time. In addition to processed products such as Tsukudani and kelp rolls, it also has an umami taste. It is widely distributed as dashi kelp because it contains abundant glutamic acid, which is the source of kelp. Makombu (makonbu) is also used as a crude drug in which the leaf stem is dried and has a gastrointestinal mucosa-protecting action and the like, and thus is mainly used in a herbal medicine for enhancing gastrointestinal function. So far, Macombus has an angiogenesis inhibitory action (Patent Document 4), a skin barrier function improving action based on involucrin production promotion (Patent Document 5), and a preventive/therapeutic effect for diabetic complications based on aldose reductase inhibition (Patent Document 6). ) Is known. However, nothing has been known so far about the effect of promoting growth of stem cells and the effect of maintaining undifferentiated state of mackerel.

JP 2004-24089 A Special table 2002-525042 gazette Japanese Patent No. 3573354 JP, 2006-22033, A JP, 2009-84195, A JP, 2008-214245, A

Pittenger M.A. F. Et al., Science, 1999, Vol. 284, pp. 143-147 Goodell M.D. A. Et al., Nat. Med. , 1997, Vol. 3, pp. 1337-1345 Zulewski H. et al. Et al., Diabetes, 2001, Vol. 50, pp. 521-533 Suzuki A. Et al., Hepatology, 2000, Vol. 32, pp. 1230-1239 Zuk P. A. Et al., Tissue Engineering, 2001, Vol. 7, pp. 211-228 Hasegawa, Y., Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 Mabuchi, Y. et al., Japanese Society for Regenerative Medicine, 2007, Vol. 6, pp. 263-268 Kitagawa Zen et al., Japanese Society of Regenerative Medicine, 2008, Vol. 7, pp. 14-18 Thomson J. A. Et al., Proc. Natl. Acad. Sci. USA, 1995, Vol. 92, pp. 7844-7848 Thomson J. A. Et al., Science, 1998, Vol. 282, pp. 1145-1147 Reubinoff B.A. E. Et al., Nature Biotech. , 2000, Vol. 18, pp. 399-404 van der Laan L.D. J. 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. Hematother. Stem Cell Res. , 2001, Vol. 10, pp. 481-492

In view of the above-mentioned circumstances, the present invention finds a new substance that can efficiently proliferate stem cells while maintaining the undifferentiated state, and provides the stem cells as an undifferentiated state maintenance agent or a growth promoter. Is an issue.

As a result of intensive studies to solve the above problems, the present inventors have found that an extract of mackerel has an excellent undifferentiated state-maintaining effect and a growth-promoting effect on stem cells, which have not been known so far. The present invention has been completed.

That is, the present invention includes the following.
(1) An agent for maintaining an undifferentiated state of a stem cell, which contains an extract of mackerel as an active ingredient.
(2) A stem cell growth promoter containing an extract of mackerel as an active ingredient.
(3) The agent according to (1) or (2), wherein the stem cells are hair follicle stem cells.
(4) A method for producing a stem cell, which comprises a step of culturing the stem cell in a medium containing an extract of mackerel.
(5) A method for maintaining an undifferentiated state of a stem cell, which comprises a step of culturing the stem cell in a medium containing an extract of mackerel.
(6) A method for promoting the proliferation of stem cells, which comprises a step of culturing the stem cells in a medium containing an extract of mackerel.
(7) The method according to any one of (4) to (6), wherein the stem cells are hair follicle stem cells.

According to the present invention, stem cells can be efficiently propagated while maintaining an undifferentiated state. Therefore, the present invention can greatly contribute to the fields of regenerative medicine and rejuvenating beauty.

Hereinafter, the present invention will be described in detail. The agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation according to the present invention contains an extract of mackerel as an active ingredient.

The mackerel (Saccharina japonica, Laminaria japonica as a synonym) used in the present invention is a seaweed belonging to the kelp family Laminaria kelpaceae (genus kelp) and is mainly distributed in southern Hokkaido, Tsugaru Strait, and northern Iwate. The mackerel grows by establishing strong roots in rocks shallower than a water depth of 20 m, and the grown leaves are bamboo leaf-shaped, with a length of 1.5-6 m and a width of 20-35 cm. Is thin and wavy. Mackonbu is the highest quality edible kelp and is mainly used for soup stock.

