JP6854073B2 - Screening method for skin barrier function enhancing drug and skin barrier function evaluation method using the amount of epidermal serine racemase and / or D-serine as an index - Google Patents

Description

The present invention relates to a method for screening a skin barrier function enhancing agent and a method for evaluating a skin barrier function in the skin.

When the barrier function of the skin is lost, the moisture of the skin is lost and foreign matter invades from the outside, which causes sensitive skin, dry skin, rough skin, dermatitis and allergies. Therefore, from the viewpoint of developing functional foods, cosmetics or pharmaceuticals, it is desired to develop a drug that enhances the barrier function of the skin. Methods such as increasing the amount of intercellular lipids and arranging the sequences are known as factors for enhancing the skin barrier function. For example, as shown in Patent Document 1, drugs that promote the production of intercellular lipids have been developed. Has been done.

Examples of the index of the barrier function of the skin include transepidermal water evaporation (TEWL) and the degree of penetration into the skin using an index substance such as a pigment. The TEWL of the skin can be measured using a device such as Vapometer, but the measurement of the amount of water has a great influence on the measurement environment such as the amount of water in the air, the flow of air, the person's body temperature, psychological state, and sweat. Since it is an index that tends to be unstable due to reception, a method that can evaluate the barrier function of the skin more stably is desired. When the penetrance of the index substance is used as an index of the barrier function of the skin, the index substance such as a pigment must be applied to the skin, which imposes a heavy burden on the skin.

In order to screen a drug having an effect of enhancing the skin barrier function from a large number of candidate drugs, an experiment can be performed in vivo using TEWL as an index of the skin barrier function, but it is greatly affected by the measurement environment. It has been difficult to screen a large number of candidate drugs for drugs that are unstable and have the effect of enhancing the skin barrier function physiologically or structurally. On the other hand, as such a screening method, a method of measuring the skin intrinsic factors involved in the recovery and enhancement of the barrier function using isolated skin and evaluating the recovery and enhancement of the barrier function can be considered, but in a small amount. There is no suitable factor that can be easily evaluated with the sample of the above, and identification of such a factor has been desired.

On the other hand, it is known that amino acids include L-form and D-form as optical isomers, the amino acids constituting the living body are mainly L-form, and the D-form amino acids are extremely such as bacterial peptidoglycan. It was thought to be a limited biological component. However, as a result of recent research, various D-amino acids exist not only in microorganisms but also in plants and mammals as free forms or as amino acid residues constituting proteins, and exhibit various physiological functions. It has been revealed that there is. D-serine and D-aspartic acid were first found as free D-amino acids in mammals. It has been found that D-serine is localized in the cerebrum and hippocampus, acts as a coagonist of NMDA receptors, and plays a role in controlling excitatory neurotransmission (Non-Patent Document 1). In addition, D-aspartic acid is localized in the testis and pineal gland, and has been found to be involved in the regulation of hormone secretion. On the other hand, recent results of D-amino acid research have revealed that D-serine, D-aspartic acid, D-alanine, D-glutamic acid, and D-proline are present in the dermis and epidermis. It has been shown to decrease with age (Non-Patent Document 2). Among these, although D-serine is the D-amino acid with the highest content in the epidermis and stratum corneum, the role of D-serine in the skin is seen to be effective in reducing UV damage in fibroblasts in the dermis. Although it has been published (Patent Document 2), it has not yet been clarified in the epidermis having a high content.

Amino acid racemase is known as an enzyme involved in the production of D-amino acid, and is classified into an enzyme that depends on pyridoxal phosphate (PLP) and a racemase that does not require a cofactor. It has been reported that PLP-dependent serine racemase is present in mammals, triggered by the report that D-amino acid is present in eukaryotic cells including mammals (Non-Patent Document 3). Has not yet been confirmed to be present with racemase activity in mammals. Since D-serine is localized in the cerebrum and hippocampus, it has been confirmed that serine racemase is expressed in the brain in mammals including humans, but expression in other tissues has hardly been reported ( Non-Patent Document 3).

Japanese Unexamined Patent Publication No. 2012-92032 WO2011 / 030903

S.H. Snyder, et al., NeuroChem Res 25, 553 (2000) Y. Tojo, Food Style 21, 2013, August, (Vo. 17, No.8) Molecular Brain Research, Vo l. 125, pp. 96-104, 2004 Collection of Experimental Protocols for the Development of Functional Cosmetic Materials, CMC Publishing, 2010, pp. 214-216

Attention has been focused on increasing the amount of intercellular lipids and arranging them in order to enhance the barrier function of the skin. On the other hand, in enhancing and repairing the barrier function of the skin, increasing the number of healthy stratum corneum itself is a more effective approach from the viewpoint of enhancing the barrier function of the skin physiologically or structurally. Conceivable. However, a drug having such an action has not been known so far, and it has been difficult to find it by screening as described above. Therefore, it has been desired to develop a screening method for a drug having an effect of enhancing the barrier function of the skin physiologically or structurally.

The present inventors conducted intensive studies on D-serine whose function in the skin is not yet known, and found that D-serine contributes to the skin barrier function. In addition, when the effect of D-serine on cultured keratinocytes was investigated, the expression of filaggrin, which plays an important role in the formation of barrier function, as well as cornulin and lepetin, which are involved in the formation of the peripheral zone (CE), was observed. Based on these findings, a drug having an effect of enhancing the barrier function of the skin by using the amount of D-serine and the expression level or activity of serine racemase, which produces D-serine from L-serine, as an index in keratinocytes. It has become possible to provide a simple and stable evaluation of the enhanced barrier function of the skin, which is less burdensome to the subject, and has led to the present invention. By using such a screening method, a drug that enhances the skin barrier function could be screened.

