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WO2016117765A1 - Modèle de tissu cutané tridimensionnel analogue à la dermatite atopique et procédé de criblage de médicament pour la dermatite atopique l'utilisant - Google Patents

Modèle de tissu cutané tridimensionnel analogue à la dermatite atopique et procédé de criblage de médicament pour la dermatite atopique l'utilisant Download PDF

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WO2016117765A1
WO2016117765A1 PCT/KR2015/004356 KR2015004356W WO2016117765A1 WO 2016117765 A1 WO2016117765 A1 WO 2016117765A1 KR 2015004356 W KR2015004356 W KR 2015004356W WO 2016117765 A1 WO2016117765 A1 WO 2016117765A1
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atopic dermatitis
filaggrin
skin tissue
expression
tissue model
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손상욱
이하나
김진희
류우인
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Korea University Research and Business Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • C12N2503/06—Screening or testing on artificial skin

Definitions

  • the present invention relates to atopic dermatitis-like three-dimensional skin tissue model and a method for screening for atopic dermatitis treatments using the same, more specifically, expression of filaggrin (Filaggrin) through the treatment of TSLP (Thymic stromal lymphopoietin) or environmental factors toluene
  • TSLP Thimic stromal lymphopoietin
  • TSLP Thimic stromal lymphopoietin
  • environmental factors toluene To prepare a three-dimensional skin tissue model showing a reduced atopic dermatitis-like findings with reduced expression of the antimicrobial peptides ⁇ -defensin or Psoriasin, and thus a candidate for atopic dermatitis treatment
  • candidates for increasing the expression of filaggrin and further increasing the expression of the antimicrobial peptide ⁇ -defensin or thyrasine are selected as a therapeutic agent for the screening method of atopic dermatitis therapeutic
  • the three-dimensional skin model is an artificial skin model (RhE, which reconstructs the human epidermis in three dimensions by inducing differentiation of keratinocytes, the major cells that form the epidermis of human skin, at the air-liquid interphase). Reconstituted human skin equivalent model).
  • RhE artificial skin model
  • Atopic dermatitis is a chronic inflammatory skin disease that is accompanied by severe itching and overall dry skin, and eczema lesions recur in certain areas.They have a genetic predisposition and allergic marches associated with allergic diseases such as food allergy, allergic asthma and allergic rhinitis.
  • allergic diseases such as food allergy, allergic asthma and allergic rhinitis.
  • the pathogenesis of atopic dermatitis today is largely divided into the pathogenesis associated with skin barriers and the pathogenesis associated with abnormal immune responses, with mutations in the filaggrin gene occurring in about 40% of patients with atopic dermatitis and with severe symptoms or even persisting in adults.
  • external allergens more easily pass through the stratum corneum and sensitize the body in people with innately damaged skin barriers, leading to atopic dermatitis.
  • the core proteins constituting the skin barrier is known to play an important role in the formation of the stratum corneum (Filaggrin), Loricrin (Loricrin), Involucrin (Involucrin).
  • filaggrin is present in the form of propyllagrin, a protein constituting kerathaialin granule in the granule layer, and is decomposed into filaggrin during the final differentiation of keratinocytes to form keratinocyte membranes.
  • the filament aggregates to form a solid, flat structure of keratinocytes, which acts as a brick in the skin barrier.
  • the filaggrin breaks down into amino acids and changes into free amino acids and pyrrolidone carboxylic acids (PCAs), which are very important for moisture.
  • Filaggrin produces citrulline, PCA, and trans-UCA through degradation processes, which perform stratum corneum hydration, normalization of stratum corneum pH, permeation barrier, firmness of the stratum corneum, antibacterial and anti-inflammatory activity.
  • TSLP thymic stromal lymphopoietin
  • the top protein of abnormal signaling is the TSLP cytokine.
  • substances such as TSLP stimulate dendritic cells to respond, causing symptoms of atopic dermatitis.
  • TSLP stimulates CD11c + dendritic cells to increase TARC (Thymus and activation-regulated chemokine) and MDC production.
  • TARC Thymus and activation-regulated chemokine
  • Antibacterial peptides are an important component of the innate immune system of the human body. They are cationic peptides that are produced in epithelial tissues to establish a first line of defense against all bacteria, including viruses, bacteria, and fungi. It activates immunity and is involved in wound repair function after living injury. The lowering of these antimicrobial peptides is due to increased inhibition of Th-2 cytokines in atopic dermatitis, and ⁇ -defensin and cathelicidine when IL-4 and IL-13 are administered to keratinocytes. increase in cathelicidin) has been reported.
