WO2022102885A1 - Technique for diagnosing stress associated with atopic dermatitis using exosome-derived mirnas - Google Patents

Technique for diagnosing stress associated with atopic dermatitis using exosome-derived mirnas Download PDF

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WO2022102885A1
WO2022102885A1 PCT/KR2021/005258 KR2021005258W WO2022102885A1 WO 2022102885 A1 WO2022102885 A1 WO 2022102885A1 KR 2021005258 W KR2021005258 W KR 2021005258W WO 2022102885 A1 WO2022102885 A1 WO 2022102885A1
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mmu
seq
mir
exosome
derived
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French (fr)
Korean (ko)
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우정민
성민경
서민수
강경구
성수은
최주희
이시준
김길수
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재단법인 대구경북첨단의료산업진흥재단
경북대학교 산학협력단
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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
    • 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
    • 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
    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a composition for diagnosing stress using the fact that the expression pattern of exosome-derived miRNA changes when stress is induced by atopic dermatitis.
  • stress refers to a non-specific biological response occurring in the body to various injuries or stimuli applied to the living body.
  • Response to external temporary stress may be a natural phenomenon, but if the response to stress persists for a long time, mental or physical damage may occur.
  • stress is not always perceptible to the subject to which the stress is applied, and in the case of animals, it is sometimes difficult to recognize that they are in a state of stress.
  • exosomes are small extracellular vesicles with a diameter of about 200 nm or less that are formed inside the cell and secreted out of the cell through multivesicles. Exosomes, which contain protein and genetic information of parental cells, have recently been suggested to be useful as biomarkers for various diseases.
  • Another object of the present invention is to provide a kit for diagnosing stress induced by atopic dermatitis, comprising the composition.
  • Another object of the present invention is to provide an information providing method for diagnosing stress induced by atopic dermatitis, comprising comparing the expression level of a specific miRNA derived from an exosome of an individual with the expression level of an exosome-derived miRNA of a control group will provide
  • the present invention provides mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3) , mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu -miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu -miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 1
  • the exosome may be a nerve-derived exo.
  • the agent may include a primer or probe that specifically binds to the one or more exosome-derived miRNAs.
  • the present invention also provides a kit for diagnosing stress induced by atopic dermatitis comprising the composition.
  • the present invention also comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-mi
  • the biological sample may be blood.
  • the exosome may be a nerve-derived exo.
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), when the expression level of one or more miRNAs selected from the group consisting of mmu-miR-466i-5p (SEQ ID NO: 8) and mmu-miR-5128 (SEQ ID NO: 9) is higher than that of the control group, the subject is in a stress state It may further include the step of determining that it is.
  • mmu-let-7i-5p SEQ ID NO: 10
  • mmu-miR-130a-3p SEQ ID NO: 11
  • mmu-miR-140-3p SEQ ID NO: 11
  • the present invention comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-mi
  • the present invention analyzed miRNAs derived from exosomes of mice induced by stress due to atopic dermatitis, and then identified a number of miRNAs whose expression patterns change before and after stress treatment, and derived that they can be used as biomarkers. , it can be used in various fields related to the relationship between skin inflammation and stress and the stress diagnosis project.
  • FIG. 1 schematically shows the experimental design for the method and each group setting for inducing atopic dermatitis in Balb/c mice.
  • Figure 2 shows whether atopic dermatitis is induced after DNCB treatment is confirmed through skin tissue.
  • 3 is a graph confirming the expression pattern of specific proteins in the hippocampus of the brain at 2 days, 6 days, and 14 days after induction of atopic dermatitis.
  • Figure 4 shows the morphological confirmation of atopic dermatitis-induced animal serum-derived exosomes (A), the size distribution of the exosomes (B), and the results of confirming the exosome surface-specific expression protein.
  • Figure 5 confirms the expression of nerve-specific markers in some serum-derived exosomes and nerve-derived exosomes.
  • FIG. 6 shows the results of comparative analysis through NGS of the miRNA expression pattern of the nerve-derived exosomes in the atopic dermatitis-induced group compared to the normal group.
