KR102218833B1 - Composition for preventing hair loss or promoting hair growth comprising epireguline or epiregulin-derived peptide as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing hair loss or promoting hair growth comprising Epiregulin or a fragment thereof as an active ingredient, and more particularly, a composition for preventing hair loss or promoting hair growth, comprising Epiregulin or a fragment thereof as an active ingredient, It relates to a composition for preventing hair loss or promoting hair growth, containing fat stem cells treated with Epiregulin as an active ingredient.
Epiregulin or a fragment thereof according to the present invention not only promotes hair growth induction in telogen of the hair root, but also significantly increases the hair growth effect in mice. The fragment may be easily utilized for preventing hair loss or promoting hair growth.
In addition, the adipose stem cells treated with Epiregulin according to the present invention have improved proliferation and mobility and inhibit aging, and are useful as a hair loss treatment or a cosmetic for preventing or improving hair loss. According to the present invention, adipose stem cells treated with Epiregulin significantly increased proliferation and migration ability, and aging was significantly suppressed, and when administered to an animal model, the hair growth induction and hair growth effect were significantly increased compared to the control group. Was confirmed.

Description

Composition for preventing hair loss or promoting hair growth comprising epireguline or epiregulin-derived peptide as an active ingredient, comprising Epiregulin or a fragment thereof as an active ingredient}

The present invention relates to a composition for preventing hair loss or promoting hair growth comprising Epiregulin or a fragment thereof as an active ingredient, and more particularly, a composition for preventing hair loss or promoting hair growth, comprising Epiregulin or a fragment thereof as an active ingredient, It relates to a composition for preventing hair loss or promoting hair growth, containing fat stem cells treated with Epiregulin as an active ingredient.

Recently, as interest in beauty has increased, interest in treatment of alopecia is also increasing. Alopecia refers to a condition in which there is no hair where the hair should normally be. Hair does not have an important physiological function that is directly related to life, but it plays a very large role from a cosmetic point of view. In addition, it has functions such as UV protection and hair protection. If hair loss is severe, there may be a problem in social life, and it can have a serious impact on the psychological level, so it is important in terms of quality of life.

Hair loss can be clinically divided into scar-related hair loss and non-scarring hair loss in which only the hair falls out. In the case of scarred hair loss, the hair follicles are destroyed and the hair does not grow again. Hair is made in a place called a hair follicle, and each hair follicle goes through a periodic phase of activity and stop. The time interval of this hair cycle varies depending on the part of the body. In the case of hair, it goes through an anagen of 2 to 6 years and a catagen of 2 to 4 weeks to enter a telogen of 3 to 4 months. do. Each hair follicle has 10 to 20 hair follicle growth cycles during its lifetime.

Currently, the most widely used surgical method as a treatment for hair loss is autologous hair transplantation, and drug treatment using Propecia and minoxidil is widely used. In the case of drug treatment, the hair growth effect appears at the time of administration, but when treatment is stopped, hair loss proceeds again. In particular, minoxidil has side effects such as sexual dysfunction.

On the other hand, Epiregulin (EPR) is a protein encoded by the EREG gene in humans, and Epiregulin is known as a ligand for epidermal growth factor receptor (EGFR), but the content related to hair growth is None known.

In addition, a method of treating hair loss using stem cells has recently been in the spotlight, and in a recent study, it was confirmed that mice whose hair cycle is telogen are converted to anagen by adipose stem cells pretreated with hypoxia or vitamin C. (Won et al., J Dermatol Sci 57:134-137,2010; Kim et al., Stem Cells Dev 23:1364-1376, 2014), it can be seen that this is due to the improvement of hair regeneration ability of adipocytes. . In addition, it is known that PDGF-A, a growth factor expressed and secreted by adipose stem cells, regulates the activity of hair follicle stem cells and induces an anagen in the hair cycle (Festa et al., Cell 146:761-771, 2011, Tomita et al., J Dermatol Sci 43:105-115, 2006). However, the function and effect of Epiregulin in adipose stem cells have not been confirmed.