  • VOCs Volatile Organic Compounds
  • VOCs are liquid or gaseous organic compounds that easily evaporate into the atmosphere due to their high vapor pressure. Recently, researches related to allergic diseases have been actively conducted.
  • the mechanism of atopic dermatitis caused by TSLP, a cytokine that is overexpressed in the lesions of toluene and atopic dermatitis patients, which is an environmental hazard among volatile organic compounds (VOCs), is presented using a three-dimensional skin model. .
  • the present inventors have made intensive efforts to develop an animal replacement model that can be used for screening methods for treating atopic dermatitis based on atopic dermatitis-like three-dimensional skin tissue model studies. Reduction of the expression level of the antimicrobial peptide gene and reduction of the expression level of the filaggrin gene according to toluene treatment was confirmed in the three-dimensional skin tissue model, and the present invention was completed.
  • An object of the present invention to provide atopic dermatitis-like three-dimensional skin tissue model and a method for screening atopic dermatitis treatment using the same.
  • the present invention comprises the steps of: (a) treating a three-dimensional skin tissue model TSLP (Thymic stromal lymphopoietin) or toluene (Toluene); (b) confirming the expression of Filaggrin and antimicrobial peptides in the treated three-dimensional skin tissue model; And (c) obtaining a three-dimensional skin tissue model with reduced expression of Filaggrin as atopic dermatitis-like three-dimensional skin tissue model compared to the control group. to provide.
  • TSLP Thimic stromal lymphopoietin
  • Toluene toluene
  • the present invention also provides an atopic dermatitis-like three-dimensional skin tissue model prepared by the above method and having reduced expression of Filaggrin compared to the control group.
  • the present invention also comprises the steps of: (a) treating atopic dermatitis candidates to the atopic dermatitis-like three-dimensional skin tissue model, and then confirming the expression of filaggrin and antimicrobial peptides in the cells of the skin tissue model; And (b) provides a method for screening atopic dermatitis therapeutics using atopic dermatitis-like three-dimensional skin tissue model comprising the step of selecting a candidate to increase the expression of the filaggrin compared to the control.
  • Figure 1 shows the change in the expression of filaggrin (Filaggrin) in keratinocytes according to the concentration of TSLP treatment
  • (A) is the change in the expression of pilaggrin (Filaggrin) according to TSLP treatment confirmed by RT-PCR
  • (B) Shows changes in pilaggrin protein expression according to TSLP treatment confirmed by Western blot
  • (C) shows changes in pilaggrin protein expression in keratinocytes identified by immunofluorescence staining.
  • Figure 2 shows the change in the expression of filaggrin (Filaggrin) in keratinocytes according to the concentration of toluene (Toluene) for each concentration
  • (A) is the change in the expression of pilaggrin (Filaggrin) RNA according to TSLP treatment confirmed through RT-PCR
  • (B) is a change in the expression of pilaggrin protein (Filaggrin) protein according to the toluene treatment confirmed by Western Bull
  • (C) is a change in the expression of Filaggrin (Filaggrin) protein in keratinocytes confirmed by immunofluorescence staining method.
  • Figure 3 shows the expression change of the filaggrin (Filaggrin) in the three-dimensional skin model according to the TSLP treatment, (A) decreases the expression of Filaggrin (TS) treatment according to the TSLP treatment, (B) ⁇ according to the TSLP treatment Decreased expression of defensin and ⁇ -defensin (Psoriasin, S100 calcium-binding protein A7).
  • Figure 4 shows the decrease in the expression of Filaggrin (Filaggrin) in the three-dimensional skin model according to toluene (Toluene) treatment.
  • TSLP Thymic stromal lymphopoietin
  • TSLP Thymic stromal lymphopoietin
  • TSLP Thymic stromal lymphopoietin
  • RNA and protein of filaggrin in keratinocytes cultured with a monolayer treated with TSLP (Thymic stromal lymphopoietin) compared to a control treated with Phosphate-Buffered Saline (PBS) The amount was confirmed to decrease TSLP concentration dependent.
  • RNA and protein of pilaggrin in keratinocytes cultured in toluene-treated monolayers were dependently reduced in toluene concentration.
