KR101933497B1 - Composition for inhibiting cellular senescenece or improving skin aging - Google Patents

Abstract

A composition for inhibiting cell senescence, a composition for improving skin aging, and a cosmetic composition for improving skin aging, which contain SPRR1A protein as an active ingredient. The composition of the present invention containing SPRR1A protein as an active ingredient has an effect of controlling the expression of aging-related factors. Accordingly, a composition comprising the SPRR1A protein can be used as the various compositions provided in the present invention.

Description

Technical Field [0001] The present invention relates to a composition for inhibiting cell senescence or for improving skin aging,

The present invention relates to a composition for inhibiting cell senescence or for improving skin aging.

Aging refers to all the physiological changes of the body that occur over time. It is a life phenomenon that occurs in a wide variety of ways depending on individuals. Aging varies in appearance and speed from one individual to another, and may vary from one organization to another.

On the other hand, when the aging phenomenon is specifically examined, the functional changes of the constituent organs and tissues occur, which can be attributed to a change in function of the cells constituting the individual, that is, cell senescence.

Another cause of cell senescence is active oxygen. The active oxygen generated during metabolism is highly reactive and can aggravate cells by irreversibly destroying cellular components such as lipids, proteins, sugars, or DNA, thereby causing oxidative stress of cells or tissues. When this oxidative stress is continuously exposed, radicals are increased in the body, collagen, elastin, and hyaluronic acid, which are connective tissues, are destroyed, ) Can happen. Furthermore, the increase of the radical oxidizes the lipid part of the cell membrane, thereby destroying the cell, which may cause diseases such as dermatitis, acne or skin cancer.

Accordingly, scorch acid,? -Tocopherol or SOD, which is known as a free radical scavenging function substance, is added to cosmetics or medicines in order to prevent aging of cells and diseases associated with skin aging. However, they are not only expensive, but also have poor chemical stability at the time of blending, so that it is difficult to expect a substantial effect. For this reason, the development of safer and more effective substances for suppressing aging remains an important issue to be solved in the fields of medicine, food, and cosmetics.

BACKGROUND OF THE INVENTION [0002] Techniques as a background of the invention have been made in order to facilitate understanding of the present invention. And should not be construed as an admission that the matters described in the technical background of the invention are present in the prior art.

Depending on the progression of cell senescence, specific genes may show differences in expression. Specifically, specific genes in aged cells may appear at lower expression levels in non-aging cells.

The inventors of the present invention have been able to recognize that the expression level of SPRR1A gene among specific genes is decreased in senescent cells. Thus, the inventors of the present invention have recognized the SPRR1A gene as a gene associated with aging of cells, furthermore, skin cell senescence, and studied the role of the SPRR1A gene.

As a result, the inventors of the present invention found that the SPRR1A gene and the SPRR1A protein regulate the factors associated with aging, and that it is possible to inhibit cellular senescence and improve skin aging by controlling the expression of factors associated with aging Respectively.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a composition for inhibiting cell senescence and a composition for improving skin aging, which contains SPRR1A protein as an active ingredient, which can regulate the expression of factors associated with aging.

Another object of the present invention is to provide a cosmetic composition for improving skin aging comprising SPRR1A protein or a specific amino acid sequence thereof as an active ingredient.

Another object to be solved by the present invention is to provide a composition for inhibiting cell senescence, which comprises transforming a recombinant vector having a gene having a specific nucleotide sequence of the SPRR1A gene and isolating and purifying the translated protein therefrom, And to provide a method for producing the composition for inhibition.

Yet another object of the present invention is to provide a method for screening a candidate compound for inhibiting cell senescence by measuring the expression level of SPRR1A gene or the level of SPRR1A protein in a cell, .

The problems of the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, there is provided a composition for inhibiting cell senescence comprising SPRR1A protein capable of regulating the expression of an aging-related factor.

As used herein, the term "aging " means any physiological change of the body that occurs over time. The cause of aging can be caused by oxidizing action by active oxygen species such as superoxide, hydrogen peroxide, hydroxyl group and singlet oxygen, disappearance of telomere, damage of gene and the like. Accordingly, it may be important not only to prevent aging based on the cause of the aging revealed, but also to suppress the progress of aging.

