KR20170050633A

KR20170050633A - Tripeptide with activities of growth and activation of skin keratinocyte stem cell and use thereof

Info

Publication number: KR20170050633A
Application number: KR1020150152415A
Authority: KR
Inventors: 최호일; 김동석; 송상용; 양현; 이정옥; 고희주; 이상화; 김도형; 여혜린
Original assignee: 주식회사 펩트론; 주식회사 엘지생활건강
Priority date: 2015-10-30
Filing date: 2015-10-30
Publication date: 2017-05-11
Also published as: KR101796678B1

Abstract

The present invention provides peptides for improving skin regeneration ability or skin elasticity, comprising tripeptide and a structure of mutants thereof. The peptide induces proliferation of epidermal keratinocyte stem cell and secretion activity of cell growth factors, thereby showing an improving effect of skin regeneration ability or a skin elasticity effect, and being able to be used in various fields of cosmetics or pharmacology.

Description

[0001] The present invention relates to tripeptides having activity of proliferating and activating keratinocyte stem cells,

The present invention relates to a peptide having the effect of proliferating human keratinocyte stem cells, or a variant thereof, and a use thereof, and more particularly, to the use of the expression of various growth factors of human keratinocyte cells as an agent for promoting proliferation and proliferation of keratinocyte stem cells And a pharmaceutical or cosmetic composition containing the growth promoting agent.

Skin is the largest organ of the human body, accounting for about 18% of our body volume, consisting of epidermis, dermis and subcutaneous fat layers. Such skin is in direct contact with the external environment and plays an important protective layer for protecting the human body from various harmful factors such as harmful microorganisms and ultraviolet rays, and at the same time, it has a biochemical function necessary for metabolism of the whole body. It is an indispensable institution.

The skin is structurally largely composed of the epidermis and the dermis, and the epidermis can be divided into a stratum corneum composed of flat hexagonal cells and a living epidermis. The living epidermis comprises four layers: a basal layer (Stratum Spinosum), a granular layer (Stratum Granulosum), and a transparent layer (stratum Iucidum). Among them, keratinocyte stem cells (KSC) of the skin present in the basal layer are known to be capable of regenerating.

The skin keratinocyte stem cells continue to divide and produce two types of cells, one is self-renewal, and the other is a transdermal embryonic stem cell. , TA cells). These cells divide several times and form keratinocyte cells, which play a central role in the skin barrier function of the mid / late part of the skin developmentally. The keratinocytes undergo differentiation and are pushed up to the skin surface, which is the last stage of dermatologic development. In this process, differentiation and keratinization of the keratinocytes are made and the keratin layer is formed, and eventually the keratinocytes are separated from the keratin layer.

The keratinocyte and metastasis-amplifying cells are usually distributed in an extremely low number of about 4 to 7% of the basal layer cells, and as the person becomes older, the ability to proliferate the keratinocyte stem cells and metastasis-amplifying cells decreases . If the ability to make keratinocytes is lowered, the skin regenerating ability is eventually decreased and the aging phenomenon such as skin elasticity deteriorates. Therefore, it is necessary to develop a composition that promotes the proliferative capacity of dermal horny stem cells.

An example of the present invention is to provide an agent for promoting proliferation of keratinocyte stem cells without stimulation to the skin, which comprises a peptide as an active ingredient.

A further aspect of the present invention is to provide a composition for promoting the expression or production of various growth factors of dermal horny stem cells, which comprises a peptide as an active ingredient.

Another aspect of the present invention is to provide a cosmetic composition comprising as an active ingredient an agent for promoting the proliferation or proliferation of various keratinocyte stem cells of the skin keratinocyte stem cell, Improvement of skin moisturizing ability, improvement of skin elasticity, improvement of skin wrinkles, and improvement of skin aging can be achieved.

Another aspect of the present invention is to provide a pharmaceutical composition comprising, as an active ingredient, an agent for promoting the proliferation or proliferation of various keratinocyte stem cells in the skin keratinocyte stem cell, Improvement of skin moisturizing ability, improvement of skin elasticity, improvement of skin wrinkles, and improvement of skin aging can be achieved.

