KR101655384B1 - Atopic Dermatitis-like 3-Dimensional Skin Tissue Model, and Method of Screening Therapeutic Agents for Atopic Dermatitis Using the Same - Google Patents
Atopic Dermatitis-like 3-Dimensional Skin Tissue Model, and Method of Screening Therapeutic Agents for Atopic Dermatitis Using the Same Download PDFInfo
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Abstract
The present invention relates to a 3D model of atopic dermatitis-like skin tissue and a method for screening a therapeutic agent for atopic dermatitis using the same. More particularly, the present invention relates to a method for screening a therapeutic agent for atopic dermatitis using filamentous fungi such as TSLP (Thymic stromal lymphopoietin) And a 3-dimensional skin tissue model showing atopic dermatitis-like similarity in which the expression of the antimicrobial peptide β-defensin or Psoriasin is further reduced. Thus, a candidate for atopic dermatitis treatment candidate A method for screening a therapeutic agent for atopic dermatitis characterized in that after treatment of a substance, a candidate substance which increases the expression of pilar green and further increases the expression of the antimicrobial peptide,? -Diphencin or cephalosporin is selected as a therapeutic agent will be.
The three-dimensional skin tissue model according to TSLP or toluene treatment of the present invention shows a decrease in gene expression of filagreen, which is an important finding in skin lesions of atopic dermatitis, and β-diphencin or cephalosporin, which is an antibacterial peptide, And can provide a new animal replacement test method that can be used for evaluation of efficacy of atopic dermatitis therapeutic agent and research on mechanism.
Description
The present invention relates to a 3D model of atopic dermatitis-like skin tissue and a method for screening a therapeutic agent for atopic dermatitis using the same. More particularly, the present invention relates to a method for screening a therapeutic agent for atopic dermatitis using filamentous fungi such as TSLP (Thymic stromal lymphopoietin) And a 3-dimensional skin tissue model showing atopic dermatitis-like similarity in which the expression of the antimicrobial peptide β-defensin or Psoriasin is further reduced. Thus, a candidate for atopic dermatitis treatment candidate A method for screening a therapeutic agent for atopic dermatitis characterized in that after treatment of a substance, a candidate substance which increases the expression of pilar green and further increases the expression of the antimicrobial peptide,? -Diphencin or cephalosporin is selected as a therapeutic agent will be.
The three-dimensional skin model is an artificial skin model (RhE, RL) that reconstructs the human epidermis three-dimensionally by inducing differentiation of keratinocytes, the main cells forming the epidermis of human skin, from the air-liquid interphase Reconstituted human skin equivalent model. Through this, it is possible to do many researches such as substitution of primary stimulation of skin. Artificial skin models such as EPISKINTM, EpiDermTM, and SkinEthicTM have already been listed in the OECD Guideline, certifying their usefulness as an alternative to skin irritation through a variety of validation studies.
Atopic dermatitis is a chronic inflammatory skin disease in which eczema lesions repeatedly recur in certain areas, accompanied by severe itching and overall dry skin. It has a genetic predisposition and is associated with allergic diseases such as food allergies, allergic asthma, and allergic rhinitis (Akiharu Kubo meat al ., J Clin Invest . 122 (2): 440-447, 2012; Hywel CW et al ., N Engl J Med , 352: 2314-2324, 2005). The pathogenesis of atopic dermatitis today is largely divided into the pathogenesis associated with skin barrier and the pathogenesis associated with the abnormal immune response. The mutation of the pilana green gene occurs in about 40% of patients with atopic dermatitis, After being reported to be common, external allergens in people with inherently impaired skin barriers are more likely to pass through the stratum corneum and cause sensitization to the body, leading to atopic dermatitis.
On the other hand, it is known that the key protein that constitutes the skin barrier plays an important role in the stratum corneum formation such as filaggrin, Loricrin, and Involucrin.
Among them, pillared green exists in the form of propylagreen, a protein that constitutes the keratohyalin granule in the granule layer, and decomposes into pillar green in the final differentiation process of the keratinocyte to form keratin membrane The filaments are agglomerated to form a hard and flat structure of the keratinocytes, which acts as a brick in the skin barrier. As it moves up to the top of the stratum corneum, the pillar green is decomposed into amino acids and transformed into free amino acids and pyrrolidone carboxylic acid (PCA), which are very important for humidification. It has been reported that degradation products of Pillar Green play the most important role in keeping skin moist (McGrath JA et al ., Trends in Molecular Medicine , 14: 20-27, 2008; Brown SJ et al ., Journal of Investigative Dermatology , 132: 751-762, 2012).