More specifically, the aging herein can mean cell senescence. Cell senescence is a phenomenon in which normal somatic cells can no longer divide after a certain number of divisions, which can contribute to the aging of individuals and tissues, and may be associated with abnormal proliferation of cells and a mechanism of inhibiting cancer formation. Cell senescence is caused by the disappearance of telomere at the end of the chromosome, increased activity of cancer genes or cancer suppressor genes, excessive oxidative stress, cytotoxic substances such as ultraviolet rays or radiation, anticancer drugs, and inflammatory reactions due to repeated division of somatic cells .

Furthermore, cell senescence not only contributes to the aging of individuals and tissues, but can also be a pathogen of a variety of different diseases. Aging cells can be observed in inflammatory lesions such as rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin injured tissue, arteriosclerotic vascular tissue, and the like. Cell senescence may also be observed in proliferative hyperplasia, hepatitis, and liver cancer. Thus, aged cells have poor cell division ability. Therefore, when aged cells accumulate, damaged tissues are not easily restored, but also secrete enzymes or inflammatory cytokines that degrade surrounding tissues and accelerate tissue damage . As a result, cell senescence can contribute to the pathogenesis of aging-related diseases.

Accordingly, development of a method for delaying cell senescence, which may cause various diseases, and further development of a substance inhibiting cell senescence may be important.

As used herein, the term "aging-associated factor" may refer to factors that are present at various levels within the cell or exhibit a change in activity, depending on the progress of cell senescence. For example, senescence-associated factors may be cell growth inhibitory proteins including SA-β-gal (senescence-associated β-galactosidase) and p53, p16 and p21, insulin-like growth factor binding proteins (IGFBPs) There may be inflammatory proteins including IL-6 (interleukin-6), IL-1 alpha (Interleukin 1 alpha), TGF-beta (transforming growth factor-beta) and interferon. Specifically, as aging progresses, the activity of SA-β-gal may increase in cells, and protein levels of p53, p16 and p21, IGFBPs, IL-6, IL-1α, TGF-β and interferon are high . Furthermore, the aging-associated factors may further comprise an aging-associated gene that encodes the aforementioned protein.

As used herein, the term "expression" may refer to the expression of a gene, and furthermore the formation of a protein. For example, the expression of an aging-related factor may be the generation of transcripts comprising mRNA by transcription of the aging-associated genes described above, or the generation of aging-related proteins by translation of an aging-associated gene.

As used herein, the term "SPRR1A protein" is one of the proline rich proline amino acid rich proline-rich proteins that make up the protein. The level of the SPRR1A protein or the expression level of the SPRR1A gene may decrease as the aging of the cells progresses. In particular, the level of SPRR1A protein or the expression level of the SPRR1A gene in senescent skin cells can be reduced compared to non-aged skin cells. The SPRR1A protein can modulate protein levels or expression levels of aging-related factors. For example, SA-β-gal, p53, p16 and p21, IGFBPs, IL-6, IL-1α, TGF-β and the like in senescent cells treated with the SPRR1A protein or overexpressing the SPRR1A gene The level of expression of the interferon gene or the level of these proteins may be less than in otherwise senescent cells. Furthermore, the level of senescence-associated factors in senescent cells treated with the SPRR1A protein or in the senescent cells over-expressing the SPRR1A gene may be lower than in normal, undigested cells. That is, treatment of SPRR1A protein or overexpression of SPRR1A gene may have an effect of inhibiting cell senescence and further improving skin aging. Accordingly, the SPRR1A protein can be used as a composition for inhibiting cell senescence and a composition for improving skin aging.

Furthermore, the SPRR1A protein comprises all SPRR1A proteins from yeast, plant or animal, preferably an animal, more preferably a human-derived SPRR1A protein.

According to one embodiment of the present invention, the SPRR1A protein may have a weight of 0.00001 wt.% To 10 wt.%, Based on the total composition weight. However, in the composition of the present invention, the effective amount of the SPRR1A protein is not particularly limited and can be variously determined depending on the degree of senescence of the cells.

Furthermore, the effective amount and effective dosage of the whole composition in various embodiments of the present invention may be varied depending on the formulation method, administration method, administration time and / or route of administration of the composition, The type and severity of the response, the type of subject being treated, the age, body weight, general health status, severity or severity of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously with the subject, , And the like, and those skilled in the art will readily determine and prescribe dosages that are effective in improving the desired aging.

According to one embodiment of the present invention, there is provided a composition for inhibiting cell senescence comprising a polypeptide having 70% or more homology with the amino acid sequence of SEQ ID NO: 1.