Accordingly, the inventors of the present invention confirmed that hair growth induction is promoted by Epiregulin or a fragment thereof after intensive research to develop a method for improving the production yield of adipose stem cells and enhancing the hair growth effect, as well as Epiregulin By this, it was confirmed that the proliferation and migration of adipose stem cells increased, aging was suppressed, and the hair regeneration ability of Epiregulin-treated fat stem cells was improved, and the present invention was completed.

An object of the present invention is to provide a composition for preventing hair loss or promoting hair growth, containing Epiregulin or a fragment thereof as an active ingredient.

Another object of the present invention is to provide a proliferation method capable of enhancing the ability to induce hair growth of adipose stem cells.

Another object of the present invention is to provide a pharmaceutical composition or cosmetic composition for preventing hair loss or promoting hair growth, containing as an active ingredient a fat stem cell with enhanced hair growth induction ability or a culture solution thereof.

Another object of the present invention is to provide a composition capable of enhancing the ability to proliferate, migrate, and inhibit aging of fat stem cells.

To achieve the above object,

The present invention provides a composition for preventing hair loss or promoting hair growth, including Epiregulin (EPR) or a fragment thereof.

In a preferred embodiment of the present invention, the Epiregulin fragment may be represented by the amino acid sequence of SEQ ID NO: 1.

According to another preferred embodiment of the present invention, the composition may be a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition.

The present invention also provides a proliferation method capable of enhancing the ability to induce hair growth of adipose stem cells, comprising the step of culturing adipose-derived stem cells in a medium to which Epiregulin is added.

The present invention also provides a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising adipose stem cells treated with Epiregulin or a culture solution thereof as an active ingredient.

The present invention also provides a cosmetic composition for preventing hair loss or promoting hair growth, containing as an active ingredient a fat stem cell cultured by treatment with Epiregulin or a culture solution thereof.

The present invention also provides a composition for promoting the proliferation or migration of adipocytes containing Epiregulin as an active ingredient.

The present invention also provides a composition for inhibiting aging of fat stem cells containing Epiregulin as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising adipose stem cells treated with Epiregulin or a culture solution thereof as an active ingredient.

The present invention also provides a cosmetic composition for preventing hair loss or promoting hair growth, containing as an active ingredient a fat stem cell cultured by treatment with Epiregulin or a culture solution thereof.

Epiregulin or a fragment thereof according to the present invention not only promotes hair growth induction in telogen of the hair root, but also significantly increases the hair growth effect in mice. The fragment may be easily utilized for preventing hair loss or promoting hair growth.

In addition, the adipose stem cells treated with Epiregulin according to the present invention have improved proliferation and mobility and inhibit aging, and are useful as a hair loss treatment or a cosmetic for preventing or improving hair loss.

The adipocytes treated with Epiregulin according to the present invention have the advantage of remarkably increasing the proliferative ability during cultivation, thereby dramatically reducing the cultivation period.

According to the present invention, adipose stem cells treated with Epiregulin significantly increased proliferation and migration ability, and aging was significantly suppressed, and when administered to an animal model, the hair growth induction and hair growth effect were significantly increased compared to the control group. Was confirmed.

1 is a view confirming the hair growth effect of Epiregulin in experimental animals.
Figure 2 is a view confirming the increase in the length of the growth period mustache of the mouse by treatment with Epiregulin.
3 shows the result of confirming the degree of cell proliferation by treating and culturing Epiregulin on dermal papilla cells (hDPC), staining with trypan blue, and counting directly.
4 is a view showing the hair growth effect of the Epiregulin fragment (A) and the degree of proliferation of dermal papilla cells in experimental animals (B).
Figure 5 shows the effect of improving the proliferation ability of adipose stem cells according to Epiregulin treatment.
6 shows the effect of improving the mobility of fat stem cells according to Epiregulin treatment through scratch analysis.
7 shows the effect of improving the mobility of adipocytes according to Epiregulin treatment by using a transwell.
8 shows the effect of improving the regenerative ability of adipose stem cells according to Epiregulin treatment through colony forming ability analysis (clonogenic assay).
9 shows the effect of inhibiting aging of fat stem cells according to Epiregulin treatment through beta-galactocidase staining.
10 is a view confirming the hair growth effect of the adipocytes treated before Epiregulin in an experimental animal.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention relates to a composition for preventing hair loss or promoting hair growth, including Epiregulin (EPR) or a fragment thereof.