In order to accomplish the above object, the present invention provides a skin stimulating agent for promoting proliferation of keratinocyte stem cells comprising a peptide as an active ingredient, an agent for promoting expression or production of various growth factors of dermal keratotic stem cells, A skin elasticity improvement, a skin wrinkle improvement, and a skin aging improvement.

Hereinafter, the present invention will be described in more detail.

The peptide according to the present invention may comprise a tripeptide of phenylalanine-histidine-arginine (F-H-R) or a mutant thereof.

The tripeptides of the invention, for example, phenylalanine-histidine-arginine (FHR) or variants thereof, include an L- or D-amino acid enantiomer containing an amino acid residue that is a single enantiomer or a combination of two enantiomeric forms . In addition, as long as the primary amino acid sequence is maintained, various modifications can be introduced into the tripeptides of the present invention. Some modifications may be used to increase the efficacy of the peptide, while other modifications may facilitate peptide manipulation. For example, functional groups that can be introduced into the peptide include hydroxyl, acetylation, ester, or amide groups. For example, the reaction for the introduction of the functional group can be carried out by acetylation of the hydroxyl group with an alkyl halide; Esterification or amidation of the carboxyl group. Carboxy-terminal amidation of peptides can provide peptides that are less sensitive to protease degradation, increase its solubility and increase therapeutic efficacy compared to its free acid form.

Modifications of the peptide according to the present invention may include, for example, at least one group selected from the group consisting of amidation of the C-terminal of the tripeptide and acetylation of the N-terminal. Specifically, the hydroxy group of the phenylalanine of the present invention may be substituted with an acetyl group, or the carboxyl group of arginine may be amidated. For example, the peptide or a variant thereof may be a compound having the formula:

[Chemical Formula 1]

X-phenylalanine-histidine-arginine-Y

In Formula 1,

X is a hydroxy group or an acetyl group,

Y is a carboxy group, -NH _2, -NHR ¹ , or -NR ¹ R ² , And R ¹ and R ² may each independently be a C 1 -C 4 alkyl group.

The synthesis of the peptides of the present invention can be carried out, for example, using a device or using genetic engineering techniques. When synthesized using a device, the desired peptide is synthesized by the Fmoc solid-phase method in an automatic peptide synthesizer (PeptrEX-R48, Peptron, Daejeon, Korea). The synthetic peptide was separated from the resin and purified by reversed-phase HPLC (reverse-phase HPLC, Prominence LC-20AB, Shimadzu, Japan) using a Shiseido Capsel pack C18 analytical RP column (Shiseido capcell pak C18 analytical RP column) &Lt; / RTI > The final peptide is identified using a mass spectrometer (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA). When a genetic engineering technique is used, the desired nucleotide sequence of the desired peptide is introduced into a protein expression vector, and then a bacterium in which protease deficient such as BL21 (λDE3) and BL21 (λDE3) pLys is deficient, Induced expression using IPTG and pure separation.

The tripeptide according to the present invention or a mutant thereof improves skin condition through proliferation of skin keratinocyte stem cells, such as improvement of skin regeneration ability, improvement of skin moisturizing ability, improvement of skin elasticity, improvement of skin wrinkles and improvement of skin aging.

As used herein, the term "skin regeneration" is a broad concept including the ability to generate new keratinocytes in response to an overall skin aging, a wound caused by skin epidermis, and the like. Specifically, It is the process that differentiation and keratinization occurs and the stratum corneum is formed by the early stage differentiation and keratinocyte which is transferred to the surface of the skin. do. Among them, the key process of skin regeneration is the proliferation of keratinocyte stem cells in the basal layer and metastasis - amplification cells which are the daughter cells of these cells. In order to restore skin damage caused by the physical and chemical environment outside the skin, it is necessary to actively proliferate keratinocyte stem cells and metastasis-amplification cells, which are in the basal layer of skin and responsible for skin regeneration. The reason for the scar remains when the skin is damaged due to age, while the scar is not left even when the skin damage occurs at a normal age. The level of proliferation of keratinocyte and metastasis-amplification cells necessary for skin regeneration is remarkable depending on the age . That is, as the aging progresses, the degree of proliferation of these cells becomes low, which leads to a decrease in skin regeneration ability, which may delay the recovery of wounds and the like and reduce skin elasticity. Therefore, the proliferation of Keratinocyte Stem Cells (KSC) can regenerate the skin or prevent skin aging.