The source of mackerel is not particularly limited, but can be obtained from the above-mentioned growing area. In the present invention, it is preferable to use whole seaweed (whole algae) as a raw material for extracting mackerel, but a part thereof, for example, a leaf part (leaf body/adult leaf), a stem part (core/middle rib) , Spores, roots and the like. In addition, the seaweed main body may be used as it is for extraction, and may be subjected to treatments such as drying, crushing, and fine cutting.

The extraction method is not particularly limited, and it can be performed by a method of stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. Examples of the extraction solvent include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol). , Glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Etc.) and the like. Among these solvents, polar solvents such as water, lower alcohols and liquid polyhydric alcohols are preferable, and water, ethanol, 1,3-butylene glycol and propylene glycol are more preferable. These solvents may be used alone or in combination of two or more. Particularly preferred extraction solvents include water or water-ethanol mixed polar solvents. It is also possible to use a solvent whose pH is adjusted by adding an acid or an alkali to the extraction solvent.

The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more with respect to the seaweed (dry weight), but in the case of concentrating or isolating after extraction. For convenience of operation, it is preferably 100 times or less. The extraction temperature and time can be appropriately selected depending on the type of solvent used, the pressure during extraction, and the like. For example, 10 to 100° C., preferably 30 to 90° C., and 30 minutes to 24 hours, preferably 1 hour to 10 hours can be exemplified.

The above extract may be used as it is as an extracted solution, but if necessary, within the range that the effect of the present invention is exerted, concentration (concentration by vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization by activated carbon, etc. It may be used after being subjected to treatments such as deodorization and ethanol precipitation. Further, the extracted solution may be concentrated to dryness, spray-dried, freeze-dried and the like and used as a dried product.

The extract of mackerel thus obtained has an effect of efficiently proliferating stem cells while maintaining the undifferentiated state at the biological level or at the culture level. Can be used as Further, the agent for maintaining an undifferentiated state of stem cells or the growth promoter according to the present invention is also used as a stem cell culture medium additive for efficiently growing stem cells while maintaining an undifferentiated state, and as a research reagent. be able to.

By applying the undifferentiated state-maintaining agent or proliferation promoter for stem cells according to the present invention to stem cells of mammals including human, it is possible to maintain the undifferentiated state of stem cells and promote the proliferation of stem cells. .. The stem cells to which the agent for maintaining an undifferentiated state of a stem cell or the growth promoter according to the present invention is applied are not particularly limited as long as they meet the purpose of the present invention. For example, embryonic stem cells (ES cells); bone marrow, blood , Somatic stem cells present in skin (epidermis, dermis, subcutaneous tissue), fat, hair follicle, brain, nerve, liver, pancreas, kidney, muscle and other tissues; stem cells artificially produced by gene transfer or the like (Artificial pluripotent stem cells: iPS cells). The undifferentiated state-maintaining agent or growth promoter for stem cells according to the present invention preferably exerts more effect on hair follicle stem cells, maintains the undifferentiated state of hair follicle stem cells in the bulge region of the hair follicle, and differentiates. It is possible to promote proliferation while suppressing the above. 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 nuclei of somatic cells, And ES cells obtained by modifying the genes on the chromosome of those ES cells using a genetic engineering technique. Such an ES cell can be produced, for example, by a method known per se, but it can be obtained from a predetermined institution, or a commercially available product can be purchased. Further, these stem cells may be any of primary culture cells, subculture cells or frozen cells.

Further, the agent for maintaining an undifferentiated state of a stem cell or a growth promoter according to the present invention can be applied to all mammalian-derived stem cells as long as they have equivalent characteristics with respect to the direction of differentiation of stem cells and the process of differentiation. It is possible. For example, the agent for maintaining an undifferentiated state of stem cells or a growth promoter according to the present invention is a mammalian stem cell such as human, monkey, mouse, rat, guinea pig, rabbit, cat, dog, horse, cow, sheep, goat, pig, etc. Can be effective against.