Epiregulin is a family of epidermal growth factors and plays an important role in the differentiation and growth of skin cells. The Epiregulin fragment may include GQCIYLSGDRC (SEQ ID NO: 1), and in order to increase the efficacy of preventing hair loss or promoting hair growth, it can be applied by modifying the amino acid sequence as necessary. The modified Epiregulin fragment may preferably include GQCIYLNNNNN (SEQ ID NO: 2), but 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or It may contain sequences that are 99% or more identical.

"Hair loss" that can be prevented or treated by the composition of the present invention is a phenomenon in which the hair is completely out of the scalp, and can prevent or treat male pattern hair loss and female pattern hair loss, and prevent or treat male pattern hair loss caused by hair follicle damage It is preferable to, but is not limited thereto.

The term "hair growth" of the present invention means hair growth from the scalp, and specifically means to induce hair to be formed by forming hair follicles in a hair loss area or a hairless area (hairless area). This is used in the art in the same sense as "hair growth" or "wool", which means that the length of the hair becomes longer (ie, hair growth).

In one embodiment of the present invention, as a result of confirming the hair generation effect by treating Epiregulin on the back of the mouse from which the hair was removed, the group treated with Epiregulin produced hair faster and more abundantly than the control group. It was found that there is a better hair growth effect (FIG. 1). In addition, as a result of tissue culture by treatment with Epiregulin, it was confirmed that the length of the mouse mustache was significantly increased in the culture solution treated with Epiregulin (FIG. 2). Furthermore, when Epiregulin was treated and cultured on dermal papilla cells, it was confirmed that the proliferation ability of dermal papilla cells was remarkably improved in the culture solution treated with Epiregulin (FIG. 3).

In another embodiment of the present invention, as a result of injecting an Epiregulin fragment into a hair removal mouse, as shown in FIG. 4A, it was found that induction of the hair growth phase was accelerated in the Epiregulin-treated rat.

In addition, as a result of confirming the proliferation effect of dermal papilla cells by the Epiregulin fragment, as shown in FIG. 4B, it was confirmed that the proliferation of dermal papilla cells significantly increased in a concentration-dependent manner.

Therefore, since it was confirmed that Epiregulin (EPR) or a fragment thereof of the present invention effectively induces the growth of hair, it can be easily used for preventing hair loss or promoting hair growth.

The composition of the present invention may be prepared as a pharmaceutical composition, quasi-drug composition, or cosmetic composition.

The site to which the pharmaceutical composition, quasi-drug composition, or cosmetic composition containing the composition of the present invention can be applied can be applied to any part of the body that requires hair growth as well as the scalp. For example, it can be used to improve the condition of hair or hair damaged by a scar caused by trauma, a wide forehead or M-type forehead for the purpose of simple cosmetic effect, eyelashes or eyebrows, and alopecia.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It doesn't work. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, mucosal administration, and eye drop administration.

The appropriate dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. Preferably, the dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg (body weight) on an adult basis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art. Or it can be made by incorporating it into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

When the composition of the present invention is prepared as a quasi-drug composition, the fat-derived stem cells exhibiting an effect of preventing hair loss or promoting hair growth may be added as it is, or may be used together with other quasi-drug or quasi-drug ingredients, and may be appropriately used according to a conventional method. have. The mixing amount of the active ingredient may be appropriately determined depending on the intended use.