Further, in the present invention, the term "skin elasticity" is expressed by elastic fibers composed of elastin existing in the dermal layer, and "skin elasticity enhancement" means that elastic fibers composed of elastin and collagen, Skin elasticity is maintained.

The tripeptide according to the present invention or its variant according to the present invention can promote the expression or production of a cell growth factor of dermal horny stem cells. IGFBP-4, IGFBP-6, IGF-I, and IGFBP-6, which are increased by both peptide-1 and peptide-2, IGF-II, G-CSF, HB-EGF, IGFBP-3, IGF-II and IGF-IGF-ISR, M-CSF, M-CSFR, TGF-beta, VEGF and VEGFR3. , NT-3, PDGF-AB, PDGF-BB, PIGF, SCF, SCFR, TGF-beta3 and peptide-2 may be b-NGF or GCSF alone.

EGF (Epidermal Growth Factor) promotes the growth of fibroblasts and keratinocytes and induces collagen biosynthesis and regeneration of skin barrier. IGF (Insulin like Growth Factor) is known to promote cell division and regenerate damaged cells. FGF (Fibroblast Growth Factor) is a generic term for a wide variety of signaling substances (cytokines) and is known to be a growth factor that is deeply involved in the proliferation and differentiation of cells and tissues. It is known that the proliferation of fibroblasts increases the synthesis of ECM (Extracellular Material) such as collagen, elastin fibers and hyaluronic acid. VEGF (Vascular Endothelial Growth Factor) is mainly derived from platelets and promotes the division of vascular endothelial cells and is known to be involved in the formation of new blood vessels. It protects blood vessels, is involved in cell differentiation and migration, and is known to affect hair growth and hair follicle strengthening. Platelet-Derived Growth Factor (PDGF) promotes proliferation of osteoblasts and osteogenic tissues involved in bone regeneration and bone regeneration, promotes endothelial cell proliferation, and produces new blood vessels And promotes fibroblast proliferation and is known to be involved in collagen production. TGF (Transforming Growth Factor) is known to play a major role in the synthesis of collagen. It promotes the production of ECM material, promotes cell division and angiogenesis, and differentiates stem cells into osteoblasts It is known to promote.

The peptide according to the present invention is a constituent component of all proteins. It activates skin regeneration without side effects on the skin. Even if a large amount of protein is used as a constituent component of a protein, it has no side effects and can be used safely in sensitive areas such as eyes. . Since the peptide according to the present invention has a small molecular weight, the skin permeability is extremely excellent. Therefore, when the cosmetic composition of the present invention is topically applied to the skin, the skin condition will be effectively improved.

In one embodiment of the present invention, the tripeptide of the present invention shows excellent cell proliferation rate against human dermal horny stem cells compared to the negative and positive control, while showing almost no cytotoxicity, and exhibits various cell growth factors, Effectively increased the secretion of the factor. As described above, the tripeptide according to the present invention has a characteristic of exhibiting an excellent skin regeneration ability and an effect of increasing skin elasticity, and thus is highly useful as a cosmetic or a drug.

In another aspect, the present invention provides a composition for improving skin condition comprising the tripeptide or a mutant thereof as an active ingredient. Specifically, the composition for improving skin regeneration ability, skin moisturizing power, skin elasticity improvement, skin wrinkle improvement, And improvement of at least one kind selected from the group consisting of improvement. The composition may be prepared in various forms, but in view of the object of the present invention, it is preferable that the composition is formulated as a skin external preparation. Such compositions may be used as pharmaceuticals or cosmetics.

Accordingly, as yet another aspect, the present invention relates to a cosmetic composition for improving skin condition comprising the tripeptide or a mutant thereof as an active ingredient.

The cosmetic composition according to the present invention can be used as a cosmetic composition in the form of a solution, an ointment for external use, a cream, a foam, a nutritional lotion, a softening water, a pack, a soft water, an emulsion, a makeup base, But are not limited to, emulsions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays It is not. In addition, the cosmetic composition of the present invention may further comprise at least one cosmetically acceptable carrier to be incorporated in a general skin cosmetic composition, and examples thereof include oil, water, a surfactant, a moisturizer, A thickening agent, a chelating agent, a coloring matter, an antiseptic, a perfume, and the like may be appropriately compounded, but the present invention is not limited thereto.