The agent for maintaining an undifferentiated state of a stem cell or the growth promoter according to the present invention may be applied to a stem cell in vitro or in vivo, and in any case, the action can be exerted. Therefore, the agent for maintaining the undifferentiated state of stem cells or the growth promoter according to the present invention, by culturing the stem cells in a medium for stem cell culture added with an effective amount thereof, or by administering to mammals including humans , Can maintain the undifferentiated state of stem cells and promote proliferation.

Stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention, since the extract of Macombus as an active ingredient has an excellent stem cell undifferentiated state maintaining action and proliferation promoting action, skin, osteoblast, cartilage, It acts on stem cells of tissues or organs in the body such as muscles, nerves, fats and livers, and is effective for treating, improving and preventing disorders or damages of the tissues or organs. Further, since stem cells decrease or function decline with age, the undifferentiated state maintenance agent or proliferation promoter of the stem cells according to the present invention is effective in reducing or reducing the function of stem cells in the above-mentioned tissues or organs in the living body. It is effective in treating, ameliorating and preventing related diseases. Here, damage or damage to tissues or organs, as diseases related to reduction or functional decline of stem cells, for example, in the skin-related, wrinkles, tarmi, spots, dullness, rough skin, thickening of skin, open pores, acne scars. , Wounds, scars, keloids, etc., and also includes damage to the scalp and hair such as thinning hair and hair loss. In bone-related cases, osteoporosis, fractures (vertebral compression fractures, femoral neck fractures, etc.), in cartilage diseases, osteoarthritis, rheumatoid arthritis, herniated discs, etc., in nerve-related areas, spinal cord injury, facial nerve paralysis. , Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, memory loss with age, blood related aplastic anemia, leukemia, cardiovascular related myocardial infarction, arteriosclerosis obliterans, etc. In relation to periodontal disease, alveolar bone damage due to alveolar pyorrhea, in ophthalmology-related retinitis pigmentosa, age-related macular degeneration, glaucoma, etc., and in liver-pancreas-related hepatitis, cirrhosis, diabetes, etc. Not limited to.

In addition, the extract of mackerel has an action of promoting the growth of hair follicle stem cells and an action of maintaining an undifferentiated state. That is, the extract of mackerel can suppress the reduction or depletion of hair follicle stem cells that are the origin of hair by promoting the differentiation of hair follicle stem cells in the bulge region of the hair follicle and promote the proliferation of hair follicle stem cells. Therefore, it can be used as an active ingredient of a prophylactic and/or improving agent for alopecia. In the present invention, "prevention and/or amelioration of alopecia" includes prevention of hair loss and thinning hair, improvement of the degree of hair loss and thinning hair (number and range), reduction of progression rate of hair loss and thinning hair, hair loss and thinning hair. Including suppression of loss of gloss and elasticity of hair. Further, the preventive and/or ameliorating effect of alopecia includes both a case where a direct action mechanism is shown on the hair and a case where a percutaneous action mechanism on the head is shown. In the present invention, alopecia refers to a condition in which a part or all of the hair comes off and a part or the whole of the scalp can be seen through due to various stresses such as aging, diseases, ultraviolet rays and overwork. .. Alopecia includes, for example, androgenetic alopecia (AGA), diffuse alopecia (FAGA), alopecia areata, senile alopecia, seborrheic alopecia, alopecia pityroides, postpartum (postpartum) alopecia. Alopecia, mechanical (compressive or traction) alopecia, congenital alopecia, alopecia after burns or trauma, drug-induced alopecia with anticancer agents, scarring alopecia (lichen planus pilaris, erythematous) Lupus, bald folliculitis, papillodermatitis, etc.), trichotyromania (hair loss), and the like, but are not limited thereto.

The content of the extract of mackerel in the agent for maintaining the undifferentiated state of stem cells or the growth promoter according to the present invention is not particularly limited, but may be, for example, 0 depending on the properties of the extract (extract, concentrate, or dried product). It can be appropriately set in the range of 0.0001 to 100% by weight, preferably 0.0001 to 10% by weight, and more preferably 0.001 to 1% by weight.

