TWI740964B - Use of promoting adipocyte differentiation and method for screening materials for promoting adipocyte differentiation - Google Patents

Abstract

The present disclosure discloses a composition comprising a material for promoting SRBC expression as an active ingredient and a method for screening materials for promoting adipocyte differentiation. The composition comprising a material for promoting SRBC expression as an active ingredient according to one aspect of the present invention promotes the differentiation of adipocytes and thus can be usefully used for skin care, scar treatment, etc. In addition, the method for screening materials for promoting adipocyte differentiation according to one aspect of the present invention allows to treat skin adipocytes with a test material and identify the level of relative expression of SRBC genes, thus enabling to screen materials for promoting adipocyte differentiation, conveniently, rapidly and effectively.

Description

Use of promoting adipocyte differentiation and screening of substances that promote adipocyte differentiation method

The present invention discloses a composition including an active ingredient that promotes the expression of SRBC or an active substance as an active ingredient and a method for screening a substance that promotes adipocyte differentiation.

The basic structure of the skin is maintained by the subcutaneous adipose tissue, and the subcutaneous adipose tissue affects the volume and strength of the skin. Therefore, compared to providing elasticity to the dermis or epidermis in the outer skin layer, increasing the volume of subcutaneous fat can maintain the volume and elasticity of the skin. At the same time, in order to increase the volume of adipose tissue, research is being conducted on substances that promote the differentiation of fat cells. The term "differentiation of adipocytes" refers to a series of processes in which preadipocytes (preadipocytes) are converted into adipocytes that continue to occur in our body during the life cycle. It is reported that the differentiation of adipocytes takes place in the cell through the complex regulation process of gene expression through external stimuli (such as hormones). Among the external signals that induce adipocyte differentiation, insulin is the most widely known hormone and is known to play a key role in the regulation of adipocyte metabolism.

[technical problem]

In one aspect, the purpose of the present invention is to provide a novel adipocyte differentiation marker.

In another aspect, the purpose of the present invention is to promote adipocyte differentiation and thereby increase skin volume.

In another aspect, the purpose of the present invention is to use a novel marker to facilitate the screening of substances that promote adipocyte differentiation.

[Technical solutions]

In one aspect, the present invention provides a composition that includes as an active ingredient a serum deprivation response factor-related gene product that binds to C-kinase. , SRBC) performance or active substance.

In another aspect, the present invention provides a skin care method, which includes the step of treating the skin with a composition that includes as an active ingredient a substance that can promote the performance or activity of SRBC.

In another aspect, the present invention provides a method for adipocyte differentiation, which includes a method for treating isolated adipocytes with a composition that includes as an active ingredient a substance that can promote the performance or activity of SRBC.

And, in another aspect, the present invention provides a method for screening a substance that promotes adipocyte differentiation, which includes the following steps: treating adipocytes with a test substance; The degree of expression of the gene product (SRBC) associated with serum deprivation response factor bound to C-kinase before and after the substance treatment.

In another aspect, the present invention provides a kit for screening substances that can promote adipocyte differentiation, which includes an adipocyte; and instructions, wherein the instructions describe the method.

[Effects of the invention]

The composition that can promote the expression or activity of SRBC as an active ingredient including one aspect of the present invention can promote the differentiation of adipocytes and therefore can be usefully applied to skin care, scar treatment, and the like. In addition, the method for screening substances that promote adipocyte differentiation in one aspect of the present invention allows a test substance to treat adipocytes and determine the relative expression level of SRBC genes, so that substances that promote adipocyte differentiation can be screened conveniently, quickly and effectively.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention is a composition that promotes adipocyte differentiation, which includes as an active ingredient a substance that can promote the expression or activity of a serum deprivation response factor related gene product (SRBC) bound to C-kinase.

In addition, in one aspect, the present invention is a composition for enhancing skin volume, which includes a substance that promotes the performance or activity of SRBC as an active ingredient.

In addition, in one aspect, the present invention is a composition for improving skin wrinkles or enhancing skin elasticity, which includes a substance that promotes the performance or activity of SRBC as an active ingredient.

The relationship between SRBC performance and adipocyte differentiation is still unknown. The inventors of this case discovered for the first time that SRBC is a marker of adipocyte differentiation. It is based on the discovery that when the expression level of SRBC increases, the peroxisome proliferator-activated receptor γ (peroxisome proliferator-activated receptor γ), which is known to be involved in adipocyte differentiation, The performance of gamma, PPAR-γ) and fatty acid synthase (FAS) are up-regulated and triglycerides increase.

