KR102215667B1 - Composition for skin regeneration and anti-aging comprising peripheral blood-derived mononuclear cells and platelet-rich plasma, and skin regeneration method using the same - Google Patents

Abstract

The present specification relates to a composition for regenerating skin and preventing aging by increasing collagen synthesis, and a method for preventing skin regeneration using the same. More specifically, it relates to a composition for skin regeneration and anti-aging, including platelet-rich plasma and peripheral blood-derived mononuclear cells, and having an effect of increasing collagen synthesis. In addition, the present specification relates to a skin regeneration method using the composition. The composition for skin regeneration and anti-aging according to an aspect of the present invention can provide an excellent skin regeneration method by showing an effect of promoting collagen synthesis in the skin.

Description

Composition for skin regeneration and anti-aging comprising peripheral blood-derived mononuclear cells and platelet-rich plasma, and skin regeneration method using the same

The present specification relates to a composition for regenerating skin and preventing aging, and a skin regeneration method using the same. More specifically, it relates to a composition for skin regeneration and anti-aging, including a mixture of platelet-concentrated plasma and peripheral blood-derived mononuclear cells, which increases collagen synthesis and inhibits collagenase, an enzyme that degrades collagen. In addition, the present specification relates to a skin regeneration method using the composition.

Collagen, a major component of the extracellular matrix, is a major matrix protein produced in fibroblasts of the skin and is present in the extracellular stroma. In addition, it is an important protein that accounts for about 30% of the total weight of a biological protein and has a solid triple helix structure. Collagen forms most of the organic substances in skin, tendons, bones and teeth, especially in bones and skin (dermis). In most other sieve structures, they exist as fibrous inclusion bodies.

In the skin, various metabolic activities decrease due to the aging process, and the total activity of skin cells decreases. Therefore, due to such aging, changes in collagen production ability and various processes occurring after collagen transcription occur, leading to a decrease in collagen production. Therefore, one of the causes of skin wrinkles is a deficiency of skin collagen (collagen). Collagen and elastin give the skin elasticity and elasticity, and when they weaken with aging, the skin is easily damaged and old.

From a histological point of view, skin aging appears as a change in the composition of extracellular matrix proteins that form a matrix in the dermis of the skin. Among the extracellular tissues of the dermis, collagen proteins impart strength and tension to the skin, which protects the skin from external irritation or force, and occupies 90% of the dermal layer, so the decrease in collagen is closely related to skin aging and wrinkle formation. There is this.

In skin aging, an enzyme called collagenase (MMP, matrix metallopretease, matrix metallopretease) is involved. Synthesis and degradation of extracellular matrix such as collagen in vivo are properly regulated, but the synthesis decreases as aging progresses. The expression of collagenase (MMP), an enzyme that breaks down collagen, is promoted, resulting in decreased skin elasticity and wrinkle formation. Collagenase is a zinc-dependent intracellular proteolytic enzyme that can degrade or restructure components of the extracellular structure of the dermis. Among them, MMP-1 acts on collagen in the dermis to segment it. Therefore, the regulation of the amount and activity of this enzyme is an important factor in wrinkles.

The most representative skin anti-wrinkle substances are retinol and its derivatives, called retinoids, and are known to have good effects in various fields such as psoriasis, aging, cancer, and acne.In addition, when applied to the skin, these retinoids are collagenase ( It is known to prevent loss of collagen by inhibiting MMP-1) and to prevent and restore endogenous and photoaging by stimulating collagen formation. However, despite these effects, it is known to cause local skin irritation reactions such as erythema, itchiness, psoriasis, and scaling when applied to the skin. Therefore, there is a need to develop a new anti-wrinkle material that is safe for the living body, inhibits collagenase (MMP-1) and has better collagen synthesis ability than the existing material.

