JP6867726B1 - Compositions for improving and / or improving skin health - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for improving and / or improving the health condition of skin. SOLUTION: The composition is for promoting and / or improving the health condition of the skin, and the composition is obtained by culturing mesenchymal stem cells in a medium supplemented with hyaluronic acid. A composition comprising clear and / or cell secretions. [Selection diagram] None

Description

The present disclosure relates to compositions for promoting and / or improving skin health.

Mesenchymal stem cells are one of the stem cells (stem cells) existing in the adult body, and have the ability to differentiate into tissues derived from mesoderm (for example, bone, cartilage, blood vessels, cardiomyocytes, etc.). Examples of mesenchymal stem cells include bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and the like.

In recent years, mesenchymal stem cells are expected to have a cosmetic effect, and in particular, the culture supernatant of the cells is used for skin care. For example, Non-Patent Document 1 discloses a cosmetic product containing a culture supernatant of mesenchymal stem cells, collagen, and the like. The culture supernatant is a composition containing the medium itself and various secretions secreted from the cells, which is obtained when the cells are cultured in the medium.

Patent Document 1 discloses that by applying a migration-imparting agent for adipose-derived stem cells to the skin, the adipose-derived stem cells migrate to the dermis layer and improve the skin condition. Patent Document 2 discloses a medium for stem cells containing hyaluronic acid.

International Publication No. 2016/194760 U.S. Patent Application Publication No. 2020/0002678

https://beauty.hotpepper.jp/kr/slnH000455287/blog/bidA031357383.html

As described above, mesenchymal stem cells are expected to be applied to cosmetics, but it is desired to further improve the effect on the target tissue (for example, skin). Therefore, it is an object of the present disclosure to provide a composition for promoting and / or improving the health condition of the skin.

As a result of diligent studies by the inventor, it was found that when the culture supernatant is obtained, the properties of the culture supernatant are significantly changed by adding hyaluronic acid to the medium and then culturing the mesenchymal stem cells. Specifically, when skin fibroblasts were cultured in the culture supernatant, it was found that they had a great effect on the gene expression of skin fibroblasts. Although this mechanism is not clear, it is known that hyaluronic acid promotes improvement of skin condition such as wound treatment. By culturing stem cells in a hyaluronic acid-containing medium, stem cells are stimulated and the skin condition is adjusted. There is a possibility of producing the substance of.

The invention of the present disclosure has been completed based on the above findings and includes the following inventions in one aspect.
(Invention 1)
A composition for promoting and / or improving the health of the skin, wherein the composition is obtained by culturing mesenchymal stem cells in a medium supplemented with hyaluronic acid, and the stem cells, culture supernatant, and /. Or a composition comprising cell secretions.
(Invention 2)
The composition of the present invention, wherein the weight average molecular weight of the hyaluronic acid is 5,000 or less.
(Invention 3)
The composition of the invention 1 or 2, wherein the concentration of the hyaluronic acid is 20 μg / ml or more and 1000 μg / ml or less.
(Invention 4)
The composition according to any one of inventions 1 to 3, which is a composition for cosmetics.
(Invention 5)
The composition according to any one of Inventions 1 to 3, which is a composition for health foods.
(Invention 6)
The composition according to any one of inventions 1 to 3, which is a composition for treating atopy.
(Invention 7)
The composition according to any one of inventions 1 to 3, which is used for treating a skin disease related to any one or more of the following genes.
(A) Col3A1,
(B) Meprin-α,
(C) Elastin,
(D) p16 ^INK4a and
(E) HAS1
(Invention 8)
The composition according to any one of inventions 1 to 3, which is used for wound healing.
(Invention 9)
The composition according to any one of inventions 1 to 3, which is a hair growth and / or hair growth promoter.
(Invention 10)
The composition according to any one of the inventions 1 to 9, wherein the composition is a culture supernatant obtained by culturing mesenchymal stem cells in a medium supplemented with hyaluronic acid and / or processing thereof. A composition comprising a substance.
(Invention 11)
The composition according to any one of the inventions 1 to 10, wherein the composition contains mesenchymal stem cells cultured in a medium supplemented with hyaluronic acid.