  • the present invention comprises the steps of: (a) treating a three-dimensional skin tissue model with TSLP (Thymic stromal lymphopoietin) or toluene; (b) confirming the expression of Filaggrin and antimicrobial peptides in the treated three-dimensional skin tissue model; And (c) obtaining a three-dimensional skin tissue model having reduced expression of pilaggrin as atopic dermatitis-like three-dimensional skin tissue model compared to the control group.
  • the control means a three-dimensional skin tissue model treated with Phosphate-Buffered Saline (PBS).
  • the cells constituting the three-dimensional skin tissue model may be characterized in that the human keratinocytes (keratinocytes), the antimicrobial peptide is ⁇ -defensin ( ⁇ -defensin) or thyrasine (Psoriasin ),
  • the expression of the filaggrin (Filaggrin) and the antimicrobial peptide may be characterized by using reverse transcription-polymerase chain reaction (Reverse Transcription-PCR), Western blot or immunohistochemical staining method .
  • the step (c) is characterized in that to obtain a three-dimensional skin tissue model with atopic dermatitis-like three-dimensional skin tissue model, the expression of pilaggrin (Filaggrin) is reduced and the antimicrobial peptide is further reduced compared to the control group can do.
  • the control means a three-dimensional skin tissue model treated with Phosphate-Buffered Saline (PBS).
  • the present invention relates to an atopic dermatitis-like three-dimensional skin tissue model prepared by the above method and having reduced expression of Filaggrin as compared to the control group.
  • the control means a three-dimensional skin tissue model treated with Phosphate-Buffered Saline (PBS).
  • PBS Phosphate-Buffered Saline
  • the cells constituting the three-dimensional skin tissue model may be characterized in that the human keratinocytes (keratinocytes), the atopic dermatitis-like three-dimensional skin tissue model of the filaggrin (Filaggrin) compared to the control group It may be characterized in that the expression is reduced, the expression of the antimicrobial peptide is further reduced, the antimicrobial peptide may be characterized in that the ⁇ -defensin ( ⁇ -defensin) or sarirasin (Psoriasin), Expression of filaggrin and antimicrobial peptides can be characterized by using reverse transcription-polymerase chain reaction (PCR), western blot or immunohistochemical staining.
  • the control means a three-dimensional skin tissue model treated with Phosphate-Buffered Saline (PBS).
  • the present invention (a) treatment of atopic dermatitis candidates to the atopic dermatitis-like three-dimensional skin tissue model, and then confirmed the expression of filaggrin (Filaggrin) and antimicrobial peptides in the cells of the skin tissue model Doing; And (b) relates to a method for screening atopic dermatitis treatment using atopic dermatitis-like three-dimensional skin tissue model comprising the step of selecting a candidate to increase the expression of the filaggrin compared to the control.
  • the control group refers to atopic dermatitis-like three-dimensional skin tissue model treated with Phosphate-Buffered Saline (PBS) or dimethyl sulfoxide (DMSO).
  • the cells constituting the three-dimensional skin tissue model may be characterized in that the human keratinocytes (keratinocytes), the antimicrobial peptide is ⁇ -defensin ( ⁇ -defensin) or thyrasine (Psoriasin ),
  • the expression of the filaggrin (Filaggrin) and the antimicrobial peptide may be characterized by using reverse transcription-polymerase chain reaction (Reverse Transcription-PCR), Western blot or immunohistochemical staining method
  • the step (b) may be characterized in that to increase the expression of the filaggrin (Filaggrin) compared to the control, and to select a candidate substance to further increase the expression of the antimicrobial peptide as a therapeutic agent.
  • the control group refers to atopic dermatitis-like three-dimensional skin tissue model treated with Phosphate-Buffered Saline (PBS) or dimethyl sulfoxide (DMSO).
  • Example 1 Changes in the expression of Filaggrin in keratinocytes cultured in monolayer following TSLP or toluene treatment
  • TSLP Thimic stromal lymphopoietin
  • HaCaT cell line (order no. 300493, CLS, Eppelheim, Germany; German Cancer Research Center, Heidelberg, Germany) (Boukamp et al., J. Cell) Biol. 106: 761-771, 1988) and treated with 0, 1, 10, 100ng / ml of TSLP in the culture for 16 hours (control was treated with Phosphate-Buffered Saline (PBS)), followed by TRIzol reagent. After addition, it was ground finely using a homogenizer (homogenizer: OMNI-inc, USA), and RNA was isolated according to the manufacturer's method.
  • PBS Phosphate-Buffered Saline
  • Filaggrin (Filaggrin) primer SEQ ID NO: 1): 5'-ggtctggacgttcagggtct-3 '
  • Filaggrin-r (reverse) SEQ ID NO: 2]: 5'- ggatgtggtgtggctgtgat-3 '
  • GAPDH primer GAPDH primer
  • 35 cycles were performed using the denaturation step (95 ° C., 30 sec), the annealing step (56 ° C., 30 sec), and the extension step (72 ° C., 40 sec) as one cycle, and amplified by PCR (Polymerase