Specifically, we have completed the following inventions:
[1] A method for screening a skin barrier function enhancing drug using the activity or expression level of serine racemase and / or the amount of D-serine as an index in keratinocytes.
[2] Below:
The process of adding a candidate drug to keratinocytes,
A step of measuring the activity or expression level of serine racemase and / or the amount of D-serine in keratinocytes.
The screening method according to item 1, further comprising a step of determining the skin barrier function enhancing action of the candidate drug from the expression level or activity of serine racemase and / or the amount of D-serine.
[3] The screening method according to item 2, further comprising a step of inducing differentiation of keratinocytes.
[4] The screening method according to item 1, wherein the screening method is performed on differentiation-induced keratinocytes.
[5] The screening method according to any one of items 1 to 4, wherein the serine racemase expression level is measured by measuring the amount of serine racemase mRNA or protein.
[6] The screening method according to any one of items 1 to 4, wherein the measurement of serine racemase activity is by measuring the conversion efficiency of a substrate or the amount of D-amino acid product.
[7] 1 or one selected from the group consisting of hydrolyzed crow barley protein, rumpuyan extract, white-spotted sardine extract, pyridoxal phosphate, and vitamin B _{6 obtained by the screening method according to items 1 to 6.} A serine racemase activator containing multiple substances as active ingredients.
[8] A method for evaluating a skin barrier function using a serine racemase activity or expression level and / or a D-serine level as an index from a skin sample isolated from human skin.
[9] Below:
A step of measuring serine racemase activity or expression level and / or D-serine level in a skin sample isolated from a subject.
The method for evaluating the skin barrier function according to item 8, which comprises a step of determining the barrier function of the target skin from the serine racemase activity or expression level and / or the D-serine amount.
[10] The method for evaluating a skin barrier function according to item 8 or 9, wherein the serine racemase expression level is measured by measuring the amount of serine racemase mRNA or protein.
[11] The method according to item 8 or 9, wherein the measurement of serine racemase activity is by measuring the conversion efficiency of a substrate by serine racemase or measuring the amount of D-amino acid product.
[12] 1 or selected from the group consisting of hydrolyzed crow barley protein, rumpuyan extract, Elephantopus moliza extract, pyridoxal phosphate, and vitamin B _{6 obtained by the screening method according to items 1 to 6.} A skin barrier enhancer containing multiple substances.
[13] One or more substances selected from the group consisting of hydrolyzed crow barley protein, rumpuyan extract, white sardine extract, pyridoxal phosphate, and vitamin B _{6 for subjects with reduced skin barrier function.} A method for enhancing skin barrier function, which comprises administering.
[14] One or more substances selected from the group consisting of hydrolyzed crow barley protein, rumpuyan extract, Elephantopus mollinus extract, pyridoxal phosphate, and vitamin B _{6 for subjects with reduced serine racemase activity.} A method for activating serine racemase, which comprises administering.
[15] for the production of serine racemase activators, hydrolyzed oat protein, Rumupuyan extract, white nosed Ige Kouzorina extract, pyridoxal phosphate, and the use of vitamin B ₆ compound.
[16] for the manufacture of a skin barrier function enhancing agent, hydrolyzed oat protein, Rumupuyan extract, white nosed Ige Kouzorina extract, pyridoxal phosphate, and the use of vitamin B ₆ compound.

By using the screening method of the present invention, it becomes possible to select a drug capable of improving or enhancing the skin barrier function. In addition, the skin barrier function evaluation method of the present invention can determine the skin barrier function at the cellular or tissue level or biochemically.

FIG. 1A shows a comparison of the amount of free L-serine in the skin of the high TEWL group and the low TEWL group. FIG. 1B shows a comparison of the amount of free D-serine in the skin of the high TEWL group and the low TEWL group. FIG. 2 is a chromatogram in which intracellular D-serine was detected before and after giving a differentiation-inducing stimulus in cultured keratinocytes. FIG. 3A is a diagram showing that the hydrolyzed oat protein solution, which is one of the candidate drugs, increased the amount of free D-serine. FIG. 3B is a diagram showing that hydrolyzed oat protein solution, which is one of the candidate drugs, increased the expression of serine racemase. FIG. 4 is a diagram showing that the amount of free D-serine was increased by adding pyridoxal phosphate (PLP), which is one of the candidate drugs, to the cultured keratinocytes. FIG. 5 is a diagram showing that D-serine increased the expression of the filaggrin gene in a three-dimensional skin model. FIG. 6 is a diagram showing that D-serine increased the expression of the cornulin gene in a three-dimensional skin model. FIG. 7 is a diagram showing that D-serine increased the expression of the lepetin gene in a three-dimensional skin model.

The present invention relates to a method for screening a skin barrier function enhancing agent, which is a combination of one or more indicators of serine racemase expression level, activity, and D-serine level in keratinocytes.

The screening method of the present invention makes it possible to obtain a drug having a skin barrier function enhancing effect. Here, the barrier function of the skin is a function of retaining water in the skin and preventing foreign substances from invading from the outside, and the stratum corneum is particularly responsible for such a function in the epidermis. The skin barrier function can be measured by the transepidermal water evaporation (TEWL), and it is considered that the higher the water evaporation, the lower the skin barrier function. Increased skin barrier function can be achieved, for example, by increasing the number of layers in the stratum corneum, maturing the keratinized outer membrane (cornified envelope) in the stratum corneum, and increasing intercellular lipids such as cholesterol and ceramide. However, it is not intended to be limited to these. Therefore, although not intended to be bound by theory, the agents selected by the screening method of the present invention enhance the expression or activity of serine racemase by application to the skin, or D. -It is considered that the amount of serine can be increased, and as a result, effects such as an increase in the number of layers in the stratum corneum can be exerted and the skin barrier function can be enhanced.

The candidate drug used in the screening method of the present invention may be any compound, for example, a compound obtained from a combinatorial library, a mixture or an extract. As the candidate drug, for example, a compound, mixture or extract library of a food material, a cosmetic material or a pharmaceutical material can be used. According to the present invention, a drug having an action of enhancing the skin barrier function can be applied to foods such as functional foods and nutritional foods, cosmetics, pharmaceuticals or quasi-drugs. Cosmetics can be applied to, for example, lotions, creams, milky lotions, gels, beauty essences, ointments, facial masks, bath salts, body soaps, shampoos, rinses, foundations, etc., but are intended to be limited to these. is not it. More preferably, it is applied to cosmetics for sensitive skin, such as lotions, creams, milky lotions, and beauty essences. If it is a drug or a quasi-drug, it can be applied to various forms such as ointments and creams for transdermal administration and forms for oral administration.

In the screening method of the present invention, the expression level or activity of serine racemase and / or the amount of D-serine is used as an index in keratinocytes. The index means that the expression level or activity of serine racemase and / or the amount of D-serine is taken into consideration. Therefore, when a candidate drug is added, the screening method of the present invention can be obtained by selecting a drug having a skin barrier function enhancing action based on the expression level or activity of serine racemase and / or the amount of D-serine. Corresponds to.

Keratinocytes are cells that make up about 95% of the epidermis. In the living body, stem cells of keratinocytes are present in the basal layer of the epidermis and move to the outer layer as they mature to form the stratum spinosum, the stratum granulosum, and the stratum granulosum. The properties of keratinocytes change with maturation, and cells are flattened in the stratum spinosum and stratum granulosum, lipids such as ceramide are released into the intercellular spaces, and enucleation occurs in the outermost stratum corneum. This series of maturation processes can be called the differentiation of keratinocytes. The cells in which enucleation has occurred lose their cell morphology and are layered to form a stratum corneum, and finally peel off as dirt from the outermost layer.