A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and further, a protein formed by folding the polypeptide may have an activity of regulating the expression of an aging-related factor possessed by SPRR1A protein. Accordingly, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and furthermore, the protein formed by folding the polypeptide may have a cell aging-inhibiting activity, and further, a skin aging-improving activity. In various embodiments of the present invention, . ≪ / RTI >

As used herein, the term "homology" may refer to a wild-type base sequence or similarity with an amino acid sequence. Accordingly, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of 70% or more identical thereto or the protein formed by the folding of the polypeptide substantially retains the cell aging-inhibiting activity and further the cell aging-improving activity . The polypeptide or protein having high homology with SEQ ID NO: 1 may have a higher cytotoxic activity. Accordingly, the composition of the present invention may include a polypeptide consisting of an amino acid sequence having an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO: 1, preferably 90% or more.

Further, a polypeptide or protein having such activity may comprise the amino acid sequence of SEQ ID NO: 1 with addition, deletion, substitution, or a combination thereof. Specifically, if the polypeptide or protein is substantially a polypeptide or protein having biological activity such as deletion mutation in the amino acid sequence of SEQ ID NO: 1, deletion or addition such as addition of functional sequence and swapping, or cell aging inhibitory activity or skin aging improving activity, ≪ / RTI >

According to one embodiment of the present invention, there is provided a composition for inhibiting cell senescence comprising a peptide having 70% or more homology with the amino acid sequence of SEQ ID NO: 2.

The polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, and further, the protein formed by folding the polypeptide may have an activity of regulating the expression of an aging-related factor possessed by the SPRR1A protein. Accordingly, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, and furthermore, the protein formed by folding the polypeptide may have a cell aging-inhibiting activity, and further, a skin aging-improving activity. In various embodiments of the present invention, . ≪ / RTI >

In addition, the polypeptide consisting of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence having 70% or more identical thereto, or the protein formed by folding of the polypeptide may be regarded as having substantially the cell aging-inhibiting activity and further the cell aging-improving activity. The polypeptide or protein having high homology with SEQ ID NO: 2 may have a higher cytotoxic activity. Accordingly, the composition of the present invention may include a polypeptide consisting of an amino acid sequence having an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO: 2, preferably 90% or more.

Furthermore, a polypeptide or protein having such activity may comprise the amino acid sequence of SEQ ID NO: 2 with addition, deletion, substitution, or a combination thereof. Specifically, if a polypeptide or protein has substantially the biological activity of inhibiting cell senescence or improving skin aging by deletion or addition such as deletion mutation, addition of functional sequence and swapping in amino acid of SEQ ID NO: 2, ≪ / RTI >

According to one embodiment of the present invention, there is provided a composition for inhibiting cell senescence, which comprises a step of treating candidate cells for cell senescence-suppressing composition and a step of measuring the expression level of SPRR1A gene or the level of SPRR1A protein in a target cell A screening method is provided.

Here, the SPRR1A protein can be used not only in a composition exhibiting various activities in various embodiments of the present invention, but also in a method for screening compositions for inhibiting cell senescence.

As used herein, the term "screening" may mean drug screening, and may include compositions for inhibiting cell senescence or compositions for inhibiting cell senescence, which have an effect of increasing the expression level of the SPRR1A gene or the level of SPRR1A protein, But is not limited to, searching for a candidate for a composition for improving aging.

As used herein, the term "subject cell" may be a skin cell or a stem cell, but not limited thereto, and all the cells in which aging may occur are target cells.

The expression level of the SPRR1A gene can be measured by reverse transcription-PCR, competitive reverse transcription-PCR, real-time reverse transcription-PCR, RNase protection analysis , RNase protection assay, Northern blotting, and DNA chip method, but the present invention is not limited thereto.

Measurement of the level of SPRR1A protein can be performed by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACSs), and protein chip methods can be used for immunoassays, But is not limited thereto.

According to one embodiment of the present invention, the measuring step may further comprise determining the candidate substance as a substance for improving skin senescence, when the level of expression of the SPRR1A gene or the level of the SPRR1A protein in the subject cell is equal to or higher than a predetermined level .

Furthermore, when the level of expression of the SPRR1A gene or the level of the SPRR1A protein in the subject cell after treatment with the aging-inhibiting composition candidate is higher than the level before the treatment, the candidate substance is a cell senescence-inhibiting substance, . ≪ / RTI >

The screening method according to an embodiment of the present invention may further include measuring an expression level or a protein level of IL-1 alpha, IL-6, P16 and P21 gene in the target cells.