In one aspect, the compositions of the present disclosure are obtained by culturing mesenchymal stem cells in a medium supplemented with hyaluronic acid. As a result, components that affect the expression of genes in skin cells (particularly skin fibroblasts, epidermal keratinocytes, etc.) are secreted from mesenchymal stem cells. Then, the component can increase or decrease the expression level of genes involved in promoting and / or improving the health condition of the skin.

It is a graph which relativized the expression level of each gene by quantitative PCR. It is a graph which relativized the expression level of each gene by quantitative PCR. It is a graph which relativized the expression level of each gene by quantitative PCR.

Hereinafter, specific embodiments for carrying out the invention of the present disclosure will be described. The following description is for facilitating the understanding of the invention. That is, it is not intended to limit the scope of the present invention.

1. 1. Compositions for Promoting and / or Improving Skin Health In one embodiment, the present disclosure relates to compositions for promoting and / or improving skin health. Such compositions are obtained by culturing mesenchymal stem cells in a medium supplemented with hyaluronic acid. More specifically, the composition can include a culture supernatant obtained by culturing mesenchymal stem cells and / or a processed product thereof. As another embodiment, the composition comprises mesenchymal stem cells and / or secretions of such cells cultured in a medium supplemented with hyaluronic acid. Alternatively, the composition may include any combination of these stem cells, culture supernatant, and / or cell secretions.

1-1. Definitions The term "to promote and / or improve skin health" as described herein includes the purpose of ameliorating certain diseases associated with the skin. Furthermore, the term is not limited to therapeutic purposes. For example, the term includes the purpose of improving the appearance (gloss, swelling, etc.) of the skin even if it does not have a specific disease.

Also, the term "enhancement and / or improvement" as described herein includes not only cosmetic enhancement and / or improvement, but also maintaining the status quo. For example, the term "enhancement and / or improvement" as described herein also includes maintaining features that would otherwise decline with age (eg, preventing the increase of wrinkles, age spots, etc.).

Furthermore, the term "obtained by culturing mesenchymal stem cells in a medium supplemented with hyaluronic acid" described herein is not limited to those obtained directly, but also those obtained indirectly. Including. Examples of the former include a culture supernatant after culturing mesenchymal stem cells and mesenchymal stem cells. Examples of the latter include processed culture supernatants, for example, those to which other components have been added, and those to which further treatment has been performed. Examples of further treatment include, for example, a culture supernatant that has been filtered, a product that has been dehydrated and concentrated, a product in which a specific component (for example, cell secretion) has been extracted from the supernatant, and the like. ..

1-2. Ingredients In one embodiment, the compositions of the present disclosure can include at least one of the following (1) and (2).
Considering (1) mesenchymal stem cells or their secretions (2) ease of handling when commercializing the culture supernatant or these processed products, (2) is preferable.

Further, in addition to the above components, other components may be added as needed. For example, other components include excipients, antibiotics, pH buffers and the like. Further, as other components, additional components (for example, antioxidants, fragrances, whitening components, moisturizing components, whitening components, etc.) for promoting and / or improving the health condition of the skin can be mentioned.

1-3. Manufacturing Methods In one embodiment, the present disclosure relates to methods of manufacturing compositions for promoting and / or improving skin health.
The manufacturing method includes at least the following steps.
-Add hyaluronic acid to the medium.
-Culture mesenchymal stem cells in the medium after addition.
-Collect the culture supernatant and / or cells after culturing.

1-3-1. Mesenchymal stem cells (derived from fat and bone marrow)
The mesenchymal stem cells are not limited to those derived from a specific tissue, and may be derived from any tissue. For example, bone marrow origin, umbilical cord blood origin, placenta origin, adipose tissue origin and the like can be mentioned. Typically, mesenchymal stem cells derived from bone marrow and / or adipose tissue can be used.

1-3-2. The hyaluronic acid added to the hyaluronic acid medium is not particularly limited, but a low molecular weight hyaluronic acid is preferable. More specifically, the weight average molecular weight is 5,000 or less, preferably 2,000 or less, more preferably 1,500 or less, and most preferably 1,000 or less. The preferred concentration used is 20 to 1000 μg / ml. The molecular weight of hyaluronic acid is calculated by a method (SEC-LS) that combines size exclusion chromatography and a light scattering detector.