The term "skin" as used herein refers to an organ that covers the outer side of a living organism and is composed of an epidermis layer, a dermis layer, and a subcutaneous fat layer. The term includes not only the tissues covering the face or the outside of the body but also the scalp and hair.

The term "volume" as used herein refers to the volume or volume on the surface or inside of the skin layer. This may mean the appearance of voluminous surface. Accordingly, the skin with increased or increased volume looks lifted, healthy and young. And, for example, when skin injury (injury), wound (wound), burns, etc. are damaged, scars or depressions caused by them, or the skin or tissue is incised due to surgery, the increase in the volume of skin tissue can result in injured tissue And the volume of the skin layer increases and provides the effect of restoring the skin or tissue to its original state.

The term "enhancement of volume" as used herein refers to an increase in the volume of the skin layer. It also refers to lifted and healthy-looking skin or injured skin tissue restored to approximate its original state due to the increase in skin volume. Accordingly, increasing the volume of the skin tissue can provide the effect that the injured skin is restored to its original state due to the increase in the volume of the drooping skin and the stretched skin surface.

In the above aspect, the composition can inhibit the movement of preadipocyte factor-1 (pref-1) into the nucleus. In addition, the composition can inhibit the performance of pref-1.

In the above aspect, the composition can increase one or more selected from peroxisome proliferator activated receptor gamma (PPAR gamma), fatty acid synthase (FAS), stearyl-CoA desaturase 1 The performance of the group consisting of (stearoyl-CoA desaturase 1, SCD-1) and adiponectin.

In a specific embodiment, the composition may include a vector, which includes a gene encoding SRBC. In this aspect, the gene may include the sequence of gene ID: 109042. In a specific embodiment, the vector may be a pcDNA 3.1 vector (Invitrogen Co., Carlsbad, CA, USA), in which a gene encoding SRBC is inserted between the Xba I position and the Bam HI position. The gene sequence of SRBC can be obtained from Pubmed's gene ID: 109042.

In the above aspect, the composition may include 0.0001 to 70 wt% based on the total weight of the composition for promoting the performance or activity of SRBC. For example, it can be no less than 0.0001 wt%, no less than 0.01 wt%, no less than 0.1 wt%, no less than 10 wt%, no less than 20 wt%, no less than 30 wt%, no Less than 40 wt%, no less than 50 wt%, or no less than 60 wt% and no more than 70 wt%, no more than 60 wt%, no more than 50 wt%, no more than 40 wt%, no More than 30 wt%, not more than 20 wt%, not more than 10 wt%, not more than 1 wt%, not more than 0.1 wt%, or not more than 0.001 wt%.

In a specific embodiment, the composition may be a composition for skin care. The composition used for skin care can be a cosmetic composition.

In a specific embodiment, the composition may be a pharmaceutical composition for restoring depressed skin.

The beauty composition may be, for example, basic cosmetics, makeup cosmetics, hair care cosmetics, body care cosmetics, and the like. The formula is not particularly limited and can be appropriately selected according to the current situation.

For example, the beauty composition can be formulated as the following but not limited to: solution, suspension, emulsion, paste, gel, cream , Lotion, powder, soap, cleansing, oil, powder foundation, emulsion foundation, wax foundation), spray, etc. More specifically, it can be formulated into: basic cosmetics, such as softening lotion, nourishing lotion, milk lotion, body lotion, nourishing cream, Massage cream, moisturizing cream, hand cream, essence, eye cream, cleansing cream, cleansing foam, Cleansing water, pack, glue, patch, oil-in-water (O/W) emulsion, water-in-oil (W/O) emulsion, etc.; coloring cosmetics , Such as lipstick, makeup base, foundation, etc.; cleaning products, such as shampoo, conditioner, body cleanser, toothpaste, mouthwash, etc.; Or hair care and beauty products, such as hair tonic, hair fixatives such as hair gel or mousse, hair growth promoter, hair dye, etc.

The cosmetic composition contains a cosmetically acceptable medium or matrix. This can be provided in any suitable formulation, such as solutions, gels, anhydrous solids or pastes, oil-in-water emulsions, suspensions, microemulsions, microcapsules, microgranules or ions (liposomes) and / Or nonionic vesicular dispersion, cream, skin lotion, lotion, powder, ointment, spray or conceal stick. These compositions can be prepared according to methods commonly used in the art.

When the formulation of the present invention is a solution or emulsion, a solvent, dissolving agent or emulsifier can be used as a carrier. Examples include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol (propylene glycol), 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan ester of sorbitan).

