KR101349300B1 - Composition for inhibiting hair loss or promoting hair growth containing vimentin - Google Patents

Abstract

The present invention relates to the use of non-mentin (vimentin) having a function of promoting the growth of dermal papilla cells, and cell migration and growth factor expression, more specifically keratinocyte (melanocyte), melanocyte ( It relates to a new use of non-mentin to promote the proliferation of dermal papilla cells, which can be differentiated into melanocytes, etc. to grow hair, and to maintain hair follicles to prevent hair loss.
Therefore, the present invention can be usefully used for hair loss inhibition and hair growth treatment effect through the growth of non-mentin.

Description

Composition for inhibiting hair loss or promoting hair growth containing non-mentin {Composition for inhibiting hair loss or promoting hair growth containing vimentin}

The present invention relates to a composition for inhibiting hair loss or promoting hair growth comprising bimentin.

Human hair is primarily responsible for the protective function to protect the scalp from external stimuli such as ultraviolet rays and aesthetic functions to express the external image of the individual. However, in modern society, hair loss is frequently caused by natural factors such as environmental pollution, strong ultraviolet rays, and physiological factors such as stress and hormonal imbalance. Therefore, the need for materials for the prevention and treatment thereof is urgently needed.

Hair follicles, the physiological organs in the body that make up the hair, form the hair during the postnatal development, the anagen phase in which the hair grows actively, the catagen phase in which the hair degenerates, and the hair fall out. The hair cycle is divided into a telogen phase, an exogen phase in which hair loss is maintained, and is involved in hair growth, maintenance, and dropout. In many studies, the activity of the hair follicles is caused by dermal papilla cells, and in particular, proliferation and differentiation of dermal papilla cells is primarily involved in the progress of the hair growth cycle and hair formation (Non Patent Literature 1). In the growth phase where hair is actively growing, vigorous proliferation and differentiation of dermal papilla cells occurs actively, and hair growth is stopped and these cells are killed in the degenerative, resting and hair loss phases in which hair loss occurs (Non-Patent Document 2). Therefore, hair growth and hair loss are closely related to the proliferation and death of dermal papilla cells. Therefore, the prolonged growth phase by inhibiting the proliferation of the dermal papilla cells or the inhibition of cell death and the shortening of the degenerative stage, resting period, and alopecia will be improved and treated. It is feed. In addition, cell division and migration near the nipple are closely related to the growth of the hair. During the growth phase, new hair is generated from the nipple, which is activated by various cytokines and hormones, resulting in cell migration to the nipple. It affects the growth of (nonpatent literature 3, nonpatent literature 4).

Also, among the activating factors of hair follicles, growth factors such as vertican, IGF-1, and VEGF are most strongly expressed in the growth phase and decreased in the degenerative and resting phases to induce the growth of dermal papilla cells and to maintain the hair growth phase. It is known to help maintain this normally (nonpatent literature 5, nonpatent literature 6).

Representative drugs minoxidil and finasteride, which are currently approved by the FDA, have side effects such as systemic hair growth and decreased sexual function, and the specific mechanism of action of minoxidil is not clear. Therefore, it is necessary to develop a biological material that promotes proliferation of dermal papilla cells in hair follicles and increases expression of growth factors while having less concern about side effects.

Botchkarev VA, Kishimoto J, Molecular control of epithelial-mesenchymal interactions during hair follicle cycling. J Iinvestig Dermatol Symp Proc 8, 46-55,2003 Botchkarev V.A, Batchkareva N.V., Nakamura M, Noggin is required for induction of the hair follicle growth phase in postnatal skin. FASEB J 15, 2205-2214, 2001 Eva M.J. Peters, Desmond J Tobin, Migration of melanoblasts into developing murine hair follicle is accompanied by transient c-kit expression, J Histochem Cytochem, 50, 6751-766, 2002 Jahoda CA, Cell movement in the hair follicle dermis-more than a two-way street? J Invest Dermatol, 1221, 1523-1747, 2003 Wight T, Versican: a versatile extracellular matrix proteoglycan in cell biology. Current Opinion Cell Biol 14, 617-623, 2002 Kulessa H, Turk G, Inhibition of Bmp signaling affects growth and differentiation in the anagen hair follicle, EMBO J 19, 6664-6674, 2000

As discussed above, growth factors related to the proliferation and migration of dermal papilla cells and hair growth are very important factors in normal hair maintenance, hair loss inhibition and hair growth. In contrast, the present inventors have completed the present invention by experimentally confirming that the non-mentin protein promotes the growth and migration of dermal papilla cells and increases the expression of growth factors such as IGF-1.