The quasi-drug composition for preventing hair loss or promoting hair growth of the present invention is not particularly limited in its formulation, and may be variously formulated in the form of a quasi-drug product known in the art to exhibit an effect of preventing hair loss or promoting hair growth. The formulated quasi-drugs are hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow hair growth agent, eyelash hair growth agent, eyelash nutrient, pet For shampoo, pet rinse, hand sanitizer, detergent soap, soap, disinfectant cleaner, wet tissue, mask, ointment, patch or filter filler, etc., including all quasi-drugs in the usual sense.

In addition, in each formulation, the quasi-drug composition for preventing hair loss or promoting hair growth may be arbitrarily selected and blended according to the formulation or purpose of use of other quasi-drugs. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, and may include, for example, a thickener, a stabilizer, a solubilizing agent, a conventional auxiliary agent such as a vitamin, a pigment and a perfume, and a carrier.

When the composition of the present invention is prepared as a cosmetic composition, the ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the fat-derived stem cells as active ingredients, such as antioxidants, stabilizers, And conventional adjuvants such as solubilizers, vitamins, pigments and fragrances, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation and spray may be formulated, but is not limited thereto.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as carrier components. I can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspending agents such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

In another aspect, the present invention relates to a proliferation method capable of enhancing the ability to induce hair growth of adipose stem cells, including the step of culturing by treating an adipocyte culture medium with Epiregulin.

Epiregulin contained in the culture medium of the present invention may be added at a concentration of 5 to 50 ng/ml as described above.

The culturing step of the present invention may be to inoculate 　 fat stem cells in a medium containing Epiregulin and culture for 48 to 72 hours.

In the present invention, the culture may be performed according to a suitable medium and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art. These various culture methods are disclosed in various literatures (eg, James et al., Biochemical Engineering , Prentice-Hall International Editions). Depending on the cell growth method, suspension culture and adherent culture are classified into batch, fed-batch, and continuous culture methods according to the culture method.

The medium used for cultivation should adequately satisfy the cultivation requirements of adipose stem cells. The medium contains various carbon sources, nitrogen sources, trace elements, and growth factors. Examples of carbon sources that can be used include glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, linoleic acid. Fatty acids such as, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources and urea such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL), and soybean meal, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Inorganic nitrogen source is included. These nitrogen sources may be used alone or in combination. The medium may contain, as personnel, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a corresponding sodium-containing salt. In addition, it may contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids and suitable precursors may be included. During cultivation, the pH can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid in an appropriate manner. In addition, during cultivation, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Preferably, the culture medium in the present invention may be a medium commonly used in the field of animal cell culture technology, and commercially available DMEM (Dulbecco's Modified Eagle Medium), DMEM-F12 (mix of DMEM and Ham's F-12 medium) , MEM (Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), α-MEM (MEM Alpha Modification), RPMI 1640 (Roswell Park Memorial Institute 1640), M199/F12 (Medium 199 and Ham's F-12 medium), IMEM (Improved MEM), MCDB 131 medium, or the like may be used, and the medium may be used as a base and additionally added Epiregulin or HB-EG as described above. In addition, if necessary, fetal bovine serum (FBS), growth factors, or differentiation-related factors may be additionally included, and antibiotics commonly used for stem cell culture, for example, penicillin, strepto Antibiotics such as streptomycin, kanamycin, ampicillin or amphotericin B may be added alone or in combination. During cultivation, a certain concentration (eg, 5%) of CO ₂ can be maintained as in normal animal cell culture conditions. The cultivation temperature is usually 20 ℃ to 45 ℃, and preferably can be maintained at 25 ℃ to 40 ℃.

After completion of the culture, centrifugation or filtration may be performed in order to separate the culture medium from the 　 fat 　 stem cells, and this step can be performed by a person skilled in the art as needed.