The cultured keratinocytes used in the present invention may be cells obtained from any of the basal layer, spiny layer, granular layer, and stratum corneum of the epidermis, and stem cells such as cutaneous epithelial stem cells and pluripotent stem cells. For example, it may be a cell whose differentiation is induced from ES cells, iPS cells, EG cells and the like. The cells obtained from the epidermis and cultured may be primary cultured cells, subcultured cells, or even cancerous or immortalized cells. In addition, the cultured keratinocytes used in the present invention may be, for example, strained keratinocytes derived from humans or animals. These cell lines of keratinocytes can be obtained from public institutions and distributors.

The culture may refer only to the desired cells, may contain other cells, or may be composed of cells and a medium. Therefore, the keratinocyte culture may refer only to keratinocytes, may contain keratinocytes and other cells, or may be composed of keratinocytes alone or keratinocytes, other cells and a medium. Examples of such cultures include monolayer cultures, mixed cultures, and three-dimensional cultures. The medium for culturing keratinocytes may be any medium as long as it enables the culture of keratinocytes. For example, various additives are added to the medium of HuMedia-KB2 (Kurabo) or Keratinocyte-SFM (Invitrogen). However, it is not intended to be limited to these. The culture conditions are usually 37 ° C. and in the _{presence of 5% CO 2,} but can be changed as long as keratinocytes grow. From the viewpoint of setting the abundance of D-serine to serine racemase activity, the medium is preferably in an L-serine-enriched state. Normally, D-amino acid is not intentionally added to the culture medium, and the abundance of L-serine with respect to D-serine in the medium is at least 1.1 times, more preferably 10 times, still more preferable. Is more than 50 times. For example, in a medium (KGM medium) in which the HuMedia-KG growth additive set is added to HuMedia-KB2 (Kurabo Industries), the abundance of L-serine with respect to D-serine is 100 times or more.

The keratinocyte culture is cultivated after sowing, and a culture having a concentration of about 50% to confluence can be used for an experiment. The collection density is preferably 70% or more, more preferably 80% or more, from the viewpoint of inducing differentiation. The step of adding the candidate drug to the keratinocyte culture may be performed by replacing the medium of the keratinocyte culture with the medium containing the candidate drug, or the candidate for the culture containing the keratinocyte and the medium. The drug may be added as is or as a diluent.

Serine racemase is a pyridoxal 5'phosphate (PLP) -dependent racemase that is a vitamin B _{6 coenzyme type.} Although not intended to be limited to theory, serine racemase converts between L-serine and D-serine through the formation of anionic intermediates by abstracting the α-hydrogen of serine. It is an enzyme that can be performed. In the conversion reaction between L-serine and D-serine, the enthalpy does not change before and after the reaction, and the reaction is driven by entropy. Therefore, in the equilibrium state, the amounts of L-serine and D-serine are equal. Since the abundance of L-serine is higher than that of D-serine in the living body, the presence of serine racemase converts L-serine in the living body into D-serine. Even under culture conditions, since the medium usually contains a large amount of L-form, L-serine is similarly converted to D-serine by the presence of serine racemase. In the present invention, the racemase may be the same as the racemase whose expression was recognized in the brain according to Non-Patent Document 3, or may be a homolog or ortholog thereof. Therefore, it refers to an antibody that can be amplified by the primer pair used in the same document and can be detected by the anti-racemase antibody described in the same document. Serine racemase can be appropriately specified by a person skilled in the art from the website of a public institution such as NCBI according to the animal species.

In one aspect of the present invention, the activity of serine racemase in keratinocytes can be used as an index to evaluate the barrier function of the skin or screen for a drug that enhances the barrier function of the skin. The activity of serine racemase may be based on the amount of D-serine produced by the conversion of L-serine contained in a normal medium when a substance that serves as a substrate for the enzyme is not added, or other substances containing D-serine. It may be based on the total amount of D-amino acids in. Further, by adding a substance other than serine that can be a substrate of serine racemase, the amount of product based on the conversion of the additive or the conversion efficiency may be measured. Since serine racemase converts the excess optical isomer to the other optical isomer until the D-form and L-form become 50:50, it is naturally possible to add the D-form in the activity measurement. In that case, the enzyme activity can also be evaluated by measuring the increase in L-serine caused by the conversion. In addition to this, for example, if cycloserine or a derivative of serine (N-methylated product, acetylated product, phosphorylated product, etc.) is added, the enzyme activity can be evaluated by examining the conversion efficiency to the corresponding optical isomer. is there. When measuring the activity of serine racemase based on the amount of D-serine, the abundance of D-serine in the skin sample isolated from the subject, or in the keratinocyte culture, i.e. in the medium, in the cells, or both. It may be determined by enzymatic parameters determined based on changes in the abundance of D-serine in skin samples or in keratinocyte cultures. This method is also applicable to all of the above-mentioned substances that serve as substrates for enzymes. As an enzymatic parameter indicating the activity of the serine racemase enzyme, it may be indicated by determining the _{V max} and K _{m values.}

In a further aspect of the present invention, the skin barrier function can be evaluated or the skin barrier function enhancing drug can be screened using the amount of D-serine in keratinocytes as an index. The amount of D-serine may be measured in skin samples isolated from the subject, or in keratinocyte cultures, i.e. in medium, cells, or both. The method for measuring the amount of D-serine can be carried out by using a known method, for example, an HPLC method using an optical isomer separation column, a gas chromatography method, an electrophoresis method, or an enzymatic method. If the serine racemase activity is measured by measuring the amount of D-serine, L-serine may be added to the keratinocyte culture to increase the amount of D-serine. When serine racemase undergoes a conversion reaction between L-serine and D-serine, the enthalpy does not change before and after the reaction, and the reaction is driven by entropy. Therefore, by adding L-serine, serine is added. Depending on the activity of racemase, the amount of D-serine can be increased, making it easier to measure the amount of D-serine.

In another embodiment, the expression level of serine racemase can be used as an index to evaluate the skin barrier function or screen for a skin barrier function enhancing drug. The expression level of serine racemase can be determined by measuring the protein of serine racemase by an immunological method using an anti-serine racemase antibody. As an immunological method, for example, Western blotting, fluorescent immunostaining, or ELISA may be used. As the anti-serine racemase antibody, a commercially available monoclonal antibody or polyclonal antibody may be used, or an antibody recovered by immunizing an experimental animal such as a rabbit or a mouse with a serine racemase protein or an antigen peptide may be used.