Accordingly, an object of the present invention is to provide a new use of non-mentin to promote the growth and migration of dermal papilla cells and to increase the expression of growth factors such as IGF-1.

As a means for solving the above problems, the present invention provides a composition for preventing hair loss or promoting hair growth comprising a non-mentin protein as an active ingredient.

As another means for solving the above problems, the present invention provides a composition for preventing hair loss or promoting hair growth comprising a fusion protein combined with a non-mentin protein and a protein delivery domain as an active ingredient.

As another means for solving the above problems, the present invention provides a composition for preventing hair loss or promoting hair growth comprising a nucleic acid encoding a non-mentin protein as an active ingredient.

As another means for solving the above problems, the present invention provides a composition for preventing hair loss or promoting hair growth comprising a nucleic acid encoding a fusion protein combined with a non-mentin protein and a protein delivery domain as an active ingredient.

As another means for solving the above problems, the present invention is a hair loss prevention or hair growth comprising at least one selected from the group consisting of non-mentin protein, protein delivery domain-non-mentin fusion protein and a nucleic acid encoding the protein as an active ingredient Provide accelerators.

As another means for solving the above problems, the present invention is a hair loss prevention or hair growth comprising at least one selected from the group consisting of non-mentin protein, protein delivery domain-non-mentin fusion protein and a nucleic acid encoding the protein as an active ingredient Promoting cosmetics for promotion.

In the present invention, non-mentin promotes the growth and migration of dermal papilla cells and expression of growth factors, and can be usefully used as a drug or cosmetic material for the purpose of inhibiting hair loss and treating hair growth with this effect.

Figure 1 shows a cleavage map of the pHis / TAT- non-mentin expression vector.
Figure 2 is transformed in Escherichia coli by introducing a non-mentin gene of human in a pHis / TAT expression vector and stained with Coomassie brilliant blue of the protein delivery domain-non-mentin recombinant protein obtained through protein expression and purification One result is shown.
3 is a cell proliferation ELISA analysis using BrdU (5-bromo-2'-deoxyuridine) for the degree of cell proliferation when the protein delivery domain-non-mentin fusion protein is treated by concentration in the dermal papilla cells isolated from the rats. It is a graph obtained through [1, control; 2, non-mentin 10 nM treatment; 3, non-mentin 100 nM treatment; 4, non-mentin 1 uM treatment; 5, non-mentin 10 uM treatment; 6, GFP 1 uM treatment].
Figure 4 shows the result of measuring the degree of cell migration when treated with protein delivery domain-non-mentin fusion protein concentration to the dermal papilla cells [wound healing assay] [1, immediately after wound healing; 2, non-mentin 10 nM treatment; 3, non-mentin 100 nM treatment; 4, non-mentin 1 uM treatment; 5, non-mentin 10 uM treatment; 6, GFP 1 uM treatment].
Figure 5 shows the results of the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the expression levels of growth factors IGF-1 and VEGF by treating the protein delivery domain-non-mentin fusion protein to the dermal papilla cells [lane 1 , Control; Lane 2, non-mentin 10 nM treatment; Lane 3, non-mentin 100 nM treatment; Lane 4, non-mentin 1 uM treatment].
Figure 6 is a photograph confirming the hair growth effect by applying the TAT- non-mentin fusion protein to the rat hair root removed.
7 is a photograph showing the hair growth effect after saline, TAT-GFP, minoxidil, TAT- non-mentin fusion protein of Example 1 were respectively treated in a mouse model from which hair root was removed.
Figure 8 was treated with saline, TAT-GFP, minoxidil, TAT- non-mentin fusion protein of Example 1 in the mouse model from which the hair root was removed, and after 23 days, the mouse head, torso, tail, etc. It is the photograph which photographed the thickness of the hair of the site (up) and the graph which shows the thickness of the hair of the torso (bottom).
9 is saline, TAT-GFP, minoxidil, TAT- non-mentin fusion protein of Example 1, respectively, in a mouse model from which hair follicles were removed.