Platelet Rich Plasma (PRP) refers to platelet-rich plasma. When blood is drawn and separated using a centrifuge, it refers to the portion that contains the most abundant platelets at the bottom of the separated plasma. Platelet-concentrated plasma autologous skin regeneration is a procedure that was originally used for wound regeneration, and regenerates aged skin using one's own blood. Under the assumption that the platelets at the bottom of the separated plasma are rich in growth factors such as PDGF and FGF, it is assumed that it has the effect of promoting cell proliferation, collagen production, hyaluronic acid production, epithelial cell growth, angiogenesis, and wound healing. It is treated by making a fine hole using a skin roller and applying it. In addition, platelet-concentrated plasma can be used in various treatment and cosmetic fields, including implants and promotion of wound healing in sinus elevation, heart surgery, orthopedics, and dermatology (chronic wounds). Platelet-concentrated plasma, which specially treats one's blood so that it becomes platelet-rich plasma, is transparent, soft and elastic by regenerating elastic fibers such as collagen by activating stem cells in the skin by growth factors in the platelets in the blood. It restores condensation and helps aging progress slowly.

In International Publication No. 2005/065269, various compositions including platelet-enriched plasma and fibroblasts for skin treatment, in particular, platelet-enriched plasma, which is a dermatologically acceptable carrier for the skin to reduce the generation of wrinkles, for example. It is disclosed for the repeated administration of.

Mononuclear cells are produced in the bone marrow and released into the bloodstream, and newly formed mononuclear cells move to the peripheral blood, and circulating mononuclear cells attach to the epithelial cells of the peripheral blood vessels and can move into various tissues, and become immature dendritic cells or macrophages. Can be differentiated. Monocytes include peripheral blood-derived mononuclear cells, bone marrow-derived mononuclear cells, or fat-derived mononuclear cells, depending on the cell of origin. Peripheral blood mononuclear cells (PBMC) refer to cells with spherical nuclei present in blood. Mononuclear cells contain immune cells such as B cells, T cells, macrophages, dendritic cells, and natural killer cells, and recently utilize the functions of these immune cells. Therefore, studies are being conducted to use it as a therapeutic agent for the prevention and treatment of diseases. In fact, research on the development of cell therapy products using autologous peripheral blood cells to cure diseases such as cancer has been actively conducted, and the number of immune cell therapy products that have already been approved for the product is increasing. However, there is no known method for increasing collagen synthesis and improving skin regeneration using autologous peripheral blood mononuclear cells.

Skin treatment or cosmetic compositions containing platelet-concentrated plasma are widely known for their skin regeneration effect, but there is a new demand for a method of preventing skin regeneration and aging by dramatically increasing collagen synthesis. Research to develop a containing composition is ongoing.

Under this background, the present inventors have found that a mixed composition containing both platelet-rich plasma (PRP) and peripheral blood-derived mononuclear cells (PBMC) enhances collagen synthesis and has skin regeneration and anti-aging effects, and has completed the present invention. .

In one aspect, it is an object of the present invention to provide a skin regeneration and anti-aging composition superior in collagen synthesis effect than a conventional composition containing platelet-concentrated plasma. It is also intended to provide a skin regeneration method using this.

In one aspect, the present invention provides a composition for skin regeneration and anti-aging, characterized in that it comprises peripheral blood-derived mononuclear cells (PBMC, Peripheral Blood Mononuclear Cell).

In another aspect, the present invention provides a composition for skin regeneration and anti-aging, further comprising platelet-enriched plasma together with peripheral blood-derived mononuclear cells.

In another aspect, the present invention provides the use of peripheral blood-derived mononuclear cells, platelet-concentrated plasma, or a combination thereof for use in the preparation of a composition for skin regeneration and anti-aging.

In another aspect, the present invention provides peripheral blood-derived mononuclear cells, platelet-enriched plasma, or a combination thereof for skin regeneration and anti-aging.

Further, in another aspect, the present invention provides a non-therapeutic cosmetic use of peripheral blood-derived mononuclear cells, platelet-concentrated plasma, or a combination thereof as an active ingredient for skin regeneration and anti-aging.

In the composition according to an aspect of the present invention, the mononuclear cells derived from human peripheral blood may be extracted from whole blood.

In the composition according to an aspect of the present invention, the peripheral blood-derived mononuclear cells may be characterized in that the autologous cells.

In the composition according to an aspect of the present invention, the platelet-rich plasma may be characterized in that it is derived from autologous blood.

In the composition according to an aspect of the present invention, the platelet-concentrated plasma is concentrated at least 5 times, and the platelet concentration is 1x10 ⁶ /μl to 5x10 ⁶ /μl.