The expression level of serine racemase may be determined by determining the amount of serine racemase mRNA in the cell. The mRNA may be measured by any method, for example, based on a quantitative PCR method. Quantitative PCR methods are usually performed as follows, but are not intended to be limited thereto. MRNA is extracted from the recovered cells, and cDNA is prepared using a poly T primer and reverse transcriptase. By using the Cyber Green method or the fluorescent probe method for the prepared cDNA, the amount of mRNA contained in the cells can be quantified.

More specifically, the screening method of the present invention is as follows:
The process of adding a candidate drug to keratinocytes,
The step of measuring the serine racemase expression level or activity and / or the D-serine level in keratinocytes, and the serine racemase expression level or activity and / or the D-serine level determine the skin barrier function enhancing action of the candidate drug. The process may be included. The screening method of the present invention may further include a step of inducing differentiation of keratinocytes.

The differentiation-inducing step is performed by giving a differentiation-inducing stimulus to keratinocytes. By giving a differentiation-inducing stimulus to keratinocytes, cell flattening, increase of fibers or granules in cells, obscuring of cell gaps, stratification (especially in the case of monolayer culture), and finally desorption Morphological changes such as nuclei and changes in proteins such as activation of keratin 1, keratin 10, involucrin, and transglutamine sase and changes in proteins occur (Journal of the Japan Skin Society 90 (12), 1089- 1101, http: //drmtl.org/data/090121089.pdf, Drug News Perspect 2004, 17 (2) p117). The differentiation-inducing stimulus may be any stimulus as long as it can cause the above-mentioned differentiation of keratinocytes. For example, a calcium ion stimulus that increases the concentration of calcium ions, Folball-12-Millistate-13- It may be stimulation with acetate (Hawley-Nelson P, Standley JR et al, Exp Cell Res, 137: 155-167, 1982), physical stimulation such as voltage or gas phase exposure, or stimulation with biological fluid. When stimulating with calcium ions, a calcium salt or a diluted solution thereof may be added so that the final concentration of calcium ions is 0.1 mM to 10 mM, or the final concentration of calcium ions is prepared in advance. It may be replaced with the prepared medium. As the calcium salt used, for example, calcium chloride, calcium hydrogen carbonate, calcium carbonate, calcium nitrate, calcium acetate and the like are used. The upper limit of the final concentration of calcium ions is preferably 5 mM or less, more preferably 2 mM or less from the viewpoint of not adversely affecting cell culture. From the viewpoint of promoting differentiation induction, the lower limit of the concentration is preferably 0.5 mM or more, more preferably 1.0 mM or more.

The differentiation induction step and the candidate drug addition step may be performed at the same time, the differentiation induction step may be followed by the candidate drug addition step, and vice versa, that is, the differentiation induction step is performed after the candidate drug addition step. The process may be performed. Although not intended to be theoretically bound, the timing of differentiation induction and candidate drug addition can be varied depending on the desired mechanism of action of the candidate drug. For example, from the viewpoint of examining a drug that antagonizes the differentiation-inducing stimulant, the addition step is preferably performed at the same time as the stimulus step or before the stimulus. Furthermore, from the viewpoint of promoting / suppressing cell changes (morphological changes, expression of various intracellular factors, etc.) after differentiation induction, the addition step is preferably performed at the same time as or after the stimulation step, and the receptor on which the differentiation induction stimulation acts. From the viewpoint of searching for a mechanism such as blocking the body or the like in advance, it is preferable that the stimulation step is performed after the addition step. When the addition step of the candidate drug and the stimulation step of inducing differentiation are not performed at the same time, a cell culture step of several minutes to several hours may be included between the addition step and the stimulation step.

A cell culture step may be further included after the stimulation of differentiation induction and the addition of the candidate drug, and before the step of measuring the expression level or activity of serine racemase and / or the amount of D-serine. The period of this cell culture step can be arbitrarily selected depending on the type and concentration of the differentiation inducer used and the type of keratinocytes, and for example, the cell culture can be performed for 6 hours to 1 week. From the viewpoint of fully expressing serine racemase, the lower limit of the culture period is preferably 12 hours or more, more preferably 1 day or more. On the other hand, the upper limit of the culture period is not particularly specified because it differs depending on the culture method such as monolayer culture, multi-layer culture, and three-dimensional culture, but from the viewpoint of maintaining healthy cell culture conditions, monolayer culture In the case of, it is preferably within one week, more preferably within three days.

The screening method of the present invention may further include a damage imparting step. The damage-imparting step may be such that the cultured keratinocytes are damaged by physical or chemical stimuli, such as ultraviolet irradiation, addition of a radical source such as an oxidizing agent, addition of a surfactant, and the like. Examples include replacement with an oligotrophic medium. It is considered that the expression of serine racemase is decreased in the keratinocytes damaged by the damage giving process. Therefore, if a decrease in racemase expression level or racemase activity and / or D-serine level is suppressed in a screening method in which a damage-imparting step is performed after the addition step of the candidate drug, the candidate drug damages the barrier function of the skin. Can be used as a preventive agent. On the other hand, when the decreased racemase expression level or racemase activity and / or the amount of D-serine increases in the screening method in which the candidate drug addition step is performed after the damage imparting step, the candidate drug restores the skin barrier function. , Can be an improvement or therapeutic agent. Candidate agents that increase the amount of racemase expression or racemase activity, and / or the amount of D-serine in a screening method that does not impart damage can be said to be a skin barrier function enhancer.

As a result of performing the screening method of the present invention using the expression level of serine racemase as an index for the cosmetic material library, hydrolyzed crow barley protein, lumpuyan extract, and white bana as agents capable of increasing the expression level of serine racemase in keratinocytes. Igakouzorina extract could be selected. When the expression level of serine racemase is increased, the serine racemase activity is also increased, the D-serine content in the stratum corneum is increased, and the skin barrier function can be renewed. Therefore, these substances can also be referred to as a serine racemase expression promoter, a serine racemase activator or a skin barrier function enhancer. The drug screened by the screening method of the present invention is not limited to the extract derived from Lumpuyan or Elephantopus mollis, and the extract of a closely related species also has a skin barrier function enhancing effect. Therefore, as the serine racemase expression promoter, serine racemase activator or skin barrier function enhancer of the present invention, the ginger (Zingiberaceae) family Zingiber genus plant and the Asteraceae (Compositae) genus Elephamtopus plant Extracts are mentioned. Therefore, by blending these substances into cosmetics, it is possible to produce cosmetics that exert a skin barrier function enhancing effect.