Pilar green produces citrulline, PCA, and trans-UCA through degradation, which performs stratum hydration, normalization of the stratum corneum, permeability barrier, integrity / cohesion of the stratum corneum, and antibacterial and anti-inflammatory actions.
Ichthyosis vulgaris, caused by a mutation in the fila green gene (FLG), is associated with very severe skin dryness. That is, the mutation of the filaggrin gene reduces the production of filaggrin protein and reduces the production of natural moisturizing factors, thereby causing dry skin. There is no difference in the expression of Loricrin or Involucrin, which account for a large percentage of epidermal differentiation proteins in non-lesional skin of patients with atopic dermatitis. However, there is a report that only the expression of toxic filaggrin is reduced, It has been actively studied since it has emerged as a pathogenesis of atopic dermatitis.
Studies on atopic dermatitis patients with no LOF mutation in the pilla green gene have been carried out. However, studies on the reduction of filla greens due to external environmental factors other than genetic factors have been conducted.
Several reports have reported that reduced expression of pilgar green by Th2 cytokines, which have been reported to be increased in patients with atopic dermatitis (Kim BE et al ., Journal of Investigative Dermatology , 131: 1272-1279, 2011; Howell MD et al ., J Allergy Clin Immunol , 120: 150-5, 2007).
Atopic dermatitis patients have high TSLP (Thymic stromal lymphopoietin) in keratinocytes. The highest protein of abnormal signal transduction is the TSLP cytokine. When allergen penetrates into the body, TSLP and other substances stimulate dendritic cells to react, which manifests as symptoms of atopic dermatitis. TSLP stimulates CD11c + dendritic cells to increase TARC (Thymus and activation-regulated chemokine) and MDC production. The concentration of these cytokines is closely related to the symptoms of atopic dermatitis (Holgate ST et al ., Trends in Immunology , 28: 248-251, 2007).
The reduction of anti-microbial peptides is evident in acute lesions in patients with atopic dermatitis. The antimicrobial peptide is an important element that constitutes the innate immune system of the human body. It is a cationic peptide, which is formed in the epithelial tissue and constitutes the first line of defense that shows broad antibacterial action against viruses, bacteria and fungi. It activates the immune system and is involved in wound repair after vital injury. This decrease in antimicrobial peptides is due to the inhibition by Th-2 cytokine which is increased in atopic dermatitis. When IL-4 and IL-13 are administered to keratinocytes, β-defensin and catechol cathelicidin) was reported to be inhibited.
In addition, Volatile Organic Compounds (VOCs) are liquid or gaseous organic compounds that are easily vaporized into the atmosphere because of their high vapor pressure. They generate photochemical reactions in the air to generate photochemical oxidizing substances such as ozone, which causes photochemical smog Recently, research on allergic diseases has been actively conducted as a substance.
In the present invention, the mechanism of atopic dermatitis caused by TSLP, a cytokine that is overexpressed in lesions of volatile organic compounds (VOCs), which are environmentally harmful substances, in toluene and atopic dermatitis patients, is proposed using a three-dimensional skin model .
The present inventors have made intensive efforts to develop an animal replacement model that can be used for a screening method for treating atopic dermatitis based on studies on atopic dermatitis-like three-dimensional skin tissue model. As a result, The reduction of the expression level of the antimicrobial peptide gene and the reduction of the expression amount of the filaggrin gene following the treatment with toluene were confirmed in a three-dimensional skin tissue model, thereby completing the present invention.
It is an object of the present invention to provide an atopic dermatitis-like three-dimensional skin tissue model and a screening method of a therapeutic agent for atopic dermatitis using the same.
(A) treating TSLP (Thymic Stromal Lymphopoietin) or toluene (Toluene) with a 3D skin tissue model; (b) identifying the expression of filaggrin and an antimicrobial peptide in the treated three-dimensional skin tissue model; And (c) obtaining a three-dimensional skin tissue model in which the expression of filaggrin is reduced as compared to a control, as an atopic dermatitis-like three-dimensional skin tissue model. to provide.