However, without being limited thereto, in the measuring step, expression levels or protein levels of various aging-related factors including p53, IGFBPs, TGF-beta and interferon can be measured.

At this time, when the level of expression of IL-6, P16 and P21 gene or the level of protein in the subject cell after treatment with the candidate substance for anti-aging composition is lower than the level before the treatment, the candidate substance has an activity of inhibiting cell senescence, It may be a substance having cell aging improving activity.

The subject cell according to an embodiment of the present invention may be an endogenous senescence or photoaged cell over time.

As used herein, the term "endogenous aging" refers to aging that occurs over time, regardless of particular environmental factors.

As used herein, the term "photo aging" refers to aging due to environmental factors such as ultraviolet light.

Further, the subject cell in the present invention may be a cell aged by smoking, drug administration, or menopause.

According to an embodiment of the present invention, a gene having a base sequence having 70% or more homology with the nucleotide sequence of SEQ ID NO: 3 or a gene having a nucleotide sequence having 70% or more homology with the nucleotide sequence of SEQ ID NO: 4 Transforming the recombinant vector with a host cell, culturing the transformed host cell to obtain a translated protein from the nucleotide sequence, and separating and purifying the protein. / RTI >

Specifically, the gene having the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 can encode a polypeptide having a cell aging-inhibiting activity, further, a skin aging-improving activity. Furthermore, a gene having a homology of 70% or more with the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 can also substantially encode a polypeptide having cytoprotective activity, furthermore, skin aging improving activity.

As used herein, the term "vector" refers to a DNA sequence operably linked to a regulatory sequence capable of regulating expression of the protein in a suitable host cell, and sequences introduced to facilitate other gene manipulations or to optimize the expression of the protein ≪ / RTI > The regulatory sequence may include a promoter to control transcription, an operator optionally added to regulate transcription, an appropriate mRNA ribosome binding site, and sequences to control transcription and termination of translation.

Specifically, a vector is a vector in which the promoter is a CAT promoter, and may include a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a virus vector such as an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector. In addition, the vector may be in a form that is integrated into the genome of the host cell.

As used herein, the term "recombinant" can be a genetic recombination, which may mean splicing genomic DNA isolated from a heterologous organism or synthesized gene. For example, a gene consisting of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a gene having 70% or more homology with these nucleotides can be recombined into a vector.

As used herein, the term "transformation" may mean that the genetic properties of an organism are altered by importing DNA from the outside. Transformation methods may include, but are not limited to, use of ribosomes, electroporation, chemicals that increase the uptake of the DNA, direct DNA injection into the host cell, particle gun impact, and micro- Can be used as a transformation method. For example, a gene comprising a nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a gene having 70% or more homology with these nucleotides may be transformed into a host cell.

As used herein, the term "host cell" may refer to a cell or plasmid used for viral infection, a vector such as phage DNA, and a bacterium used for propagation of the insertion gene.

The transformable host cell may be a prokaryotic host cell such as Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. Cells, but are not limited thereto. It is also possible to use fungi (for example Aspergillus), yeast (for example, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora crassa) and the like can be used as host cells. In addition, cells derived from higher eukaryotes, including insect cells, plant cells, mammals and the like, can be used as host cells. However, a variety of cells can be used as host cells without being limited thereto.

As used herein, the term "translation" refers to the process by which an mRNA produced by transcription of DNA produces a polypeptide of a protein according to its genetic code. The protein translated in the present invention may comprise a polypeptide translated from the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Furthermore, a polypeptide translated from a nucleotide sequence having homology of at least 70% with these sequences, and further, a protein produced by folding the polypeptide can be included.

As used herein, the term "separation and purification" may mean separating and purifying the translated protein or polypeptide thereof as an active ingredient. The separation and purification process can be carried out by any method known in the art without limitation, and chromatographic-based purification methods can be preferably used.

According to one embodiment of the present invention, there is provided a composition for improving skin aging comprising SPRR1A protein capable of regulating expression of an aging-related factor.

As used herein, the term "improvement of skin aging" may mean improvement of the skin damaged by aging, and hence regeneration. Thus, the composition for improving skin aging comprising SPRR1A protein of the present invention can improve skin damaged by aging. For example, treatment of the SPRR1A protein with aged skin of an individual may reduce the expression of factors associated with aging, thereby improving aged skin.