The present invention also relates to a method of stimulating stem cell growth while culturing stem cells in a medium containing an extract of mackerel, while maintaining the undifferentiated state of the stem cells. In other words, the method according to the present invention comprises a step of culturing stem cells in a medium containing an extract of Macombus, a method for producing stem cells, a method for maintaining an undifferentiated state of stem cells, or a method for promoting proliferation of stem cells. You can

In the method according to the present invention, culture of stem cells may be carried out by using a medium generally used for maintaining and proliferating undifferentiated state of stem cells. For example, a basic medium containing components necessary for survival and proliferation of stem cells (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins and fatty acids), specifically, Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium(MEM), RPMI1640, Basal Medium Eagle(BME), Ham's F12, Ham's F12K(Kaighn's modified of Ham's F12), Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12(D-MEM/F-12), Glasgow Inimum Essential Medium (Glasgow MEM), Hank's balanced salt solution, MCDB153 medium and the like can be mentioned. In addition, growth medium [epithelial cell growth factor (EGF), cholera toxin, basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), Stem Cell Factor (SCF), etc.], hormone (insulin , Hydrocortisone, etc.) are preferably added. In addition, if necessary, the medium may include tumor necrosis factor (TNF), vitamins, interleukins, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement. , Antibiotics, etc. may be contained.

In addition to the above components, the medium preferably contains bovine pituitary extract (BPE), serum or serum substitute at a content of 0.1 to 20% (v/v). Any known serum or serum substitute can be used. For example, the serum includes fetal bovine serum (FBS, FCS) and the like, and the serum substitute includes KSR and the like. However, since the components of serum differ depending on the lot, and the effect varies, it is preferable to use serum after lot checking.

Commercially available media include mesenchymal stem cell basal medium made by Invitrogen, mesenchymal stem cell basal medium made by Sanko Junyaku, MF medium made by TOYOBO, Hank's balanced salt solution made by Sigma, etc. Can be used. In addition, when the stem cells are hair follicle stem cells, a commercially available medium for epithelial cells containing components necessary for culturing epithelial cells (for example, insulin, hydrocortisone, epidermal growth factor, etc.) may be used. Keratinocyte-SFM medium (Thermo Fisher Scientific), Humedia-KG2 medium (KURABO), CnT-PR medium (CellnTec), MCDB153 medium (Sigma), serum-free medium for normal human keratinocytes (DS Pharma Biomedical) Manufactured by the company) and the like.

The incubator used for culturing the stem cells is not particularly limited as long as it can cultivate the stem cells, and examples thereof include a flask, a petri dish, a dish, a plate, a chamber slide, a tube, a tray, a culture bag, and a roller bottle. ..

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

The concentration of Macombus extract added to the medium used for stem cell culture may be appropriately determined according to the content of Macombus extract in the undifferentiated state maintaining agent or proliferation promoter for stem cells according to the present invention. Although it is possible, the concentration is, for example, 10 to 10000 μg/mL, preferably 100 to 5000 μg/mL in terms of the dried product of Macombus. Further, these extracts may be added to the medium periodically during the culture period of the stem cells.

The culture conditions of the stem cells may be the usual conditions used for culturing the stem cells, 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 1-10%, preferably about 2-5%. The medium is preferably exchanged once every 2 to 3 days, more preferably every day. The culturing conditions can be appropriately changed and set within a range in which the stem cells can survive and proliferate.

The undifferentiated state maintenance of the stem cells, for example, compared with the stem cells cultured in the absence of the undifferentiated state maintenance agent of the stem cells according to the present invention, cultured in the presence of the undifferentiated state maintenance agent of the stem cells of the present invention It can be evaluated whether the expression level of the stem cell undifferentiated marker gene is significantly maintained at the mRNA level or the protein level at the same level as the expression level at the start of culture in the stem cells. Examples of the stem cell undifferentiated marker gene include Nanog gene (Cell Res. 2007 Jan; 17(1):42-9. Review. Nanog and transcriptional networks in embryonic stem cell pluripotonics. A., etc.). .. Examples of the hair follicle stem cell undifferentiated marker gene include KRT15 (Keratin, type I cytoskeletal 15) and the like.

