WO2012057567A2 - Klotho 유전자를 포함하는 피부 활성물질 탐색용 키트 및 이를 이용하여 피부 활성물질을 탐색하는 방법 - Google Patents
Klotho 유전자를 포함하는 피부 활성물질 탐색용 키트 및 이를 이용하여 피부 활성물질을 탐색하는 방법 Download PDFInfo
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- WO2012057567A2 WO2012057567A2 PCT/KR2011/008132 KR2011008132W WO2012057567A2 WO 2012057567 A2 WO2012057567 A2 WO 2012057567A2 KR 2011008132 W KR2011008132 W KR 2011008132W WO 2012057567 A2 WO2012057567 A2 WO 2012057567A2
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- WIPO (PCT)
- Prior art keywords
- skin
- klotho
- expression
- screening
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/148—Screening for cosmetic compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the present invention relates to a kit for screening skin active materials and a method for searching for skin active materials using the same, and more specifically, to express klotho gene expression in skin cells by using a search kit including an ldotho gene known as a longevity gene.
- the present invention relates to a skin active material search kit that searches for skin active substances such as anti-aging substances and whitening moles by separating and purifying candidate substances, and a method of searching for skin active substances using the same.
- an object of the present invention is to provide a search kit for screening a skin active substance through the expression of klotho gene in skin cells and a method for searching for a skin active substance using the same.
- the present invention comprises a Klotho protein gene, a primer for amplifying the Klotho protein gene of SEQ ID NO: 1.2, a probe of SEQ ID NO: 3, and skin cells.
- a search kit for searching for skin active substances by screening for substances that increase the expression level of Klotho protein in the skin cells is provided.
- the present invention provides a method for searching the skin active material using the search kit.
- the dermal actives search kit according to the present invention is an anti-aging medulla through the expression of klotho gene in skin cells. Skin actives such as whitening substances and moisturizing moles could be screened quickly and easily.
- La human embryonic kidney cells (lffiK293). Keratinocytes (NI1K) and Fibroblasts (NI1K).
- HEK293 human non 1 st kidney cells. This graph shows the results of quantitative real-time PCR confirming Klotho protein expression levels in keratinocytes (NHK) and fibroblasts (NHF).
- FIG. 3 is a graph showing quantitative real-time PCR results confirming Klotho protein expression levels on days 2 and 8 after treatment of retinoic acid (RA) and retinol (R0L: retinol) on keratinocytes.
- RA retinoic acid
- R0L retinol
- Figure 4 is a graph showing the decrease in Klotho gene expression level in human neonatal epithelial keratinocytes (HFJ0) knocked down Klotho gene.
- FIG. 5 is a graph showing the results of confirming the change in the expression level of MMP-9 in human neonatal epithelial keratinocytes (HEK) knocked down Klotho gene.
- Figure 6 shows the result of confirming the change in MMP-9 activity by treatment of recombinant Klotho protein to human neonatal epithelial keratinocytes (HEK).
- Figure 7 lug / ml recombinant Klotho protein in human neoplastic keratinocytes (HEK). The result of confirming the change of HAS2 mRNA expression level after 8 days after 0-.5ug / ml treatment was 8 days.
- Figure 8 is a graph showing the results of confirming the change in the expression level of 1) 21 in human neonatal dermal fibroblasts (HDF) knocked down the Klotho gene.
- the present invention relates to a method that can detect the expression level of Klotho protein in skin cells, and through this screening a substance capable of inducing the expression of Klotho protein, a longevity gene in skin cells, can search for skin active substances .
- the skin active substance is an anti-aging substance that improves skin wrinkles or elasticity or improves rough skin, brightens or blemishes skin tone. It may include both whitening and moisturizing substances that improve skin pigmentation such as freckles, skin blemishes and sunburn.
- correction Sheet (Rule 91) ISA / KR Klotho protein gene.
- An anti-aging substance can be searched for by screening a substance consisting of a probe of SEQ ID NO: 3 (Table 1 saliva-crude) and skin cells, which increase the expression level of Klotho protein in the skin cells.
- the skin cells may be normal keratinocytes (NHK), fibroblasts (NHF: normal human fibroblasts) or melanocytes (N11M: norma 1 human melanocyte)- It is not limited to this.
- NHK normal keratinocytes
- NHL normal human fibroblasts
- N11M norma 1 human melanocyte
- the present invention relates to a method of searching for skin active substances through a step of confirming whether Klotho protein is expressed after treating candidate skin cells, and separating and purifying the material rate to increase Klotho protein expression.