  • the present invention provides mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3), mmu-let-7e-5p ( SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu-miR-466i-5p (SEQ ID NO: 7) 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 11) 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-mi
  • miRNA refers to an approximately 22 nt untranslated RNA that acts as a post-transcriptional repressor through base binding with the 3' untranslated region (UTR) region of mRNA.
  • exosome miRNAs can be carried out by extracting exosomes from a sample and detecting exosome miRNAs in the extract. Detection of these exosomes miRNAs can be measured by hybridization and amplification reactions, but is not limited thereto and can be easily carried out using various techniques known in the art.
  • the exosome may be a nerve-derived exosome.
  • exosomal miRNA a marker whose expression level is decreased in a sample of a biological sample
  • exosome miRNA which is a marker whose expression level is increased, in a stress state induced by atopic dermatitis. It refers to a molecule that can be used for detection of a marker by checking the expression level of
  • the agent may include a primer or probe that specifically binds to the one or more exosome-derived miRNAs.
  • the detection of a nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers hybridized to a nucleic acid molecule or a complement of the nucleic acid molecule.
  • detection of exosome miRNAs using primers can be performed by amplifying the gene sequence using an amplification method such as PCR and then confirming whether the gene is amplified by a method known in the art.
  • a primer is a nucleic acid sequence having a short free 3' hydroxyl group.
  • a short nucleic acid sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand copying.
  • Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures.
  • stress can be diagnosed by performing PCR amplification using the sense and antisense primers specifically binding to the one or more miRNAs to check the expression level. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.
  • the probe refers to a nucleic acid fragment, such as RNA or DNA, corresponding to several bases to several tens of bases as long and is labeled.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.
  • stress can be diagnosed by performing hybridization using one or more of the above miRNAs and complementary probes and checking the expression level. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.
  • primers or probes can be appropriately designed by those skilled in the art based on known sequences.
  • primers or probes can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods.
  • Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoros. amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
  • the expression level of miRNA can be measured according to a method commonly used in the art, for example, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting), or gene chip, and the like are included, but are not limited thereto.
  • RT-PCR reverse transcriptase polymerase reaction
  • Competitive RT-PCR competitive reverse transcriptase polymerase reaction
  • Real-time RT-PCR real-time reverse transcriptase polymerase reaction
  • RNase protection assay RNase protection assay
  • Northern blotting Northern blotting
  • gene chip and the like are included, but are not limited thereto.
  • the present invention provides a kit for diagnosing stress induced by atopic dermatitis comprising the composition.
  • the kit may include not only an agent for measuring the expression level of the exosome miRNA, but also tools, reagents, etc. commonly used in the art that are used to be suitable for use as a stress diagnosis kit.
  • the tool or reagent examples include, but are not limited to, a suitable carrier, a labeling material capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like.
  • the labeling material is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.
  • the carrier includes a soluble carrier and an insoluble carrier
  • an example of the soluble carrier is a physiologically acceptable buffer known in the art, for example, PBS
  • an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly It may be acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.
  • kit of the present invention includes the above-described composition as a component, redundant descriptions are omitted in order to avoid excessive complexity of the present specification.
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-mi
  • the “comparison with the expression level of the corresponding exosome-derived miRNA in the control sample” means that the comparison target between the subject and the control group is the same type of miRNA derived from the same type of exosome.
  • mmu-let-7a-5p SEQ ID NO: 1
  • mmu-let-7b-5p SEQ ID NO: 2
  • mmu-let-7c-5p SEQ ID NO: 3
  • mmu-let-7e-5p SEQ ID NO: 4
  • mmu-miR-126a-5p SEQ ID NO: 5
  • mmu-miR-3473b SEQ ID NO: 6
  • mmu-miR-3473e SEQ ID NO: 7
  • determining that the subject is in a stress state may further include.
  • mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p (SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16) ), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a-5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 19) No.
  • the term "low expression” used while referring to the expression level of the exosome miRNA is a biomarker (exosome-derived miRNA in the present invention) indicating an abnormal process, disease, or other condition in an individual or a symptom thereof.