1-3-3. Medium The medium is not particularly limited, and a medium known in the art (for example, DMEM, F12, RPMI, etc.) can be used. Examples of the medium include Invitrogen's mesenchymal stem cell basal medium, Sanko Pure Medicine's mesenchymal stem cell basal medium, TOYOBO's MF medium, Biomimetic Sympathies' sf-DOT (registered trademark), and the like. Be done.

1-3-4. Culturing conditions The culturing conditions are not particularly limited and may be any conditions known in the art. For example, the temperature may be 35 ° C to 40 ° C, typically 37 ° C. If appropriate, the incubator may be controlled so _{that CO 2 is 5%.} The culture period is not particularly limited, but it is preferable to culture for at least 24 hours, preferably 48 hours or more. The upper limit is not particularly limited, but is 168 hours or less. The following description is not intended to limit the scope of the invention, but it is believed that hyaluronic acid stimulation alters mesenchymal stem cells to secrete some useful components. Therefore, a certain culture period is required because it takes a certain period of time for the mesenchymal stem cells to change after stimulation with hyaluronic acid.

In a preferred embodiment, mesenchymal stem cells are first grown on a dish and then cultured in hyaluronic acid-supplemented medium. For example, the confluent is reached from the semi-confluent (for example, a state in which 80% or more of the cells are occupied in the field of view under a microscope), and then the culture is performed in a medium supplemented with hyaluronic acid. In this case, hyaluronic acid serves as a physiological stimulus to the mesenchymal stem cells, rather than as a scaffold that exists between the cells and the dish. From this point of view, the present invention according to a preferred embodiment is different in the technical idea as compared with Patent Document 2 in which hyaluronic acid is used as a cell scaffold.

In a more preferred embodiment, the hyaluronic acid added to the medium is a low molecular weight hyaluronic acid. In general, the extracellular matrix that serves as a scaffold for cells is often a polymerized polymer such as collagen or fibronectin. Hyaluronic acid is no exception, and when used as a scaffold, it is common to use high molecular weight hyaluronic acid. From this point of view, the present invention according to a more preferable embodiment is different in the technical idea as compared with Patent Document 2 in which hyaluronic acid is used as a cell scaffold.

In addition, low-molecular-weight hyaluronic acid has a high inhibitory function on cell death, and is considered to have a stronger tendency to play a role as a trigger for signal transduction than high-molecular-weight hyaluronic acid. Then, it is considered that a beneficial substance is secreted extracellularly by triggering a beneficial signal transduction. The present invention according to a preferred embodiment utilizes a beneficial substance secreted extracellularly, and has a different technical idea from Patent Document 2 which attempts to utilize an intracellular component.

In a more preferred embodiment, a culture supernatant (also referred to as a culture solution) containing a large amount of factors secreted from the cells is used instead of the cell crushed extract (extract) used in Patent Document 2. If a cell crushed extract is used, for example, when the content is applied to the skin, it is expected that many proteins and nucleic acids that are not normally released to the outside of the cell will be exposed to the skin. To. If antibodies against this protein or nucleic acid are produced, it may lead to autoimmune diseases. Specifically, it is already known that anti-DNA double-stranded antibody and anti-histone antibody are produced in large amounts in patients with systemic lupus erythematosus, and anti-centromere antibodies are produced in large numbers in patients with systemic scleroderma. Therefore, it is thought that autoimmune diseases develop when these antibodies attack cells throughout the body. Therefore, if a crushed cell extract is used, there is a risk that the intracellular component will permeate into the blood vessel and an antibody against the component will be produced when the skin is torn, so that it is cultivated. It is desirable to use Qing, and the present invention according to a more preferable embodiment is different from Patent Document 2 in terms of technical idea.

1-3-5. Other processing (filter, etc.)
After culturing, the culture supernatant and / or cells may be collected and subjected to further treatment. For example, in the case of the culture supernatant, it may be treated with a filter or concentrated. In the case of cells, they may be transferred to a DMSO-added medium for cryopreservation.

1-3-6. Processing into Dosage Form Also, in one embodiment, the compositions of the present disclosure may be processed into a particular shape for a product of a particular application. For example, the composition may be a solid, a liquid, or a semi-solid (gel). In the case of a solid, it may be processed into a powder or a tablet. When the component of the composition is cells, the cells may be suspended in a preservation solution or the like.