In the composition according to an aspect of the present invention, the composition may be characterized in that it increases the synthesis of collagen or inhibits collagenase activity.

In another aspect, the present invention provides a skin regeneration method comprising the step of applying the composition to the skin.

In the skin regeneration method according to an aspect of the present invention, the step of applying to the skin may include at least one of applying to the skin, fine drilling, and applying heat.

In another aspect, the present invention comprises the steps of separating mononuclear cells derived from human peripheral blood through a density gradient centrifugation method, washing with a phosphate buffer solution to separate peripheral blood-derived mononuclear cells (PBMC); Whole blood is centrifuged to recover the plasma component, the supernatant, the recovered supernatant is centrifuged to recover the platelet-deficient plasma (PPP, Platelet Poor Plasma) portion, and the remaining portion is resuspended to obtain concentrated platelet-concentrated plasma (PRP). Manufacturing steps; And it provides a method for producing a composition for skin regeneration and anti-aging comprising the step of suspending the peripheral blood-derived mononuclear cells in platelet-concentrated plasma.

In the manufacturing method according to an aspect of the present invention, the platelet-enriched plasma may be characterized in that it has a platelet concentration that is 5 times or more concentrated compared to a normal platelet concentration.

The composition comprising platelet-enriched plasma and peripheral blood-derived mononuclear cells according to the present specification is expected to provide an excellent method for skin regeneration by enhancing collagen synthesis and inhibiting collagen-degrading enzymes.

1 is a measurement of the collagen synthesis degree of mononuclear cells derived from peripheral blood suspended in a phosphate buffer solution mixed with human fibroblasts in a ratio of 1:10, 1:50, and 1:100 to the number of human fibroblasts. This is a graph showing the results.
2 is a mixture of peripheral blood-derived mononuclear cells suspended in platelet-enriched plasma with human fibroblasts at a ratio of 1:10, 1:50, and 1:100 to the number of human fibroblasts, and platelet-enriched plasma and human fibers. It is a graph showing the result of measuring the degree of collagen synthesis by comparing blast cells mixed culture.
3 is a collagenase (MMP-) obtained by mixing and culturing peripheral blood-derived mononuclear cells suspended in platelet-enriched plasma with human fibroblasts at a ratio of 1:10, 1:50, and 1:100 to the number of human fibroblasts. 1) A graph showing the results of measuring the degree of inhibition of activity.

The present specification provides a composition comprising mononuclear cells, for example, mononuclear cells derived from peripheral blood, and further comprising platelet rich plasma (PRP). The composition of the present specification is useful for skin regeneration and anti-aging. Do. As the mononuclear cells of the present specification, mononuclear cells derived from peripheral blood may be preferably used.

In addition, the present specification comprises the steps of separating mononuclear cells derived from human peripheral blood through a density gradient centrifugation method, and separating mononuclear cells (PBMC) derived from peripheral blood by washing with a phosphate buffer solution; Whole blood is centrifuged to recover the plasma component, the supernatant, the recovered supernatant is centrifuged to recover the platelet-deficient plasma (PPP, Platelet Poor Plasma) portion, and the remaining portion is resuspended to obtain concentrated platelet-concentrated plasma (PRP). Manufacturing steps; And it provides a method for producing a composition for skin regeneration and anti-aging comprising the step of suspending the peripheral blood-derived mononuclear cells in platelet-concentrated plasma. Preferably, the platelet-enriched plasma is characterized by having a platelet concentration that is 5 times or more concentrated compared to the normal platelet concentration.

Monocytes mediate acute and chronic inflammation, promote the repair of damaged tissues by phagocytosis and fibrin degradation, induce angiogenesis (blood vessel formation), and regulate extracellular matrix production. In the composition according to an aspect of the present invention, mononuclear cells derived from bone marrow, fat derived or peripheral blood may be used, and preferably include mononuclear cells derived from peripheral blood. The composition according to an aspect of the present invention dramatically increases collagen synthesis, thereby exhibiting effects of skin regeneration and anti-aging.