The present invention is characterized by the fact that non-mentin can promote the proliferation of papillary cells and increase the expression of growth factors, thereby affecting the hair cycle and preventing and treating hair loss diseases.

In the present invention, the effect of promoting non-mentin dermal papilla cell growth. In other words, non-mentin significantly increases dermal papilla cell proliferation. The papilla cells experimental group treated with the protein delivery domain-non-mentin fusion protein showed a significant effect on the dermal papilla cell proliferation compared to their control group, and showed an increase in cell proliferation efficiency up to 300% or more compared to the control group (see FIG. 2). When measuring the papillary cell proliferation effect of each non-mentin protein concentration, showed the greatest effect at 1 uM concentration (see Figure 2).

The present invention shows the cell migration activation effect of non-mentin dermal papilla cells. In other words, non-mentin significantly increases cell migration of dermal papilla cells. The dermal papilla cells treated with the protein transfer domain-non-mentin fusion protein showed a significant effect on the dermal papilla cell migration compared to their control group (see FIG. 3).

The present invention shows the effect of promoting the expression of growth factor IGF-1 in non-mentin dermal papilla cells. In other words, non-mentin increases the expression of growth factor IGF-1 in dermal papilla cells. The dermal papilla cells treated with the protein transfer domain-non-mentin fusion protein showed an increase in IGF-1 expression compared to their controls (see FIG. 4).

Accordingly, the present invention provides a composition for preventing hair loss or promoting hair growth comprising non-mentin protein as an active ingredient.

The non-mentin protein included in the pharmaceutical composition of the present invention may be derived from a mammal including a human, and according to the individual to be treated, the origin of the non-mentin may be appropriately selected. Preferably, the non-mentin of the present invention may be derived from a human, and the amino acid sequence of the non-mentin derived from humans is known (SEQ ID NO: 1: GenBank acession no. NP — 003371.2).

 In addition, the non-mentin protein included in the pharmaceutical composition of the present invention may include both natural or recombinant non-mentin proteins or proteins having physiological activities substantially equivalent thereto. Proteins having substantially equivalent physiological activity include functional equivalents and / or functional derivatives of naturally occurring proteins or recombinant non-mentin proteins.

The "functional equivalent" is an amino acid sequence variant in which some or all of the naturally occurring protein amino acids are substituted, or a part of the amino acids are deleted or added, and at least 70%, preferably 80% or more, more preferably, with the non-mentin protein. For example, it refers to a protein having a sequence homology of 90% or more and exhibiting substantially homogeneous physiological activity with non-mentin protein.

The "functional derivative" refers to a protein in which some chemical structures have been modified to increase or decrease physicochemical properties while maintaining the basic skeleton and physiological activity of the non-mentin protein. For example, functional derivatives include proteins whose structure has been altered to alter the stability, shelf life, volatility or solubility of the non-mentin protein.

In one embodiment of the present invention was used a non-mentin protein represented by SEQ ID NO: 1.

In addition, the present invention provides a composition for preventing hair loss or promoting hair growth comprising a fusion protein combined with a non-mentin protein and a protein delivery domain as an active ingredient.

The protein delivery domain (PTD) may include a trans-activating transcriptional activator (TAT) derived from human immunodeficiency virus type I (HIV-1); Antp (Antennapedia or penetratin) peptide, which is an antennapedia Homeodomain of Drosophila; Mutant phenotype protein 1 (Mph-1) of the mouse transcription factor, VP22 (viral protein 22) of Herpes simplex virus (HSV-1); Human protamine P4 (HP4) from herring protamine; Transportan, MAP (Model amphipathic peptide), TP10 (Transportan-10), CTP (Cardiac targeting peptide), K5-FGF (K5-fibroblast growth factor, AAVALLPAVLLALLP [SEQ ID NO: 11]), HAP-1 (Huntingtin) associated protein 1, SFHQFARATLAS [SEQ ID NO: 12]), 293P-1 (SNNNVRPIHIWP [SEQ ID NO: 13]), CADY (cysteamidation PTD, Ac-GLWRALWRLLRSLWRLLWRA-Cya [SEQ ID NO: 14]), PF6 (PepFect6, Stearyl-AGYLLGK ( εNH) INLKALAALAKKIL-NH ₂ ), arginine rich peptide (RXR), poly-arginine (R _n (n = 6-12)), polylysine, or modified peptides thereof (e.g. And, one selected from the group consisting of peptides modified 47-57 amino acid residues of the TAT protein, etc.), but any one that can deliver the protein into the cell can be used. In one embodiment of the present invention TAT- non-mentin fusion protein (SEQ ID NO: 2) was used.