The present invention also provides an atopic dermatitis-like three-dimensional skin tissue model produced by the above method and having reduced expression of filaggrin compared to a control.
(A) treating the atopic dermatitis-like three-dimensional skin tissue model with a candidate for atopic dermatitis therapeutic agent, and then confirming the expression of filaggrin and an antimicrobial peptide in cells of the skin tissue model; And (b) selecting a candidate substance that increases the expression of the filaggrin as compared to a control group, as a therapeutic agent. The present invention also provides a method for screening a therapeutic agent for atopic dermatitis using a three-dimensional skin tissue model similar to atopic dermatitis.
The three-dimensional skin tissue model according to TSLP or toluene treatment of the present invention shows a decrease in gene expression of filagreen, which is an important finding in skin lesions of atopic dermatitis, and β-diphencin or cephalosporin, which is an antibacterial peptide, And can provide a new animal replacement test method that can be used for evaluation of efficacy of atopic dermatitis therapeutic agent and research on mechanism.
FIG. 1 shows changes in the expression of filaggrin in keratinocytes following TSLP treatment by concentration, (A) changes in filaggrin RNA expression by TSLP treatment confirmed by RT-PCR, (B) (Filaggrin) protein expression change by TSLP treatment confirmed by Western blotting, and (C) shows the change of filaggrin protein expression in keratinocyte determined by immunofluorescence staining.
FIG. 2 shows changes in the expression of filaggrin in keratinocytes following treatment with toluene by concentration. (A) shows changes in filaggrin RNA expression by TSLP treatment confirmed by RT-PCR, (B) shows changes in the expression of filaggrin protein by toluene treatment as determined by Western Bullet, and (C) shows changes in expression of filaggrin protein in keratinocytes as determined by immunofluorescence staining.
FIG. 3 shows the expression of filaggrin in a three-dimensional skin model according to TSLP treatment. FIG. 3 (A) shows the decrease of filaggrin expression by TSLP treatment, FIG. 3 - defensin and Psoriasin, S100 calcium-binding protein A7 (S100A7 gene product).
Figure 4 shows the decrease in expression of filaggrin in a three-dimensional skin model following treatment with toluene (Toluene).
In the present invention, TSLP (Thymic stromal lymphopoietin) or TSLP (Thymic stromal lymphopoietin) -treated TSLP (Thymic stromal lymphopoietin), which is expressed in patients with atopic dermatitis, Dependent peptidoglycoprotein and antimicrobial peptide and confirmed that the protein reduction of the filaggrin protein is reduced when the toluene treatment is applied to the atopic dermatitis-like three-dimensional skin tissue A new animal replacement test method using the model was developed.
In one embodiment of the present invention, the expression of RNA and protein of filaggrin in keratinocytes cultured in a monolayer treated with TSLP (Thymic stromal lymphopoietin) compared with a control group treated with PBS (Phosphate-Buffered Saline) Lt; / RTI > concentration was decreased in a TSLP concentration-dependent manner.
In another embodiment of the present invention, the expression level of RNA and protein in keratinocyte filaggrin cultured in a toluene-treated monolayer was decreased dependently on the concentration of toluene in comparison with the control group treated with PBS.
In another embodiment of the present invention, it has been confirmed that the reduction of the amount of filaggrin and antimicrobial peptide expressed by the TSLP treatment occurs in the three-dimensional skin model, and the expression of filaggrin in the keratinocyte cultured as a monolayer .
In another embodiment of the present invention, it has been confirmed that the reduction of the amount of filaggrin expression by the toluene treatment occurs in the three-dimensional skin model, which is similar to the trend of the expression of pilar green in the keratinocyte cultured as a monolayer .
Accordingly, in one aspect, the present invention provides a method of treating a three-dimensional skin tissue model comprising: (a) treating TSLP (Thymic stromal lymphopoietin) or toluene (Toluene) (b) identifying the expression of filaggrin and an antimicrobial peptide in the treated three-dimensional skin tissue model; And (c) obtaining a three-dimensional skin tissue model in which the expression of filaggrin is reduced as compared to a control, as an atopic dermatitis-like three-dimensional skin tissue model. . Here, the control group refers to a three-dimensional skin tissue model treated with PBS (Phosphate-Buffered Saline).