Platelet rich plasma (PRP) is a platelet concentrate and contains platelets at a higher level than the platelet concentration normally found in blood. For example, the platelet concentration is 2 times, 5 times, 10 times or 100 times or more than the concentration in normal blood. Preferably, platelet-enriched plasma contained in the composition according to an aspect of the present invention is concentrated by at least 5 times. Platelet-enriched plasma contains elements other than platelets, such as plasma, growth factors, and white blood cells. Platelet-rich plasma can be obtained from a variety of animals, preferably from humans. Platelet-enriched plasma can be obtained from autologous or allogenic, but is preferably obtained from autologous.

According to an aspect of the present invention, the dosage of peripheral blood-derived mononuclear cells and platelet-enriched plasma is based on the number of cells, the number of fibroblasts in the skin to be administered: the ratio of the number of cells in the peripheral blood-derived mononuclear cells and platelet-enriched plasma Range is 1:8 or more, 1:10 or more, 1:15 or more, 1:20 or more, 1:30 or more, 1:40 or more, 1:50 or more, 1:70 or more, 1:80 or more, 1:100 Or more, or 1:110 or more. In another aspect, the ratio range is 1:120 or less, 1:110 or less, 1:100 or less, 1:80 or less, 1:70 or less, 1:50 or less, 1:40 or less, 1:30 or less, 1:20 Hereinafter, it may be 1:15 or less, or 1:10 or less. Preferably, the ratio range may be 1:10 to 1:100.

The composition according to the present invention may be a pharmaceutical composition, a cosmetic composition, a food composition, and the like. In addition, the composition according to an aspect of the present invention can be applied to all animals, including humans, dogs, chickens, pigs, cows, sheep, guinea pigs or monkeys.

The composition according to an aspect of the present invention may be administered orally, rectal, transdermal, intradermal, intravenous, intramuscular, intraperitoneal, intramedullary, intrathecal or subcutaneous.

Formulations for oral administration may be tablets, pills, soft or hard capsules, granules, powders, liquids, or emulsions, but are not limited thereto. Formulations for parenteral administration may be injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays, but are not limited thereto.

The pharmaceutical composition according to an aspect of the present invention may include additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavors or sweeteners, if necessary. The pharmaceutical composition according to an aspect of the present invention may be prepared by a conventional method in the art.

The active ingredient of the pharmaceutical composition according to an aspect of the present invention will vary depending on the age, sex, weight, pathological condition and severity of the subject to be administered, the route of administration, or the judgment of the prescriber. Determination of the applied amount based on these factors is within the level of those skilled in the art, and its daily dose is, for example, 0.01 µg/kg/day to 10 g/kg/day, specifically 0.1 µg/kg/day to 1 g/kg/day. , More specifically, it may be from 0.5 μg/kg/day to 100 mg/kg/day, but if there is a difference in effect according to the dose, it can be appropriately adjusted. The pharmaceutical composition according to an aspect of the present invention may be administered once to three times a day, but is not limited thereto.

The health functional food composition according to an aspect of the present invention may be prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candy, syrup, and beverages, but is not limited thereto. When using the health functional food composition according to an aspect of the present invention as a food additive, the health functional food composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. have. Examples of foods to which the health functional food composition can be added include meat, bread, candies, snacks, noodles, dairy products, vitamin complexes, beverages, tea, drinks, etc., but are not limited thereto, and in the usual sense Includes all health foods. In addition, it may include ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients and seasoning agents.

The cosmetic composition according to an aspect of the present invention is not particularly limited in its formulation, and softening lotion, astringent lotion, nutritional lotion, eye cream, nutrition cream, massage cream, cleansing cream, cleansing foam, cleansing water, powder, essence , May have a formulation such as a pack.

The cosmetic composition according to an aspect of the present invention may be prepared in various forms according to a conventional cosmetic preparation method. For example, the cosmetic composition may be prepared in the form of a cosmetic product, lotion, cream, lotion, etc. containing the peripheral blood-derived mononuclear cells and/or platelet-concentrated plasma, which is a conventional cleansing solution, astringent solution, and moisturizing solution. It can be diluted and used. In addition, the cosmetic composition may contain conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the cosmetic composition field.

The formulation of the composition according to an aspect of the present invention is not particularly limited, but may be formulated into, for example, tablets, granules, powders, liquids, solid preparations, injections, and the like. In addition to the active ingredient, each formulation may be appropriately selected and blended by a person skilled in the art according to the formulation or purpose of use without difficulty, and synergistic effects may occur when applied simultaneously with other ingredients.