In addition, as a result of performing the screening method of the present invention using serine racemase activity as an index for a cosmetic material library, pyridoxal phosphate (PLP: active vitamin B ₆ ) could be selected in keratinocytes. When serine racemase activity is enhanced, the D-serine content in the stratum corneum increases and the skin barrier function can be renewed. Therefore, pyridoxal phosphate can be called a serine racemase activator or a skin barrier function enhancer.

The hydrolyzed oat protein used in the present invention is a hydrolyzed product of a protein obtained from oat (Avene satiba). The oat protein is extracted from, for example, seeds, leaves, roots, bran and the like, with seeds being particularly preferred. The oats are preferably dried and crushed immediately after collection. Drying may be carried out in the sun or using a commonly used dryer. Proteins are hydrolyzed by acid or enzyme treatment during or after extraction. Oats are edible plants from West Asia to Europe and North America. The extraction solvent used in the present invention may be any solvent as long as it is usually used for extraction, and in particular, alcohols such as methanol and ethanol, hydrous alcohols, and organic solvents such as acetone and ethyl acetate ester may be used alone or in combination. It can also be extracted with water.

The amount of the hydrolyzed oat protein in the present invention can be arbitrarily selected by those skilled in the art as long as it exerts its effect. For example, the blending amount is 0.00005 to 20.0% by mass as a dried product in the total amount of the external preparation. The upper limit of the blending amount is preferably 10% by mass or less, more preferably 5% by mass or less, from the viewpoint of formulation and effect. The lower limit of the blending amount is preferably 0.0001% by mass or more, more preferably 0.0005% by mass or more, from the viewpoint of exerting a sufficient skin barrier function enhancing effect.

As the plant of the genus Zingiber of the family Zingiberaceae used in the present invention, Lempuyang (scientific name: Zingiber aromaticum Mal.) Is suitable. Rumpuyan is a plant that grows especially in dry grasslands and pastures in Indonesia. The extract used in the present invention is obtained by immersing the whole plant, such as the leaves of the plant, stems including rhizomes, and fruits, together with an extraction solvent, optionally heating and refluxing, filtering, and concentrating. The extraction solvent used in the present invention may be any solvent as long as it is usually used for extraction, and in particular, alcohols such as methanol and ethanol, hydrous alcohols, and organic solvents such as acetone and ethyl acetate ester may be used alone or in combination. It can also be extracted with water.

The amount of the extract of the Zingiber genus plant of the family Zingiberaceae in the present invention can be arbitrarily selected by those skilled in the art as long as it exerts its effect. For example, the blending amount is 0.00005 to 20.0% by mass as a dried product in the total amount of the external preparation. The upper limit of the blending amount is preferably 10% by mass or less, more preferably 5% by mass or less, from the viewpoint of formulation and effect. The lower limit of the blending amount is preferably 0.0001% by mass or more, more preferably 0.0005% by mass or more, from the viewpoint of exerting a sufficient skin barrier function enhancing effect.

As the plant of the genus Elephamtopus of the family Asteraceae used in the present invention, Elephantopus mollis (scientific name: Elephantopus mollis) is suitable. Elephantopus mollis is a plant native to Venezuela in South America and widely naturalized in tropical Asia. The extract of the Asteraceae Elephamtopus genus plant used in the present invention is obtained by immersing or heating and refluxing the leaves, stems, flowers, bark, seeds or fruits, whole plant plants, etc. of the above plant together with an extraction solvent. After that, it is obtained by filtering and concentrating. The extraction solvent used in the present invention may be any solvent as long as it is usually used for extraction, and in particular, alcohols such as methanol and ethanol, hydrous alcohols, and organic solvents such as acetone and ethyl acetate ester may be used alone or in combination. Can be done.

The amount of the extract of the plant belonging to the genus Elephamtopus of the family Asteraceae in the present invention can be arbitrarily selected by those skilled in the art as long as it exerts its effect. For example, the blending amount is 0.00005 to 20.0% by mass as a dried product in the total amount of the external preparation. The upper limit of the blending amount is preferably 10% by mass or less, more preferably 5% by mass or less, from the viewpoint of formulation and effect. The lower limit of the blending amount is preferably 0.0001% by mass or more, more preferably 0.0005% by mass or more, from the viewpoint of exerting a sufficient skin barrier function enhancing effect.

The pyridoxal phosphate used in the present invention is a substance used as a coenzyme of serine racemase. Pyridoxal phosphate is an active form of pyridoxal known as vitamin B ₆ compound. Although not intended to be limited to theory, pyridoxal phosphate can increase the activity of serine racemase as a coenzyme, resulting in D-serine acting on keratinocytes to act as a skin barrier. It is considered that the expression of genes involved in the function, such as filaggrin, cornulin, and lepetin, is enhanced, thereby enhancing the skin barrier function. Vitamin B ₆ contains pyridoxine and pyridoxamine in addition to pyridoxal, and is known to be a component to be added to cosmetics. However, it is a water-soluble vitamin, and its absorption rate and stability when administered transdermally. May have a problem. _{Therefore, vitamin B 6} derivatives having increased fat solubility for the purpose of improving transdermal absorption _{are known. For example, vitamin B 6} derivatives such as pyridoxine trishexyldecanoate, pyridoxine triparmitate, pyridoxine dipalmitate, and pyridoxine dicaprylate are also known. It included in the vitamin B ₆ class. In addition, pyridoxine cyclic phosphoric acid and pyridoxine hydrochloride are known for the purpose of improving stability. Not only pyridoxal phosphate, but also B vitamins ₆ are used as serine racemase activators or skin function enhancers. The blending amount of these is, for example, 0.000001 to 10% by mass. The upper limit of the blending amount is preferably 10% by mass or less, more preferably 1% by mass or less, from the viewpoint of formulation and effect. The lower limit of the blending amount is preferably 0.000001% by mass or more, more preferably 0.00001% by mass or more, from the viewpoint of exerting a sufficient skin barrier function enhancing effect.