The PTD-non-mentin fusion protein of the present invention obtains DNA fragments from non-mentin genes by PCR and then introduces them into plasmid vectors (such as prokaryotic expression vector pET or eukaryotic expression vector pcDNA) containing PTD. After transforming the cells to express the protein, it can be prepared through an appropriate protein purification process.

The present invention also includes a composition for preventing hair loss or promoting hair growth comprising a nucleic acid encoding a non-mentin protein or a nucleic acid encoding a fusion protein in which a non-mentin protein and a protein delivery domain are bound.

Nucleic acids encoding the non-mentin protein or PTD-non-mentin fusion protein include DNA or RNA. Preferably, the nucleic acid encoding the non-mentin protein may be derived from a mammal, more preferably a human. The human nonmentin gene is known (SEQ ID NO: 3, GenBank accession no. BC000163). The nucleic acid encoding the PTD-non-mentin fusion protein includes a gene represented by SEQ ID NO: 4.

The nucleic acid is introduced into an expression vector, such as a plasmid or viral vector, by known methods and then the expression vector is expressed phenotypicly by infection or transduction by various methods known in the art. Can be introduced.

The plasmid expression vector is a method of delivering plasmid DNA directly to human cells by FDA's approved gene transfer method for human use (Nabel, E. G., et al., Science, 249: 1285-1288, 1990). Plasmid DNA has the advantage that it can be homogeneously purified unlike viral vectors. As the plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used. For example, but not limited to, pET, pcDNA, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91/08291), pVL1392 (PharMingen) and the like are representative. In an embodiment of the present invention, the pHis / TAT plasmid prepared by the present team (Kwon JH et al., 2007, Biochem Biophys Res Commun., 363, 399-404) was used.

Plasmid expression vectors comprising the nucleic acid are methods known in the art, such as, but not limited to, transient transfection, microinjection, transduction, cell fusion, Calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroinvasion It can be introduced into tumor cells by electroporation, gene guns and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267: 963-). 967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988). Preferably transient transfection methods can be used.

In addition, viral expression vectors including the nucleic acid include, but are not limited to, retroviruses, adenoviruses, adenoviruses, herpes viruses, avidoxviruses, and the like.

The retroviral vector is constructed such that all of the viral genes have been removed or altered so that non-viral proteins are made in the cells infected by the viral vector. The main advantages of retroviral vectors for gene therapy are to deliver large quantities of genes in cloned cells, to correctly integrate genes transferred into cellular DNA, and to not cause subsequent infections after gene transfection (Miller, AD, Nature). , 1992, 357: 455-460). FDA-certified retroviral vectors were prepared using PA317 amphoteric retrovirus package cells (Miller, A.D. and Buttimore, C., Molec. Cell Biol., 6: 2895-2902, 1986).

Non-retroviral vectors include adenoviruses as mentioned above (Rosenfeld, MA, et al., Cell, 68: 143-155, 1992; Jaffe, HA et al., Nature Genetics, 1: 372- 378, 1992; Lemarchand, P. et al., Proc. Natl. Acad. Sci. USA, 89: 6482-6486, 1992). The main advantage of adenoviruses is the ability to carry large quantities of DNA fragments (36 kb genome) and to infect very high reverse non-cloned cells. Herpes viruses can also be usefully used for human gene therapy (Wolfe, JH, et al., Nature Genetics, 1: 379-384, 1992). In addition, suitable known viral vectors can be used in the compositions of the present invention.

The composition for inhibiting hair loss and promoting hair growth according to the present invention may be prepared as a pharmaceutical composition or a cosmetic composition, and may be prepared as a medicine or cosmetics, and when prepared as a medicine, orally or intravenously, subcutaneously, intranasally or intraperitoneally. It is administered quadrature. Oral administration also includes sublingual application. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.