The composition according to the present invention preferably contains at least 0.1%, at most about 20%, preferably at most about 5% and more preferably at most 1% (w/w) of peripheral blood-derived mononuclear cells and/or platelet enriched plasma. Contains. The choice of an appropriate concentration depends on factors such as, for example, the preferred dosage, frequency and method of delivery of the active ingredient.

In the present specification, various transformations may be applied and various embodiments may be provided. Hereinafter, an aspect of the present invention will be described in more detail. However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing an aspect of the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

The terms used in the present specification are intended only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The term in which the number is omitted before the noun is not intended to limit the quantity, but rather indicates the existence of one or more of the mentioned noun items. The terms “comprising”, “having”, and “including” are interpreted as open terms (ie, meaning “including but not limited to”).

Mentioning a range of values is simply because it is an easy way instead of referring individually to each individual number falling within that range, and each individual number, unless otherwise stated, is as if individually stated in the specification. Is incorporated herein as such. End values of all ranges are included within that range and can be combined independently.

All methods mentioned herein may be performed in any suitable order unless otherwise specified or clearly contradicted by context. The use of any one and all embodiments or exemplary language (e.g., "such as") is only intended to better describe the invention and, unless included in the claims, is intended to limit the scope of the invention. I am not trying to limit it. No language in the specification is to be construed as essential to the practice of the present invention with any unclaimed element. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs.

Preferred embodiments of the invention include the most optimal mode known to the inventor for carrying out the invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the preceding description. The inventors expect those skilled in the art to make appropriate use of such variations, and the inventors expect the invention to be practiced in a manner different from that described herein. Accordingly, the present invention includes equivalents and all variations of the subject matter of the invention mentioned in the appended claims, as permitted by the patent law. Moreover, within all possible variations, any combination of the above-mentioned components is included in the present invention unless otherwise indicated herein or clearly contradicted by context. While the present invention has been specifically shown and described with reference to exemplary embodiments, those skilled in the art will appreciate that various changes may be made in form and detail without departing from the spirit and scope of the invention as defined by the following claims.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples and experimental examples. However, the following examples and experimental examples are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1]

Materials and methods of experiment

Fibroblast culture

Human fibroblasts (CCD-986sk) obtained from ATCC were added to DMEM/High modified (Hyclone) medium with 10% FBS (Gibco), 1% Penicillin streptomycin (Gibco), and 1 mM Sodium Pyruvate (Gibco). After culturing under conditions of ℃, 5% CO ₂ and passaged 1:2 or 1:3 using Trypsin-EDTA solution (Gibco), the 3rd to 5th generation cells were used in the experiment.

Isolation of mononuclear cells derived from peripheral blood

Human peripheral blood mononuclear cells (hPBMC) were extracted from 10 to 100 cc of whole blood. Mononuclear cells derived from human peripheral blood were isolated through density gradient centrifugation using reagents Lymphoprep or Ficoll-Paque, washed twice with phosphate buffered saline (PBS), and used for experiments.

Platelet-rich plasma (PRP, Platelet Rich Plasma) manufacturing

10 ~ 100 cc of whole blood is centrifuged at a rate of 150g ~ 200g for 5 ~ 10 minutes to recover the plasma component, the supernatant. The recovered supernatant is centrifuged at a rate of 800g to 1000g for 10 to 15 minutes to recover about 1/3 of the platelet-deficient plasma (PPP, Platelet Poor Plasma), and resuspend the remaining 2/3 to make platelet-concentrated plasma. It was prepared and used in the experiment. The normal platelet concentration in blood is 1.5 ~ 4 x 10 ⁵ /µl, and platelet-concentrated plasma is more than 5 times concentrated. In this experiment, 1 ~ 5 x 10 ⁶ /µl was used.

[Example 2]

Each experiment was conducted to find out that collagen synthesis in fibroblasts was increased when platelet-enriched plasma was treated together rather than when only peripheral blood-derived mononuclear cells were treated.