2. Uses In one embodiment, the compositions of the present disclosure can be used for a variety of uses. For example, the compositions of the present disclosure can be applied to cosmetics, health foods, therapeutic agents and the like. As mentioned above, it is considered that the stimulation of hyaluronic acid changes the mesenchymal stem cells and secretes some useful components. Therefore, if at least one of the culture supernatant and the cells after culturing is contained in the composition, a useful component is contained. Moreover, in the case of cells after culturing, useful components can be secreted extracellularly. Then, the useful component can have a good effect on tissues such as skin (for example, skin fibroblasts, epidermal keratinocytes, etc.).

2-1. Cosmetic Compositions In one embodiment, the compositions of the present disclosure can be applied to cosmetics and are particularly suitable for skin cosmetics. The administration form is not particularly limited, but in the case of skin cosmetic use, a form suitable for application to the skin is preferable. For example, the compositions of the present disclosure are preferably in the form of a paste, liquid, or gel.

Therefore, in one embodiment, the compositions of the present disclosure may be formulated with appropriate additional ingredients, depending on the mode of administration. For example, the compositions of the present disclosure may contain ingredients commonly found in skin cosmetics. Examples of such ingredients include moisturizing ingredients, surfactants, emollient ingredients, barrier improving ingredients, anti-aging ingredients, whitening ingredients, cell activating ingredients, anti-inflammatory (irritant relief, anti-allergic) ingredients, antioxidant ingredients, UV protection ingredients, etc. Antibacterial ingredient, blood circulation promoting ingredient, astringent ingredient, keratin exfoliating ingredient, stabilizing ingredient (preservative, chelate, pH adjustment, etc.), warming and cooling ingredient, fragrance, coloring agent, solvent, antioxidant, surface treatment agent, scrubbing agent, Examples include vegetable fats and oils and plant extracts.

2-2. Composition for health food (administration form, other ingredients)
In one embodiment, the composition of the present disclosure can be applied to health foods, and in particular, may be a composition for health foods having a useful effect on the skin. The form of administration is not particularly limited, but may be encapsulated in an appropriate capsule, enteric-coated capsule, or the like in consideration of digestion and absorption in the digestive organs. In the case of a composition for health food, the administration form is oral ingestion. Therefore, for example, the composition of the present disclosure is preferably in the form of a solid, a liquid, or a gel. More specifically, the composition may be in a form that can be ingested by chewing and / or swallowing. Therefore, the composition may be in the form of a liquid or gel such as a jelly or a beverage, as well as a solid substance such as a tablet or a biscuit of a supplement.

Therefore, in one embodiment, the compositions of the present disclosure may be formulated with appropriate additional ingredients, depending on the mode of administration. For example, the compositions of the present disclosure may contain ingredients commonly found in health foods. Examples of such components include preservatives, preservatives, flavors, pH regulators, antioxidants, fungicides, sweeteners, colorants and the like.

2-3. Composition for atopy treatment In one embodiment, the composition of the present disclosure can be applied to the treatment of atopy, and in particular, the composition for atopy treatment may have a useful effect on the skin. The form of administration is not particularly limited, and examples thereof include application, subcutaneous injection, and oral administration. Therefore, for example, the composition of the present disclosure is preferably in the form of a solid, a liquid, a paste, or a gel.

Therefore, in one embodiment, the compositions of the present disclosure may be formulated with appropriate additional ingredients, depending on the mode of administration. Examples of such components include excipients, carriers, pH buffers, transfection accelerators, other atopic therapeutic agents and the like.

2-4. Compositions for Other Uses In one embodiment, the compositions of the present disclosure can be applied to other uses (eg, for wound healing, for treating skin diseases, for hair growth and / or for hair growth promoters, etc.). .. In particular, the compositions of the present disclosure may be useful in diseases associated with increased or decreased expression of at least one protein selected from:
(A) Col3A1,
(B) Meprin-α,
(C) Elastin,
(D) p16 ^INK4a and
(E) HAS1

(A) Col3A1, also called alpha 1 of type III collagen, is one of the components of the triple helix of collagen fibers, and histologically, it is a protein that constitutes the skin. The decrease in the protein is known to contribute to the signs of skin aging. In addition to this, Col3A1 can also be involved in wound healing (including healing of surgical wounds).

