Method of evaluating skin barrier function, method of screening material for enhancing skin barrier function using the evaluating method, the material for enhancing skin barrier function, and cosmetic containing the material for enhancing skin barrier function

a skin barrier function and function technology, applied in the direction of drugs, instruments, drug compositions, etc., can solve the problems of skin barrier dysfunction, affecting the function of skin barrier, and affecting the so as to improve the appearance and appearance of skin barrier, enhance the function of skin barrier, and improve the effect of skin barrier function

Inactive Publication Date: 2010-03-18
POLA CHEM INDS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0031]According to the present invention, it is possible to provide a technology for developing an excellent material for enhancing skin barrier function, and to develop and provide a cosmetic having excellent ability to enhance the skin barrier function based on the technology.

Problems solved by technology

In fact, there is a high incidence of the skin barrier dysfunction, so it is required to develop means for preventing a disease in an early stage from becoming severe or means for ameliorating an actual symptom.
However, in many cases, expression of the skin barrier dysfunction described above is liable to be recognized after developments of severe symptoms such as an occurrence of skin roughness and a rapid increase in amount of transepidermal water loss.
There are some proposals on animal models for skin barrier dysfunction (see, for example, Patent Documents 5 and 6), but in all the proposals, animals must be used and much time and efforts are required.
Further, there is a room for doubt about its reproducibility.
Moreover, in such screening using animal models, the number of test samples to be evaluated is limited.
There has not been known at all a method of evaluating skin barrier function using culture cells.
However, there has been also known that the excessive calcium ion impairs the skin barrier function (see, for example, Non-patent Documents 1, 2, and 3).
Moreover, a technology for adjusting the concentration of a metal ion such as a calcium ion in the skin has not been known at all.

Method used

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  • Method of evaluating skin barrier function, method of screening material for enhancing skin barrier function using the evaluating method, the material for enhancing skin barrier function, and cosmetic containing the material for enhancing skin barrier function
  • Method of evaluating skin barrier function, method of screening material for enhancing skin barrier function using the evaluating method, the material for enhancing skin barrier function, and cosmetic containing the material for enhancing skin barrier function
  • Method of evaluating skin barrier function, method of screening material for enhancing skin barrier function using the evaluating method, the material for enhancing skin barrier function, and cosmetic containing the material for enhancing skin barrier function

Examples

Experimental program
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Effect test

example 1

[0054]An ion permeability test using normal human epidermal cells was performed based on a method of evaluating skin barrier function of the present invention.

[0055]1) Cell Inoculation and Ion Permeability Experiment

[0056]Frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurabo Industries Ltd.) were thawed and cultured in a 0.15 mM Ca-containing culture medium (Humedia-KG2; manufactured by Kurabo Industries Ltd.) at 37° C. in 50% carbon dioxide atmosphere. A Transwell (registered trademark) manufactured by Corning Incorporated (diameter 12 mm, polyethylene terephthalate, 0.4 μm pore) was placed on a Millicell (registered trademark) tissue culture plate (manufactured by Millipore), and the above-mentioned culture medium was added in amounts of 1.5 ml to the lower layer and 0.5 ml to the upper layer. Then, the normal human epidermal keratinocytes (NHEK) were inoculated at 1×105 / cm2 and cultured for additional 72 hours. After the cells were confirmed to be confluent, ...

example 2

[0071]In the same way as in Example 1, calcium was selected as a metal ion to be added to compare calcium permeability coefficients and magnesium permeability coefficients in a low-calcium medium cultured cells (shown as Ca− in FIG. 3) (0.15 mM) and in a high-calcium medium cultured cells (shown as Ca+ in FIG. 3) (1.45 mM). That is, frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurabo Industries Ltd.) were thawed and cultured in a 0.15 mM Ca-containing culture medium (Humedia-KG2; manufactured by Kurabo Industries Ltd.) at 37° C. in 50% carbon dioxide atmosphere. A Transwell (registered trademark) manufactured by Corning Incorporated (diameter 12 mm, polyethylene terephthalate, 0.4 μm pore) was placed on a Millicell (registered trademark) tissue culture plate (manufactured by Millipore), and the above-mentioned culture medium was added in amounts of 1.5 ml to the lower layer and 0.5 ml to the upper layer. Then, the normal human epidermal keratinocytes (NHEK) we...

example 3

[0072]TERs and metal ion permeability coefficients in the case where claudin-1, one of tight junction proteins serving as adhesion factors for epidermal cells, was suppressed with siRNA were measured in accordance with the method of Example 1. That is, frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurabo Industries Ltd.) were thawed and cultured in a 0.15 mM Ca-containing culture medium (Humedia-KG2; manufactured by Kurabo Industries Ltd.) at 37° C. in 50% carbon dioxide atmosphere. Thereafter, siRNA was transfected into the cultured cells. A Transwell (registered trademark) manufactured by Corning Incorporated (diameter 12 mm, polyethylene terephthalate, 0.4 μm pore) was placed on a Millicell (registered trademark) tissue culture plate (manufactured by Millipore), and the above-mentioned culture medium was added in amounts of 1.5 ml to the lower layer and 0.5 ml to the upper layer. Then, the human epidermal keratinocytes transfected with siRNA were inoculated ...

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Abstract

A method for developing an excellent material for enhancing skin barrier function, and providing a cosmetic which is developed based on the method and has excellent ability to enhance skin barrier function is described. The method evaluates skin barrier function by the steps of 1) culturing normal human epidermal cells in wells having a semipermeable membrane with two sides, in presence and absence of a test substance; 2) adding a medium not containing metal ions to a first side of the semipermeable membrane in each well and a medium containing the metal ions to a second side of the semipermeable membrane in each well, provided that the cells are adhered to the first side of the semipermeable membrane; 3) culturing the cells; 4) measuring the concentration of the metal ions in each medium; and 5) calculating permeability of the metal ions in the epidermal cell layer based on the measured concentrations of the metal ions in each media. The test substance is effective for enhancing skin barrier function when the permeability is reduced in the medium in which the test substance is added compared to the permeability in the medium in which no test substance is added. The larger the level of reduced permeability, the higher the suitability of the test substance as a material for enhancing skin barrier function.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of evaluating skin barrier function, a method of screening material for enhancing skin barrier function using the evaluating method, the material for enhancing skin barrier function, and a cosmetic containing the material for enhancing skin barrier function.BACKGROUND ART[0002]At present days, patients who suffer from skin diseases due to skin barrier dysfunction, such as a high incidence of atopic dermatitis, have increased rapidly. This is considered to be caused by an excessive increase in stress (see, for example, Patent Documents 1, 2, 3, and 4). In fact, there is a high incidence of the skin barrier dysfunction, so it is required to develop means for preventing a disease in an early stage from becoming severe or means for ameliorating an actual symptom.[0003]However, in many cases, expression of the skin barrier dysfunction described above is liable to be recognized after developments of severe symptoms such as an ...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): A61K8/97C12Q1/02A61Q19/00
CPCG01N33/84G01N33/5044A61P3/14A61P17/00
InventorKURODA, SHOHEIYAMAMOTO, TAKUYASAEKI, YUKOKURASAWA, MASUMI
OwnerPOLA CHEM INDS