In the present invention, the cells constituting the three-dimensional skin tissue model may be keratinocytes. The antimicrobial peptide may be β-defensin or Psoriasin, The expression of the filaggrin and the antibacterial peptide may be confirmed by reverse transcription-PCR (PCR), Western blotting or immunohistochemical staining , The step (c) is characterized in that a three-dimensional skin tissue model in which the expression of filaggrin is reduced and the expression of the antibacterial peptide is further reduced as compared with the control group is obtained as an atopic dermatitis-like three-dimensional skin tissue model can do. Here, the control group refers to a three-dimensional skin tissue model treated with PBS (Phosphate-Buffered Saline).
In another aspect, the present invention relates to an atopic dermatitis-like three-dimensional skin tissue model produced by the above method and having reduced expression of filaggrin as compared to a control. Here, the control group refers to a three-dimensional skin tissue model treated with PBS (Phosphate-Buffered Saline).
In the present invention, the cells constituting the three-dimensional skin tissue model may be characterized by being keratinocytes of human, and the atopic dermatitis-like three-dimensional skin tissue model may be characterized by a filaggrin The antimicrobial peptide may be characterized in that the expression is decreased and the expression of the antimicrobial peptide is further reduced. The antimicrobial peptide may be β-defensin or Psoriasin, Expression of filaggrin and antimicrobial peptides can be characterized using reverse transcription-PCR, Western blot or immunohistochemical staining. Here, the control group refers to a three-dimensional skin tissue model treated with PBS (Phosphate-Buffered Saline).
In another aspect, the present invention provides a method for treating atopic dermatitis, comprising the steps of: (a) treating the atopic dermatitis-like three-dimensional skin tissue model with a candidate substance for treating atopic dermatitis, and then detecting the expression of filaggrin and an antimicrobial peptide in cells of the skin tissue model; ; And (b) selecting a candidate substance that increases the expression of the filaggrin as compared with a control group. The present invention also relates to a method for screening a therapeutic agent for atopic dermatitis using a three-dimensional skin tissue model similar to atopic dermatitis. Here, the control group refers to a three-dimensional skin tissue model similar to atopic dermatitis treated with PBS (Phosphate-Buffered Saline) or DMSO (dimethyl sulfoxide).
In the present invention, the cells constituting the three-dimensional skin tissue model may be characterized as keratinocytes of human. The antimicrobial peptide may be β-defensin or Psoriasin The expression of the filaggrin and the antibacterial peptide may be confirmed by reverse transcription-PCR (PCR), Western blotting or immunohistochemical staining , The step (b) may be characterized by selecting a candidate substance that increases the expression of filaggrin and increases the expression of the antibacterial peptide more than the control group. Here, the control group refers to a three-dimensional skin tissue model similar to atopic dermatitis treated with PBS (Phosphate-Buffered Saline) or DMSO (dimethyl sulfoxide).
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
Example
One:
TSLP
Or pyrazine in keratinocytes cultured in a monolayer with toluene treatment (
Filaggrin's
Expression change
One) TSLP process
In order to confirm the expression of filaggrin expressed by TSLP (Thymic stromal lymphopoietin), TSLP was treated at the concentration of keratinocytes cultured in a monolayer by concentration, RNA and proteins were extracted from the cells, And the change of the expression level was confirmed.
(HaCaT cell line; order No. 300493, CLS, Eppelheim, Germany; German Cancer Research Center, Heidelberg, Germany) (Boukamp et al. al . , J. Cell Biol . 10, and 100 ng / ml of TSLP were treated for 16 hours (treated with PBS (Phosphate-Buffered Saline)) and cultured with TRIzol reagent After homogenizing with a homogenizer (OMNI-inc, USA), the RNA was isolated according to the manufacturer's instructions. CDNA of each tissue was prepared using the reverse transcriptase (Super-bio, Korea). (SEQ ID NO: 1): 5'-ggtctggacgttcagggtct-3 ', Filaggrin-r (reverse) [SEQ ID NO: 2]: 5'- (SEQ ID NO: 3): 5'-AGCAATGCCTCCTGCACCACCAAC-3 ', GAPDH-r (reverse) [SEQ ID NO: 4]: 5'-CCGGAGGGGCCCCCCCAGTCT-3') primer (GAPDH- 35 cycles were performed with one cycle consisting of denaturation step (95 ° C., 30 seconds), annealing step (56 ° C., 30 seconds) and extension step (72 ° C., 40 seconds), and amplified by PCR (Polymerase Chain Reaction) . The amplified sample was observed for its expression pattern on 1% agarose gel (FIG. 1A).