When the formulation of the present invention is a suspension, liquid diluents (such as water, ethanol or propylene glycol), suspending agents (such as ethoxylated isostearyl alcohol), polyoxyethylene sorbitol (polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth tragacanth) as a carrier.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone ), bentonite, silica, talc or zinc oxide as the carrier.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier. In particular, when the formulation is a spray, it may further include a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether.

In a specific embodiment of the present invention, the cosmetic composition may further contain a thickening agent. The thickener contained in the cosmetic composition of the present invention can be methyl cellulose, carboxymethyl cellulose, carboxymethyl hydroxyguanine, and hydroxymethyl cellulose. (hydroxymethyl cellulose), hydroxyethyl cellulose, carboxyvinyl polymer, polyquaternium, cetearyl alcohol, stearic acid , Carrageenan and so on. Preferably, one or more of carboxymethyl cellulose, carboxyvinyl polymer and polyquaternary ammonium salt can be used. Optimally, carboxyvinyl polymers can be used.

In a specific embodiment of the present invention, the cosmetic composition can contain various suitable bases and additives as required, and the type and quantity of the cosmetic composition can be easily determined by those skilled in the art. The cosmetic composition may contain acceptable additives as needed. For example, it may include preservatives, colorants, additives, etc. commonly used in the art.

Specifically, the preservative may be phenoxyethanol, 1,2-hexanediol, etc., and synthetic fragrances may be used.

In addition, in a specific embodiment of the present invention, the cosmetic composition may include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polypeptides, and polysaccharides. Polysaccharide, sphingolipid and seaweed extract. In addition, it may further include oils and fats, humectants, emollients, surfactants, organic or inorganic pigments, organic powders, ultraviolet absorbers, preservatives, sterilizers, antioxidants , Plant extracts, pH adjusters, ethanol, colorants, fragrances, blood circulation stimulants, coolants, antiperspirants, purified water, etc.

However, the ingredients that can be included in the beauty composition are not limited thereto. Moreover, the quantity of these components can be determined within a range that does not adversely affect the purpose and effects of the present invention.

The pharmaceutical composition of one aspect of the present invention can be various oral or parenteral formulations. When the composition is formulated, use commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc., and by combining at least one compound It is prepared by mixing with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc can be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc., and various excipients (such as wetting agents, sweeteners, flavoring agents, preservatives) and water and liquid paraffins commonly used as simple diluents can be added . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), injectable esters (such as ethyl oleate), etc. can be used as non-aqueous solvents or suspensions. Witepsol, macrogol, tween 61, cacao butter, laurinum, glycerogelatin, etc. can be used as suppository bases.

In one aspect of the present invention, the composition can be administered parenterally or orally depending on the purpose. It can be divided into one to several divided doses so that the daily dose is 0.1-500 mg per kilogram of body weight, preferably 1-100 mg. The dose to the patient can be changed according to the patient's weight, age, sex, health status, diet, application frequency, application method, discharge rate, disease severity, etc.

The pharmaceutical composition of one aspect of the present invention can be formulated into any form suitable for pharmaceutical preparations, including oral preparations (such as powders, granules, tablets, soft or hard capsules, suspensions, emulsions, syrups) and Aerosols, injections and sterile injection solutions.

The composition of one aspect of the present invention can be administered to mammals (such as rats, mice, livestock, humans, etc.) via various routes (such as parenteral, oral, etc.), and all administration methods are predictable. For example, it can be administered orally, transdermally, intravenously, intramuscularly, or via subcutaneous injection.

The health food composition preparation of one aspect of the present invention is not particularly limited, but the composition can be formulated into, for example, liquid preparations (such as tablets, granules, powders and drinks), caramels, colloids ( gels), bars, etc. The food composition of the present invention may include various nutrients, vitamins, minerals (electrolytes), flavoring agents (such as synthetic flavoring agents and natural flavoring agents), coloring agents and improving agents ( Cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickening agent, pH regulator, stabilizer, preservative, glycerin, ethanol , Carbonizing agent used in carbonated beverages, etc. In the food composition of each preparation, in addition to the active ingredient, those skilled in the art can appropriately select and add according to the purpose of the composition Other commonly used ingredients. Adding other ingredients, especially compositions containing probiotics, may produce synergistic effects.

In another aspect, the present invention is a skin care method, which includes treating the skin with the composition. In a specific embodiment, the composition can be applied transdermally to wrinkled skin, scarred skin, etc., to achieve a skin care effect.