  • the human keratinocyte line (HaCaT cell line) was cultured and treated with 0, 1, 10, 100ng / ml of TSLP in the culture solution for 16 hours (the control group was PBS (Phosphate).
  • -Buffered Saline), RIPA buffer (Elpis, Korea) were treated with the cells and allowed to stand on ice for 30 minutes, and then centrifuged for 13000rpm and 30 minutes in a refrigerated centrifuge. was loaded onto an SDS-PAGE gel.
  • the protein contained in the loaded gel was then transferred to PVDF and then blocked with 5% Bovine Serum Albumin (BSA) for 1 hour, followed by a 1: 1000 dilution of the Filaggrin antibody (Santa Cruz, USA). Then incubated. Then, after the HRP-attached secondary antibody reaction and washing process, the band of the protein was confirmed using the ECL solution (FIG. 1B).
  • BSA Bovine Serum Albumin
  • cytoperm / cytofix (BD, USA) was treated in the cultured human HaCaT cell line according to the experimental method provided to fix the cells and filaggrin (Filaggrin).
  • PBS Phosphate-Buffered Saline
  • Cy3 attached secondary antibody 100 for 1 hour at 4 ° C.
  • RNA and protein of filaggrin in keratinocytes were reduced in a TSLP concentration-dependent manner compared to the control group treated with PBS compared to the physiological stromal lymphopoietin (TSLP) treated monolayer.
  • TSLP physiological stromal lymphopoietin
  • toluene was treated to each concentration of keratinocytes cultured in a monolayer, and the change in the expression level of the filaggrin at the RNA and protein levels was confirmed.
  • the human keratinocyte line (HaCaT cell line) was cultured and treated with 0, 12.5, 25, 50 nM of toluene in the culture solution for 24 hours (the control group was PBS (Phosphate-Buffered). Saline)), TRIzol reagent was added and finely ground using a homogenizer (homogenizer) and RNA was isolated according to the manufacturer's method. The isolated RNA was prepared cDNA of each tissue using a reverse transcriptase (Super-bio, Korea).
  • Filaggrin primer (Filaggrin-f (SEQ ID NO: 1): 5'-ggtctggacgttcagggtct-3 '
  • Filaggrin-r (reverse) [SEQ ID NO: 2]: 5'-ggatgtggtgtggctgtgat using the prepared cDNA as a template -3 ') and GAPDH primers (GAPDH-f (forward) [SEQ ID NO: 3]: 5'-AGCAATGCCTCCTGCACCACCAAC-3'
  • 35 cycles were carried out using a denaturation step (95 ° C., 30 seconds), annealing step (56 ° C., 30 seconds), and an extension step (72 ° C., 40 seconds) as one cycle, and amplified by PCR.
  • the amplified samples were observed for expression in 1% agarose gel (FI
  • the human keratinocyte line (HaCaT cell line) was cultured and treated with 0, 12.5, 25 and 50 nM of toluene in the culture solution for 24 hours (the control group was PBS (Phosphate-Buffered). Saline)) and RIPA buffer (Elpis, Korea) were treated with the cells and allowed to stand on ice for 30 minutes, followed by centrifugation at 13000 rpm for 30 minutes in a refrigerated centrifuge. Loading on SDS-PAGE gels.
  • the proteins contained in the loaded gels were then transferred to PVDF and blocked for 1 hour with 5% BSA, followed by incubation with 1: 1000 dilution of the Filaggrin antibody (Santa Cruz, USA). After the HRP-attached secondary antibody reaction and washing process, the band of the protein was confirmed using the ECL solution (FIG. 2B).
  • cytoperm / cytofix (BD, USA) was treated in the cultured human HaCaT cell line according to the experimental method provided to fix the cells and filaggrin (Filaggrin).
  • RNA and protein of pilaggrin in keratinocytes cultured in toluene-treated monolayers decreased in a toluene concentration-dependent manner compared to the control group treated with PBS.
  • the three-dimensional skin model used in Examples 2 and 3 is a normal human epidermal keratinocyte derived from Neonatal-foreskin tissue or adult breast tissue.
  • Human Epithelial Keratinocytes (NHEK) consists of 8 to 15 cell layers, which have a basal-, spinous-, granular- and cornified-layer Similar to in vivo epithelial tissue (Carlson et al., Curr Protoc Cell Biol., Chapter 19: Unit 19.9. Doi: 10.1002 / 0471143030.cb1909s41, 2008).
  • the three-dimensional skin model has metabolic and cell division activity, pro-filaggrin, K1 / K10 cytokeratin pair, and involucrin, which are mature epithelial specific differentiation markers. ) And type 1 epidermal transglutaminase.
  • the three-dimensional skin model maintains normal epidermal morphology for up to three weeks in Dulbecco's Modified Eagle's Medium (DMEM) medium containing epidermal growth factor, insulin, hydrocortisone, and antibiotics. Can be cultured in the meantime.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the three-dimensional skin model was cultured using a culture method of MatTek, followed by administration (treatment) of TSLP (Thymic stromal lymphopoietin) or toluene, and the expression changes of filaggrin (Filaggrin) and antimicrobial peptides were confirmed.