(B) Meprin-α is a subunit of Zn protease. And it has a function of cleaving the propeptide of type III collagen. It is known that the ratio of type III collagen / type I collagen decreases with aging, and Meprin is also known to decrease with aging. Furthermore, it has been reported that the decrease in the ratio of type III collagen / type I collagen is suppressed by preventing the decrease in meprin due to aging (https://www.jstage.jst.go.jp/article). / sccj / 47/4 / 47_278 / _article / -char / ja /).

(C) Elastin is a fiber having a role of supporting collagen fibers. Elastin decreases with aging, and this phenomenon causes wrinkles. It is also known to be blended as an ingredient in cosmetics and / or supplements. In addition to this, elastin can also be involved in wound healing (including healing of surgical wounds). Furthermore, the hair growth and / or hair growth agent may contain an elastin component, and elastin can be involved in hair growth.

(D) p16 ^INK4a is known to have a function of inhibiting the cell cycle, and further, it is known that aging progresses when the expression level of this protein increases.

(E) HAS1 is called hyaluronan synthase 1 and is one of the enzymes having a function of synthesizing hyaluronic acid. It is known that the expression level of HAS1 and hyaluronic acid are decreased in the skin of atopic patients. It is known that this hyaluronic acid is also blended as an ingredient in cosmetics and / or supplements. In addition to this, HAS1 can also be involved in wound healing (including healing of surgical wounds) via synthesized hyaluronic acid. In addition, HAS1 can also be involved in hair growth and / or hair growth via synthesized hyaluronic acid.

3. 3. Example
3-1. Example 1 (primary culture of mesenchymal stem cells)
First, adipose tissue-derived mesenchymal stem cells were prepared. Specifically, subcutaneous adipose tissue was collected from patients scheduled to receive regenerative medicine using adipose tissue-derived mesenchymal stem cells. The subcutaneous adipose tissue serves as a raw material necessary for the preparation of cells for administration. The surplus after the subcutaneous adipose tissue was separated was used for primary culture. In addition, consent for research use was obtained from the patient in advance.

The subcutaneous adipose tissue was subjected to centrifugation (400 × g for 5 minutes) and separated into three layers. Specifically, the lipid fraction, the adipose tissue fraction, and the aqueous fraction were separated in order from the upper layer. The upper and lower layers were discarded, leaving the adipose tissue fraction in the middle layer. To the remaining adipose tissue fraction, 4 times the amount of 0.15% collagenase enzyme solution per tissue weight was added. It was permeated at 37 ° C. for 1 hour and treated with an enzyme. After the adipose tissue was dispersed by enzymatic treatment, the adipose tissue was subjected to centrifugation (400 × g for 5 minutes). As a stromal vascular cell fraction containing mesenchymal stem cells, the precipitated fraction was suspended in 30 mL of PBS (-) solution. Then, the suspension was passed through a cell strainer (mesh size 70 μm diameter), and the tissue residue and the like captured by the cell strainer were discarded. Then, the liquid passing fraction was subjected to centrifugation again (400 × g for 5 minutes), and the precipitated fraction was suspended in 6 mL of serum-free culture solution sf-DOT (Biomimetic Sympathies). The entire cell suspension was seeded in a T25 flask (CellBIND; Corning, 3289) and allowed to stand in _{an incubator (37 ° C., 5% CO 2) to start primary culture.}

3-2. Example 2 (passage culture, P0⇒P1⇒P2)
Total medium replacement was performed once every 3 days. The supernatant was discarded and cells growing on the bottom of the flask were selectively grown. To the cells in the T-25 flask that had grown to semi-confluent, 2 mL of an enzyme solution (TrypLE Express; Thermo Fisher Scientific, 12604021) was added, and the cells were detached from the bottom of the flask (standing at 37 ° C. for 5 minutes). ). Cells were diluted with PBS (−) and subjected to centrifugation (400 × g for 5 minutes). The precipitated cells were suspended in the culture medium sf-DOT, and a part of the precipitated cells was separated and the number of cells was counted by the trypan blue staining method. Cells suspended in sf-DOT were seeded in a new T75 flask (CellBIND; Corning, 3290 _{) and allowed to stand in an incubator (37 ° C., 5% CO 2} ) for subculture (P0 → P1). ). After that, subculture was carried out in the same procedure to obtain the required number of cells (P1 → P2).