The composition comprising the non-mentin protein, PTD-non-mentin fusion protein or nucleic acid encoding the protein as an active ingredient according to the present invention may be prepared and administered in the form of various formulations by further including a pharmaceutically acceptable carrier. have. The term 'pharmaceutically acceptable' refers to a composition which is physiologically acceptable and does not cause an allergic reaction or the like, when administered to humans, usually gastrointestinal disorders, dizziness, and the like. Pharmaceutically acceptable carriers may include binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavors in the case of oral administration, and in the case of injections, buffers, preservatives, analgesics Solubilizers, isotonic agents and stabilizers can be used in combination, and in the case of topical administration agents, bases, excipients, lubricants and preservatives can be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, tpokey, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, may be prepared in unit dosage ampoules or in multiple dosage forms. Can be. In addition, the pharmaceutical compositions of the present invention may be prepared according to techniques commonly used in the form of various formulations.

Hair loss prevention or hair growth promoting composition according to the invention may be administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. Effective dose levels are well known in factors such as disease type, severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, drugs used concurrently, and other medical fields in a patient. It can be determined according to the factor. The composition of the present invention may be administered alone or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by a person skilled in the art. In particular, the active ingredient preferably comprises 0.01 to 30 parts by weight, more preferably 0.1 to 20 parts by weight based on 100 parts by weight of the total hair loss prevention or hair growth promoting composition.

Therefore, the present invention may include a hair loss prevention or hair growth promoting agent comprising at least one selected from the group consisting of a non-mentin protein, a protein delivery domain-non-mentin fusion protein, and a nucleic acid encoding the protein.

In addition, the present invention may include a cosmetic for preventing hair loss or promoting hair growth comprising at least one selected from the group consisting of a non-mentin protein, a protein delivery domain-non-mentin fusion protein, and a nucleic acid encoding the protein.

The cosmetics of the present invention further contain a cosmetically acceptable carrier to be prepared in conventional formulations such as creams, lotions, tonics, sprays, aerosols, oils, solutions, suspensions, gels, ointments, emulsions, waxes, pastes and the like. Can be. The composition of the present invention usually contains a base, and other components generally used as cosmetic raw materials may be appropriately blended as necessary. The base of the composition of the present invention may be used as long as it is commonly used, examples thereof include purified water, mineral water, ethanol, glycerin, squalene, 1,3-propylene glycol, 1,3-butylene glycol, castor oil, Tsubaki oil and liquid petrolatum. Examples of other ingredients to be incorporated into the composition of the present invention include, but are not limited to, surfactants, emulsifiers, thickeners, preservatives (e.g., p-hydroxybenzoyl methyl), antioxidants, fragrances and the like.

In addition to the above components, the cosmetics of the present invention may include components that may serve to supply nutrients to hair follicles or hair growth promoting auxiliary components that are commonly used. For example, vitamins, amino acids, animal and vegetable oils, sodium chloride And the like, but are not limited to these. Their amount is preferably 0.0001 to 10% by weight, in particular 0.01 to 1% by weight of the total composition. In this case, the vitamins include, but are not limited to, vitamin A, vitamin B1, vitamin B2, niacin (nicotinic acid), vitamin C, vitamin E, sodium pantothenate, potassium pantothenate and biotin H (vitamin H). Amino acids include, but are not limited to, waveguides. Animal and vegetable oils include, but are not limited to, horse oil, egg oil, olive oil, camellia oil, rapeseed oil, sesame oil, germ oil, and the like.

The cosmetic of the present invention can be used by a method such as applying or spreading directly on the hair or the scalp. The hair to which the composition of the present invention is applied includes hair follicles and hair follicles, hair and eyelashes and eyelashes, beard, armpits, pubic hair, hair follicles and hair follicles. Therefore, the present invention hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow hair regrowth agent, eyelash regrowth agent, eyelash nutrient, pet shampoo , Pet rinse, and the like.