In another aspect, the present invention relates to a pharmaceutical composition for improving skin condition comprising the tripeptide or a mutant thereof as an active ingredient. The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. Furthermore, in another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a skin disease caused by the promotion of dermal erythropoietic stem cells, which comprises the di-peptide or a mutant thereof as an active ingredient. For example, but not limited to, the above-mentioned " skin diseases caused by promotion of proliferation of keratinocyte stem cells " include wrinkles, melisma, freckles, striae distensae, .

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral preparations, suppositories and sterilized injection solutions according to a conventional method Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. In the case of the composition according to the present invention, the tripeptide as an active ingredient is preferably in the form of an external preparation for skin. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. The administration may be carried out once a day or divided into several doses.

The cosmetic or pharmaceutical composition according to the present invention may further comprise at least one substance having a skin regeneration or skin elasticity improving function. Furthermore, the cosmetic or pharmaceutical composition according to the present invention may contain the proliferation promoting agent of the keratinocyte stem cell in the range of 10 to 4,000 ppm.

The present invention also provides a method for improving the skin condition of an individual by administering to a subject in need thereof a skin condition improving composition comprising a tripeptide or a mutant thereof.

The subject refers to mammals, including dogs, pigs, horses, cows, etc., including humans, who want to improve or prevent poor skin conditions such as wrinkles, aging, low elasticity, scarring, .

The skin conditions such as skin wrinkles, skin aging, skin elasticity, scratches, scars and the like, diseases, diseases or abnormalities arising therefrom of the above-mentioned subject by the above method of the present invention can be effectively used in the object of the skin condition improving composition of the present invention It will of course be understood by those skilled in the art that the object of the present invention can be achieved by the administration, improvement, improvement, prevention, or treatment, through the above-described contents and embodiments described below.

In the present invention, "administering" means introducing a predetermined substance into an individual by any appropriate method, and the administration route of the composition of the present invention is either oral or parenteral &Lt; / RTI > The composition of the present invention may also be administered by any device capable of transferring the active substance to a target subject, for example, a cell.

The composition of the present invention induces the proliferation of dermal keratinocyte cells, thereby exhibiting an improvement in skin condition such as improvement in skin regeneration ability, improvement in skin moisturizing ability, improvement in skin elasticity, improvement in skin wrinkles and skin aging, .

Hereinafter, the present invention will be described more specifically by way of examples and test examples, but the scope of the present invention is not limited thereto.

[ Example One]

Analysis of human skin keratinocyte proliferation effect

1-1: Peptide synthesis

In this example, the synthesis was carried out according to the general peptide solid phase synthesis method (Wang C. Chan, Perter D. White, "Fmoc Solid Phase Peptide Synthesis", Oxford) in order to synthesize various cell permeation peptide derivatives. More specifically, coupling was performed one by one from the C-terminus using an automatic synthesizer (ASP48S, Peptron, Inc.) using Fmoc-SPPS (9-Fluorenyl methyl oxycarbonyl solid phase peptide synthesis) method. All monomeric materials used in the synthesis of peptide derivatives are protected with Fmoc at the N-terminus and amino acids protected with trityl (Trt), t-butyloxycarbonyl (Boc), t-butyl Respectively. As the coupling reagent, HBTU (2- (1H-Benzotirazloe-1-yl) -1,1,3,3-tetramethyluronium hexafluorophophate / HOBt (Hydroxybenzo triazloe) / NMM (N-methylmorpholine) was used.

(1) Protected amino acid (8 eq.) And coupling agent HBTU (8 eq.) / NMM (16 eq.) Were dissolved in DMF (dimethyl formamide) and reacted at room temperature for 2 hours.

(2) To remove Fmoc, 20% (v / v) Piperidine / DMF was added and reacted twice at room temperature for 5 minutes.

The peptide derivatives were produced by repeating the reactions (1) and (2). Then, acetylation was carried out at the N-terminal portion using acetic anhydride and diisopropylethylamine (DIEA).