In the step of determining the skin barrier function enhancing action of a candidate drug from the serine racemase expression level or activity or D-serine amount, the measured serine racemase expression level or activity and / or the D-serine amount is preset. It can be determined by using a table showing the relationship between the expression level or activity, and / or the D-serine level, and the barrier function enhancing action. Such a table, as a control, shows the amount of racemase expression or activity and / or the amount of D-serine, the amount of serine racemase expression or activity, and / or D measured by conducting the same experiment without the candidate drug. -It can be prepared using the amount of racemase expression or activity and / or the amount of D-serine measured by performing the same experiment with a control agent known to increase the amount of serine. In another aspect, a threshold value can be set in advance from the comparison with the control, and the skin barrier function enhancing effect of the candidate drug can be determined by whether or not the threshold value is statistically significantly higher than the threshold value. A person skilled in the art can appropriately set such a threshold value according to the desired skin barrier function enhancing action. As such a threshold value, for example, a threshold value of magnification can be arbitrarily set with respect to the expression level or activity of serine racemase and / or the amount of D-serine in a drug-free control, but for example, at 1.1 times or more. Values can also be set with statistically significant and trend differences. The statistically significant difference means when p <0.05, and the tendency difference means when p <0.1. In another aspect, as a control, the expression level or activity was higher than that of the racemase expression level or activity and / or the D-serine level measured by conducting the same experiment without the candidate drug. In some cases, it may be determined that the skin has a barrier function enhancing effect, and in that case, it can be determined that the higher the expression level or activity and / or the D-serine amount, the more the skin barrier function enhancing effect. ..

In another aspect, the present invention may relate to a method of evaluating, determining, or differentiating a skin barrier function using the serine racemase expression level or activity and / or the D-serine level as an index. Such evaluation, determination, or differentiation may be performed by measuring the D-serine content, serine racemase activity, or serine racemase expression level in a skin sample, for example, a stratum corneum sample obtained by tape stripping from a subject. .. The evaluation, judgment, or identification method of the skin barrier function is usually performed by measuring TEWL using a Vapomater or the like, but TEWL depends on the condition of the skin, the numerical value changes due to washing or friction, and further, skin diseases. In patients, there is not necessarily a correlation between the numbers and skin health. The evaluation, determination, or differentiation method of the present invention can measure the skin barrier function at the cellular level inherent in the skin by using the serine racemase activity or expression level and / or the D-serine level as an index. Is. Furthermore, since the evaluation method of the present invention determines the health of the skin barrier function at the cellular level, it makes it possible to predict the future skin barrier function rather than the current skin barrier function. Evaluation, determination, or differentiation of skin barrier function is determined using a preset chart or threshold value showing the relationship between the serine racemase expression level or activity and / or D-serine level and the barrier function enhancing action. be able to. Such charts or thresholds are the expression levels or activities obtained in skin samples collected and isolated from a group of subjects having healthy skin, a group of subjects having rough skin, and a group of subjects by age. , And / or can be set based on the amount of D-serine.

It was found that the addition of D-serine to cultured cells increased the expression of some genes. Among them, it was found that the expression of filaggrin, which plays an important role in the formation of barrier function, and cornulin and lepetin, which contribute to the formation of the cornified cell envelope (CE), are increased. Although not intended to be limited to theory, serine racemase activators or expression promoters screened according to the present invention can increase the D-serine content in keratinocytes, and D-serine can be found in keratinocytes. , It is considered that the skin barrier function is enhanced by enhancing the expression of proteins involved in the barrier function. Therefore, D-serine itself can also be used as a skin barrier function enhancer. In addition, D-serine can be said to be an expression promoter for the filaggrin gene, the cornulin gene, and the lepetin gene, and can also be called a keratinization promoter.

Filaggrin is a basic protein rich in histidine, arginine, serine, glycine, glutamic acid, etc., and has a function of depositing and aggregating on keratin fibers. As a result, the keratin fibers inside the keratinocytes aggregate, and the entire cell is flattened, changing from the stratum granulosum to the stratum corneum and becoming keratinized. Filaggrin is expressed as a precursor called profilaggrin in the granule cells of the epidermis, and when the granule cells are keratinized, it is decomposed into filaggrin by the action of proteolytic enzymes. Decreased filaggrin is considered to be a major cause of ichthyosis and atopic dermatitis, and these symptoms show a marked decrease in skin barrier function.

Cornulin and lepetin are proteins that make up the cornified cell envelope that lines the cell membrane of keratinocytes. Cornulin and lepetin are genes located at the chromosome position of 1q21, like proteins such as involucrin and loricrin, which are other constituents of the peripheral zone, and are expressed in the outermost layer of the epidermis at the final stage of keratinization. .. The proteins that make up the peripheral zone, along with keratin aggregated by the action of filaggrin, are involved in the barrier function of the skin.

According to the screening method of the present invention, the skin barrier function enhancer or the serine racemase activator may be incorporated into cosmetics or pharmaceuticals. Such cosmetics are used in subjects with reduced skin barrier function, such as subjects with symptoms such as atopic dermatitis, ichthyosis, psoriasis, and rough skin. In addition, when incorporated into a pharmaceutical product, the pharmaceutical product can be used as a pharmaceutical composition for the treatment of atopic dermatitis, ichthyosis, psoriasis and the like.

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to this.

Example 1: Verification of the relationship between the D-serine content in the stratum corneum and the barrier function The subjects were 57 volunteers whose test sites were healthy, and after washing the test site by a conventional method, the test site wet with washing was wiped, and the temperature was kept constant. The mixture was allowed to stand for 30 minutes under constant humidity conditions (room temperature 22 ° C., humidity 45%). Then, the transdermal water evaporation amount (TEWL) was measured by Vapometer (Delfin). The measurement was performed 3 times, and the average value was used for the subsequent analysis. Next, the stratum corneum at the position where TEWL was measured was peeled off twice using an adhesive tape, and the second sheet was extracted with amino acids in a 95% aqueous methanol solution. The optical isomers of the extracted amino acids were analyzed according to a previously reported method (reference: Yurika Miyoshi et al., Journal of Chromatography B, 877 (2009) 2506-2512). The outline is shown below. The extracted amino acids were fluorescently derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in borate buffer (pH 8.0) and trifluoroacetic acid. After making it acidic, it was subjected to two-dimensional micro HPLC (Shiseido). The first dimension is a micromonolith ODS column (inner diameter 0.53 mm, overall length 500, 750, 1000 mm, Shiseido), and the second dimension optical resolution is an optical resolution column with an inner diameter of 1.5 mm suitable for each amino acid. A mobile phase was used. NBD-amino acids were detected by fluorescence emission at 530 nm (excitation wavelength 470 nm). The results are shown in Table 1 and FIG.

In order to verify the relationship between the D-serine (D-Ser) content in the stratum corneum and the barrier function of the skin, each subject was arranged in order of TEWL value, and the top 17 subjects (barrier function subgroup) and the bottom 17 subjects (barrier function). The D-serine content in the stratum corneum of the upper functional group) was compared. Significant differences between the two groups were verified by non-paired Student's t-test. As a result, it was shown that humans with a high skin barrier had a significantly higher D-serine content. On the other hand, the results of the comparison of L-serine showed that the lower barrier function group tended to have a lower L-serine content and the upper barrier function group tended to have a higher L-serine content, but the significant difference was There wasn't. Therefore, it can be said that the high D-serine content in the upper barrier function group is not due to the amount of the precursor L-serine, but to the influence of racemase expression and activity.

