JP6072404B2 - Screening or evaluation method for epidermal barrier function - Google Patents

Description

  The present invention relates to a screening or evaluation method for an epidermal barrier function regulator.

  When the epidermal barrier function is disrupted, it is considered that the skin is likely to dry, and that the abnormality of the immune system is caused and the onset and symptoms of atopic dermatitis are likely to progress. In addition, since skin is a part that attracts attention in terms of beauty and health, it is important to determine what kind of substance is involved in the adjustment of the epidermal barrier function.

In the screening of such epidermal barrier function regulators, model animals have been used in the past, but in order to evaluate many test substances, a three-dimensional skin model is used as an alternative to animal experiments from the viewpoint of animal welfare. Have been used (Non-Patent Documents 1 and 2).
For example, a method for evaluating the influence of ultraviolet rays using a three-dimensional cultured skin model capable of producing melanin obtained by seeding pigment cells or melanocytes (Patent Document 1); using human normal epidermal cells For example, a screening method for a skin barrier function-enhancing material (Patent Document 2) using the permeability of metal ion, particularly calcium, as an index is known.

  However, the expression mechanism of the epidermal barrier function is complex, so the knowledge of which point to focus on when screening and evaluating epidermal barrier function regulators using a three-dimensional skin model is still not sufficient. It is hard to say that it is obtained.

JP 2010-193822 A JP 2010-511376 A

Perkins M et al, Toxicological Science, 48, 218-229, 1999 Cannon CL, Toxicology in vitro, 8, 889-91, 1994

  In view of such a situation, the present invention provides an evaluation tool related to skin barrier function adjustment in a three-dimensional skin model and a method of using the evaluation tool.

  As a result of intensive studies, the inventors have changed the epidermal barrier function of the three-dimensional skin model, and the expression level of ABCA12 and the distribution state of glucosylceramide change. Therefore, these can be used as screening and evaluation tools. The present invention has been completed by finding out what can be done.

That is, the present invention relates to the following [1] to [ 4 ].
[1] A comparison process that compares the reaction when a test substance is added to a three-dimensional skin model (excluding those collected from experimental animals) and the reaction when no test substance is added, and each Compare ABCA12 distribution of expression levels and glucosylceramide in the reaction as an indicator, and a ABCA12 expression level when ABCA12 expression levels upon addition of the test substance after addition of known epidermal barrier dysfunction agent was not added test substance And when the test substance is added compared to the state observed to aggregate around the cell nucleus of granule cells, which is the distribution state of glucosylceramide when the test substance is not added If the distribution of glucosylceramide Ru continuously observed in the granular layer upper portion, the test substance has an improving effect epidermal barrier function, addition of the test substance When the expression level of ABCA12 is decreased compared to the expression level of ABCA12 when the test substance is not added, and around the nucleus of the granule cell, which is the distribution state of glucosylceramide when the test substance is added compared aggregated in a state of being observed in a case where distribution of glucosylceramide when not adding the test substance Ru are continuously observed in the granular layer upper portion, the test substance is epidermal barrier dysfunction A screening method characterized by selecting a substance having an epidermal barrier function improving action or a substance having an epidermal barrier function disorder action from the test substance group by performing at least a determination step for determining that the substance has an action , or commentary the test substance you and evaluating whether having improved action or epidermal barrier dysfunction act epidermal barrier function Method.
[2] After the addition of the test substance, within ~ 2 months after the addition, methods towards the [1], wherein a plurality of times said comparison step and determining step.

[3] and three-dimensional skin model for which the test substance is added to a three-dimensional skin model without the addition of test substance (except those collected from the experimental animals) with the control and which is, of the three-dimensional skin model 3D skin model after adding a known epidermal barrier dysfunction agent to the 3D skin model and the control , using as an index the contrast process for comparing the reaction with the control reaction, and the ABCA12 expression level and glucosylceramide distribution in each reaction The ABCA12 expression level in the case is increased compared with the ABCA12 expression level in the control, and is compared with the state observed by aggregation around the cell nucleus of the granule cell, which is the distribution state of the control glucosylceramide. If distribution of glucosylceramide original skin model Ru continuously observed in the granular layer upper part is the three-dimensional skin Recovered Dell epidermal barrier function, of granule cells in the case ABCA12 expression level in the three-dimensional skin model was reduced compared to ABCA12 expression level in a control, and the distribution state of glucosylceramide three-dimensional skin model If distribution of glucosylceramide control as compared to the state which has been observed in aggregate around the cell nucleus Ru continuously observed in the granular layer upper portion, epidermal barrier function of the three-dimensional skin model is impaired A screening method characterized by selecting a three-dimensional skin model suitable for evaluating the epidermal barrier function with high sensitivity from a group of three-dimensional skin models by performing at least the determination step of determining evaluation how to characterized in that the skin model to assess whether it is appropriate to sensitively evaluate the epidermal barrier function.
[4] After adding the test substance to the three-dimensional skin model, within ~ 2 months after the addition, methods towards the [3], wherein a plurality of times said comparison step and determining step.

  According to the present invention, it is possible to provide an evaluation tool related to epidermal barrier function adjustment in a three-dimensional skin model and a method of using the evaluation tool.

Cross-sectional image observation of 3D skin model. Expression state of ABCA12 in a three-dimensional skin model. Expression staining image of glucosylceramide in a three-dimensional skin model. Cross-sectional image observation of SLS-added and non-added three-dimensional skin model (1, 7, 21 day follow-up observation). Expression state of ABCA12 in a three-dimensional skin model with and without SLS (1, 7, 21 day follow-up). Expression staining images of glucosylceramide in a three-dimensional skin model with and without SLS (1, 7 and 21 day follow-up). Cross-sectional image observation of a three-dimensional skin model with and without calcein added (7, 21 days follow-up observation).