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

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

Further, the promotion of the proliferation of stem cells, for example, of the stem cells cultured in the presence of the growth promoting agent for stem cells of the present invention, as compared to the stem cells cultured in the absence of the growth promoting agent for stem cells of the present invention. It can be evaluated by whether or not the cell number is significantly increased. The cell number can be measured, for example, by the MTT method or the WST method using a commercially available cell number measurement kit. 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 after culturing for a predetermined time in the presence of the stem cell growth promoter of the present invention is a It can be determined that the proliferation of stem cells could be promoted when the relative ratio (control) when cultured in the presence was higher.

The stem cells prepared by the method according to the present invention described above can be used as a transplant material (cell transplant) and can be carried out by the same method as conventional bone marrow transplant or cord blood transplant.

According to the above-mentioned undifferentiated state maintaining agent or growth promoting agent for stem cells according to the present invention or the method according to the present invention, an extract of mackerel alone, or separately or mixed with a medium, the stem cells It can also be provided as a reagent kit for maintaining a differentiated state or promoting proliferation. The kit can include an instruction manual and the like as necessary. Alternatively, the mackerel extract can be mixed with a medium and provided as a medium for maintaining an undifferentiated state of stem cells or promoting proliferation.

When the agent for maintaining the undifferentiated state of the stem cells or the growth promoter according to the present invention is administered in vivo, it is also possible to administer it as it is, together with appropriate additives within a range that does not impair the effects of the present invention. It can be provided by being mixed with various compositions such as cosmetics, quasi drugs, pharmaceuticals, foods and drinks. The drug of the present invention includes a drug used for animals, that is, a veterinary drug.

When the agent for maintaining an undifferentiated state of stem cells or a growth promoter according to the present invention is mixed with cosmetics or quasi drugs, the dosage form is an aqueous solution system, a solubilization system, an emulsion system, a powder system, a powder dispersion system, Any of an oil liquid system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, a water-oil-powder three-layer system and the like may be used. In addition, the cosmetics and quasi-drugs are selected from various components, additives, bases, etc., which are usually used in the composition for external use for skin, depending on the type thereof, together with the agent for maintaining the undifferentiated state of stem cells or the growth promoter. However, they can be appropriately mixed and manufactured according to a method known in the art. The form may be any of liquid, emulsion, cream, gel, paste, spray and the like. As the compounding ingredients of the external composition for skin, for example, oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons ( Liquid paraffin, squalene, squalane, vaseline, 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, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid) Acids, etc.), saccharides (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and hydrolysates of proteins, amino acids and salts thereof, vitamins, plant/animal extract components, various surface activities Examples include agents, humectants, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, and fragrances.

The types of cosmetics and quasi-drugs include, for example, lotions, emulsions, gels, beauty essences, general creams, sunscreen creams, packs, masks, facial cleansers, toilet soaps, foundations, bath powders, body lotions, Body shampoo, hair shampoo, hair conditioner, hair restorer and the like can be mentioned.

When the agent for maintaining the undifferentiated state of stem cells or the growth promoter according to the present invention is added to a drug, it is mixed with a pharmacologically and pharmaceutically acceptable additive, and is suitable for application to the affected area. Can be formulated into various preparations. The pharmacologically and pharmaceutically acceptable additives include base materials for preparations, carriers, excipients, diluents, binders, lubricants, coatings, which 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 , Coloring agents, sweeteners, corrigents, flavors and the like may be appropriately added to prepare various preparation forms that can be systemically or locally administered orally or parenterally by various known methods. When the pharmaceutical product of the present invention is provided in each of the above-mentioned forms, it can be produced by a production method usually used by those skilled in the art, for example, the production method shown in the general rules for formulation [2] each formulation of the Japanese Pharmacopoeia.