  • a biomarker exosome-derived miRNA in the present invention
  • high expression used while referring to the expression level of the exosomal miRNA in the present specification is when the biomarker indicates or is a symptom of an abnormal process, disease, or other condition within the subject, from a healthy or normal subject or a subject for comparison Refers to a value or level of a biomarker in a biological sample that is higher than a value or level range of a biomarker detected in the obtained biological sample.
  • biological sample refers to any sample obtained from an individual in which the expression of miRNA of the present invention can be detected.
  • the "individual” may correspond without limitation as long as it is a subject showing a similar pattern to the atopic induced group of the present invention when stress is induced by atopic dermatitis, and may preferably be a mammal.
  • the biological sample is any one selected from the group consisting of blood, saliva, biopsy, skin tissue, liquid culture, feces and urine, but is not particularly limited thereto.
  • it is blood, and it can be prepared by processing by a method commonly used in the art.
  • the exosome may be a nerve-derived exosome.
  • the present invention comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
  • step (b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-mi
  • Example 1-1 laboratory animal
  • mice were housed in conditions of temperature, humidity, and controlled room lighting (24 ⁇ 2 °C, 50 ⁇ 20 % relative humidity, 12/12 h light/dark cycle with lights on at 07:00). In addition, mice were given unrestricted access to food and water.
  • the mice and reagents used in the present invention are commercially available.
  • Example 1-2 Atopic dermatitis model induced by DNCB
  • Atopic dermatitis model was induced using 1-Chloro-2,4-dinitrobenzene (DNCB) (Sigma).
  • DNCB 1-Chloro-2,4-dinitrobenzene
  • FIG. 1 The protocol for inducing atopic dermatitis in each group is shown in FIG. 1 .
  • mice were divided into control groups, DNCB 2 days, DNCB 6 days and DNCB 14 days, and then the dorsal hair was removed.
  • DNCB day 2 group mice were treated once with 200 ⁇ L of 1% DNCB diluted acetone and olive oil (4:1) and sacrificed 24 hours later.
  • mice were treated twice with 200 ⁇ L of 1% DNCB on days 0 and 3, followed by sacrifice on day 6.
  • mice in the DNCB 14-day group were sensitized with 200 ⁇ L of 1% DNCB on days 0 and 3, then treated with 2% DNCB daily from days 6 to 13, and sacrificed on day 14.
  • Brains were dissected and fixed in 10% neutral buffered formalin. The tissue was then processed and embedded in paraffin. Brain paraffin block sections were cut to a thickness of 4 ⁇ m at the level of the hippocampus.
  • Hematoxylin and eosin (H&E) staining was performed using a DAKO CoverStainer (Agilent, Santa Clara, CA, USA). Immunohistochemistry (IHC) was performed to confirm the expression of the neuroinflammatory marker protein. Brain sections were prepared for anti-BDNF (dilution 1:500; Abcam, Abcam, Cambridge, UK), COX2 (dilution 1:250; Abcam, Abcam, Cambridge, UK), GFAP (dilution 1:500; Abcam, Cambridge, UK). Stained with primary antibody. Next, the labeled polymer DAKO EnVisionTM + System-HRP (Agilent, Santa Clara, CA, USA) was sectioned immunohistochemically according to the manufacturer's instructions.
  • IHC Immunohistochemistry
  • brain sections were scanned using a Pannoramic SCAN II (3DHISTECH Kft., Budapest, Hungary). Micrographs were taken with a CaseViewer (3DHISTECH Kft., Budapest, Hungary). Finally, hippocampal neuroinflammatory markers were quantified with Imaged J software (NIH, Bethesda, MD, USA).
  • ExoQuick solution (System Biosciences, Palo Alto, CA, USA) was used to isolate exosomes from serum by modifying the manufacturer's instructions. First, 3 mL serum was pelleted through centrifugation at 3,500 ⁇ g at 4 °C for 15 min to remove cell debris. Next, 3 mL of debris-deleted serum and 885 ⁇ L of ExoQuick solution were mixed. After centrifugation of the mixture at 3,500 ⁇ g for 10 min, the pellet was resuspended in 200 ⁇ l PBS.