[Three-dimensional skin model]
The three-dimensional skin model used in the method of the present invention is not particularly limited, and examples thereof include three-dimensional cultured skin and organ cultured skin. In addition, the organ culture skin can include those in which the skin containing the organ or the organ in the organ is taken out and cultured, and those derived from this include those derived from epidermis cells described later. it can.
As the three-dimensional skin model, a commercially available one can be used. Commercially available products include, for example, Epiderm (trademark: manufactured by MatTek Corporation), EPISKIN (registered trademark: Skin)
Ehic Laboratories).
The three-dimensional skin model can also be created by a known method. For example, it is also possible to use what was produced by Japanese Patent Application No. 2010-252818 description and a well-known method (For example, Unexamined-Japanese-Patent No. 2005-168398 etc.).

Examples of the method for producing the three-dimensional skin model include, for example, seeding epidermal keratinocytes on a liquid-permeable membrane of a culture insert and, if necessary, producing by combining immersion culture, gas-liquid culture, and the like. It is done. The epidermal cells to be seeded at this time are preferably those grown (initially) to about 5 × 10 ^{2 to} 5 × 10 ⁶ cells / cm ² . In addition, dermal fibroblasts can be added to create a three-dimensional skin model.

The epidermal cells are not particularly limited, and examples thereof include those derived from mammals such as humans, pigs, mice, rats, guinea pigs, hamsters, cows, and rabbits.
Further, the epidermal cells may be collected from epidermal keratinocytes of the skin (preferably human newborn foreskin), epithelial cells of the oral mucosa, corneal cells of the eye, and the like. Among these, when considering the human skin condition, those derived from humans are preferred.
The liquid-permeable membrane is not particularly limited as long as the seeded epidermal cells are adhered or fixed, and the epidermal cells can be grown thereon and can serve as a support. For example, base materials, such as a polycarbonate, a polyethylene terephthalate, a polystyrene, are mentioned. Further, the base film may be coated with an extracellular matrix such as collagen, laminin, fibronectin or the like, or a substance that assists cell adhesion such as poly L-lysine.
A culture vessel such as a culture insert for producing a three-dimensional skin model may be used. Examples of the support used in addition to the culture insert include collagen gel, collagen sponge, acellularized dermis from which epidermal cells have been removed, and those incorporating dermal fibroblasts as feeder cells.

As an example of the three-dimensional skin model preparation method, a more detailed description will be given below, but the method is not limited thereto.
Examples thereof include a three-dimensional cultured skin preparation method including at least a growth culture step and a differentiation induction step.
For example, the growth culture step includes culturing in a culture growth medium under 5% CO ₂ concentration for about 1 to 14 days until epidermal cells become confluent on the bottom surface of the culture insert.
In the differentiation induction step, the culture growth medium is changed to a cell differentiation medium, and epidermal cells are cultured (immersed) for about 6 to 48 hours in the culture medium. ) And culturing for about 0.5 to 3 days to induce differentiation of cultured epidermal cells.

Note that the three-dimensional cultured skin preparation method may include an initial growth step before the growth culture step.
As an initial growth step, the prepared cell suspension of epidermal cells 10 ³ to 10 ⁵ cells / mL is subconfluent with respect to a petri dish in a culture growth medium under a wet condition of 5% CO _2. For example, it is allowed to grow until it becomes confluent.

Here, as the culture growth medium, for example, MCDB153 or a commercial product based on this may be used. The calcium concentration of this medium should just be about 0.05-0.1 mM.
In addition, as the cell differentiation medium, for example, a commercial product such as a medium containing about 1.2 to 1.5 mM calcium may be used.
The culture temperature at this time should just be the temperature which can be culture | cultivated corresponding to origin, for example, in the case of human origin, 20 degreeC-40 degreeC, Preferably it should just be about 37 degreeC.
The oxygen concentration is preferably cultured in an atmosphere of 3 to 25%, more preferably 18 to 22%.

[Screening or evaluation method of epidermal barrier function regulator]
After adding a test substance to the three-dimensional skin model, the epidermal barrier function of the test substance using as an index a screening and evaluation tool of ABCA12 expression level and / or distribution state of glucosylceramide (hereinafter referred to as “evaluation tool”). Determine the adjustment.
Specifically, the screening or evaluation method of the present invention includes a step of checking the reaction of the evaluation tool by the test substance and a determination step of determining the adjustment of the epidermal barrier function of the test substance.
At this time, it is preferable to make the determination by comparing with the response of the evaluation tool by the control (for example, addition of a control substance), because more accurate screening or evaluation can be performed.

As an example, (1) a comparison process for comparing the response of the evaluation tool when the test substance is added to the three-dimensional skin model and the response of the evaluation tool when the control substance is added to the three-dimensional skin model, and this control A screening or evaluation method including a determination step of determining adjustment of the epidermal barrier function that the test substance exerts on the three-dimensional skin model using the reaction by the substance as a determination criterion is included.
In addition, (2) a comparison process for comparing the response of the evaluation tool before or after the addition of the test substance with the response of the evaluation tool after the addition of the test substance, A screening or evaluation method including a determination step of determining adjustment of the epidermal barrier function to the original skin model. Here, the term “no test substance added” includes a case where PBS (Phosphate Buffered Saline) containing no test substance is added.
In the comparison process, the reaction measurement process of the evaluation tool with the test substance and the reaction measurement process of the evaluation tool with the known epidermal barrier function regulator (control substance) may be performed simultaneously in parallel. It may be done separately. A reaction result of a known epidermal barrier function regulator may be obtained in advance or later, and the obtained reaction result by the control substance may be compared with the reaction result by the test substance.
As the control substance, a known epidermal barrier function adjusting agent, preferably a substance whose function is well known, is preferably used because it is easy to determine the epidermal barrier function adjustment of the test substance.
In order to improve accuracy, it is desirable to use the control substance under the same culture conditions as when the test substance is used.