Example 2: Changes in D-serine content associated with induction of keratinocyte differentiation Cell culture and drug treatment Human normal keratinocytes were placed in a 24-well multiplate so that the cell density was 2.5 × 10 ⁴ cells / well. Was cultivated in KGM medium (Kurabou) under the conditions of 37 ° C. and 5% CO _2. After 3 days, the cells were replaced with KGM medium supplemented with 1.8 mM calcium chloride and 10 mM L-serine (test group) or KGM medium supplemented with only 10 mM L-serine (control group), and cultured for another 2 days.

Sample preparation for HPLC and analysis of free D-serine by HPLC After washing the cells with PBS, add 500 μL of 5% trichloroacetic acid aqueous solution to suspend the cells well, collect them, sonicate them on ice for 30 minutes, and then centrifuge them. The supernatant was obtained. Amino acids were extracted by adding 20 times the amount of methanol to this supernatant. The optical isomers of the extracted amino acids were analyzed according to a previously reported method (reference: Yurika Miyoshi et al., Journal of Chromatography B, 877 (2009) 2506-2512). The outline is shown below. The extracted amino acids are fluorescently derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in borate buffer (pH 8.0) and trifluoroacetic acid. After making it acidic, it was subjected to two-dimensional micro HPLC (Shiseido). A micromonolith ODS column (inner diameter 0.53 mm, overall length 750 mm, Shiseido) is used for the first dimension, and an optically resolution column with an inner diameter of 1.5 mm and a mobile phase suitable for each amino acid are used for the second dimension optical resolution. There was. NBD-amino acids were detected by fluorescence emission at 530 nm (excitation wavelength 470 nm). The results of the test group and the control group are shown in FIG.

Example 3: Screening of candidate drugs Cell culture and drug treatment Human normal keratinocytes were seeded on a 24-well multiplate so that the cell density was 2.5 × 10 ⁴ cells / well, and KGM medium (Krabou) was used. ), The cells were cultured under the conditions of 37 ° C. and 5% CO _2. After 3 days, the medium was replaced with KGM medium containing 1.8 mM calcium chloride, 10 mM L-serine and a diluted solution of the candidate drug, and the cells were cultured for another 2 days. Seven kinds of drugs were used as candidate drugs, and 0.5% hydrolyzed oat protein solution was used as one of them.

Sample preparation for HPLC and analysis of free D-serine by HPLC After washing the cells with PBS, add 500 μL of 5% trichloroacetic acid aqueous solution to suspend the cells well, collect them, sonicate them on ice for 30 minutes, and then centrifuge them. The supernatant was obtained. Amino acids were extracted by adding 20 times the amount of methanol to this supernatant. The optical isomers of the extracted amino acids were analyzed according to a previously reported method (reference: Yurika Miyoshi et al., Journal of Chromatography B, 877 (2009) 2506-2512). The outline is shown below. The extracted amino acids are fluorescently derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in borate buffer (pH 8.0) and trifluoroacetic acid. After making it acidic, it was subjected to two-dimensional micro HPLC (Shiseido). A micromonolith ODS column (inner diameter 0.53 mm, overall length 750 mm, Shiseido) is used for the first dimension, and an optically resolution column with an inner diameter of 1.5 mm and a mobile phase suitable for each amino acid are used for the second dimension optical resolution. There was. NBD-amino acids were detected by fluorescence emission at 530 nm (excitation wavelength 470 nm). The results are shown in Table 2 and FIG. 3A below.

Quantitative PCR (RT-PCR)
After washing the cells with PBS, mRNA was extracted from the cells using MagNA Pure LC (Roche Diagnostics). Next, cDNA was synthesized using a kit of reverse transcriptase SuperScriptVILO (Invitrogen). Using this cDNA as a template, real-time PCR was performed using the fluorescent dye CyberGreen with Light Cycloner (Roche Diagnostics). In the PCR reaction, the enzyme was activated at 95 ° C. for 10 minutes, and then the amplification reaction at 95 ° C. for 15 seconds, 60 ° C. for 10 seconds, and 72 ° C. for 10 seconds was repeated 30 times. The gene expression level was calculated as a relative value from the number of cycles until a certain amount of PCR product was obtained. In addition to the serine racemase gene, RT-PCR was also performed on the G3PDH (glyceraldehyde-3-phosphate) gene as an internal standard, and the drug was evaluated by correcting the relative expression level of serine racemase with the relative expression level of G3PDH. As the primers used in RT-PCR, those described in Non-Patent Document 3 and Non-Patent Document 4 were selected and used. The results are shown in Table 2 and FIG. 3B below.

Among the candidate drugs used in this screening, hydrolyzed oat protein solution was shown to enhance the expression of serine racemase in the differentiation-induced keratinocytes. Furthermore, it was also shown that the hydrolyzed oat protein solution increased the D-serine content in the differentiation-induced keratinocytes. Based on these results, the hydrolyzed oat protein solution can be selected as an effective drug for enhancing the skin barrier function.

Example 4: Screening of candidate drugs Cell culture and drug treatment Human normal keratinocytes were seeded on a 24-well multiplate so that the cell density was 2.5 × 10 ⁴ cells / well, and KGM medium (Krabou) was used. ), The cells were cultured under the conditions of 37 ° C. and 5% CO _2. After 3 days, the medium was replaced with KGM medium containing 1.8 mM calcium chloride, 10 mM L-serine and a diluted solution of the candidate drug, and the cells were cultured for another 2 days. Twelve kinds of drugs were used as candidate drugs, and among them, 0.5% Lumpuyan extract and 0.5% Elephantopus mollis extract were included.

Quantitative PCR (RT-PCR)
After washing the cells with PBS, mRNA was extracted from the cells using MagNA Pure LC (Roche Diagnostics). Next, cDNA was synthesized using a kit of reverse transcriptase SuperScriptVILO (Invitrogen). Using this cDNA as a template, real-time PCR was performed using the fluorescent dye CyberGreen with Light Cycloner (Roche Diagnostics). In the PCR reaction, the enzyme was activated at 95 ° C. for 10 minutes, and then the amplification reaction at 95 ° C. for 15 seconds, 60 ° C. for 10 seconds, and 72 ° C. for 10 seconds was repeated 30 times. The gene expression level was calculated as a relative value from the number of cycles until a certain amount of PCR product was obtained. In addition to the serine racemase gene, RT-PCR was also performed on the G3PDH (glyceraldehyde-3-phosphate) gene as an internal standard, and the drug was evaluated by correcting the relative expression level of serine racemase with the relative expression level of G3PDH. As the primers used in RT-PCR, those described in Non-Patent Document 3 and Non-Patent Document 4 were selected and used. The results are shown in Table 3 below.