- Fibroblasts (NHF), NHM—promoblac Normal Human Epidermal Melanocytes (NHEM) juvenile foreskin, gall-derived delanocyte-forming cells, Promocell), NHHHDP ( ⁇ -dark pigment; hookin-derived melanocytes, Invitrogen) and NHM—MP (NHM) RT-PCR was used to detect the presence of Klotho mRNA in cDNA of moderate pigments (Asian-derived melanocytes, Invitrogen). At this time, PCR reaction conditions were performed by repeating the cycle of denaturation for 30 seconds at 94 ° C, annealing for 30 seconds at 551: and expansion for 60 seconds at 72 ° C.
- HEK Human neonatal epithelial keratinocytes (HEK: Lonza. NHEK—Neo-Neonatal Normal Human Epidermal Kerat inocytes.Pooled) in 6-wel medium (KBM ⁇ gold, Lc za)
- HEK Human neonatal epithelial keratinocytes
- HEK Human neonatal epithelial keratinocytes
- Human Epidermal Kerat inocytes Pooled were dispensed in 6—well with 5xl () 4 in medium (KBM-gold) and incubated at 37 ° C for 24 hours, followed by preparation of Klotho protein (R & D systems, Recombinant Human Klotho). lug / ml was incubated for 8 days. As a control, HEK without Klotho protein was used.
- the cultured HEK culture was loaded on gelatin gel, and the results are shown in FIG. 6 through zymography.
- hyaluronic acid synthase2 a hyaluronic acid synthase known as skin moisturizing factor
- human neonatal epithelial keratinocytes Longza, NHEK-Neo-Neonatal Normal
- Human Epidermal Kerat inocytes was treated with recombinant Klotho protein to observe HAS mRNA expression.
- HEK Human neonatal keratinocytes
- RM was extracted from the cultured HEK using RNeasy mini ki qiagen), and cDNA was synthesized by RT-PCR using Superscript III kit (invitrogen). Quantitative real-time PCR was performed using a probe for HAS2 (TaqMan® Gene Expression Assays) to observe the expression of HAS2 mRNA and the results are shown in FIG. 7.
- HAS2 mRNA expression was decreased in the control group Reference Example 4, which was untreated on Day 8, than Reference Example 1, which was not treated on Day 2.
- Reference Example 5 the recombinant Klotho protein treatment group on day 8, it can be seen that the expression of MS2 mRNA was significantly increased compared to Reference Example 1 on the second day untreated control group.
- the decrease in the expression of MS2 mRNA in Reference Example 4 than in Reference Example 1 appears to occur during HEK differentiation and keratinization, and it can be seen that the recovery of reduced HAS2 at 8 days by recombinant klotho protein treatment, It can be seen that klotho protein plays a role in restoring the epidermis's moisturizing ability.
- Test Example 7 Increased expression of p21 in human neonatal dermal islet cells (HDF) knocked down Klotho gene
- RNAiMAX small interfering RNA
- GIBC0 0PTI-MEM
- RNAiMAX transfection reagent
- SEQ ID NO: 1 and 2 is the DNA sequence of the primer for amplification of Klotho protein gene.