In order to confirm the change of protein expression level of pilar green, a human keratinocyte cell line (HaCaT cell line) was cultured and treated with TSLP of 0, 1, 10, 100 ng / ml for 16 hours (control group: PBS (Buffered Saline) and RIPA buffer (Elpis, Korea). The cells were incubated in ice for 30 minutes, centrifuged at 13000 rpm for 30 minutes in a refrigerated centrifuge, Was loaded onto an SDS-PAGE gel. Protein contained in the loaded gel was then transferred to PVDF, blocked with 5% BSA (Bovine Serum Albumin) for 1 hour, diluted 1: 1000 with Filaggrin antibody (Santa Cruz, USA) Followed by incubation. Next, after the secondary antibody reaction and washing process with HRP, the protein band was confirmed using ECL solution (FIG. 1B).
In order to confirm the change of protein expression level of pilar green within the cells, cytoperm / cytofix (BD, USA) was applied to the cultured human keratinocyte line (HaCaT cell line) according to the provided method to fix the cells and to fix filaggrin ) Antibody diluted 1: 100 in PBS (Phosphate-Buffered Saline), reacted at 4 ° C for 1 hour, washed with PBS, diluted 1: 100 with Cy3-conjugated secondary antibody and incubated at 4 ° C for 1 hour After the reaction, the cells were washed with PBS, and expression of pilgar green was confirmed under a fluorescence microscope (Fig. 1C).
As a result, it was confirmed that the amount of RNA and protein expressed in keratinocyte filaggrin cultured in a single layer treated with TSLP (Thymic stromal lymphopoietin) was decreased in a TSLP concentration-dependent manner, compared to the control group treated with PBS.
2) Toluene ( Toluene ) process
In order to confirm the expression of filaggrin by the treatment with toluene (Toluene), the keratinocytes cultured in the monolayer were treated with toluene at different concentrations, and the expression level of the filaggrin at the RNA and protein level was examined.
In order to confirm the change of RNA expression level of pilar green, the human keratinocyte line (HaCaT cell line) was cultured and treated with 0, 12.5, 25, 50 nM toluene for 24 hours (the control group was phosphate-buffered Saline), TRIzol reagent was added, and the mixture was finely ground using a homogenizer. RNA was isolated according to the manufacturer's instructions. CDNA of each tissue was prepared using the reverse transcriptase (Super-bio, Korea). Filaggrin-f (forward) [SEQ ID NO: 1]: 5'-ggtctggacgttcagggtct-3 ', Filaggrin-r (reverse) [SEQ ID NO: 2]: 5'-ggatgtggtgtggctgtgat 3 ') and a GAPDH primer (GAPDH-f (forward) [SEQ ID NO: 3]: 5'-AGCAATGCCTCCTGCACCACCAAC-3', GAPDH-r (reverse) [SEQ ID NO: 4]: 5'- CCGGAGGGGCCATCCACAGTCT- 35 cycles were performed with one cycle consisting of denaturation step (95 ° C., 30 seconds), annealing step (56 ° C., 30 seconds) and extension step (72 ° C., 40 seconds). The amplified samples were observed for their expression pattern on 1% agarose gel (FIG. 2A).
(HaCaT cell line) was cultured in the culture medium for 24 hours (0, 12.5, 25, and 50 nM toluene was treated with phosphate buffered saline (PBS) Saline) and RIPA buffer (Elpis, Korea) were added to the cells. The cells were allowed to stand for 30 minutes on ice, centrifuged at 13000 rpm for 30 minutes in a refrigerated centrifuge, and 50 μg of protein SDS-PAGE gel. Protein contained in the loaded gel was then transferred to PVDF, blocked with 5% BSA for 1 hour, diluted 1: 1000 with Filaggrin antibody (Santa Cruz, USA) and incubated. After the secondary antibody reaction with HRP and the washing process, the protein band was confirmed using ECL solution (FIG. 2B).