  As an example of more specific determination of epidermal barrier function adjustment, the response of the evaluation tool when using a known epidermal barrier function inhibitor such as sodium lauryl sulfate (SLS), and the evaluation tool when using a test substance In the case where the responses are almost identical, it is determined that the test substance has an inhibitory effect on the epidermal barrier function.

The test substance is not particularly limited and is a known or novel substance (for example, an extract, an organic compound, etc.); a blend or composition of these substances; a pharmaceutical, a cosmetic, a skin external preparation, a quasi-drug And the like (for example, known epidermal barrier function regulators) and the like used for the purpose. By using the evaluation tool of the present invention, knowledge about the epidermal barrier function of this test substance with respect to a three-dimensional skin model (particularly, three-dimensional cultured skin) can be obtained. This enables screening or evaluation of the epidermal barrier function regulator.
In addition, as the control substance, it is preferable to use a substance whose skin barrier function adjustment is already known so as to be a criterion for determination according to the screening or evaluation of the target skin barrier function regulator.

In general, the “epidermal barrier function” is said to be a function of the epidermis that prevents invasion of substances, microorganisms, and the like from outside the body and transpiration of moisture from the body through the skin.
Here, if the epidermis barrier function is “good”, a beautiful and healthy state is maintained, and specifically, there is a state in which the skin has high moisture retention and is well-textured. When the epidermis barrier function is “abnormal (decreased)”, it means that the skin is not in a healthy state, and examples thereof include dry skin and rough skin caused by the skin.

  If an evaluation tool for easily determining the above-mentioned skin barrier function can be found, it is possible to easily search for the mechanism of the skin barrier function, and use this evaluation tool to adjust the skin barrier function. Substances and substances containing the same (for example, materials and raw materials such as extracted / purified products and organic synthetic products; products using raw materials and raw materials such as cosmetics, quasi-drugs and topical skin preparations) (hereinafter referred to as “ Screening and evaluation of “skin barrier function regulator” are also facilitated.

  Here, the “skin barrier function regulator (substance related to skin barrier function regulation)” may be any substance that has some effect on the skin barrier function and contains it. For example, substances that have a positive effect (including maintaining the skin barrier function) include an epidermal barrier function improving agent, an epidermal barrier function promoter, an epidermal barrier function maintaining agent, an epidermal barrier function restoring agent, and an epidermal barrier function. Inhibitors that block the substances exerted can be mentioned. Examples of substances that have a bad influence include epidermal barrier dysfunction agents and epidermal barrier function recovery inhibitors.

And, as shown in the following [Examples], even when this form observation is performed when a substance capable of inducing epidermal barrier dysfunction is added to the three-dimensional cultured skin, the change in epidermal barrier function is observed. Could not be judged easily. (See FIG. 1).
Furthermore, changes in the epidermal barrier function can be easily determined by using the gene expression levels of Keratin 14, Keratin 10, and Involucrin, which are commonly used when evaluating epidermal function, as an index, or by morphological observation using these stains. could not.
In contrast, when a substance capable of inducing epidermal barrier dysfunction was added to the three-dimensional cultured skin and cultured, attention was paid to glucosylceramide. As a result of immunostaining, there was a change in the distribution state of this glucosylceramide. It was recognized that this glucosylceramide and ABCA12 gene expression level had a correlation (FIGS. 2 and 3).

Incidentally, in the process of epithelial cell metabolism, lamellar granules containing glucosylceramide are formed in the granule layer. When this granule layer moves to the stratum corneum, lamellar granules are secreted and glucosylceramide is metabolized to ceramide. This ceramide plays a role in maintaining the epidermal barrier function as an intercellular lipid. Therefore, it was considered that the distribution state of glucosylceramide and the level of ABCA12 expression directly affected the epidermal barrier function.
According to conventional knowledge, ABCA12 has been reported as a lipid transport protein for lamellar granules in the epidermis. However, when an external stimulus is applied to the epidermal barrier function (for example, the barrier dysfunction is induced from the outside). It was completely unclear how they were involved.

Thus, it was found that the epidermal barrier function can be easily determined (evaluated) using the distribution state of glucosylceramide and the expression level of ABCA12 as an index.
Therefore, the method of the present invention uses ABCA12 expression level and / or glucosylceramide distribution as an evaluation tool.
That is, after adding a test substance to a three-dimensional skin model, it is possible to screen or evaluate an epidermal barrier function regulator by using ABCA12 expression level as an index and / or indexing the distribution state of glucosylceramide. is there.
Further, the number of times of addition is not particularly limited, and may be one or more times depending on the purpose. In the case of multiple times, the same or different test substances may be added. Moreover, when another test substance is further added, these test substances may be added simultaneously or separately.

The timing for determining the adjustment of the epidermal barrier function of the test substance is not particularly limited, but it is preferable that the test substance be performed at regular intervals or after a lapse of a certain period within the culture period after addition of the test substance. For example, it is preferable to perform measurement and determination within a culture period immediately after the addition of the test substance to 2 months (preferably 1 day to 4 weeks).
More specifically, in culture for 1 day to 2 months (preferably culture for 1 day to 4 weeks), for example, irregular intervals such as the 1st day, 1st week and 3rd week, etc. Measurement is performed at regular intervals; measurement is performed after 3 days to 2 weeks have elapsed after the addition, preferably after 5 to 9 days.

In addition, as shown in [Examples] below, after adding a known epidermal barrier function regulator (eg, epidermal barrier dysfunction agent (SLS, etc.)), after about 3 weeks, the expression level of ABCA12 and glucosylceramide It was confirmed that the distribution state of was almost the same as that without addition (control) (see FIGS. 4 to 7). Specifically, it was confirmed with an ABCA12 expression level and glucosylceramide distribution evaluation tool that a certain period of time passed after the addition of SLS and the state of the three-dimensional cultured skin was restored to a normal state. Therefore, using the evaluation tool, after adding the test substance to the 3D epidermis model, the same or another test substance is added again, and when the test substance's epidermal barrier function is restored to normal, It is possible to evaluate when returning to the state.
In addition, by using these evaluation tools, it is possible to grasp the recovery period of the epidermal barrier function. It is also possible to evaluate the test substance during long-term use or continuous use by adding it once or multiple times.