The composition for improving skin aging according to an embodiment of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium But are not limited to, silicates, celluloses, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. These may be used alone or in combination of two or more. Furthermore, when the composition for improving skin aging according to an embodiment of the present invention is formulated, it can be prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient.

According to the administration method, the composition for improving skin aging according to an embodiment of the present invention can be prepared into a preparation easy to administer.

For example, where the composition is prepared as a solid dosage form for oral administration, solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid dosage forms may contain one or more excipients For example, starch, calcium carbonate, sucrose, lactose or gelatin may be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have.

In addition, when the composition is prepared as a preparation for parenteral administration, the preparation for parenteral administration may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external agent, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

Accordingly, the compositions in various embodiments of the present invention may be administered in a variety of ways. That is, the composition can be administered by oral administration or parenteral administration method in which the composition can reach the desired site, and preferably, it can be administered by a parenteral administration method. For example, the method of parenteral administration may be intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, and a method of applying, spraying or inhalation the above composition may be used But are not limited to.

More preferably, the composition may be administered transdermally during parenteral administration, and more preferably, the composition may be administered by a local administration method comprising applying the composition to the skin of an individual.

According to one embodiment of the present invention there is provided a polypeptide comprising a SPRR1A protein, a polypeptide having 70% or more homology with the amino acid sequence of SEQ ID NO: 1, and a polypeptide having 70% or more homology with the amino acid sequence of SEQ ID NO: 2 At least one cosmetic composition for improving skin aging is provided.

Further, the cosmetic composition for improving skin aging according to the present invention comprises a protein produced by folding of a polypeptide having 70% or more homology with the amino acid sequence of SEQ ID NO: 1, a protein having 70% or more homology with the amino acid sequence of SEQ ID NO: 2 And may further comprise a protein produced by folding of the polypeptide.

The composition according to an embodiment of the present invention may be used as a gel, a lotion, a powder, a cleansing oil, a foundation, a spray, a solution, an ointment for external use, a cream, a foam, a nutrition lotion, At least one formulation selected from the group consisting of emulsifiable concentrates, buffers, liquid cleansers, bath salts, sunscreen creams, sun oil, suspensions, emulsions and pastes. However, it is not limited thereto.

The cosmetic composition according to another embodiment of the present invention may be manufactured by, but not limited to, a semi-solid preparation such as an external ointment, lotion and the like.

The composition according to an embodiment of the present invention may further comprise a cosmetically acceptable carrier.

At this time, the cosmetically acceptable carrier included in the cosmetic composition according to another embodiment of the present invention varies according to the formulation of the cosmetic composition. Furthermore, the carrier in the present invention can use one carrier or a mixture of two or more carriers.

For example, when the cosmetic composition according to another embodiment of the present invention is ointment, paste, cream or gel, the carrier may be an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, a polyethylene glycol, , Bentonite, silica, talc, zinc oxide, and the like.

When the formulation of the cosmetic composition according to another embodiment of the present invention is a powder or a spray, the carrier may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and the like, But are not limited to, propellants such as chlorofluorohydrocarbons, propane / butane or dimethyl ether.

When the formulation of the cosmetic composition according to another embodiment of the present invention is an interface-active agent-containing cleansing, the carrier is selected from the group consisting of aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, But are not limited to, fatty alcohols, fatty alcohols, glycerides, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters, .

When the formulation of the cosmetic composition according to another embodiment of the present invention is a solution or emulsion, a solvent, a dissolving agent or an emulsifying agent may be used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, Benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil and the like can be used, and in particular, it is possible to use cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, Glycol or sorbitan fatty acid esters, and the like, but are not limited thereto.

When the formulation of the cosmetic composition according to another embodiment of the present invention is a suspension, the carrier may be a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like, but are not limited thereto.

In the case that the formulation of the composition according to another embodiment of the present invention is a soap, the carrier may be an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, an isethionate, a lanolin derivative, an aliphatic alcohol, Esters, glycerol, sugars, and the like.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention.

The present invention provides a composition containing SPRR1A protein as an active ingredient, and has the effect of inhibiting cell senescence or improving skin aging using the SPRR1A protein.

Further, the present invention has an effect of improving skin aging by providing a cosmetic composition containing SPRR1A protein as an active ingredient.

The present invention can provide a method for producing a composition for inhibiting cell senescence capable of producing a composition for inhibiting cell senescence by transforming a recombinant vector having a gene having a specific nucleotide sequence of a gene and purifying the translated protein from the recombinant vector It is effective.