  • TSLP physiological stromal lymphopoietin
  • a three-dimensional skin model consisting of normal human epithelial keratinocytes (The EpiDerm TM Skin Model, EPI-100, MatTek) was developed using MatTek's culture method. After culturing, 100ng / ml TSLP was treated in the culture solution for 16 hours (control was treated with Phosphate-Buffered Saline (PBS)), and a three-dimensional skin model was taken (the three-dimensional skin model treated with TSLP was obtained in Example 3). Used for the screening of atopic dermatitis treatments), fixed in 4% formaldehyde (Sigma, USA), and then embedded in paraffin to prepare slides.
  • PBS Phosphate-Buffered Saline
  • the tissue slides were subjected to deparinization using xylene and dehydration using alcohol, and then the slides were immersed in a citrate buffer for 10 minutes in a microwave oven, and then cooled at room temperature for 1 hour.
  • the slides were washed with distilled water and allowed to react with 5% goat serum at room temperature for 1 hour, followed by anti-Plaggrin, anti- ⁇ -defensin, and anti-Psoriasin antibodies. Incubation was by diluting to 1:50. After reacting the HRP-attached secondary antibody, it was developed using a DAB kit to confirm the change in the expression of Filaggrin in the tissue.
  • the amount of expression of ⁇ -defensin and psoriasin in the three-dimensional skin model of TSLP-treated filaggrin and antibacterial peptides compared to the control group to which PBS was administered was TSLP. It was found to decrease in a concentration dependent manner, which was similar to the trend of filaggrin expression in the keratinocytes cultured with monolayers.
  • a three-dimensional skin model (The EpiDerm TM Skin Model, EPI-100, MatTek) was incubated by MatTek's culture method, and 50 ⁇ M of toluene was treated in the culture solution for 24 hours.
  • Control was treated with Phosphate-Buffered Saline (PBS)
  • PBS Phosphate-Buffered Saline
  • a three-dimensional skin model was taken (toluene-treated three-dimensional skin model is used for the screening of the atopic dermatitis treatment agent of Example 3) 4% formaldehyde (Sigma, USA), and then embedded in paraffin to make a slide.
  • the tissue slides were subjected to deparinization using xylene and dehydration using alcohol, and then the slides were immersed in a citrate buffer for 10 minutes in a microwave oven, and then cooled at room temperature for 1 hour.
  • the slides were washed with distilled water, reacted with 5% goat serum for 1 hour at room temperature, and then incubated with 1:50 dilution of the Filaggrin antibody.
  • HRP-attached secondary antibody was reacted by color development using DAB kit to confirm the change in the expression of Filaggrin (Filaggrin) in the tissue.
  • Example 1 Statistical analysis in Example 1 and Example 2 was used for the test of significance between the two groups using the t-test test, which is effective when * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 compared to the control group Considered as a difference. All data are expressed as mean ⁇ measurement standard error (SEM).
  • atopic dermatitis-like three-dimensional skin tissue model obtained by administering TSLP or toluene to the three-dimensional skin tissue model (The EpiDerm TM Skin Model, EPI-100, MatTek) in the same manner as in Example 2, the control group was treated with PBS ( Phosphate-Buffered Saline (DMSO) or dimethyl sulfoxide (DMSO) were treated, and the experimental group treated with atopic dermatitis candidates, followed by filaggrin and antimicrobial peptides ( ⁇ -defensin or Psoriasin). Expression was analyzed using Reverse Transcription-PCR, Western blot and / or immunohistochemical staining.
  • PBS Phosphate-Buffered Saline
  • DMSO dimethyl sulfoxide
  • candidate atopic dermatitis treatment PBS (Phosphate-Buffered Saline) or dimethyl sulfoxide (DMSO) treated in atopic dermatitis-like three-dimensional skin tissue model was treated at an appropriate concentration and exposure time by conventional methods in the art (Thomas K. Petersen, Basic & Clinical Pharmacology & Toxicology , 99: 104-115, 2006).
  • Candidates that increase filaggrin and antimicrobial peptide expression in the experimental group compared to the control group were evaluated and selected as potential atopic dermatitis treatments.
  • a three-dimensional skin tissue model according to the TSLP or toluene treatment of the present invention shows a decrease in the gene expression of pilagrin and the antimicrobial peptide ⁇ -defensin or thyrasine, an important finding in skin lesions of atopic dermatitis. It is of great value and can provide a new animal replacement test method that can be utilized for evaluating the efficacy of mechanisms for treating atopic dermatitis and researching mechanisms.