3-3. Example 3 (Preparation of culture supernatant)
First, adipose tissue-derived mesenchymal stem cells were cultured in sf-DOT as described above. Adipose tissue-derived mesenchymal stem cells were seeded in the ^{same medium at 3000 cells / cm 2 per T75 flask.} On the third day when the semiconfluent was reached, it was washed once with PBS (−). Then, the following two media were prepared for the culture supernatant.
(I) Basic medium (DMEM / F12; SIGMA, D8900),
(Ii) Basic medium (DMEM / F12; SIGMA, D8900) + low molecular weight hyaluronic acid HA4 (100 μg / ml added)

The cells were cultured in the media of (i) and (ii) for another 3 days, and the supernatants of each were collected. The collected culture supernatant was filtered through a 0.2 μm PES syringe filter (25 mm GD / X syringe filter (PES 0.2 μm sterilized); 6896-2502; GE Healthcare Japan). The culture supernatant after filtration was stored frozen at −28 ° C. until used for analysis.

The following three types of culture supernatants were prepared using the culture supernatants obtained above.
CM1: Culture supernatant derived from the above (i) H-CM: Culture supernatant derived from the above (ii) CM2: Culture supernatant derived from the above (i), low molecular weight hyaluronic acid HA4 (100 μg / ml) Addition

3-4. Example 4 (Treatment of skin fibroblasts using culture supernatant)
Normal human dermal fibroblasts (Normal Human Dermal Fibroblast (NHDF); Takara, C-12302) were prepared. The cells were passaged and maintained on Fibroblast medium (Sciencell, 2301). ^{NHDF was seeded at 3000 cells / cm 2} in each well of a 12-well plate (Corning, 3336) and cultured overnight.

3-5. Example 5 (Treatment of epidermal keratinocytes using culture supernatant)
Normal human epidermal keratinocytes (Normal Human Epidermal Keratinocyte (NHEK); Takara, C-12006) were prepared. The cells were passaged and maintained with Keratinocyte-SFM (Thermo Fisher Scientific, 17005042). ^{NHEK was seeded at 10000 cells / cm 2} in each well of a 12-well plate (Corning, 3336) and cultured overnight.

The next day (16 to 24 hours after sowing), the medium was replaced with various supernatants (CM1, CM2, H-CM), and the cells were further cultured for 3 days (about 72 hours). After culturing, Total RNA was extracted from NHDF and NHEK using the LiliaPrep RNA Miniprep system (Promega, Z6012). After extraction, cDNA synthesis (PrimeScript RT Master Mix; Takara, RR036A) was performed using 500 ng of RNA, and further quantitative PCR (Thunderbird Sybr qPCR Mix; TOYOBO, QPS-201X5) was performed.

The mixture for cDNA synthesis was prepared according to the following composition.
5xPrimeScript RT Master Mix 2 μl (final concentration 1 x)
Total RNA 500 ng
RNase free H ₂ O Adjusted to a total of 10 μl

The above mixture was treated with Applied Biosystems' Veriti 96 well Thermal Cycler under the following conditions.
37 ℃ 15 minutes ↓
85 ℃ 5 seconds ↓
4 ℃ ∞

The synthesized cDNA (10 μl) was diluted 10-fold with 90 μl TE (10 mM Tris-HCl pH 8.0 + 1 mM EDTA pH 8.0). The dilution was subjected to quantitative PCR.

3-6. Example 6 (Analysis of gene expression in skin fibroblasts and epidermal keratinocytes)
More specifically, the dilutions were mixed under the following conditions.
2 × THUNDERBIRD Probe qPCR Mix 10 μl
5 mM Forward Primer 0.4 μl
5 mM Reverse Primer 0.4 μl
H ₂ O 8.2 μl
cDNA (10-fold dilution) 1 μl

The above mixture was used to amplify the cDNA using CFX-Connect from Bio-Rad. The conditions of the PCR cycle were as follows.
1.95 ° C for 1 minute (initial denaturation)
2.95 ° C for 15 seconds (denaturation)
3.60 ° C for 30 seconds (elongation)
(Repeat steps 2 and 3 40 times, and detect a fluorescent signal after each step 3)
Raise the temperature from 4.65 ° C to 95 ° C in 0.5 ° C increments, hold the temperature for 5 seconds, and then detect the fluorescence signal.