Human fibroblasts were placed in 6 x 10 ⁴ /well in a 6-well plate and cultured for 16 to 18 hours. The culture medium was removed and washed twice with phosphate buffered saline (PBS). Human peripheral blood-derived mononuclear cells were suspended in phosphate buffered solution or platelet-concentrated plasma (PRP), and PBS+PBMC or PRP+PBMC mixture was added to human fibroblasts cultured in a 6-well plate 1:10, 1:50, The mixture was cultured for 24 hours at a ratio of 1:100 [number of cells]. The culture medium secreted by human fibroblasts was collected and the amount of collagen was measured using the ELISA (Procollagen type I peptide EIA kit, Takara) method. For ELISA, put 100 µl of Antibody-POD conjugated solution into a 96-well and incubate at 37°C for 3 hours. After removing the culture medium from the well and washing 3 times with a phosphate buffer solution, 100 µl of a color developing reagent (TMBZ) was added, incubated for 15 minutes at room temperature, and 100 µl of 1N sulfuric acid was added, followed by measurement with an ELISA reader at 450 nm.

Peripheral blood-derived mononuclear cells suspended in a phosphate buffer solution were mixed and cultured with human fibroblasts cultured in 6-well plates at a cell number ratio of 1:10, 1:50, and 1:100. As the mononuclear cell concentration increased (dose-dependent), collagen synthesis of human fibroblasts increased to 8%, 44%, and 60%, respectively (see FIG. 1).

As a result of mixing cultured group of mononuclear cells derived from peripheral blood suspended in platelet-enriched plasma with human fibroblasts at a cell number ratio of 1:10, 1:50, and 1:100, platelet enriched compared to the platelet-enriched plasma alone treatment group. The plasma and peripheral blood-derived mononuclear cell mixture treatment group further induced an increase in collagen synthesis in human fibroblasts by 22%, 33%, and 46%, respectively. In addition, the increase in collagen synthesis was increased (dose-dependent) as the concentration of mononuclear cells derived from peripheral blood in the mixed treatment group increased (see FIG. 2).

Through the above experimental results, it can be seen that the composition according to an aspect of the present invention exhibits an activity of enhancing collagen synthesis in fibroblasts. Through this, it can be seen that the composition according to an aspect of the present invention can have skin regeneration and anti-aging effects through collagen synthesis.

[Example 3]

Inhibition of collagenase (matrix metalloproteinase-1, MMP-1) activity by peripheral blood-derived mononuclear cells and platelet-enriched plasma

In order to find out whether a mixture of peripheral blood-derived mononuclear cells and platelet-concentrated plasma has an effect of inhibiting the activity of collagenase (MMP-1), which is known to reduce collagen concentration by decomposing collagen, an experiment was conducted.

Human fibroblasts were placed in 6 x 10 ⁴ /well in a 6-well plate and cultured for 16 to 18 hours. The culture medium was removed and washed twice with phosphate buffered saline (PBS). Human peripheral blood-derived mononuclear cells were suspended in phosphate buffered solution or platelet-concentrated plasma (PRP), and PBS+PBMC or PRP+PBMC mixture was added to human fibroblasts cultured in a 6-well plate 1:10, 1:50, The mixture was cultured for 24 hours at a ratio of 1:100 cells. The culture medium secreted by human fibroblasts was collected and the amount of collagenase was measured using an ELISA (Human MMP-1 ELISA kit, RayBiotech) method. The ELISA method is to add 100 µl of each 1/2 diluted culture solution and standard solution to a 96-well coated with Antibody-Human MMP-1, react at room temperature for 2.5 hours, and wash 4 times with 400 µl of washing buffer. 100 µl of Biotinylated anti-Human MMP-1 was added to a 96-well, reacted at room temperature for 1 hour, and washed 4 times with 400 µl of washing buffer. After that, 100 µl of HRP-Streptavidin solution was added to a 96-well, reacted for 45 minutes, and washed 4 times with 400 µl of washing buffer. Finally, 100 µl of a color developing reagent (TMB) was added, incubated for 30 minutes at room temperature, and 50 µl of 0.2N sulfuric acid was added, and then measured at 450 nm with an ELISA reader.