The form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include tablets, sugar-coated tablets, capsules, troches, granules, powders, solutions, pills, emulsions, syrups, suspensions, and elixirs. Agents such as agents, injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, suppositories, ointments, lotions, eye drops, sprays, A skin absorbent, a transmucosal absorbent, a parenteral agent such as a patch, and the like can be mentioned. Alternatively, it may be a dry product to be redissolved when used, and in the case of an injectable preparation, it is provided in a unit dose ampoule or a multi-dose container.

An agent for maintaining an undifferentiated state of a stem cell according to the present invention or a growth promoter is a topical preparation suitable when used as a medicine for treating, ameliorating, and preventing the skin-related damages and diseases, for example, Examples include ointments, creams, gels, liquids, patches (patches, plasters), foams, sprays, sprays and the like. Ointment refers to a homogeneous semisolid external preparation, and includes oily and ointment, emulsion ointment and water-soluble ointment. The gel agent refers to an external preparation in which a water-insoluble component hydrate compound is suspended in an aqueous liquid. The liquid agent refers to a liquid external preparation, and includes lotions, suspensions, emulsions, liniments and the like.

The pharmaceutical agent of the present invention functions as a prophylactic agent for suppressing the onset of the above-mentioned diseases and/or as a therapeutic agent for improving a normal condition. The active ingredient of the pharmaceutical agent of the present invention is derived from a natural product, and is highly safe and has no side effects. Therefore, when used as a pharmaceutical agent for treating, ameliorating, and preventing the above-mentioned diseases, human, mouse, rat, rabbit. It can be orally or parenterally administered in a wide range of doses to mammals such as dogs, cats and the like.

The content of the agent for maintaining the undifferentiated state of stem cells or the growth promoter in the cosmetics, pharmaceuticals, and quasi-drugs of the present invention is not particularly limited, but it is a dried product of mackerel extract based on the total weight of the formulation (composition). It is preferably 0.001 to 30% by weight, more preferably 0.01 to 10% by weight. The above amount is merely an example, and may be appropriately set/adjusted in consideration of the type and form of the composition, a general amount used, efficacy, and the like. The method of adding the active ingredient in the formulation may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

Further, the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation according to the present invention can be incorporated into food and drink. Further, in the present invention, food and drink, in addition to general food and drink, foods that can be ingested for the purpose of maintaining or promoting health other than pharmaceuticals, for example, health foods, functional foods, health functional foods, or special uses Used to include food. Health foods include foods provided under the names such as dietary supplements, health supplements, and supplements. Foods with health claims are defined by the Food Sanitation Act or Health Promotion Act, and can display the effects of specific health, the function of nutritional components, the reduction of disease risk, etc., based on scientific evidence Includes foods with functional labeling that can display the contents notified to the Commissioner of the Consumer Affairs Agency regarding the functionality. In addition, the special-purpose foods include foods for the sick, foods for the elderly, foods for infants, foods for pregnant women, and the like, which indicate that they are suitable for a specific target person or a patient having a specific disease. When the stem cells are hair follicle stem cells, the present agent can be preferably used as a health food for preventing or improving hair loss and thinning hair. Here, the indication of the effect of specific health attached to food and drink and the function of nutritional components, etc., is displayed on products such as containers, packaging, instructions, package inserts, product leaflets and pamphlets, newspapers and magazines, etc. It can be an advertisement for the product.

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

Types of food and drink include breads, noodles, confectionery, dairy products, processed fish and animal products, oils and fats and processed foods, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks , Milk drinks, etc.), concentrated stock solutions of the drinks, adjustment powders, and the like, but are not limited thereto.

The food or drink of the present invention may be appropriately blended with additives usually used depending on its type. As the additive, any additive that is acceptable under the Food Sanitation Law can be used, and for example, sweeteners such as glucose, sucrose, fructose, isomerized 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 turbidity agents and preservatives.