In order to confirm the change of protein expression level of pilar green within the cells, cytoperm / cytofix (BD, USA) was applied to the cultured human keratinocyte line (HaCaT cell line) according to the provided method to fix the cells and to fix filaggrin ) Antibody The antibody was diluted 1: 100 in PBS, reacted at 4 ° C for 1 hour, washed with PBS, diluted 1: 100 with Cy3-conjugated antibody, reacted at 4 ° C for 1 hour, And then the expression of pillar green was confirmed under a fluorescence microscope (Fig. 2C).
As a result, it was confirmed that the amount of RNA and protein expressed in keratinocyte filaggrin cultured in the toluene-treated monolayer decreased in dependence on the concentration of toluene, compared with the control group treated with PBS.
Example
2:
TSLP
Or in the three-dimensional skin model with toluene administration
Filaggrin) and
Expression changes of antimicrobial peptides
Three-dimensional skin model
The three-dimensional skin model (EpiDermTM Skin Model, EPI-100, MatTek) used in Examples 2 and 3 is a normal human epidermal keratinocyte from Neonatal-foreskin tissue or adult breast tissue Human Epithelial Keratinocytes: NHEK), which have basal-, spinous-, granular-, and cornified-layer structures. in vivo (in vivo epithelial tissue (Carlson et < RTI ID = 0.0 > al . , Curr Protoc Cell Biol. , Chapter 19: Unit 19.9. doi: 10.1002 / 0471143030.cb1909s41, 2008). In addition, the three-dimensional skin model has metabolic and mitotic activity, and is characterized by pro-filaggrin, a K1 / K10 cytokeratin pair, involucrin ) And type I epidermal transglutaminase. ≪ / RTI > The three-dimensional skin model is maintained in a normal DMEM (Dulbecco's Modified Eagle's Medium) medium containing epidermal growth factor, insulin, hydrocortisone, antibiotics, etc. for up to 3 weeks in an epidermal morphology .
The three-dimensional skin model was cultured using a MatTek culture method, and then TSLP (Thymic stromal lymphopoietin) or toluene was administered (treated) as described below, and the expression of filaggrin and antimicrobial peptides was examined.
One) TSLP process
Carried out within (in the Example 1 in vitro In order to confirm whether the reduction of filaggrin expression by TSLP (Thymic stromal lymphopoietin) was observed in 3-D skin model, TSLP treatment was applied to 3-D skin model, Were identified by immunohistochemical staining.
(EpiDerm ™ Skin Model, EPI-100, MatTek) composed of human normal epithelial keratinocytes was cultured by MatTek's culture method in order to confirm the change of protein expression level of pilar green The TSLP-treated three-dimensional skin model was treated with 100 ng / ml of TSLP for 16 hours (treated with PBS (Phosphate-Buffered Saline) (For screening of atopic dermatitis), fixed in 4% formaldehyde (Sigma, USA) and embedded in paraffin to make slides. After the tissue slides were dephosphorylated using xylene and dehydrated using alcohol, the slides were immersed in a citrate buffer, and the slides were rotated in a microwave oven for 10 minutes, followed by cooling at room temperature for one hour. The slides were washed with distilled water and reacted with 5% goat serum for 1 hour at room temperature. The cells were treated with anti-filaggrin, anti-β-defensin and anti-threerine (Psoriasin) 1:50 and incubated. HRP-conjugated secondary antibody was reacted and DAB kit was used for color development to confirm the expression of filaggrin in the tissues.
As a result, the expression level of filaggrin and antimicrobial peptides such as β-defensin and Psoriasin in the three-dimensional skin model administered with TSLP compared to the control group administered with PBS became TSLP Dependent manner, and this was similar to the trend of pilar green expression in the keratinocyte cultured as a monolayer.
2) Toluene (T olive ) process
Carried out within (in the Example 1 in vitro In order to confirm that the reduction of filaggrin expression by toluene was observed in the 3 - dimensional skin model, toluene was applied to the 3 - dimensional skin model, and the expression of the filaggrin was analyzed by immunohistochemical staining Respectively.