In one embodiment of the present invention, in order to confirm the activation of adipose stem cells by Epiregulin, it was confirmed that the proliferation and migration of adipose stem cells increased after Epiregulin treatment (FIGS. 5 to 7 ).

In another embodiment of the present invention, the regenerative ability and aging inhibitory ability of Epiregulin-treated fat stem cells were confirmed (FIGS. 8 and 9 ).

In another embodiment of the present invention, the effect of generating hair was confirmed by transplanting 　 fat stem cells treated with Epiregulin on the back of a mouse from which hair was removed. As a result, it was found that the group transplanted with Epiregulin activated 　 adipose stem cells had a faster and more abundant hair than the control group, and thus had a better hair 　 proliferation effect (FIG. 10).

Accordingly, in another aspect, the present invention relates to a pharmaceutical composition for preventing, treating, or promoting hair growth, comprising, as an active ingredient, adipocytes cultured by treating Epiregulin, or a culture solution thereof.

The pharmaceutical composition may be used for cell transplantation.

In another aspect, the present invention relates to a quasi-drug composition for preventing hair loss or improving hair growth, containing Epiregulin-treated 　 fat stem cells or a culture medium thereof as an active ingredient.

In another aspect, the present invention relates to a cosmetic composition for preventing hair loss or improving hair growth, containing Epiregulin-treated fat stem cells or a culture solution thereof as an active ingredient.

As for the pharmaceutical composition, quasi-drug composition, and cosmetic composition, the above description may be applied in the same manner.

The present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Checking the hair growth effect of Epiregulin

After removing the hair on the back of a 6-week-old male C3H rat using a pet hair epilator and a hair removal cream, 100 ng/ml of Epiregulin (Peprotec, Cat No. 100-04) was added 100 μl to the rat for 12 days. During the subcutaneous injection. On the 16th day, a razor was used to push the newly grown hair on the back of a rat, and the degree of hair regeneration was examined through weight measurement.

As a result, as shown in Fig. 1, it was found that the induction of the hair growth phase was accelerated in the mice treated with Epiregulin.

Confirmation of the effect of tissue culture in Epiregulin-treated medium

Normal mouse growth stage mustache (anagen vibrissal) was isolated using a scalpel and tweezers. The isolated mice mustaches were cultured with 5-20 ng of Epiregulin in quantitative medium (2 mM L-glutamine, 10 μg/ml insulin, 10 ng/ml hydrocortisone, Penicillin and Williams E medium without serum). . Individual hair follicles were photographed 72 hours after the start of incubation (Edmund Optics Ltd, UK). The change in hair length was calculated from the photograph and expressed as the mean±SE of 8-10 hair follicles.

As a result, as shown in FIG. 2, in the group treated with Epiregulin than in the control group, the length of the isolated mice's mustache was increased.

Confirmation of the proliferation effect of dermal papilla cells by treatment with Epiregulin

Follicle Dermal Papilla cell Growth media (Promocell, Heidelberg, Germany) medium containing 1% Antibiotic and Antimycotic Hyclone sv30079.91 for human dermal papilla cells (hDPC) Cells were prepared by inoculating and culturing in a 5% carbon dioxide incubator at 37°C. The hDPC prepared in 12-well was inoculated into the culture medium at a concentration of ^{5×10 3 /well.} After incubation overnight, 5 to 20 ng of Epiregulin was treated and further cultured for 48 hours. Then, 500 μl of trypsin is added, the cells are floated in each well, and the same amount of medium is added to neutralize it, and the medium mixed with the cells is collected in a 1.5 ml tube. Here, only 50 μl was taken, 50 μl of trypan blue was added, stained, and the number of cells was calculated using the formula to check the degree of cell proliferation.

As a result, it was confirmed that the proliferation of dermal papilla cells was increased when Epiregulin was treated as shown in FIG. 3.