When the food or drink of the present invention is a general food or drink, it can be manufactured by including a step of adding an extract of mackerel in the usual manufacturing process of the food or drink. Further, in the case of health food, it may be in accordance with the above-mentioned method for producing a pharmaceutical product. For example, in the case of a tablet-like supplement, an additive such as an excipient is added to and mixed with an extract of mackerel, and a tableting machine, etc. It can be manufactured by applying pressure under molding. In addition, other materials (for example, vitamins such as vitamin C, vitamin B ₂ , and vitamin B ₆ , minerals such as calcium, dietary fiber, etc.) can be added as necessary.

The amount of mackerel extract in the food and drink of the present invention may be an amount capable of exerting an undifferentiated state maintaining effect and a growth promoting effect on stem cells, but the general intake of the target food and drink, the form of the food and drink It may be appropriately set in consideration of efficacy, effect, taste, taste, cost, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to these examples.

[Example 1] Production example of mackerel extract An extract of mackerel was produced as follows. In Production Examples 1 to 4, seaweed whole algae was used as the extraction material.

(Production Example 1) Production of hot water extract of mackerel 200 mL of water was added to 10 g of dried mackerel, and the mixture was extracted at 95 to 100°C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.0 g of a hot water extract of mackerel.

(Production Example 2) Production of 50% ethanol extract of Macombus 10 g of the dried product of Macombus was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. The obtained extract was filtered and then concentrated to dryness with an evaporator to obtain 0.3 g of Macombus 50% ethanol extract.

(Production Example 3) Production of mackerel ethanol extract 10 g of dried mackerel ethanol was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. The obtained extract was filtered and then concentrated to dryness with an evaporator to obtain 0.1 g of an ethanol extract of mackerel.

(Production Example 4) Production of Mackerel 1,3-butylene glycol extract 10 g of dried Macombus was soaked in 200 mL of 1,3-butylene glycol at room temperature for 7 days for extraction. The obtained extract was filtered to obtain 190 g of 1,3-butylene glycol extract of mackerel.

[Example 2] Growth-promoting effect of mackerel extract on hair follicle stem cells and effect of maintaining undifferentiated state The growth-promoting effect of mackerel extract prepared in Example 1 on stem cells and maintaining undifferentiated state are described below. An experimental example of the effect and the result are shown.

(Experimental Example 1) Evaluation of proliferation promoting effect of mackerel extract on hair follicle stem cells (1) Culturing of human hair follicle stem cells Human hair was collected without hair and the bulge region of hair follicle tissue was included using a scalpel or the like. The tissue was harvested. After washing with PBS(-), trypsin (BD Biosciences) treatment was performed. Then, cells were isolated and collected using a cell strainer (FALCON). The collected cells were seeded on a culture plate and maintained until they became confluent using KG2 medium (KURABO). The cells that became confluent were collected, replated on a culture plate, and then engrafted and expanded cells were used as hair follicle stem cells in the following test.

(2) Evaluation of proliferation promoting activity of mackerel extract on hair follicle stem cells The mackerel extract was allowed to act on the hair follicle stem cells to evaluate how the mackerel extract was involved in the proliferation of hair follicle stem cells. Hair follicle stem cells cultured in KG2 medium (manufactured by KURABO) were seeded on a 24-well culture plate at 1×10 ⁴ cells each, and the final concentration of the test substance (Macomb extract of Production Examples 1 to 4) was 1000 μg/ It was added so that the amount became ml, and the cells were cultured for 3 days.

The cell proliferation rate of hair follicle stem cells after 3 days was detected by MTT assay. The MTT assay was carried out using a commercially available MTT assay kit (manufactured by Cosmo Bio) according to the attached protocol. The total number of cells when the test substance was not added was used as a control, and the increase/decrease (%) in the number of cells when the test substance was added was calculated when the control was 100 (%), and the effect of promoting stem cell proliferation was evaluated. The results of these tests are shown in Table 1 below.

As shown in Table 1, all the extracts of mackerel (Production Examples 1 to 4) had a remarkable effect of promoting proliferation on hair follicle stem cells. In addition to the stem cells used in this experimental example, when a similar test was performed on embryonic stem cells (ES cells), a remarkable effect of promoting stem cell proliferation was observed.