That is, by adding a known epidermal barrier function regulator and a test substance to a three-dimensional cultured skin one or more times simultaneously or separately, screening of the epidermal barrier function regulator from the test substance and the epidermal barrier function of the test substance Can be evaluated.
For example, if a known epidermal barrier function modifier is a substance that adversely affects the epidermal barrier function, and if it is positively evaluated that the epidermal barrier function has been maintained, improved or recovered after the addition of the test substance, the test substance can be recovered. It can be determined as an agent for controlling the skin barrier function, such as an agent; an epidermal barrier function promoter; an inhibitor for blocking the action of the skin barrier function inhibitor.
On the other hand, if the known epidermal barrier function regulator is a substance that has a good effect on the epidermal barrier function, and the test substance is negatively evaluated that the epidermal barrier function is maintained, decreased or improved after the test substance is added, It can be determined as an epidermal barrier function regulator, such as an inhibitor that inhibits the action of an epidermal barrier function promoter.
In addition, the addition timing of a known epidermal barrier function regulator and a test substance may be either simultaneous or separate. When evaluating the recovery of the epidermal barrier function, a test substance can be added to a three-dimensional cultured skin model that has adversely affected the epidermal barrier function with a drug or the like.
Thus, even if the state of the epidermal barrier function of the three-dimensional skin model is changed by a drug or the like, the change state and the subsequent progress state are indicated with the ABCA12 expression level as an index and / or the distribution state of glucosylceramide as an index. By doing so, there is an advantage that it is easy to screen and evaluate the epidermal barrier function regulator.

Therefore, using the evaluation tool of the present invention, a known epidermal barrier function regulator is added, and before changing the epidermal barrier function, in the middle of the change or after being changed, the test substance is added, and ABCA12 It is possible to evaluate the epidermal barrier function by comparing the expression level, the distribution state of glucosylceramide, and the period until recovery. It is also possible to screen a desired epidermal barrier function regulator from a test substance.
As a specific example, a known epidermal barrier function regulator is added to a three-dimensional cultured skin once or a plurality of times, followed by culturing with a test substance added once or a plurality of times, and the ABCA12 expression level and / or Alternatively, the skin barrier function of these test substances can be evaluated using the distribution state of glucosylceramide as an index.
Controls at this time include those with no known epidermal barrier function regulator and test substance added; those with a known epidermal barrier function regulator added and no test substance added; those with only PBS added, etc. Can be selected as appropriate.
Further, after adding the test substance, a period in which the above-mentioned control and ABCA12 expression level and / or glucosylceramide distribution state are almost equivalent is obtained, and this is used as a recovery period, and the epidermal barrier function is evaluated by the length of this period. It is also possible.

As described above, after a certain period of time, the state of the three-dimensional skin model recovers to almost the same state as before the addition of a known epidermal barrier function regulator and / or test substance or a control (such as PBS). After the substance is added, the same or different test substance may be added to evaluate the ability of the test substance to adjust the epidermal barrier function.
In the case of the same test substance, it is possible to evaluate the epidermal barrier function (for example, decrease in sensitivity) when repeated. In the case of another test substance (different test substances), it is possible to evaluate the epidermal barrier function such as synergistic effects and reciprocal effects between the test substances.
The culture method after adding the above-mentioned test substance or epidermal barrier function regulator may be performed by a known culture method for maintaining a three-dimensional skin model.

[ABCA12 expression level]
When determining the ABCA12 expression level as an index, for example, the epidermal barrier function may be determined based on the expression level of ABCA12 (preferably, the expression level of a gene such as mRNA or the expression level of a polypeptide or the like).
When the expression level of ABCA12 is maintained or increased compared to the control expression level of ABCA12, the epidermal barrier function is good; when it is decreased, the epidermal barrier function is abnormal (decreased). It is possible to determine that there is. At this time, the state in which the skin barrier function is good includes maintaining the skin barrier function.
Examples of the “control” used as a judgment criterion at this time include a state before addition of the test substance and a state where it is not added; a state where the control substance is added: a state where a solvent is added, and the like.

By determining in this way, it is possible to acquire a desired epidermal barrier function regulator, and it is also possible to perform evaluation. For example, when ABCA12 expression level is greatly increased, it can be determined as an agent (substance) having a strong pharmacological action for improving the epidermal barrier function. For example, when the PBS containing no test substance is taken as a control and this is taken as 100%, it is preferable to set the determination criterion as 120% or more, preferably 150% or more.
Moreover, when ABCA12 expression level is decreasing very much, it can determine with the agent (substance) which has strong pharmacological action of epidermal barrier function inhibition. For example, when the control is 100%, it is preferable to set the determination standard as 50% or less, preferably 20% or less.
Moreover, when ABCA12 expression level hardly changes, it can determine with an agent (substance) without an epidermis barrier function adjustment effect | action. For example, when the control is 100%, ± 20%, preferably ± 10% is suitable.

In addition, the test substance can be evaluated by comparing the recovery amount of the expression level of ABCA12.
For example, an epidermal barrier dysfunction agent is added to a three-dimensional skin model; a test substance is added simultaneously or after a certain period of time, and the expression level of ABCA12 is measured. At this time, with respect to the expression level of ABCA12, when the value of only the barrier dysfunction agent is set to 100%, a criterion is set that there is an effect of recovering the epidermal barrier function if the expression is 120% or more, preferably 150% or more. It is preferable to do this. In addition, when the value of the substance that does not contain a barrier dysfunction agent (control: PBS) (the culture period is the same) is 100%, the closer it is (preferably 80% or more, more than 90%), A determination criterion may be set.
In addition, the test substance can be evaluated by comparing the period until the ABCA12 expression level recovers. For example, an epidermal barrier dysfunction agent is added to a three-dimensional skin model; a test substance is added in parallel or after a certain period of time, and the period (days, hours) until the ABCA12 expression level recovers is measured. In this case, when the recovery period (number of days) when the test substance is not added is 100%, it can be evaluated as an agent for recovering epidermal barrier function when the period is shortened by 10% or more, preferably 30% or more. is there.