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

Example  1: Protein Delivery Domain Vimentin  Fusion protein manufacturing

The protein delivery domain-non-mentin protein used in the present invention was prepared by recombinant protein expression and purification using a pHis / TAT expression vector (Kwon JH et al., 2007, Biochem Biophys Res Commun., 363, 399-404). Was done. And by the cDNA of the person as a template using two primers 5'-ATCTAC GGATCC ACCAGGTCCGTGTCCTCG-3 ' ( SEQ ID NO: 5, underlined BamHI site), 5'-AGTGTG GTCGAC TTATTCAAGGTCATCGTC-3' ( SEQ ID NO: 6, SalI site underlined) PCR fragments were obtained by DNA fragments (SEQ ID NO: 3), cut into respective restriction enzymes (BamHI / SalI), and put into a pHis / TAT expression vector to prepare a recombinant expression vector pHis / TAT-Vimentin [FIG. 1], which was E. coli BL21 (DE3). ) Was transduced. After incubating for a certain time for protein expression, IPTG (isopropyl-β-D-thio-galactoside) was added and further incubated for 4 hours. The cultured Escherichia coli was recovered by centrifugation, crushed by an ultrasonic mill, and protein-transfer domain-nonmentin fusion protein (SEQ ID NO: 2) was isolated by Ni-NTA affinity column chromatography. Purified protein was confirmed by staining with Coomassie brilliant blue (Fig. 2).

Example  2: By vimentin  by Dermal papilla cells  Proliferative effect

The dermal papilla cells obtained from the back skin of SD-rat were identified morphologically, and were identified as the dermal papilla cells by measuring ALP activity (alkaline phosphatase activity), vertican and nexin expression as biological markers. The cells were cultured in DMEM medium containing 10% FBS and antibiotics (penicillin streptomycin). On day 3 of culture, the cells were transferred to 96-well plates in a culture dish. After attaching the cells, the protein delivery domain-non-mentin protein was added to the medium by concentration, and then cultured for 2 hours, and then changed to a new medium, and further cultured for 72 hours under conditions of 5% CO ₂ and 37 ℃. Each papillary cell group treated with non-mentin protein concentration was cultured for 24 hours, and cell proliferation was performed using BrdU enzyme immunoassay kit (Millipore). After completion of the reaction, it was confirmed that the amount of dermal papilla cells increased significantly with concentration compared to the control group by reading with a 450 nm absorbance meter [FIG. 3]. At this time, the TAT-GFP protein treatment prepared in the same manner as the TAT-non-mentin protein preparation is a control to show that the hair growth effect is due to the entering non-mentin, not the effect of TAT.

Example  3: By vimentin  by Dermal papilla cells  Moving effect

A wound healing assay was performed to observe the effect on the movement of dermal papilla cells.

The dermal papilla cells were incubated in 6-well plates with DMEM medium containing 10% FBS, treated with non-mentin for 2 hours in DMEM medium containing 5% FBS, and wound with a pipette tip. The cells were further incubated for 24 hours under conditions of 5% CO₂ and 37 ° C in DMEM medium and observed under a microscope. As a result, it was confirmed that as the concentration of the TAT- non-mentin fusion protein increases, the flow of all the flow cells increases [FIG. 4].

Example  4: By vimentin  Growth factor expression promoting effect

After 24 hours treatment with dermal papilla cells mRNA was extracted using the mRNA extraction kit (Geneol). CDNA was synthesized using reverse transcriptase (Gendepot, M-MLV) and PCR was performed using Taq polymerase.

Primers used for PCR to confirm the expression of IGF-1 were 5-TCAACAAGCCCACAGCAACAGGGTAT-3 (SEQ ID NO: 7), 5-ACTCGTGCAGAGCAAAGGAT-3 (SEQ ID NO: 8). Primers used for PCR to confirm the expression of VEGF were 5 '-GACCCTGGTGGACATCTTCCAGGA-3' (SEQ ID NO: 9), 5'-GGTGAGAGGTCTAGTTCCCGA-3 '(SEQ ID NO: 10).

The result of the PCR reaction was confirmed by electrophoresis on 1.3% agarose gel, through which the expression levels of growth factors IGF-1 and VEGF were confirmed. As a result, it was confirmed that the expression of IGF-1 and VEGF is promoted as the concentration of the TAT- non-mentin fusion protein is higher [Fig. 5].

Example  5: hair loss inhibition and hair growth promoting effect

3 minutes after applying hair removal cream (Veet, Oxy Rekit Benckiser, Korea) to rats, the hair was removed with a spatula to remove hair and hair roots and then wiped off with 70% ethanol. The surface of the epidermis of the rat was pushed to remove dead skin cells, and then further dead skin cells were removed using a transparent tape. Here, 10 μg and 20 μg of the TAT-non-mentin fusion protein of Example 1 were applied, and then beated for 30 seconds to be absorbed. After two days, hair growth could be observed with the naked eye, and the result after 4 days of treatment with the TAT- non-mentin fusion protein of Example 1 was photographed.