(Experimental Example 2) Evaluation of effect of mackerel extract on maintaining undifferentiated state on hair follicle stem cells How mackerel extract is allowed to act on hair follicle stem cells and how mackerel extract is involved in undifferentiated state of hair follicle stem cells Was evaluated. Hair follicle stem cells cultured in KG2 medium (manufactured by KURABO) were seeded on a 24-well culture plate at 1×10 ⁴ cells each, and the final concentration of the test substance (Macomb extract of Production Examples 1 to 4) was 1000 μg/ The culture was performed by adding 3 ml of the mackerel extract, and the hair follicle stem cells were collected 3 days after the addition of the mackerel extract, and the undifferentiated state of the hair follicle stem cells was expressed by the gene expression level of KRT15 Analyzed.

The gene expression analysis of KRT15 was performed as follows. The hair follicle stem cells after the addition of the mackerel extract were washed twice with PBS(-), and then RNA was extracted from the cells by Trizol Reagent (Invitrogen). Using 2-STEP real-time PCR kit (Applied Biosystems), after reverse transcribing the extracted RNA into cDNA, ABI7300 (Applied Biosystems), by using the following primer set real-time PCR (95 ℃: 15 seconds , 60° C.: 30 seconds, 40 cycles), and the gene expression level of KRT15 was measured. Other operations were performed according to the prescribed method.

KRT15 (hair follicle stem cell undifferentiation marker) primer set:
5'- GATGCTGCTTGACATAAAGACACG-3' (SEQ ID NO: 1)
5'- ACCTGTCCATCCACTGACTCTTC-3' (SEQ ID NO: 2)
18s rRNA (internal standard) primer set:
5'-CCGAGCCGCCTGGATAC-3' (SEQ ID NO: 3)
5'-CAGTTCCGAAAACCAACAAAATAGA-3' (SEQ ID NO: 4)

The effect of maintaining the undifferentiated state of hair follicle stem cells was calculated by calculating the relative expression level of KRT15 gene as a ratio of the expression level of KRT15 mRNA in hair follicle stem cells at the start of culture to the expression level of 18s ribosomal RNA (18srRNA) which is an internal standard ( The value of (KRT15 gene expression level/18s rRNA gene expression level) was set as 100% in an undifferentiated state, and the value of KRT15 gene relative expression level in hair follicle stem cells after 3 days of culture was calculated and evaluated. The results of these tests are shown in Table 2 below.

As shown in Table 2, the mackerel extracts (Production Examples 1 to 4) exhibited a remarkable effect of maintaining the undifferentiated state on hair follicle stem cells. In addition to the stem cells used in this experimental example, when a similar test was performed on embryonic stem cells (ES cells), a remarkable effect of maintaining the undifferentiated state of stem cells was observed.

As shown in each of the above experimental examples, by applying the extract of mackerel to the stem cells, compared to conventional techniques, to promote the proliferation of stem cells while maintaining the undifferentiated state, simply and efficiently. Became possible.

As an application example of the present invention, application to regenerative medicine and rejuvenating 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 rejuvenation. Furthermore, after transplantation of stem cells or to the stem cells present in the tissue, the extract of mackerel according to the present invention is directly injected or orally administered, applied, by applying by application, etc., the stem cells, It is possible to grow while maintaining an undifferentiated state.
That is, the present invention can be used as a method for producing stem cells and/or an agent for maintaining an undifferentiated state of stem cells or a growth promoter in regenerative medicine and rejuvenation.

Claims (7)

An agent for maintaining an undifferentiated state of a stem cell, which contains an extract of mackerel as an active ingredient. A stem cell growth promoter containing an extract of mackerel as an active ingredient. The agent according to claim 1 or 2, wherein the stem cells are hair follicle stem cells. A method for producing a stem cell, comprising the step of culturing the stem cell in a medium containing an extract of Mackerel. A method for maintaining an undifferentiated state of a stem cell, comprising the step of culturing the stem cell in a medium containing an extract of Mackerel. A method for promoting the growth of stem cells, which comprises the step of culturing the stem cells in a medium containing an extract of mackerel. The method according to any one of claims 4 to 6, wherein the stem cells are hair follicle stem cells.