In the above cycle, the PCR product was detected, and at the same time, the unity of the PCR product was confirmed by Melting curve.

As an internal standard (housekeeping gene have the same expression in all cells and conditions) was used GAPDH (G lycer a ldehyde 3- p hosphate d e h ydrogenase).
The primer sequences for detecting each gene were as follows.
GAPDH Forward primer: 5'--agccacatcgctcagacac --3'
GAPDH Reverse primer: 5'--gcctaatacgaccaaatcc --3'
Col3A1 Forward primer: 5'--ctggaccccagggtcttc --3'
Col3A1 Reverse primer: 5'--gaccatctgatccagggtttc --3'
Meprin-α Forward primer: 5'-gcaccacaactcacactctttt --3'
Meprin-α Reverse primer: 5'-ttccacagatgtttgccttc --3'
Elastin Forward primer: 5'--ggaggtgttcccggagtc --3'
Elastin Reverse primer: 5'-ggtccccactccgtacttg --3'
p16 ^INK4a Forward primer: 5'-gtggacctggctgaggag --3'
p16 ^INK4a Reverse primer: 5'--ctttcaatcggggatgtctg --3'
HAS1 Forward Primer: 5'--acgtgcggatccttaaccc --3'
HAS1 Reverse Primer: 3'--agggcctagaggaccgctgat --3'

All primers were purchased from Fasmac Co., Ltd. (reverse phase column purification grade).
The expression level of each gene indicates that the expression level of each gene obtained by quantitative PCR is further divided by the expression level of GAPDH and standardized. Furthermore, the expression level under the control condition in which the supernatant is not treated is standardized so as to be "1". It was confirmed by Melting curve that all the PCR products amplified using the above primers were single (that is, the same primer did not amplify multiple types of sequences).

The results are shown in FIGS. 1 and 2. In the skin fibroblasts treated with H-CM, it was confirmed that the expression level of the verified gene changed in a favorable direction. That is, the expression level of genes (Col3A1, Meprin-α, Elastin) that are considered to be desirable to increase increased. On the other hand, the expression level of the ^{gene (p16 INK4a} ), which is considered to be desirable to decrease, decreased. It was also confirmed that the expression level of the verified gene (specifically, the expression level of HAS1) changed in a favorable direction also in the epidermal keratinized cells treated with H-CM.

It should be noted that such a change in the gene expression level by the H-CM treatment was more remarkable than the change in the gene expression level by the CM2 treatment. As described above, CM2 is a condition in which HA4 is added to the medium after treating the adipose tissue-derived mesenchymal stem cells with the medium. On the other hand, H-CM is a condition in which HA4 is added to the medium before the adipose tissue-derived mesenchymal stem cells are treated with the medium.

Therefore, it is considered that the adipose tissue-derived mesenchymal stem cells were stimulated by HA4 and secreted some useful substance extracellularly. It is considered that the presence of the useful substance satisfactorily changed the expression levels of genes in skin fibroblasts and epidermal keratinocytes.

Skin fibroblasts and epidermal keratinocytes are cells that are present in the skin tissue. Therefore, changes in the expression level of these genes are considered to have a positive effect on the health of the skin.

Considering the difference in conditions between H-CM and CM2 and the difference in the results, when the cells treated with H-CM are co-cultured with, for example, any one of skin fibroblasts and epidermal keratinocytes. Even if there is, it is presumed that the same effect can be obtained. Therefore, when treated under the conditions of H-CM, it is presumed that not only the culture supernatant but also the cells themselves after the culture have a good effect on the health condition of the skin. In order to verify these points, the experiment of Example 7 was carried out as shown below.