Peptide separation at Resin uses TFA (Trifluoroacetic acid) / EDT (1,2-ethanedithiol) / Thioanisole / TIS (Triisopropylsilane) / H ₂ O (mixing ratio (by weight) = 90 / 2.5 / 2.5 / 2.5 / 2.5) Respectively. The thus obtained mixed solution is subjected to an excess treatment of refrigerated diethyl ether solvent to produce a precipitate. The resulting precipitate was centrifuged to complete precipitation, excess trifluoroacetic acid, thianyzole, and ethanedithiol were firstly removed, and then the same procedure was repeated twice to obtain a solidified precipitate.

The resulting precipitate was dissolved in a water-containing solution containing 0.1% (v / v) Trifluoroacetic acid using a high performance liquid chromatographic apparatus (Shimadzu Prominence HPLC, Japan) using a C18 column (250 mm × 22 mm, 10 μm, Vydac Everest, USA) acetonitrile liner gradient (Acetonitrile concentration: 10 ~ 75% (v / v)). The molecular weight of the purified peptide derivative was confirmed using LC / MS (Agilient HP 1100 series), and the pure purified fractions were lyophilized to obtain the following peptides as TFA (Trifluoroacetic acid) salt in the form of a white powder.

Specifically, the sequences and molecular weights of the synthesized peptide derivatives are shown below.

Peptide-1: Phe-His-Arg :

Rt = 8.533 min (various concentration gradient from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA over 30 minutes);

MS (ESI) 458.24 m / e, [M + H] < ⁺ > = 458

Peptide-2: Acetyl-Phe-His-Arg-NH2 :

Rt = 6.617 min (various concentration gradient from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA over 30 min);

MS (ESI) 499.27 m / e, [M + H] < ⁺ > = 499

1-2: Culture of human skin keratinocyte stem cells

Human skin keratinocytes stem cells were cultured in a CO ₂ incubator _{(CO 2 Incubator) 37 ° C} , 5% CO 2 conditions. Human dermal keratinocytes were purchased from CellnTec, Inc., USA. The cell culture solution was used for cell culture by adding the additive (CnT-57) to 500 ml of the basic culture solution (CnT-BM) according to the guidelines of CellnTec,

1-3: Analysis of proliferation effect of human skin keratinocyte

Peptides 1 and 2 obtained in Example 1-1 were each treated for 20 hours at 20 ppm for 72 hours to measure the degree of cell proliferation using CCK-8 kit (WST-8; Dojindo, Japan) Respectively.

Analytical methods were quantified by absorbance analysis at 450 nm using an enzyme immunoassay (ELx808; BioTek, USA). The percentages of human dermal stem cell proliferation (%) are shown in Table 1 below, and Table 1 shows the results of recording the proliferation rate (%) of human keratinocyte stem cells in human dermal horny stem cells. The negative control group was untreated and the positive control group was treated with Rutin (quercetin-3-O-rutinoside) .

The cell proliferation rate (%) is a value obtained by multiplying the cell proliferation rate according to each treatment sample based on the proliferation rate of the negative control group.

Sample type Growth rate Negative control group 1.00 + 0.019 Positive control group 1.21 ± 0.051 Peptide 1 1.70 + - 0.264 Peptide 2 1.66 + 0.140

The proliferation rate of human dermal keratinocyte stem cells by peptide-1 and peptide-2 was 1.70 times (peptide-1) and 1.66 times (peptide-2), respectively, Lt; RTI ID = 0.0 > cell proliferation < / RTI > Thus, it has been confirmed that the peptides according to the present invention exhibit excellent activity as growth promoting agents of human dermal horny stem cells.

[ Example 2]

Toxicity analysis of human skin keratinocyte stem cells in two peptides

Human dermal keratinocyte cells were cultured in the same manner as described in Example 1, and each peptide was treated at a concentration of 100, 200, 400 ppm in order to determine the cytotoxicity of peptide-1 and peptide-2. The degree of cytotoxicity was quantified by absorbance analysis using enzyme immunoassay (570 nm; BioTek) after pretreatment using MTT method (Sigma Aldrich, St Louis, USA). Table 2 shows the toxicity of peptides-1 and peptide-2 in human skin keratinocyte cells. Negative control group was untreated and positive control group was Doxorubicin.