  • Neuronal exosomes were isolated by referring and modifying the conventional literature (Plasma extracellular vesicles enriched for neuronal origin: A potential window into brain pathologic processes. Front Neurosci. 2017 May 22;11:278.). Total exosomes containing 200 ⁇ L PBS were incubated with 4 ⁇ L of anti-CD171 antibody (Bioss Antibodies, Beijing, China) for 1 hour at 4° C. in a rotating mixer. After addition of 15 ⁇ L PierceTM streptavidin + UltralinkTM resin (Thermo Fisher Scientific) and 25 ⁇ L PBS, the mixture was incubated in a rotating mixer at 4° C. for 1 hour and then centrifuged at 500 ⁇ g at 4° C. for 10 minutes.
  • Anti-CD171 antibody Bioss Antibodies, Beijing, China
  • the supernatant was removed from the sample and the pellet was resuspended in 200 ⁇ L 0.1M Glycine-HCl (Biosesang, Seongnam, Korea). After mixing for 10 seconds and vortexing for 30 seconds, the mixture was pelleted by centrifugation at 4,500 x g for 10 minutes at 4 °C. The supernatant was transferred to a new tube and then 15 ⁇ L Tris-HCl (Biosesang, Seongnam, Korea) and 25 ⁇ L PBS were added.
  • Exosomes isolated from serum were resuspended in cold distilled water. After loading the exosome suspension onto a formvar carbon coated grid (Ted Pella Inc.), the suspension was fixed in 2% paraformaldehyde for 10 minutes, the solution was removed, and the sample was dried. Grids were recorded using a bioTEM (Hitachi HT7700).
  • Example 1-7 Nanoparticle tracking analysis (NTA)
  • NTA was performed using a PMX120 (Particle Metrix) nanosight instrument according to the manufacturer's instructions.
  • tEV Total exosomes
  • nEV neuronal exosomes isolated from serum were each lysed using M-PER, HaltTM Protease and Phosphatase Inihhitor Cocktail (Thermo Scientific). Concentrations of exosome lysates were determined using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Next, 20 ⁇ g of exosome lysates were separated on a BoltTM 4-12% Bis-Tris Plus gel (Invitrogen, Carlsbad, CA, USA) and polyvinylidene fluoride (PVDF) membrane (Invitrogen, Carlsbad, CA, USA).
  • BoltTM 4-12% Bis-Tris Plus gel Invitrogen, Carlsbad, CA, USA
  • PVDF polyvinylidene fluoride
  • the membrane was blocked with 5% skim milk supplemented with TBS-T for 1 h at room temperature.
  • TSG101 NovusBio, USA
  • CD171 Santa Cruz Biotechnology, Santa Cruz, CA, USA
  • TUJ1 Abcam, Cambridge, UK
  • NSE Abcam, Cambridge, UK
  • NeuN Abcam, Cambridge, UK
  • HRP horseradish peroxidase
  • a 600 ⁇ L sample was prepared by collecting neuronal exosomes isolated from serum. The concentration of exosomes was measured using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. As a result of BCA analysis, the concentrations of each sample were 1.59 mg/mL (control) and 2.34 mg/mL (DNCB 14 days). Exosome smRNA isolation and library preparation were performed in Macrogen (Seoul, Korea) using the SMARTer smRNA-Seq Kit (Clontech Laboratories Inc., Mountain View, USA) according to the manufacturer's instructions. MiRNA sequencing was then performed using a HiSeq 2500 system according to HiSeq 2500 System Instruction Manual Document # 15035786 v02 HCS 2.2.70.
  • H&E staining and IHC were performed to analyze the expression level of neuroinflammatory markers due to stress induced by atopic dermatitis.
  • Histopathological staining no significant difference was found in the hippocampus of DNCB 2 days, DNCB 6 days, and DNCB 14 days in the photomicrograph compared to the control group (FIG. 3A).