3-7. Example 7 (Co-culture of skin fibroblasts and mesenchymal stem cells)
Adipose tissue-derived mesenchymal stem cells were prepared by the same procedure as in Examples 1 and 2. Adipose tissue-derived mesenchymal stem cells were seeded at ^{3000 cells / cm 2 per T75 flask.} On the third day when the semiconfluent was reached, it was washed once with PBS (−). Then, the following two media were prepared for the culture supernatant.
(I) Basic medium (DMEM / F12; SIGMA, D8900),
(Ii) Basic medium (DMEM / F12; SIGMA, D8900) + low molecular weight hyaluronic acid HA4 (100 μg / ml added)

The cells were cultured in these media (i) and (ii) for an additional 3 days, and each cell was collected (MSC and MSC-H, respectively).

In parallel with the above, normal human dermal fibroblasts (Normal Human Dermal Fibroblast (NHDF); Takara, C-12302) were prepared. The cells were passaged and maintained on Fibroblast medium (Sciencell, 2301). 100,000 cells were seeded in each well of the NHDF 6-well plate (CellBIND; Corning, 3335). A transwell (24 mm insert for 6-well plate, 0.4 μm pore size; Corning, 3412) was placed therein. An appropriate amount of Fibroblast medium (Sciencell, 2301) was also poured into the transwell, and a control in which no cells were seeded (NHDF only), MSC seeded (NHDF-MSC), and MSC-H were seeded (NHDF-). MSC-H) was prepared. The number of seeded MSCs and MSC-Hs is 100,000. The transwell used is coated with PLL (Poly-L-Lysine; Cultrex, 3438-100-01) in advance so that the mesenchymal stem cells can adhere to the transwell. The coating was performed overnight at 37 ° C. Then, the excess PLL was washed and removed with sterile water.

Adipose tissue-derived mesenchymal stem cells and normal human skin fibroblasts were co-cultured for 3 days. Normal human skin fibroblasts were placed on the lower side, and adipose tissue-derived mesenchymal stem cells were placed on the upper side. Since the transwell has many holes of 0.4 μm, which are much smaller than the cells, the adipose tissue-derived mesenchymal stem cells in the upper part of the transwell move to the lower part and directly with normal human skin fibroblasts. Rather than exerting its effect on contact, adipose tissue-derived mesenchymal stem cell secretions were allowed to act on normal human skin fibroblasts through a small hole of 0.4 μm.

Then, RNA of normal human skin fibroblasts was recovered, and the expression level was confirmed by the same method as in Example 6 above. The results are shown in FIG. Comparing the results of co-culture with MSC and co-culture with MSC-H, it was confirmed that the result of co-culture with MSC-H changed in a better direction with respect to gene expression. .. That is, the expression level of genes (Col3A1, Elastin) that are considered to be desirable to increase in normal human skin fibroblasts increased. On the other hand, the expression level of the ^{gene (p16 INK4a} ), which is considered to be desirable to decrease, decreased. These results indicate that not only the culture supernatant but also the stem cells themselves have a positive effect on the health of the skin.

The specific embodiments have been described above. The above embodiment is merely a specific example, and the present invention is not limited to the above embodiment. For example, the technical features disclosed in one of the above embodiments can be applied to other embodiments. Further, unless otherwise specified, for a specific method, it is possible to replace some steps with the order of other steps, and an additional step may be added between the two specific steps. The scope of the present invention is defined by the claims.

Claims (8)

A composition for promoting and / or improving the health of the skin, wherein the composition is obtained by culturing mesenchymal stem cells in a serum-free medium supplemented with hyaluronic acid, a culture supernatant, and the like. And / or a composition comprising cell secretions. The composition according to claim 1, wherein the weight average molecular weight of the hyaluronic acid is 5,000 or less. The composition according to claim 1 or 2, wherein the concentration of the hyaluronic acid is 20 μg / ml or more and 1000 μg / ml or less. The composition according to any one of claims 1 to 3, which is a composition for cosmetics. The composition according to any one of claims 1 to 3, which is a composition for health foods. The composition according to any one of claims 1 to 3, which is used for wound healing. The composition according to any one of claims 1 to 6 , wherein the composition is a culture supernatant obtained by culturing mesenchymal stem cells in a medium supplemented with hyaluronic acid and / or a culture supernatant thereof. A composition comprising a work piece. The composition according to any one of claims 1 to 7 , wherein the composition contains mesenchymal stem cells cultured in a medium supplemented with hyaluronic acid.

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