Furthermore, the present invention provides a method for measuring the expression level of the SPRR1A gene or the level of the SPRR1A protein in a subject cell, and is capable of screening a composition for inhibiting cell senescence.

The effects according to the present invention are not limited by the contents exemplified above, and more various effects are included in the specification.

1 is an experimental result showing the change in the expression of aging-related factors in aged cells after overexpression of the SPRR1A gene in senescent cells.
FIG. 2A shows experimental results showing the relative expression levels of aging-related factors changed in HaCaT cells after overexpression of the SPRR1A gene in HaCaT cells.
FIG. 2B is a graph showing the relative expression levels of senescence-related factors in rat mesenchymal stem cells after overexpression of SPRR1A gene in mesenchymal stem cells of rats.
FIG. 2C shows experimental results showing an increase in SPRR1A protein as a result of overexpression of the SPRR1A gene.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention, and the manner of achieving them, will be apparent from and elucidated with reference to the embodiments described hereinafter in conjunction with the accompanying drawings. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

Hereinafter, evaluation of the composition in various examples including the SPRR1A protein was applied to rat mesenchymal stem cells or human HaCaT cell skin, and that of the aging-related factors IL-1 alpha, p16, p21, IL-6 and Although the measurement of the expression level of SA-beta -gal is exemplarily described, the present invention is not limited thereto. The composition of the present invention can inhibit cell senescence by controlling the expression level of various aging-related factors in various cells, It can show the effect of aging improvement. Accordingly, the composition provided by the present invention can be used for various purposes, for various cells, and for various individuals.

Example 1 Analysis of Expression Levels of Aging-Related Factors Altered by Overexpression of SPRR1A Gene in Aging Cells

1 is an experimental result showing the change in the expression of aging-related factors in aged cells after overexpression of the SPRR1A gene in senescent cells.

To observe changes in the expression of senescence-associated factors in senescent cells, mesenchymal stem cells are first isolated from bone marrow from 18 to 26 months of age. Isolated mesenchymal stem cells may be aged cells.

Then, the isolated mesenchymal stem cells are cultured in vitro and 2 μg of SPRR1A DNA is transformed into mesenchymal stem cells using Lipofectamine. At this time, the mesenchymal stem cells to which SPRR1A DNA was not transfected are used as a control group.

Lastly, transfected mesenchymal stem cells are cultured for 3 days, and SA-β-gal analysis is performed to stain at pH 6.0. Its stained results can indicate the expression level of SA-β-gal in mesenchymal stem cells.

Referring to FIG. 1, blue-stained SA-β-gal can be identified. At this time, the higher the blue light, the higher the expression level of SA-β-gal. Specifically, in contrast to the control group in which the DNA of SPRR1A was not injected, the mesenchymal stem cells transfected with the DNA of SPRR1A were found to be bluish. In other words, the senescent mesenchymal stem cells over-expressing SPRR1A showed that the expression level of SA-β-gal, one of the senescence-related factors, was decreased in aged mesenchymal stem cells. Thus, by overexpressing the SPRR1A gene in senescent cells, and further treating a higher level of the SPRR1A protein, the expression of the senescence-associated factor can be regulated. As a result, the composition comprising the SPRR1A protein provided in the various embodiments of the present invention is capable of inhibiting aging of cells and further improving the aging of skin cells.

FIG. 2A shows experimental results showing the relative expression levels of aging-related factors changed in HaCaT cells after overexpression of the SPRR1A gene in HaCaT cells.

First, in order to observe a change in expression of aging-related factors in aged cells, human keratinocyte HaCaT cells are prepared. Here, HaCaT cells are skin-related cells, and HaCaT cells used are aged cells over time.

Then, HaCaT cells were cultured in a 6-well plate, and when cells were grown up to 70% of the well volume, SPRR1A DNA was transfected into each cell using lipofectamine. At this time, the injected DNA was human-derived SPRR1A DNA, and the amount of injected DNA was 0.02 μg, 0.05 μg, 0.1 μg, 0.2 μg, and 0.5 μg. Furthermore, HaCaT cells transformed with a pure pCS2 vector that has not been recombined with other genes are used as a control. Thus, each well contains HaCaT cells transfected with SPRR1A DNA at five masses and control HaCaT cells.

Next, transfected HaCaT cells are cultured for 2 days to separate the RNA, and the separated RNA is converted into cDNA.