Here, ABCA12 expression level (preferably gene expression level such as mRNA, expression level of polypeptide, etc.), which is the above-described evaluation tool for skin barrier function, is measured by ABCA12 gene or protein encoded thereby ( Amino acid sequence) may be used.
Examples of these genes include the human ABCA12 gene (26154, NCBI).
At this time, a base sequence (DNA, RNA, etc.) relating to these genes, a protein (amino acid sequence) encoded by this gene, and each of these fragments (polynucleotide fragments, polypeptide fragments, etc.) may be used as indicators.
Among these, it is preferable to use, for example, the base sequences shown in SEQ ID NOs: 1 and 2 (the ABCA12 gene-derived primer) as an index because the ABCA12 expression level can be stably and accurately measured. In order to correct this expression level, it is preferable to use a housekeeping gene.

  The base sequence or amino acid sequence may be partially modified by treatment with a mutagen, random mutation, specific site mutation, deletion or insertion, unless the accuracy and sensitivity of ABCA12 expression level measurement are reduced. May have changed. For example, a base sequence or amino acid sequence in which one or several (for example, 2 to 3) bases or amino acids are substituted, deleted, or added can be used. The addition includes addition to both ends. Here, “one or several” means 1 to 9, preferably 1 to 5, more preferably about 1 to 3.

  Examples of the method for measuring the ABCA12 expression level include those using the above-described ABCA12 gene, the protein (amino acid sequence) encoded by the gene, and fragments thereof and using a known measurement method. As this known measurement method, for example, measurement method using mRNA such as PCR method, isothermal amplification method, Northern blotting method; measurement using antibody such as flow cytometry (FACScan), immunoblotting, Western blotting, antibody staining method Law, etc., and these may be combined as appropriate. At this time, a labeling substance such as a fluorescent substance, a luminescent substance or a radioactive substance may be used as necessary.

The method for measuring mRNA is not particularly limited. For example, a primer set or a DNA fragment designed based on the base sequence of the gene to be measured may be used.
An example of a method for measuring the amount of mRNA will be described below.
Total mRNA may be extracted from the three-dimensional skin model according to a known method, and may be further purified to mRNA alone if necessary. In this step, a commercially available kit may be used.
Examples of the total mRNA extraction method include guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, guanidine thiocyanate / phenol / chloroform method, and the like.
Using the obtained total RNA or purified mRNA as a template, cDNA is synthesized by reverse transcription reaction from this, and a part of the gene to be measured by PCR using two kinds of DNA fragments complementary to the nucleotide sequence to be measured as primers. By amplifying the sequence and detecting the amplified cDNA, this cDNA reflects the above-mentioned gene expression level (mRNA level) of the three-dimensional skin model. The detection method at this time is not particularly limited as long as it is a known method, and examples thereof include a method using SYBR green and a method using a fluorescent probe, and it is desirable to quantify by real-time PCR. In this step, a commercially available kit may be used.

The primer to be used can be designed and synthesized so that the base sequence of the gene to be measured can be amplified by a nucleic acid amplification method such as the PCR method. For this nucleic acid amplification reaction, a known method may be used.
For example, in the PCR method, one of the two primers corresponds to the double-stranded DNA plus strand of the gene to be measured, the other primer pairs with the minus strand of the double-stranded DNA, and is extended by one primer. The primer can be selected such that the other primer is paired with the extended strand. The length of the primer is, for example, at least 10 bases, preferably 15 to 30 bases.
Specifically, the primer can be designed and chemically synthesized based on the above-described base sequence or the base sequence registered by the accession number of each gene from a database such as Genbank. Primers may be adjusted according to a known method.

[Glucosylceramide distribution]
When determining the distribution state of glucosylceramide as an index, for example, the skin barrier function adjustment may be determined by a change in the distribution state of glucosylceramide. More specifically, it is possible to observe a change in the position of glucosylceramide by a known technique such as microscopic observation by immunohistochemistry.
As a “control” used as a criterion at this time, the test substance and the state before addition of the test substance or epidermal barrier function regulator or a state where it is not added; the condition where PBS containing no test substance or epidermal barrier function regulator is added; Examples include a state in which a control substance is added; a state in which a solvent is added, and the like.
When glucosylceramide is continuously observed in the upper part of the granule layer, the epidermal barrier function is in a normal state or in a good state; when glucosylceramide is observed to be discontinuously localized, or When a state of aggregation around the cell nucleus of the granule cell is observed, it can be determined that the epidermal barrier function is lowered.
In addition, using a known epidermal barrier function regulator as a reference substance means that the reaction (distribution state) by this is compared with the reaction (distribution state) by the test substance to determine the epidermal barrier function regulating action of the test substance. Is easy to perform.
In addition, before or during or after changing the epidermal barrier function of the three-dimensional skin, the test substance can be evaluated and evaluated by adding the test substance and comparing the resulting glucosylceramide distribution with the control. It is.
By determining in this way, it is possible to acquire a desired epidermal barrier function regulator, and it is also possible to perform evaluation. For example, when glucosylceramide is very localized, it can be determined as an agent (substance) having a strong pharmacological action that inhibits epidermal barrier function. Moreover, when the distribution state of glucosylceramide does not change so much, it can be determined that the agent (substance) does not have an action to adjust the epidermal barrier function.
In addition, after adding a substance having an adverse effect such as an inhibitor of the epidermal barrier function to change the epidermal barrier function of the three-dimensional skin model, the test substance is added, and thus the recovery state of the distribution of glucosylceramide is compared with the control. In comparison, in the case of almost coincidence (the above-mentioned epidermal barrier function is in a normal state, a normal state, a good state), it is possible to determine this test substance as an epidermal barrier function restoring agent or the like. .