A mixture of mononuclear cells derived from peripheral blood suspended with platelet-enriched plasma was mixed and cultured with human fibroblasts cultured in a 6-well plate at a ratio of 1:10, 1:50, and 1:100. As a result, platelet-enriched plasma alone was treated. It was found that the platelet-enriched plasma and peripheral blood-derived mononuclear cells mixed treatment group inhibited the activity of collagenase (MMP-1) in human fibroblasts by 2.7%, 4.5%, and 4.8%, respectively (see FIG. 3). .

Through the above experimental results, it can be seen that the composition according to an aspect of the present invention exhibits an activity that inhibits the activity of collagenase (MMP-1) that disrupts tissue in fibroblasts. Through this, it can be seen that the composition according to an aspect of the present invention can have a skin tissue regeneration effect and an anti-aging effect.

When the results of the above experimental examples are summarized, it can be seen that the composition comprising platelet-enriched plasma and peripheral blood-derived mononuclear cells according to an aspect of the present invention has an effect of enhancing collagen synthesis and inhibiting collagenase activity. It is believed that it can be used to bring about the effect of skin tissue regeneration and anti-aging, thereby enabling the development of skin regeneration methods and skin regeneration drugs or cosmetics.

Claims (16)

As an active ingredient, a composition for skin regeneration or skin aging improvement, comprising a peripheral blood-derived mononuclear cell (PBMC, Peripheral Blood Mononuclear Cell) and platelet-rich plasma (PRP, Platelet Rich Plasma),
The platelet-concentrated plasma was concentrated by centrifuging whole blood to recover a plasma component, a supernatant, and centrifuging the recovered supernatant to recover a platelet-deficient plasma (PPP, Platelet Poor Plasma) portion, and resuspend the remaining portion to be concentrated,
The skin aging improvement is skin elasticity improvement or skin wrinkle improvement, skin regeneration or skin aging improvement composition. The composition for skin regeneration or skin aging according to claim 1, wherein the peripheral blood-derived mononuclear cells are human peripheral blood-derived mononuclear cells. The composition for skin regeneration or skin aging according to claim 2, wherein the mononuclear cells derived from human peripheral blood are extracted from whole blood. The composition for skin regeneration or skin aging improvement according to claim 2, wherein the mononuclear cells derived from human peripheral blood are autologous cells. The composition for skin regeneration or skin aging according to claim 1, wherein the platelet-rich plasma is derived from autologous blood. The method of claim 1, wherein the dose of the peripheral blood-derived mononuclear cells and platelet-enriched plasma is based on the number of cells, the number of fibroblasts of the skin to be administered: the ratio of the number of cells of the peripheral blood-derived mononuclear cells and platelet-enriched plasma A 1: 8 to 1: 120 is the amount of, skin regeneration or skin aging improvement composition. The composition for skin regeneration or skin aging improvement according to claim 1, wherein the platelet-concentrated plasma is concentrated at least 5 times to have a platelet concentration of 1x10 ⁶ /μl to 5x10 ⁶ /μl. The composition for skin regeneration or skin aging according to claim 1, wherein the composition increases the synthesis of collagen or inhibits collagenase activity. The composition of claim 1, wherein the composition is transdermally, intradermally, subcutaneously, intramuscularly or vascularly administered. The composition for skin regeneration or skin aging improvement according to any one of claims 1 to 9, wherein the composition is applied to the skin through at least one of application to the skin, microperforation, and thermal application. As a method for producing a composition for skin regeneration or skin aging improvement according to any one of claims 1 to 9,
The above method,
(a) separating mononuclear cells derived from human peripheral blood through a density gradient centrifugation method, washing with a phosphate buffer solution to separate peripheral blood-derived mononuclear cells (PBMC);
(b) Whole blood is centrifuged to recover the plasma component, the supernatant, and the recovered supernatant is centrifuged to recover the platelet-deficient plasma (PPP) portion, and the remaining portion is resuspended to concentrate platelet-concentrated plasma ( Preparing PRP); And
(c) suspending peripheral blood-derived mononuclear cells in platelet-enriched plasma
Including,
The skin aging improvement is skin elasticity improvement or skin wrinkle improvement, a method of preparing a composition for skin regeneration or skin aging improvement. 12. The method of claim 11, wherein the platelet-enriched plasma has a platelet concentration that is 5 times or more concentrated than a normal platelet concentration. [10] The composition according to any one of claims 1 to 9, wherein the composition is a cosmetic composition.
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