Confirmation of hair growth effect and proliferation effect of dermal papilla cells by fragments of Epiregulin

4-1: check the hair growth effect

In the present invention, in order to confirm the hair growth effect by the Epiregulin fragment, 11 amino acids represented by GQCIYLSGDRC (SEQ ID NO: 1) were synthesized by requesting peptron, and then the experiment was performed in the same manner as in Example 1.

As a result, as shown in Fig. 4A, it was found that the induction of the hair growth phase was accelerated in the mice treated with Epiregulin.

4-2: Breast milk Confirmation of cell proliferation effect

In the present invention, in order to confirm the hair growth effect by the Epiregulin fragment, an experiment was performed in the same manner as in Example 3.

As a result, as shown in FIG. 4B, it was confirmed that the proliferation of dermal papilla cells was significantly increased in a concentration-dependent manner.

Isolation and culture of adipose-derived stem cells from adipose tissue

Human adipose-derived stem cells were obtained from subcutaneous fat by liposuction of a known method (Kim et al. J Dermatol Sci 53:96-102, 2009). Specifically, human adipose tissue supplied from the hospital was washed twice with PBS (a buffer commonly used), and then 0.075% collagenase was added and incubated by shaking for 50 minutes in an incubator at 37°C. Next, a medium mixed with PBS and α-MEM medium in half was added to the culture medium, centrifuged at 1200 rpm, and the supernatant was removed, and then only the material settled into a pellet was used. PBS was added to the separated pellet again, mixed vigorously, filtered through a 100 μm nylon mesh, centrifuged, and cultured by mixing only the pellet in a medium. The obtained adipocytes were cultured in α-MEM medium (Hyclone, Thermo Scientific) containing 10% FBS (GIBCO, Invitrogen) and 1% penicillin/streptomycin (GIBCO) at 37° C., 5% CO ₂ and 7 Cells of passage ~9 were used. Through flow cytometry, positive expression of CD44, CD73, CD90, CD105, HLA-1 and PODXL and negative expression characteristics of CD34 and CD45 were confirmed (Kim et al. Cell Death Dis 4:e588, 2013; Kim et al., Cell Biol Int 38:32-40, 2014), and the multipotency to differentiate into adipocytes, bone cells and chondrocytes was confirmed (Song et al. Stem Cells Dev 17:451-461, 2008).

Confirmation of the effect of improving the proliferation capacity of fat stem cells by treatment with Epiregulin

The adipose stem cells obtained in Example 1 were observed under a microscope, and the cells were floated with trypsin and centrifuged. The supernatant was discarded and only cells were left in the lower layer. 1 X PBS was added to release the cells well and stained with trypan blue. The number of cells was calculated using the formula to confirm the degree of cell proliferation.

As a result, as shown in FIG. 5, it was confirmed that the proliferation of adipose stem cells was remarkably increased compared to the control group when cultured by treatment with Epiregulin.

Confirmation of effect of improving migration ability of fat stem cells by treatment with Epiregulin

In order to confirm the improvement of the migration ability of adipose stem cells according to the Epiregulin treatment, a migration ability test using a scratch assay and a transwell was performed.

Specifically, for the scratch analysis, the same number of cells were cultured until completely filled in a 6-well dish, and then scratch was generated using an appropriate tool, and a medium without FBS and a concentration of 5 ng and 20 ng of Epiregulin were used. Separated by the added medium, observed for a certain period of time, and took pictures to analyze the results.

As a result, as shown in FIG. 6, it was confirmed that the number of cell migration increased after the adipose stem cells were treated with Epiregulin.

On the other hand, in the mobility test using a transwell, the same number of cells is planted in a 10mm plate and the original medium is discarded when 40-50% of the cells are grown the next day. After that, a normal medium with FBS and a composition medium containing epiregulin at a concentration of 5 ng and 20 ng, respectively, were added again and cultured for 2-3 days. This is the step of transforming cells into cells with the ability to grow and move by giving a molecular mechanism by Epiregulin. Thereafter, the composition medium was discarded and cultured again for 16 hours in a medium without FBS, and the same number of cells was seeded in a transwell. At this time, the medium entering the transwell should always be free of FBS. 700ul of normal medium containing FBS was added to the lower chamber, and after 24 hours incubation, the cells were stained with crystal dye to analyze the number of cells that migrated the transwell membrane.