Finally, the relative expression levels of senescence-related factors are measured from the translated cDNA using real-time PCR. At this time, the relative expression levels of aging-related factors were normalized by tubulin. At this time, the aging-related factors that observed expression levels were p16, p21 and IL-6.

Referring to FIG. 2A, the graph shows the expression levels of p21, p16 and IL-16 by SPRR1A DNA injection mass in HaCaT cells. Specifically, the expression levels of p21, p16 and IL-16 were reduced to about 2-fold or more in the HaCaT cells transfected with SPRR1A DNA as compared with the control. In particular, the expression levels of p16 and IL-6 were greatly decreased, and the expression levels of p21, p16 and IL-16 in HaCaT cells transfected with 0.1 μg of SPRR1A DNA were significantly reduced compared to other masses. As a result, when SPRR1A DNA is injected into HaCaT cells and further into skin cells at a dose of 0.1 μg, the effect of inhibiting aging and improving aging can be greater than that of SPRR1A DNA of different mass. However, it is not limited thereto. For example, SPRR1A DNA can be injected at a mass of 0.05 μg to 0.15 μg for the effect of aging suppression and the effect of improving aging.

FIG. 2B is a graph showing the relative expression levels of senescence-related factors in rat mesenchymal stem cells after overexpression of SPRR1A gene in mesenchymal stem cells of rats.

First, in order to observe the change in the expression of aging-related factors in aged cells, mouse mesenchymal stem cells are prepared. At this time, the mesenchymal stem cells of the rats used are aged cells over time.

Then, the mouse mesenchymal stem cells were cultured in a 6-well plate. When cells were grown up to 70% of the well volume, SPRR1A DNA was transfected into each cell using lipofectamine . At this time, DNA of SPRR1A derived from mouse was used for the injected DNA, and the amounts of DNA injected were 0.1 μg, 0.2 μg, 0.5 μg, and 1.0 μg. Further, mesenchymal stem cells not transfected with SPRR1A DNA are used as a control group. Thus, each well contains mesenchymal stem cells transfected with SPRR1A DNA in four masses and control mesenchymal stem cells.

Next, the transformed mesenchymal stem cells are cultured for 2 days to separate the RNA, and then the separated RNA is converted into cDNA.

Finally, the relative expression levels of senescence-related factors are measured from the translated cDNA using real-time PCR. At this time, the relative expression levels of aging-related factors were normalized by tubulin. At this time, the aging-related factors that observed expression levels were IL-1 alpha, p16, p21, and IL-6.

Referring to FIG. 2B, the graph shows the expression levels of IL-1α, p21, p16 and IL-16 by SPRR1A DNA injection mass in mesenchymal stem cells. Specifically, the expression levels of IL-1α, p21, p16 and IL-16 were reduced to about 2-fold or more as compared with the control group. In particular, the level of expression of p16 and IL-6 was greatly decreased. The expression level of IL-1α, p21, p16 and IL-16 in mesenchymal stem cells transfected with 0.1 μg of SPRR1A DNA was significantly reduced . As a result, when the SPRR1A DNA was injected into the mesenchymal stem cells at a mass of 0.1 μg, the effect of inhibiting aging and improving the aging could be greater than that of SPRR1A DNA of different mass. However, it is not limited thereto. For example, SPRR1A DNA can be injected at a mass of 0.05 μg to 0.15 μg for the effect of aging suppression and the effect of improving aging.

FIG. 2C shows experimental results showing an increase in SPRR1A protein as a result of overexpression of the SPRR1A gene.

More specifically, FIG. 2C shows the result of measuring the level of SPRR1A protein in cells due to overexpression of SPRR1A gene following transfection of SPRR1A DNA using Western blotting. At this time, cells in which SPRR1A DNA was not transfected were set as a control group. As a result, it was confirmed that the level of SPRR1A protein was higher in all of the experimental groups to which PRR1A DNA was transfected as compared with the control group. In particular, it can be seen that there is a large difference in protein level between the control and the cells injected with 0.1 μg of SPRR1A DNA, 0.1 μg of SPRR1A DNA and 0.2 μg of SPRR1A DNA. Thus, overexpression of the SPRR1A gene in the cell can induce a high level of expression of the SPRR1A protein.