The cell viability (%) is a value obtained by multiplying the cell proliferation rate according to each treatment sample based on the survival rate 100 of the negative control group.

Sample type Sample content Cell survival rate [%] Negative control group 0 100 ± 13.4 Positive control group 20 ppm 10.3 ± 0.7 Peptide 1

100 ppm 103.5 ± 1.6 200 ppm 196.9 ± 13.4 400 ppm 134.4 ± 2.9 Peptide 2

100 ppm 97.0 ± 4.2 200 ppm 185.8 ± 8.1 400 ppm 113.8 ± 1.7

The death of human dermal keratinocyte stem cells by peptide-1 and peptide-2 was not observed in the concentration range of 100, 200 and 400 ppm, and the cell survival rate was equal to or higher than that of the negative control, and peptide-1 and peptide- All were identified. This was 5 times, 10 times, and 20 times higher than the 20 ppm concentration of cell proliferation (Table 1).

[ Example 3]

Expression analysis of human skin keratinocyte cell growth factor in two peptides

Human keratinocytes were cultured in the same manner as described in Test Example 1, and then each peptide was treated at a concentration of 20 ppm for analysis of cell growth factor expression by peptide-1 and peptide-2, and after 72 hours (2 ml) were used.

The use of the cell growth factor anti-body (Ab) array (ab134002; Abcam, Cambridge, UK) was followed by the supplier's guidelines and the quantification of the results was performed using ImageJ software (NIH, Bethesda, And the degree of cell growth factor expression was confirmed. Table 3 shows the cell growth factors increased by peptide-1 and peptide-2 treatment in the culture medium of human dermal keratinocyte. The negative control group was a vehicle and the positive control group was a routine (Rutin).

No. cell
Growth factor The content of cell growth factor (multiple of negative control) Positive control group Peptide 1 Peptide-2 One AR 1.07 ± 0.01 1.73 ± 0.03 3.13 ± 1.03 2 bFGF 1.27 + 0.04 1.56 ± 0.00 2.13 ± 0.18 3 b-NGF 1.70 + 0.06 1.30 ± 0.03 1.58 + - 0.74 4 EGF 1.98 + 0.02 1.41 + 0.07 1.24 ± 0.01 5 EGFR 1.24 + 0.04 2.11 + 0.02 2.03 ± 0.13 6 FGF-4 1.34 ± 0.01 2.15 + 0.08 6.08 ± 0.52 7 FGF-6 1.26 + - 0.01 2.34 ± 0.03 3.12 ± 0.08 8 FGF-7 1.22 ± 0.01 2.20 ± 0.02 3.06 ± 0.14 9 GCSF 0.97 + 0.08 0.84 0.12 1.57 + - 0.10 10 GDNF 1.04 0.08 1.52 + - 0.24 0.45 + 0.64 11 GM-CSF 1.41 + 0.04 2.10 ± 0.15 1.19 ± 0.11 12 HB-EGF 1.40 + 0.07 1.57 ± 0.01 1.30 ± 0.07 13 IGFBP-2 0.51 + 0.04 1.27 + 0.04 1.45 ± 0.20 14 IGFBP-3 1.19 ± 0.01 1.48 ± 0.05 0.21 0.30 15 IGFBP-4 1.08 ± 0.05 1.43 ± 0.00 1.48 ± 1.35 16 IGFBP-6 0.35 + 0.03 1.46 ± 0.18 1.52 0.30 17 IGF-I 1.17 + 0.08 2.08 + 0.03 2.19 ± 1.15 18 IGF-ISR 1.16 ± 0.01 2.20 ± 0.07 2.58 ± 0.25 19 IGF-II 1.33 + - 0.10 1.39 + 0.02 0.98 ± 0.01 20 M-CSF 1.17 ± 0.05 1.34 + 0.03 1.17 + 0.08 21 M-CSFR 1.22 ± 0.05 1.61 + 0.07 1.34 + 0.04 22 NT-3 1.58 ± 0.53 1.96 + 0.10 0.66 + 0.02 23 PDGF-AA 0.39 + 0.03 1.19 ± 0.12 1.40 ± 0.15 24 PDGF-AB 1.04 + 0.03 1.50 + 0.03 0.48 ± 0.68 25 PDGF-BB 0.94 + - 0.01 1.76 + 0.04 0.40 + - 0.57 26 PIGF 1.27 ± 0.06 2.06 + 0.04 1.19 0.30 27 SCF 1.35 ± 0.01 2.16 ± 0.29 0.79 + - 0.23 28 SCFR 1.16 ± 0.01 1.57 ± 0.05 0.41 + 0.11 29 TGF-a 1.43 + - 0.12 1.26 + - 0.01 1.18 ± 0.01 30 TGF-beta 1.52 + - 0.34 1.61 ± 0.00 2.23 ± 0.00 31 TGF-β2 1.15 ± 0.00 1.75 ± 0.05 1.42 + 0.04 32 TGF-β3 1.18 ± 0.02 1.60 + - 0.01 0.34 + 0.26 33 VEGF 0.64 ± 0.06 1.66 + - 0.12 1.58 ± 0.05 34 VEGFR3 1.02 + 0.02 1.70 + 0.06 1.50 0.20 35 VEGF-D 1.02 + 0.02 1.65 ± 0.00 1.59 ± 0.19