In another aspect, the present invention is a method for adipocyte differentiation, which includes the step of treating and isolating adipocytes with the composition. In a specific embodiment, an adipocyte line is isolated from a subject (ie, a supplier) in need of skin care, and the isolated adipocyte line is treated with a substance that promotes SRBC performance or activity to differentiate adipocytes, and then the differentiation The fat cell line is administered to the supplier. In this case, because the subject’s own cells are used, the scar can be removed without the advantage of side effects such as immune response.

In another aspect, the present invention provides a method for screening a substance that promotes adipocyte differentiation, which includes the following steps: treating adipocytes with a test substance; The degree of SRBC performance before and after.

The term "relative degree of performance" as used herein refers to the degree of SRBC performance in adipocytes treated with a test substance compared to the degree of SRBC performance in adipocytes not treated with a test substance. The degree of performance may cover the quantity and nature of performance. In addition, the degree of expression may encompass, for example, the degree of expression of protein.

In the above aspect, the method may further include the following steps: when the expression level of SRBC in the adipocytes after treatment with a test substance is higher than the expression level of SRBC in the adipocytes before treatment with the test substance, it is determined that the test substance is Substances that promote the differentiation of fat cells or increase the volume of the skin. For example, if the performance level of SRBC in adipocytes treated with a test substance is higher than that in adipocytes not treated with the test substance, it can be determined that the administered test substance will increase the performance of SRBC. As mentioned above, if an administered test substance increases the degree of SRBC performance, it can be determined that it is a substance that promotes adipocyte differentiation.

In a specific embodiment, the degree of SRBC gene expression can be judged according to known techniques, such as but not limited to reverse transcription polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), western transduction Western blotting or immune blotting.

In a specific embodiment, in the step of determining that a test substance is a substance that promotes the differentiation of adipocytes or enhances skin volume, the degree of SRBC expression in the adipocytes after treatment with the test substance is at least 1.1 times higher than that of the adipocytes. The degree of SRBC expression in the adipocytes before the test substance is processed, and the test substance can be judged as a substance that promotes the differentiation of adipocytes or increases the volume of the skin. After treatment with a test substance, the degree of SRBC expression in the adipocytes may be, for example, but not limited to, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times. Times, at least 1.9 times, or at least 2 times higher than the former treatment with the test substance. The performance level is a statistically significant measurement result. The term "statistically significant" refers to the situation where there is a significant difference based on biostatistics, and it includes the situation where the p-value is less than 0.05 in the quantitative analysis. In another aspect, the present invention is a kit for screening substances that promote adipocyte differentiation, which includes adipocytes and instructions, wherein the instructions describe the method.

Hereinafter, the composition and effects of the present invention will be described in more detail with reference to examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples in any way.

[Example] [Example 1] Preparation of SRBC-overexpressing cell line [Example 1-1] Cell culture was performed using a 6-well plate in a 5% CO ₂ incubator containing 10% calf serum (Gibco BRL, NY, USA) and Dulbecco's modified Eagle's medium (DMEM) (Lonza, MD, USA) with 5% penicillin-streptomycin and culturing mouse 3T3-L1 fibroblasts (ATCC, CL-173) for 2 days. It proliferates without differentiation.

[Example 1-2] Cell differentiation In Example 1-1, the undifferentiated adipocytes were cultured in a 6-well plate to 95% confluence. Then, in a 5% CO ₂ incubator containing 10% fetal bovine serum (FBS) (PAA, Austria), 10 μg/ml insulin (Sigma-Aldrich, St. Louis, USA), 0.5 mM 3 -Isobutyl-1-methylxantine (3-isobutyl-1-methylxantine, IBMX) (Sigma-Aldrich, St. Louis, USA) and 1 μM dexamethasone (DEX) (Sigma-Aldrich, St. Louis, USA) . Louis, USA) in DMEM for 2 days. After that, they were cultured in DMEM supplemented with 10% FBS and 10 μg/ml insulin for another 3 days. Then, the cells were cultured in DMEM containing 10% FBS for only 2 days to obtain differentiated adipocytes.

[Example 1-3] RNA extraction and cDNA synthesis TRIzol (Gibco BRL, NY, USA) was used to extract RNA and reverse transcription system (Reverse Transcription System) (Promega Co, WI, USA) was used to synthesize cDNA.

[Example 1-4] Real-time quantitative RT-PCR (RT-qPCR) TaqMan® probe (Life technologies, CA, USA) and master mix (Life technologies, CA, USA) were added to Example 1-3 Synthesized cDNA, the performance of each gene was analyzed using Rotor-gene 3000 (Corbett Research, AUS).