As a result of the above Example 1, overexpression of the SPRR1A gene, that is, treatment of the SPRR1A protein may have an effect of inhibiting cell senescence and improving skin aging. Accordingly, the SPRR1A protein can be used as a composition for inhibiting cell senescence, a composition for improving skin aging, and a cosmetic for improving skin aging. The present invention may be effective to prevent diseases associated with cell senescence or diseases associated with skin senescence by providing the aforementioned composition. Furthermore, when SPRR1A DNA is directly transfected into senescent cells, the effect of inhibiting cell senescence and improving skin aging can be maximized at a mass of 0.1 μg. The treatment method of SPRR1A is not limited to the method of directly injecting the SPRR1A DNA in Example 1 into cells, but can be treated in various ways.

In addition, the polypeptide comprising the amino acid sequence of SEQ ID NO: 1, the peptide consisting of the amino acid sequence of SEQ ID NO: 2, and the proteins formed by folding these polypeptides may have an activity of regulating the expression of an aging-related factor possessed by the SPRR1A protein. Thus, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, the peptide consisting of the amino acid sequence of SEQ ID NO: 2, and the proteins formed by folding these polypeptides may have cell aging inhibitory activity and further skin aging improving activity , And can be used as a composition for inhibiting aging, a composition for improving cell senescence, and a cosmetic composition for improving cell senescence in various embodiments of the present invention.

In addition, the present invention can further provide a method for producing a composition for inhibiting cell senescence showing various effects. Furthermore, the present invention provides a method for screening a composition for inhibiting cell senescence, which can increase the expression level of SPRR1A, thereby reducing the expression level of aging-related factors. have. Furthermore, the present invention can further provide a composition for inhibiting cell senescence, which can directly reduce the expression level of aging-related factors by providing a method for screening a composition for inhibiting cell senescence.

Although the embodiments of the present invention have been described in detail with reference to the accompanying drawings, it is to be understood that the present invention is not limited to those embodiments and various changes and modifications may be made without departing from the scope of the present invention. . Therefore, the embodiments disclosed in the present invention are intended to illustrate rather than limit the scope of the present invention, and the scope of the technical idea of the present invention is not limited by these embodiments. Therefore, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas within the scope of equivalents should be construed as falling within the scope of the present invention.

<110> INDUSTRY-ACDEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY <120> COMPOSITION FOR INHIBITING CELLULAR SENESCENECE OR IMPROVING SKIN          AGING COMPRISING SPRR1A PROTEIN <130> 16PD5990KR <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 29 <212> PRT <213> Homo sapiens <400> 1 Gln Gln Lys Gln Pro Cys Thr Pro Pro Pro Gln Pro Gln Gln Gln Gln   1 5 10 15 Val Lys Gln Pro Cys Gln Pro Pro Pro Gln Glu Pro Cys              20 25 <210> 2 <211> 55 <212> PRT <213> Homo sapiens <400> 2 Pro Lys Thr Lys Glu Pro Cys His Pro Lys Val Pro Glu Pro Cys His   1 5 10 15 Pro Lys Val Pro Glu Pro Cys Gln Pro Lys Val Pro Glu Pro Cys Gln              20 25 30 Pro Lys Val Pro Glu Pro Cys Pro Ser Thr Val Thr Pro Ala Pro Ala          35 40 45 Gln Gln Lys Thr Lys Gln Lys      50 55 <210> 3 <211> 87 <212> DNA <213> Homo sapiens <400> 3 cagcagaagc agccttgcac cccaccccct cagcctcagc agcagcaggt gaaacaacct 60 tgccagcctc caccccagga accatgc 87 <210> 4 <211> 165 <212> DNA <213> Homo sapiens <400> 4 cccaaaacca aggagccctg ccaccccaag gtgcctgagc cctgccaccc caaagtgcct 60 gagccctgcc agcccaaggt tccagagccc tgccagccca aggtgcctga gccctgccct 120 tcaacggtca ctccagcacc agcccagcag aagaccaagc agaag 165

Claims (13)

delete delete delete delete Treating the candidate cell with a cell aging-inhibiting composition candidate; And
Measuring the level of expression of the SPRR1A gene or the level of SPRR1A protein in the subject cell,
The subject cell is a photoaged cell,
Wherein the measuring step comprises measuring the expression levels of SA-beta-gal, IL-l [alpha], p16, p21 and IL- The method further comprising the step of measuring the level of the composition for inhibiting cell senescence. 6. The method of claim 5,
Wherein the measuring step further comprises the step of determining the candidate substance as a cell senescence inhibiting substance when the level of expression of SPRR1A gene or the level of SPRR1A protein in the cell of interest is equal to or higher than a predetermined level . delete delete delete delete delete delete delete

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