As a more specific example, after a three-dimensional skin model (for example, three-dimensional cultured skin) is frozen by a known method, a thin film section (about 3 to 10 μm) is prepared, and this is used as a slide glass. Adhere to etc. On this section, an anti-glucosylceramide antibody is added, and staining is performed by a direct method or an indirect method.
The direct method is a method of labeling (for example, visualization labeling) the antibody itself against glucosylceramide, and the indirect method is a method of visualizing labeling in two or more steps by setting the reaction system to 2-3 steps. Here, examples of the visualization label include enzyme antibody methods, fluorescent antibody methods (FITC, rhodamine, Texas red, etc.), metals (gold, silver, ferritin, etc.), and methods using isotopes. At this time, examples of the secondary antibody to be used include fluorescent biotin and FITC-labeled antibody.
In addition, preparation of observation specimens such as frozen specimens in cross-sectional image observation of a three-dimensional skin model may be performed according to a known method.

As described above, according to the method of the present invention, it is possible to screen or evaluate substances relating to the epidermal barrier function using a three-dimensional skin model and products using the same.
And when ABCA12 expression level is determined as an index, it is advantageous in that it can be quantified and evaluated. On the other hand, when the distribution state of glucosylceramide is determined as an index, it is advantageous in that it can be visually evaluated. For this reason, it is advantageous to use both as indices because it can be examined from various perspectives with high sensitivity and precision, and screening and evaluation can be performed.
Based on such knowledge, it becomes possible to infer the influence of substances on human skin. Moreover, since a three-dimensional skin model is used, it is possible to easily obtain a lot of knowledge, and a skin external preparation, a quasi-drug and a cosmetic that use or use the skin barrier function regulator obtained thereby. Can also be developed. In particular, cosmetics containing substances that improve the skin barrier function are advantageous because of high market needs.

In addition, when the skin barrier function adjustment of the test substance is known, that is, when a known skin barrier function regulator is used, knowledge about the skin barrier function of the three-dimensional skin model is obtained. This makes it possible to construct a three-dimensional skin model (particularly a three-dimensional cultured skin model) in consideration of known data such as experimental data related to living skin such as humans and laboratory animals, that is, biological skin.
Incidentally, it is considered that the action mechanism of the three-dimensional skin model shows a reaction similar to that of living skin. For this reason, it is considered possible to apply to living skin of animals such as humans based on the experimental results using a three-dimensional skin model. However, there are also situations where the mechanism of action of the three-dimensional skin model does not completely match the mechanism of action of living skin. Therefore, if the relationship between the three-dimensional skin model and the biological skin can be made clearer, it can be applied to the biological skin more easily. For example, when a test substance that has found a new use with the evaluation tool of the present invention is applied to living skin, factors such as correction of experimental results of a three-dimensional skin model (for example, three-dimensional cultured skin) ), The test substance can be evaluated more easily and with high accuracy in screening, clinical trials and the like without using model animals as much as possible. It is also possible to obtain a safer and more effective skin barrier function regulator.
In addition, even if it is a substance for which known skin barrier function adjustment is known, a new skin barrier function adjustment different from that, that is, a search for a new application, can be performed by the evaluation tool of the present invention.

[3D skin model screening or evaluation method]
Moreover, since it was possible to obtain a simple evaluation tool as described above, this evaluation tool and a skin barrier function regulator (for example, a known skin barrier function regulator or a test substance whose skin barrier function has been revealed by the above). Can be used to screen a three-dimensional skin model or to evaluate the epidermal barrier function. As a result, it is also possible to obtain a three-dimensional skin model suitable for screening or evaluation of an epidermal barrier function regulator (particularly, individual uses such as functional improvement and functional disorder).
That is, after adding one or more epidermal barrier function regulators simultaneously or separately to the target three-dimensional skin model, the ABCA12 gene expression level is used as an index and / or the distribution state of glucosylceramide is indexed It is possible to provide a screening or evaluation method for a three-dimensional skin model. In addition, when adding several skin barrier function regulators, you may add each simultaneously or separately as desired.
In addition, when the epidermal barrier function modifier is a substance that adversely affects the epidermal barrier function, it is also possible to evaluate the recovery period of the three-dimensional skin model. At this time, it can be evaluated that the shorter the recovery period of the three-dimensional skin model is, the higher the recovery power is. On the other hand, the longer the recovery period of the three-dimensional skin model is, the lower the recovery power can be evaluated.
Examples of the target three-dimensional skin model include a new one produced by a new method; a commercially available product, a known one obtained by a known method, and one obtained by chemical treatment or ultraviolet treatment.
At this time, it is desirable to screen or evaluate a three-dimensional skin model in comparison with the control skin. As the control skin, a commercially available three-dimensional cultured skin or a known three-dimensional cultured skin obtained by a known technique may be used.
Note that the evaluation tool, the three-dimensional skin model culture method, and the like may be performed in accordance with, for example, the above-described [screening or evaluation method of epidermal barrier function regulator].

  As an example, after adding an epidermis barrier function regulator to a target three-dimensional skin model, culturing is performed under the culture conditions described above. At this time, the control skin is similarly cultured after the addition of the epidermal barrier function regulator. Then, using the above-described evaluation tool, the target three-dimensional skin model is screened or evaluated against the control skin.

For example, when the expression level of ABCA12 is used as an index, the expression level in the target three-dimensional skin model is compared with the expression level in the control skin. Good three-dimensional skin model; in the case of few, it can be evaluated as a three-dimensional skin model with poor epidermal barrier function.
If the expression level of ABCA12 is large, it can be evaluated as a three-dimensional skin model suitable for evaluation of the epidermal barrier function, and if not, it can be evaluated as not suitable for evaluation of the barrier function.