(EpiDerm ™ Skin Model, EPI-100, MatTek) was cultured by MatTek culture method and 50 μM of toluene was added to the culture solution for 24 hours to confirm the change in protein expression level of pilar green (The control group was treated with PBS (Phosphate-Buffered Saline)) and a 3-dimensional skin model was taken (the toluene-treated three-dimensional skin model was used for screening of the atopic dermatitis treatment agent of Example 3) USA), and embedded in paraffin to prepare slides. After the tissue slides were dephosphorylated using xylene and dehydrated using alcohol, the slides were immersed in a citrate buffer, and the slides were rotated in a microwave oven for 10 minutes, followed by cooling at room temperature for one hour. The slide was washed with distilled water, reacted with 5% goat serum for 1 hour at room temperature, and diluted with Filaggrin antibody at 1:50 and incubated. HRP-conjugated secondary antibody was reacted and DAB kit was used for color development to confirm the expression of filaggrin in the tissues.
As a result, it was confirmed that the expression level of filaggrin in the three-dimensional skin model administered with toluene was decreased in dependence on the concentration of toluene, compared with the control group administered with PBS, and the expression of fila green in the keratinocyte cultured as a monolayer .
Statistical processing
The statistical analysis in Example 1 and Example 2 was performed using a t-test as a significance test between the two groups, and it was found that when compared with the control group, * P <0.05, ** P <0.01, *** P < Respectively. All data are expressed as mean ± standard error of measurement (SEM).
Example 3: Screening method of atopic dermatitis treatment using atopic dermatitis-like 3-dimensional skin tissue model
Using an atopic dermatitis-like three-dimensional skin tissue model obtained by administering TSLP or toluene to a three-dimensional skin tissue model (The EpiDermTM Skin Model, EPI-100, MatTek) in the same manner as in Example 2, Phosphate-Buffered Saline (DMSO) or DMSO (dimethyl sulfoxide). The experimental group was treated with candidate substances for treating atopic dermatitis and then treated with pilar green and antimicrobial peptides (β-defensin or Psoriasin) Expression was analyzed using reverse transcription-PCR, Western blot and / or immunohistochemistry. Here, atopic dermatitis treatment candidate, PBS (Phosphate-Buffered Saline) or DMSO (Dimethyl sulfoxide), which has been treated with the atopic dermatitis-like three-dimensional skin tissue model, has been treated at a proper concentration and exposure time by a conventional method in the art K. Petersen, Basic & Clinical Pharmacology & amp ; Toxicology , 99: 104-115, 2006). Compared with the control group, candidates that increase the expression of pilgar and antimicrobial peptides in the experimental group were evaluated and selected as potent atopic dermatitis treatments.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> Korea University Research And Business Foundation <120> Atopic Dermatitis-like 3-Dimensional Skin Tissue Model, and Method of Screening Therapeutic Agents for Atopic Dermatitis Using the Same <130> P14-B382 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Filaggrin-f <400> 1 ggtctggacg ttcagggtct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Filaggrin-r <400> 2 ggatgtggtg tggctgtgat 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-f <400> 3 agcaatgcct cctgcaccac caac 24 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-r <400> 4 ccggaggggc catccacagt ct 22
Claims (15)
(a) treating TSLP (Thymic stromal lymphopoietin) or toluene (Toluene) on a three-dimensional skin tissue model composed of human keratinocytes;
(b) confirming the expression of filaggrin and the antimicrobial peptide β-defensin or Psoriasin in the treated three-dimensional skin tissue model; And
(c) A three-dimensional skin tissue model with reduced expression of filaggrin and antimicrobial peptides, β-defensin or Psoriasin, compared to the control group was used for atopic dermatitis-like three-dimensional skin tissue model Lt; / RTI >
(a) treating atopic dermatitis candidate agents of the atopic dermatitis-like three-dimensional skin tissue model prepared by the method of (1), and then treating the cells of the skin tissue model with filaggrin and an antimicrobial peptide, beta-defensin) or Psoriasin (Psoriasin); And
(b) selecting a candidate substance that increases the expression of the filaggrin and the antimicrobial peptide β-defensin or Psoriasin compared to the control group.
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