FGF-5, FGF-6, and FGF-7 were secreted from fibroblast growth factors, especially peptides-1 and peptides-2. Respectively.

IGFBP-4, IGFBP-6, IGF-I, and IGFBP-4, which are increased by both peptide-1 and peptide-2 IGF-II, G-CSF, HB-EGF, IGFBP-3, and IGF-II, Growth factors increased by NT-3, PDGF-AB, PDGF-BB, PIGF, SCF, SCFR, TGF-beta3 and peptide-2 were b-NGF and GCSF.

Claims

A tripeptide of phenylalanine-histidine-arginine (FHR) or a mutant thereof, wherein the mutant includes at least one substituent selected from the group consisting of C-terminal amidation and N-terminal acetylation of the tripeptide Growth promoting agent for keratinocyte stem cells.

The proliferation promoting agent according to claim 1, wherein the tripeptide or a mutant thereof promotes growth factor expression of dermal keratinocyte.

The method of claim 1, wherein the growth factor is selected from the group consisting of AR, bFGF, EGFR, FGF-4, FGF-6, FGF-7, IGFBP-2, IGFBP-4, IGFBP- CSF, M-CSFR, TGF-beta, VEGF, VEGFR3, GDNF, GM-CSF, HB-EGF, IGFBP-3, IGF-II, NT-3, PDGF- TGF-beta3, b-NGF, and GCSF.

4. The method of claim 3, wherein the growth factor is selected from the group consisting of AR, bFGF, EGFR, FGF-4, FGF-6, FGF-7, IGFBP-2, IGFBP-4, IGFBP- CSF, M-CSFR, TGF-beta, VEGF and VEGFR3.

2. The growth promoter according to claim 1, wherein said mutant is amidated at the C-terminus of the tripeptide and acetylated at the N-terminus.

A cosmetic composition for skin condition improvement comprising the proliferation promoting agent of keratinocyte stem cell according to any one of claims 1 to 5 as an active ingredient.

The cosmetic composition according to claim 6, wherein the skin condition improvement is at least one selected from the group consisting of improvement of skin regeneration ability, improvement of skin moisturizing power, improvement of skin elasticity, improvement of skin wrinkles and improvement of skin aging.

7. The cosmetic composition of claim 6, further comprising a cosmetically acceptable carrier.

[Claim 7] The cosmetic composition according to claim 6, wherein the proliferation promoting agent of the keratinocyte stem cell is contained in the range of 10 to 4,000 ppm.

The cosmetic composition according to claim 6, wherein the cosmetic composition is at least one selected from the group consisting of a solution, an external ointment, a cream, a foam, a nutritional lotion, a softening water, a pack, Wherein the formulation has a formulation selected from the group consisting of an oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing oil, a powdered foundation, an emulsion foundation, a wax foundation, Cosmetic composition.

A pharmaceutical composition for preventing or treating a skin disease caused by promoting proliferation of dermal horny stem cells comprising the proliferation promoting agent of dermal keratinocyte according to any one of claims 1 to 5 as an active ingredient.

12. The pharmaceutical composition according to claim 11, wherein the composition further comprises a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, wherein the composition is an external preparation for skin.