As a result, as shown in FIG. 7, it was confirmed that the ability to move fat stem cells was improved in a concentration-dependent manner after Epiregulin treatment.

Confirmation of self-renewal effect of fat stem cells by Epiregulin treatment

Adipose stem cells 1 X 10 ⁴ cells were seeded, and after 24 hours, Epiregulin (5,20 ng) was treated, respectively, and stained with crystal violet reagent 10 days later. It was mixed with new medium and replaced. After that, a picture was taken and the number of colonies having 50 or more cells was counted and shown in FIG. 8.

As a result, as shown in FIG. 8, after treatment with Epiregulin, the self-renewal effect was confirmed from the rapid increase in colonies of fat stem cells compared to the control group.

Confirmation of aging inhibitory effect of fat stem cells by Epiregulin treatment

In a 6-well culture dish, passage 6 fat stem cells were inoculated at a concentration of ^{1 X 10 5.} After 24 hours incubation, Epiregulin was treated with 5 and 20ng respectively, and after incubation for 4 days, the passage was increased again, and Epiregulin was continuously treated up to passage 12. After passage 12, cells were stained and analyzed using an X-Gal kit (Sigma-Aldrich, MO, USA).

As a result, as shown in FIG. 9, it was confirmed that aging of the fat stem cells cultured by treatment with Epiregulin was suppressed.

Confirmation of hair growth effect of pre-treatment fat stem cells in experimental animals

After removing the hair on the back of a 6-week-old male C3H rat using a pet hair epilator and a hair removal cream, adipose stem cells treated with 20 ng of Epiregulin for 2 days were injected subcutaneously ^{into the rat by 3 X 10 4 each.} After 12 to 16 days, the degree of hair regeneration was examined by pushing the newly grown hair on the back of the rat and weighing it.

As a result, as shown in FIG. 10, it was found that the induction of the hair growth phase was accelerated in the mice subcutaneously injected with Epiregulin-pretreated fat stem cells.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Composition for preventing hair loss or promoting hair growth comprising epireguline or epiregulin-derived peptide as an active ingredient <130> 1066820 <150> KR 10-2018-0158063 <151> 2018-12-10 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> epiregulin-derived peptide <400> 1 Gly Gln Cys Ile Tyr Leu Ser Gly Asp Arg Cys 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> epiregulin-derived peptide <400> 2 Gly Gln Cys Ile Tyr Leu Asn Asn Asn Asn Asn 1 5 10

Claims (8)

Epiregulin (Epiregulin; EPR) or a fragment derived from Epiregulin represented by the amino acid sequence of SEQ ID NO: 1,
A pharmaceutical composition for preventing hair loss or promoting hair growth, characterized in that hair growth is induced by promoting the proliferation of dermal papilla cells and increasing hair induction ability by the Epiregulin or the fragment derived from Epiregulin.
delete Epiregulin (Epiregulin; EPR) or a fragment derived from Epiregulin represented by the amino acid sequence of SEQ ID NO: 1,
A quasi-drug composition for preventing hair loss or promoting hair growth, characterized in that hair growth is induced by promoting proliferation of dermal papilla cells and increasing hair induction ability by the Epiregulin or the fragment derived from Epiregulin.
Epiregulin (Epiregulin; EPR) or a fragment derived from Epiregulin represented by the amino acid sequence of SEQ ID NO: 1,
A cosmetic composition for preventing hair loss or promoting hair growth, characterized in that hair growth is induced by promoting the proliferation of dermal papilla cells and increasing the ability to induce hair by the Epiregulin or the fragment derived from Epiregulin. delete delete delete delete

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