In addition, for example, when the distribution state of glucosylceramide is indicated, when the glucosylceramide is observed more continuously in the upper part of the granule layer, the epidermal barrier function is improved. It can be determined (evaluated) that it is a three-dimensional skin model. In addition, when glucosylceramide is observed in a more localized manner, or when a large number of aggregated states are observed around the cell nucleus of granule cells, the target is a tertiary that has a reduced epidermal barrier function. It can be determined (evaluated) that it is an original skin model.
In addition, what has a continuous distribution of glucosylceramide can be evaluated as a three-dimensional skin model suitable for epidermal barrier function evaluation, and can be evaluated as not suitable for barrier function evaluation otherwise.

In addition, it is preferable to observe within 2 months immediately after the addition of the skin barrier function regulator. Thereby, the state of the target three-dimensional skin model can be observed over time as compared with the state of the control product. As a result, it is possible to obtain more knowledge of the three-dimensional skin model such as the long-term skin barrier function reaction of the object and the skin barrier function of the object.
Furthermore, the same or different epidermal barrier function regulator and / or test substance may be added simultaneously or separately. Thereby, the resilience and the like of the three-dimensional skin model can be evaluated.

  The method of the present invention may be realized by an apparatus including a program for performing this process, an information recording medium for storing the program, a CPU that operates according to the program, and the like.

  Hereinafter, examples and the like will be given to specifically describe the present invention, but the present invention is not limited to these examples.

<Construction of 3D skin model>
A cell suspension of 1 × 10 ⁴ cells / mL of epidermal keratinocytes (derived from human newborn foreskin) is prepared in a cell growth medium (calcium concentration 0.07 mM), and 10 mL is added to a 100 mm petri dish. Thereafter, the cells were grown in a gas atmosphere of 5% carbon dioxide concentration at 37 ° C. under humidity until the cells became sub-confluent or confluent on the culture dish. The growth medium was changed every 2-3 days.
The grown cells were detached from the petri dish using trypsin and collected. The collected cells were adjusted to 5 × 10 ⁵ cells / mL in a growth medium, and an appropriate amount was added to a cell culture insert (material: polycarbonate, pore size: 0.4 μm) according to the size of the insert. After the necessary number of inserts were placed in a 60 mm petri dish, the growth medium was also added to the outside of the insert until the same level as the liquid level in the insert. After seeding the cells, growth medium was used until confluent with respect to the bottom of the insert, and the cells were cultured until the cells were confluent.
After the cells became confluent, the medium was changed to a differentiation medium (calcium concentration: 1.2 mM) and cultured for 16 hours to induce differentiation. Thereafter, the medium in the insert was sucked out and exposed to air to obtain a three-dimensional cultured skin.
<SLS exposure>
The obtained three-dimensional skin model was washed twice with PBS, and then 20 μL of each concentration of SLS solution was added to the top of the three-dimensional skin model. After 30 minutes, the solution was removed, and the upper part of the three-dimensional skin model was washed twice with PBS, and the culture was continued.
Three-dimensional skin models after exposure to SLS were collected after 1 day, 1 week, and 3 weeks, and the following examination was performed.

<Observation of cross-sectional image of 3D skin model>
A three-dimensional skin model was collected and fixed with a 4% paraformaldehyde solution, and then a frozen specimen was prepared with an OCT compound. Using a microtome, a frozen specimen was cut into sections having a thickness of about 6 μm, stained with hematoxylin and eosin, and a tissue section image was obtained by microscopic observation. (Figure 1)

<Expression state of ABCA12 in three-dimensional skin model>
RNA was extracted from the three-dimensional skin model using SV Total RNA Isolation System kit (PROMEGA). After adjusting the obtained RNA concentration to be constant, cDNA was prepared using PrimeScript 1st strand cDNA Synthesis kit (TaKaRa). The obtained cDNA was subjected to quantitative PCR using ABCA12 primer [SEQ ID NO: 1 and SEQ ID NO: 2] and GAPDH primer (forward: [SEQ ID NO: 3], reverse: [SEQ ID NO: 4]) as housekeeping genes, respectively. The amount of PCR produced was measured. (Figure 2)
When measurement was performed using the index of ABCA12 gene, it was recognized that the epidermal barrier function (skin barrier function) was decreased in comparison with the normal condition.

<Image of staining state of glucosylceramide in a three-dimensional skin model>
Using a microtome, a frozen specimen is cut into a slice of about 6 μm thickness and attached to a glass slide. An anti-glucosylceramide antibody (rabbit) was added on the section, and a primary antibody reaction was performed in a humid box at room temperature for 1 hour. After washing, a secondary antibody (Alexa Fluor 488 anti-rabbit IgG (manufactured by Invitrogen)) and DAPI Nucleic Acid Stain (manufactured by invitrogen) diluted 200-fold were added onto the sample and allowed to stand at room temperature for 1 hour. The secondary antibody reaction was performed in a wet box. After completion of the reaction, it was washed with PBS and observed (FIG. 3).
When observed with this glucosylceramide index, localization was observed when the epidermal barrier function (skin barrier function) was lowered, and in the normal state, glucosylceramide was a granule layer. It was recognized that it was continuous in a strip shape at the top.