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- Health & Medical Sciences (AREA)
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- Biomedical Technology (AREA)
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- Analytical Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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- General Health & Medical Sciences (AREA)
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- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Cosmetics (AREA)
Abstract
Description
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11836659.0A EP2634262B1 (en) | 2010-10-29 | 2011-10-28 | Kit for screening for skin-activating substances and comprising the klotho gene, and method for screening for skin-activating substances using same |
JP2013536527A JP6186279B2 (ja) | 2010-10-29 | 2011-10-28 | Klotho遺伝子を含む皮膚活性物質探索用キット及びこれを利用して皮膚活性物質を探索する方法 |
US13/881,312 US20130252247A1 (en) | 2010-10-29 | 2011-10-28 | Kit for screening for skin-activating substances and comprising the klotho gene, and method for screening for skin-activating substances using the same |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2010-0107000 | 2010-10-29 | ||
KR20100107000 | 2010-10-29 | ||
KR10-2011-0107409 | 2011-10-20 | ||
KR1020110107409A KR101907567B1 (ko) | 2010-10-29 | 2011-10-20 | Klotho 유전자를 포함하는 피부 활성물질 탐색용 키트 및 이를 이용하여 피부 활성물질을 탐색하는 방법 |
Publications (2)
Publication Number | Publication Date |
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WO2012057567A2 true WO2012057567A2 (ko) | 2012-05-03 |
WO2012057567A3 WO2012057567A3 (ko) | 2012-09-07 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/KR2011/008132 WO2012057567A2 (ko) | 2010-10-29 | 2011-10-28 | Klotho 유전자를 포함하는 피부 활성물질 탐색용 키트 및 이를 이용하여 피부 활성물질을 탐색하는 방법 |
Country Status (6)
Country | Link |
---|---|
US (1) | US20130252247A1 (ko) |
EP (1) | EP2634262B1 (ko) |
JP (1) | JP6186279B2 (ko) |
KR (1) | KR101907567B1 (ko) |
CN (1) | CN102465182A (ko) |
WO (1) | WO2012057567A2 (ko) |
Cited By (1)
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EP2975410A4 (en) * | 2013-03-13 | 2016-08-24 | Amorepacific Corp | PROCESS FOR SCREENING AFTER A SUBSTANCE WITH LIGHTENING EFFECT |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
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KR102012166B1 (ko) * | 2012-11-20 | 2019-08-20 | (주)아모레퍼시픽 | 피부 미백용 물질을 스크리닝하는 방법 |
KR102045247B1 (ko) * | 2013-01-08 | 2019-11-15 | (주)아모레퍼시픽 | 피부 면역세포를 이용하는 피부 활성물질을 탐색하는 방법 및 이를 이용한 키트 |
KR102053775B1 (ko) * | 2013-07-23 | 2019-12-09 | (주)아모레퍼시픽 | 미토콘드리아 역동성을 이용한 미백 물질 스크리닝 방법 및 이를 이용한 키트 |
CN106868089B (zh) * | 2015-12-11 | 2020-02-28 | 上海家化联合股份有限公司 | 一种利用皮肤成纤维细胞钾离子通道评价抗衰老功效的方法 |
KR102692717B1 (ko) * | 2016-09-23 | 2024-08-08 | (주)아모레퍼시픽 | Tnfrsf14 억제 물질을 포함하는 피부 미백용 조성물 및 tnfrsf14 억제 물질의 스크리닝 방법 |
CN114622024A (zh) * | 2020-12-08 | 2022-06-14 | 复旦大学 | 用于皮肤分型的标志物组合及其应用 |
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AU2003211453A1 (en) * | 2002-02-28 | 2003-09-09 | Nihon Kefir Co., Ltd. | External compositions for the skin |
JP2003334088A (ja) * | 2002-05-22 | 2003-11-25 | Pharma Design Inc | ヒト由来の新規Klotho様タンパク質及びその遺伝子 |
JP2006508666A (ja) * | 2002-12-10 | 2006-03-16 | オタワ ヘルス リサーチ インスティテュート | カスパーゼ3活性調節による幹細胞分化の調節 |
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2011
- 2011-05-27 CN CN2011101423796A patent/CN102465182A/zh active Pending
- 2011-10-20 KR KR1020110107409A patent/KR101907567B1/ko active IP Right Grant
- 2011-10-28 WO PCT/KR2011/008132 patent/WO2012057567A2/ko active Application Filing
- 2011-10-28 US US13/881,312 patent/US20130252247A1/en not_active Abandoned
- 2011-10-28 JP JP2013536527A patent/JP6186279B2/ja not_active Expired - Fee Related
- 2011-10-28 EP EP11836659.0A patent/EP2634262B1/en not_active Not-in-force
Non-Patent Citations (1)
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NATURE, vol. 390, 1997, pages 45 - 51 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2975410A4 (en) * | 2013-03-13 | 2016-08-24 | Amorepacific Corp | PROCESS FOR SCREENING AFTER A SUBSTANCE WITH LIGHTENING EFFECT |
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JP2013541960A (ja) | 2013-11-21 |
JP6186279B2 (ja) | 2017-08-23 |
KR20120049126A (ko) | 2012-05-16 |
WO2012057567A3 (ko) | 2012-09-07 |
EP2634262A2 (en) | 2013-09-04 |
KR101907567B1 (ko) | 2018-10-15 |
EP2634262A4 (en) | 2014-04-16 |
EP2634262B1 (en) | 2017-05-31 |
CN102465182A (zh) | 2012-05-23 |
EP2634262A9 (en) | 2014-11-19 |
US20130252247A1 (en) | 2013-09-26 |
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