Example  6: Confirmation of hair growth promoting effect and hair follicle activation using mouse

The skin of the mouse (C57BL / 6 mouse) was applied with a depilatory cream (Veet, Oxy Rekitbenzer, Korea) and after 3 minutes, the spatula was scraped off to remove hair and hair roots, and then wiped with buffer PBS. The surface of the epidermis of the rat was pushed to remove dead skin cells, and then further dead skin cells were removed using a transparent tape. 20 μg of the TAT-non-mentin fusion protein of Example 1 was applied thereto, and then beated for 30 seconds to be absorbed. At this time, the saline (saline) treatment group, TAT-GFP treatment group, minoxidil (Minoxidil) 1% 50μ treatment group was also tested at the same time.

After three weeks, the difference in hair growth between the experimental groups was visually observed, and FIG. 7 shows photographs of the results after 23 days of treatment. In addition, Figure 8 is a photograph taken by taking the thickness of the hair in the middle of each group of mice, the thickness of the TAT- non-mentin fusion protein treatment group of Example 1 was the thickest, hair follicles through H & E staining after sacrifice As a result of confirming the hair follicle, it was confirmed that the number of rounded activated hair follicles was increased in the TAT-non-mentin fusion protein treatment group of Example 1 [FIG. 9].

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Claims (20)

Hair loss prevention or hair growth promoting pharmaceutical composition comprising a non-mentin protein as an active ingredient.
The method of claim 1,
Composition wherein the non-mentin protein is derived from a mammal.
3. The method of claim 2,
Composition characterized in that the mammal is a human.
The method of claim 1,
The non-mentin protein is a composition, characterized in that the natural non-mentin protein or recombinant non-mentin protein.
delete The method of claim 1,
The non-mentin protein is a composition characterized in that represented by SEQ.
A pharmaceutical composition for preventing hair loss or promoting hair growth, comprising a fusion protein combined with a non-mentin protein and a protein delivery domain.
The method of claim 7, wherein
The protein delivery domain is a group consisting of TAT, Antp peptide, VP22, HP4, Transportan, MAP, TP10, K5-FGF, HAP-1, 293P-1, CADY, PF6, RXR, polyarginine or polylysine At least one selected from the composition.
The method of claim 7, wherein
The fusion protein is a composition, characterized in that represented by SEQ ID NO: 2.
Hair loss prevention or hair growth promoting pharmaceutical composition comprising a nucleic acid encoding a non-mentin protein as an active ingredient.
11. The method of claim 10,
The nucleic acid encoding the non-mentin protein is characterized in that SEQ ID NO: 3.
A pharmaceutical composition for preventing hair loss or promoting hair growth, comprising a nucleic acid encoding a fusion protein to which a non-mentin protein and a protein delivery domain are bound.
13. The method according to claim 10 or 12,
The nucleic acid is contained in the expression vector composition.
The method of claim 13,
Wherein said expression vector is a plasmid or a viral vector.
15. The method of claim 14,
The plasmid is selected from the group consisting of pET, pcDNA, pRK5, pSV16B, pVL1392 (PharMingen) and pHis / TAT.
15. The method of claim 14,
Wherein said viral vector is selected from retroviral vectors, adenovirus vectors, and herpesvirus vectors.
13. The method of claim 12,
The nucleic acid composition is characterized in that represented by SEQ ID NO: 4.
Hair loss prevention or hair growth promoting agent comprising at least one selected from the group consisting of a non-mentin protein, a protein delivery domain-non-mentin fusion protein and a nucleic acid encoding the protein.
Non-mentin protein, protein delivery domain-non-mentin fusion protein and cosmetics for preventing hair loss or hair growth promoting, characterized in that it comprises at least one selected from the group consisting of nucleic acids encoding the protein as an active ingredient.
The method of claim 19,
The cosmetics include hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow regrowth, eyelash regrowth, eyelash nutrient, pet shampoo or A rinse for hair loss prevention or hair growth cosmetics.

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