<Expression state of ABCA12 and glucosylceramide in a three-dimensional skin model>
As described above, a three-dimensional skin model was prepared, and the passage of 3 weeks was confirmed by comparing 0.01% SLS addition with no addition (PBS: control).
It was confirmed that the SCA exposure temporarily reduced and further increased the expression level of ABCA12 mRNA in the three-dimensional skin model, and thereafter became almost equivalent. Thereby, after adding an epidermis barrier function inhibitor, the temporary fall of the ABCA12 expression level of a three-dimensional skin model, and also the recovery | restoration to a steady state were confirmed.
In addition, the glucosylceramide distribution of the three-dimensional skin model was changed by exposure to SLS, and a glucosylceramide distribution including nuclei (DAPI-stained portion) was observed instead of a band-like uniform distribution. Thereafter, on the 21st day, it was observed that the powder was uniformly distributed to the upper part of the granule layer. Thus, after the addition of the epidermal barrier function inhibitor, the distribution of the glucosylceramide in the three-dimensional skin model is not a continuous band, and after that, the nucleus (DAPI) is taken in, and the nucleus (DAPI) is further captured. ) Became a lower layer, and the distribution of glucosylceramide was continuously connected, that is, recovery to a steady state was confirmed.

<Calcein dye penetration test>
As for the method using a calcein dye, although the detailed states such as ABCA12, glucosylceramide, and lipid transport are unknown, it can be estimated whether the barrier function is exhibited. For this reason, it was confirmed whether this method and the evaluation and tendency of the above-mentioned result correspond.
A three-dimensional skin model was constructed as described above, and 0.01% SLS was exposed to the surface of the skin model for 30 minutes after 3 weeks. Dye permeation was observed on day 1, day 7 and day 21 (optional) after exposure.
The upper part of the skin model used for the experiment was washed twice with PBS. 100 μL of 10 mM Calcein solution was added from the upper part of the skin model and allowed to stand. The Calcein solution was aspirated and washed 3 times with PBS. Tissue was fixed using 4% paraformaldehyde solution. After fixation, the tissue was washed twice with PBS, and the tissue was frozen and embedded with an embedding agent. Ultrathin sections were prepared, and tissue fragments were observed under a fluorescence microscope (see FIG. 7).
With calcein dye, it was understood that the barrier function was lowered on the 7th day after exposure to SLS and that the barrier function was restored on the 21st day. Moreover, this was consistent with the evaluation of the barrier function due to changes in ABCA12 mRNA expression level and glucosylceramide distribution. When the calcein dye permeation test is used, it is desirable to use the evaluation tool of the present invention in combination.

From the above, by measuring and observing changes in the expression level of ABCA12 and glucosylceramide distribution, it was confirmed that the barrier function was reduced and recovered by exposure to SLS. Moreover, the temporary fall of ABCA12 expression level by SLS exposure, and also the recovery | restoration to a steady state have been confirmed. It was confirmed that the distribution of glucosylceramide was changed with the change in the expression level of ABCA12 and then recovered.
In addition, the relationship between the epidermal barrier function and lipid transport in the conventional cosmetic region (three-dimensional skin model) was unknown, but from the above results, lipid transport is involved in the epidermal barrier function of the three-dimensional skin model. I think it suggests the possibility.

Claims (4)

Three-dimensional skin model (except those collected from the experimental animals), the in comparison step, and each reaction contrasting reaction and upon addition of the test substance, and a reaction when not adding the test substance ABCA12 the expression level and glucosyl index distribution of ceramide, ABCA12 expression levels upon addition of the test substance after addition of known epidermal barrier dysfunction agent as compared to ABCA12 expression level when not adding the test substance Glucosyl when the test substance is added compared to the state observed when it is increased and is aggregated around the cell nucleus of granule cells, which is the distribution state of glucosylceramide when the test substance is not added If distribution of ceramide Ru continuously observed in the granular layer upper portion, when the test substance has an improving effect epidermal barrier function was determined by adding test substance The expression level of ABCA12 in the sample decreases when compared with the expression level of ABCA12 when no test substance is added, and aggregates around the nucleus of the granule cell, which is the distribution state of glucosylceramide when the test substance is added and if the distribution of glucosylceramide when not adding the test substance as compared to the state they were observed Ru continuously observed in the granular layer upper part is the test substance is epidermal barrier dysfunction act A determination step for determining that the
By doing at least
A screening method characterized by selecting a substance having an epidermal barrier function improving action or a substance having an epidermal barrier dysfunction action from a group of test substances , or the test substance has an epidermal barrier function improving action or an epidermal barrier dysfunction action evaluation how to and evaluating whether having. After addition of the test substance, within ~ 2 months after the addition, methods who according to claim 1, characterized in that a plurality of times said comparison step and determining step. A control that is a 3D skin model to which the test substance is added (excluding those collected from experimental animals) and a 3D skin model to which the test substance is not added (excluding those collected from experimental animals) using the door,
Contrast step of comparing the control reaction as the reaction of the three-dimensional skin model, and as an index distribution of ABCA12 expression levels and glucosylceramide in each reaction, the addition of known skin barrier dysfunction agent to the three-dimensional skin model and the control A state in which the expression level of ABCA12 in the three-dimensional skin model was later increased as compared to the expression level of ABCA12 in the control and was observed aggregated around the cell nucleus of the granule cell, which is the distribution state of glucosylceramide in the control in distribution is granular layer upper portion of glucosylceramide three-dimensional skin model in comparison case that will be continuously observed, the epidermal barrier function of the three-dimensional skin model is recovered and, ABCA12 expression level in the three-dimensional skin model Is reduced compared to ABCA12 expression level in the control, and three-dimensional skin If the model distribution control of glucosylceramide compared with the state they were observed to aggregate in the cell nucleus around the granule cell is a distribution state of glucosylceramide of Ru is continuously observed in the granular layer upper portion, said A determination process for determining that the epidermal barrier function of the three-dimensional skin model is impaired ,
By doing at least
A screening method characterized by selecting a three-dimensional skin model suitable for sensitively evaluating the epidermal barrier function from among the three-dimensional skin model group , or the three-dimensional skin model evaluates the epidermal barrier function with high sensitivity. evaluation how to and evaluating whether appropriate or not to. After adding the test substance to the three-dimensional skin model, within ~ 2 months after the addition, methods who claim 3, wherein a plurality of times said comparison step and determining step.