[Example 1-5] The SRBC cDNA of the cell line that produces overexpression was synthesized in the mouse fibroblasts cultured in Example 1-1, using a primer (5'-GGAATTCCAATGGGGGAGAGC-3', 5'- GGGTACCGGCTGCGCTCTCTAT-3'). The synthesized cDNA was inserted into pcDNA3.1 vector (Invitrogen Co., Carlsbad, CA, USA) between XbaI and BamHI to transfect mouse 3T3-L1 fibroblasts. The transfected cells were then selected with puromycin-containing medium (3 μg/ml).

[Example 2] Oil Red O Staining and imaging preparation of over-expressing cell lines Oil Red O (ORO) (Sigma-Aldrich, St. Louis, USA) dissolved in 60% propylene glycol ( The stock solution of propylene glycol (PG) (Santa Cruz Biotechnology, Inc., CA, USA) was used to determine the amount of triacylglycerol (TG) of differentiated fibroblasts in mice. Undifferentiated cells and cells at different stages of differentiation were obtained from Examples 1 and 2. They were washed twice with cold phosphate-buffered saline (PBS) (Welgene, Daegu, Korea) and then treated with 3.7% formaldehyde (Sigma-Aldrich). , St. Louis, USA) fixed 1 hour. Then, the cells were washed twice with cold PBS and stained with the pre-prepared ORO stock solution for 30 minutes. After washing with running water, the stained cells were imaged with Olympus IX71 (Tokyo, Japan). After imaging, dissolve ORO with 100% PG and measure the absorbance at 450 nm to quantify the amount of TG.

The results show that when SRBC is overexpressed, adipocytes will differentiate at a faster rate than normal (Figures 1 and 2).

The present invention describes specific embodiments, and it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention as defined by the scope of the appended application.

Figure 1 shows the difference between normal cells and SRBC overexpressing cells, which are based on changes in the expression of adipocyte differentiation-related factors.

Figure 2 shows the observation results of the difference between naked normal cells and SRBC overexpressing cells using Oil Red O fat staining.

Figure 3 shows the results of measuring triacylglycerol (TG) synthesized when SRBC is overexpressed.

<110> South Korea Amorepacific Co., Ltd.

<120> The use of promoting the differentiation of adipocytes and the method of screening substances that promote the differentiation of adipocytes

<140> 106119776

<141> 2017-06-14

<150> KR 10-2016-0081790

<151> 2016-06-29

<150> KR 10-2017-0072314

<151> 2017-06-09

<160> 2

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400>

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400>

Claims (12)

A use of a vector containing a gene encoding a serum deprivation response factor related gene product (SRBC) gene coded with C-kinase as a composition for promoting the differentiation of adipocytes. A use of a vector including a gene encoding a serum deprivation response factor related gene product (SRBC) gene coded with C-kinase as a composition for the preparation of a skin volume-enhancing composition. A use of a vector containing a gene encoding a serum deprivation response factor related gene product (SRBC) gene coded with C-kinase as a composition for improving skin wrinkles or increasing skin elasticity. The use according to any one of claims 1 to 3, wherein the composition inhibits the movement of preadipocyte factor-1 (pref-1) to the nucleus or inhibits its expression. The use according to any one of claims 1 to 3, wherein the composition is a skin care composition. The use according to claim 1 or 2, wherein the composition is a pharmaceutical composition for restoring sunken skin. The use according to any one of claims 1 to 3, wherein the composition includes 0.0001 to 70 wt% of the vector containing the SRBC gene encoding based on the total weight of the composition. A method for adipocyte differentiation, which comprises the step of treating and isolating adipocytes with the composition described in any one of claims 1 to 3. A method for screening substances that promote adipocyte differentiation, which includes the following steps: Treating adipocytes with a test substance; and judging the relative expression levels of gene products (SRBC) associated with serum deprivation response factors bound to C-kinase in the adipocytes treated with the test substance before and after treatment with the test substance. The method according to claim 9, wherein the method further comprises a step of determining when the expression level of SRBC in the adipocytes after treatment with the test substance is higher than the expression level of SRBC in the adipocytes before treatment with the test substance The test substance is a substance that promotes the differentiation of fat cells or increases the volume of the skin. The method according to claim 10, wherein in the step of confirming that the test substance is a substance that promotes differentiation of adipocytes or enhances skin volume, the degree of SRBC expression in the adipocytes after treatment with the test substance is at least 1.1 times higher The degree of SRBC expression in the adipocytes before treatment with the test substance is determined to be a substance that promotes the differentiation of adipocytes or increases skin volume. A kit for screening substances used to promote adipocyte differentiation, comprising adipocytes; and instructions, wherein the instructions describe the method according to any one of claims 9 to 11.

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