Among the candidate drugs used in this screening, Lumpuyan extract and Elephantopus mollis extract were shown to enhance the expression of serine racemase in the differentiation-induced keratinocytes. Based on this result, Lumpuyan extract and Shirobanai gakouzorina extract can be selected as effective agents for enhancing the skin barrier function.

Example 5: Screening of candidate drugs
Cell culture and drug treatment Human normal keratinocytes were seeded on a 24-well multiplate so that the cell density was 2.5 × 10 ⁴ cells / well, and the conditions were 37 ° C. and 5% CO _{2 in KGM medium (Kurabou).} Cultured below. After 3 days, the medium was replaced with KGM medium containing 1.8 mM calcium chloride and cultured for 2 days, and then replaced with KGM medium containing 1.8 mM calcium chloride and a diluted solution of pyridoxal phosphate (PLP) as a candidate drug for another 2 days. It was cultured.

Sample preparation for HPLC and free D-serine analysis by HPLC After washing cells cultured in pyridoxal phosphate-added medium with PBS, 500 μL of 5% trichloroacetic acid aqueous solution was added to suspend and collect the cells well, and the cells were collected on ice for 30 minutes. After ultrasonic treatment, a supernatant was obtained by centrifugation. Amino acids were extracted by adding 20 times the amount of methanol to this supernatant. The optical isomers of the extracted amino acids were analyzed according to a previously reported method (reference: Yurika Miyoshi et al., Journal of Chromatography B, 877 (2009) 2506-2512). The outline is shown below. The extracted amino acids are fluorescently derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in borate buffer (pH 8.0) and trifluoroacetic acid. After making it acidic, it was subjected to two-dimensional micro HPLC (Shiseido). A micromonolith ODS column (inner diameter 0.53 mm, overall length 750 mm, Shiseido) is used for the first dimension, and an optically resolution column with an inner diameter of 1.5 mm and a mobile phase suitable for each amino acid are used for the second dimension optical resolution. There was. NBD-amino acids were detected by fluorescence emission at 530 nm (excitation wavelength 470 nm). The results of the concentration for D-serine are shown in FIG. 4 below. PLP, which is a coenzyme of serine racemase, is a drug capable of activating serine racemase, and can enhance the skin barrier function through activation of serine racemase.

Example 6: Effect of D-serine on the skin
Three-dimensional culture of epidermal keratinocytes Human normal keratinocytes were seeded on a polycarbonate insert (Millipore) with a diameter of 12 mm and a pore size of 0.4 μm, and CnT-Prime and Epithelial Culture Medium (CELLnTEC Advanced Cell Systems AG) were placed inside and outside the insert. In addition, after culturing in a 5% CO ₂ atmosphere at 37 ° C. for 3 days, the cells were replaced with Cnt-prime 3D Barrier medium (CELLnTEC Advanced Cell Systems AG) and cultured for 24 hours. Then, the medium inside the insert was removed, and the cell surface was exposed to air by adding a medium containing D-Ser at a concentration of 100 μM outside the insert, or Cnt-prime 3D Barrier medium without D-Ser as a control. It was cultured in the state. After changing the medium once every two days and culturing for 7 days, the membrane of the insert was cut out with a scalpel and immersed in a Trizol reagent (Invitrogen) to extract total RNA.

内部標準であるミトコンドリアリボソームタンパク質Ｌ１９遺伝子（MRPL19）の発現量で補正して各群の標的遺伝子の相対発現量を求めた（図５〜７）。これらの遺伝子では、Ｄ−Ｓｅｒの添加により、遺伝子発現が有意差をもって増加した。 Gene expression analysis by real-time PCR method
For total RNA extracted using Trizol reagent (Invitrogen), RNA concentration was measured using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific), and then cDNA was prepared using SuperScriptVILO (Invitrogen). Using this cDNA as a template in the Step One Real-Time PCR System (Applied Bioscience) using the Platinum SYBER Green qPCR SuperMix-UDG (Invitrogen), filaggrin (FLG), cornulin (CRNN), repetin (RPTN) genes, In addition, the relative expression level of the mitochondrial ribosome protein L19 (MRPL19) as an internal standard gene was measured. The primers used are:

The relative expression level of the target gene in each group was determined by adjusting for the expression level of the mitochondrial ribosomal protein L19 gene (MRPL19), which is an internal standard (FIGS. 5 to 7). In these genes, the addition of D-Ser significantly increased gene expression.

Claims (10)

A method for screening a skin barrier function enhancing drug using the activity or expression level of serine racemase and / or the amount of D-serine as an index in keratinocytes. below:
The process of adding a candidate drug to keratinocytes,
A step of measuring the activity or expression level of serine racemase and / or the amount of D-serine in keratinocytes.
The screening method according to claim 1, further comprising a step of determining the skin barrier function enhancing action of the candidate drug from the expression level or activity of serine racemase and / or the amount of D-serine. The screening method according to claim 2, further comprising a step of inducing differentiation of keratinocytes. The screening method according to claim 1, wherein the screening method is performed on differentiation-induced keratinocytes. The screening method according to any one of claims 1 to 4, wherein the serine racemase expression level is measured by measuring the amount of serine racemase mRNA or protein. The screening method according to any one of claims 1 to 4, wherein the measurement of serine racemase activity is by measuring the conversion efficiency of a substrate by serine racemase or measuring the amount of D-amino acid product. A method for evaluating skin barrier function using serine racemase activity or expression level and / or D-serine level as an index in a stratum corneum sample isolated from human skin. below:
A step of measuring serine racemase activity or expression level and / or D-serine level in a stratum corneum sample isolated from a subject.
The method for evaluating a skin barrier function according to claim 7, which comprises a step of determining the barrier function of the target skin from the serine racemase activity or expression level and / or the D-serine amount. The method for evaluating a skin barrier function according to claim 7 or 8, wherein the serine racemase expression level is measured by measuring the amount of serine racemase mRNA or protein. The method for evaluating the skin barrier function according to claim 7 or 8, wherein the measurement of serine racemase activity is by measuring the conversion efficiency of a substrate by serine racemase or measuring the amount of D-amino acid product.

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