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Abstract

La présente invention concerne un modèle de tissu cutané tridimensionnel analogue à la dermatite atopique et un procédé de criblage d'un médicament pour la dermatite atopique l'utilisant et, plus précisément, un procédé de criblage d'un médicament pour la dermatite atopique, le procédé étant caractérisé par : la construction d'un modèle de tissu cutané tridimensionnel qui présente des constatations similaires à celles de la dermatite atopique ayant une réduction de l'expression de la filaggrine et une réduction en outre de l'expression du peptide antibactérien β-défensine ou psoriasine, par un traitement comprenant de la lymphopoïétine stromale thymique (TSLP) ou du toluène, un facteur environnemental; le traitement du modèle de tissu cutané tridimensionnel par des médicaments candidats pour la dermatite atopique; et ensuite la sélection, en tant que médicament, d'un candidat qui augmente l'expression de la filaggrine et augmente en outre l'expression du peptide antibactérien β-défensine ou psoriasine. Le modèle de tissu cutané tridimensionnel traité avec de la TSLP ou du toluène, selon la présente invention, présente une grande valeur en tant que modèle de tissu cutané analogue à la dermatite atopique par le fait que ce dernier présente les réductions dans les expressions génétiques de la filaggrine et du peptide antibactérien β-défensine ou psoriasine, qui correspondent à des constatations importantes dans la lésion cutanée de la dermatite atopique; et le modèle de tissu cutané tridimensionnel permet de fournir un nouveau procédé d'essai de remplacement des animaux pouvant être utilisé dans des évaluation d'efficacité des médicaments pour la dermatite atopique, les mécanismes de recherche de ce derniers, et similaires.
PCT/KR2015/004356 2015-01-19 2015-04-29 Modèle de tissu cutané tridimensionnel analogue à la dermatite atopique et procédé de criblage de médicament pour la dermatite atopique l'utilisant Ceased WO2016117765A1 (fr)

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KR10-2015-0008892 2015-01-19
KR1020150008892A KR101655384B1 (ko) 2015-01-19 2015-01-19 아토피 피부염 유사 3차원 피부조직 모델 및 이를 이용한 아토피 피부염 치료제의 스크리닝 방법

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WO2019004378A1 (fr) * 2017-06-29 2019-01-03 富士フイルム株式会社 Chambre de transplantation et dispositif de transplantation

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JP2005110602A (ja) * 2003-10-09 2005-04-28 Sumitomo Pharmaceut Co Ltd アトピー性皮膚炎の疾患マーカー及びその利用
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KR20120088895A (ko) * 2010-11-19 2012-08-09 (주)아모레퍼시픽 피부 세포 분화 촉진 물질의 스크리닝 방법

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JP2005110602A (ja) * 2003-10-09 2005-04-28 Sumitomo Pharmaceut Co Ltd アトピー性皮膚炎の疾患マーカー及びその利用
KR20100098476A (ko) * 2009-02-28 2010-09-07 대한민국 (식품의약품안전청장) 아토피성 피부염 질환모델 동물 및 그의 작제방법
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019004378A1 (fr) * 2017-06-29 2019-01-03 富士フイルム株式会社 Chambre de transplantation et dispositif de transplantation

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KR20160089214A (ko) 2016-07-27

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