  • IHC staining was performed and quantified, no significant change in BDNF expression was observed in the hippocampus (Fig.
  • NGS Next-generation sequencing
  • miRNAs upregulated in DNCB for 14 days compared to controls were mmu-let-7a-5p, mmu-let-7b-5p, mmu-let-7c-5p, mmu-let-7e-5p, mmu-miR- 126a-5p, mmu-miR-3473b, mmu-miR-3473e, mmu-miR-466i-5p and mmu-miR-5128 (FIG. 7).
  • Down-regulated miRNAs are mmu-let-7i-5p, mmu-miR-130a-3p, mmu-miR-140-3p, mmu-miR-142a-3p, mmu-miR-16-5p, mmu-miR-17 -5p, mmu-miR-185-5p, mmu-miR-19b-3p, mmu-miR-24-3p, mmu-miR-27a-5p, mmu-miR-29a-3p, mmu-miR-301a-3p , mmu-miR-451a, mmu-miR-669a-3p, mmu-miR-669o-3p and mmu-miR-93-5p (Fig. 8).
  • the sequence of the miRNA is shown in Table 1 below. And some of these miRNAs were identified as miRNAs showing expression changes in depression-related studies. According to previous studies that atopic dermatitis induces psychological stress and stress induces depression, NGS data suggest that these miRNAs can be used as psychological stress biomarkers.
  • the present invention analyzed miRNAs derived from exosomes of mice induced by stress due to atopic dermatitis, and then identified a number of miRNAs whose expression patterns change before and after stress treatment, and derived that they can be used as biomarkers. , it can be used in various fields related to the relationship between skin inflammation and stress and the stress diagnosis project.

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Abstract

The present invention comprises analyzing miRNAs derived from exosomes of a mouse in which stress was induced by atopic dermatitis, and then confirming that the expression patterns of a plurality of the miRNAs changed after the stress-inducing treatment compared to before the treatment, thereby deriving that the miRNAs may be used as a biomarker, and thus the present invention may be used in various related fields such as skin inflammation and stress correlation, and stress diagnosis businesses.

Description

엑소좀 유래 miRNA를 이용한 아토피성 피부염 상관 스트레스 진단기술Atopic dermatitis correlation stress diagnosis technology using exosome-derived miRNA
본 발명은 아토피성 피부염에 의해 스트레스 유도된 경우, 엑소좀 유래 miRNA의 발현 양상이 변화한다는 점을 이용한 스트레스 진단용 조성물 등에 관한 것이다.The present invention relates to a composition for diagnosing stress using the fact that the expression pattern of exosome-derived miRNA changes when stress is induced by atopic dermatitis.
일반적으로 스트레스(Stress)란, 생체에 가해지는 여러 상해 또는 자극에 대하여 체내에서 일어나는 비특이적인 생물반응을 의미한다. 외부의 일시적인 스트레스에 대한 반응은 자연스런 현상일 수 있지만, 스트레스에 대한 반응이 오랫동안 지속될 경우에는 정신적 또는 신체적으로 손상을 입게 될 수 있다. 특히, 스트레스는 스트레스가 가해지는 대상이 언제나 자각할 수 있는 것은 아니고 동물의 경우 스트레스 상태에 있는 것을 인지하기 어려운 경우도 있으므로 스트레스 상태인지 객관적으로 진단할 수 있는 방법이 필요하다.In general, stress refers to a non-specific biological response occurring in the body to various injuries or stimuli applied to the living body. Response to external temporary stress may be a natural phenomenon, but if the response to stress persists for a long time, mental or physical damage may occur. In particular, stress is not always perceptible to the subject to which the stress is applied, and in the case of animals, it is sometimes difficult to recognize that they are in a state of stress.
한편, 엑소좀은 세포 내부에서 형성되어 다중소낭체를 통해 세포 외부로 분비되는 직경 약 200 nm 이하의 작은 세포외소포체이다. 모세포의 단백질과 유전정보를 포함하고 있는 엑소좀은 최근 다양한 질환의 바이오마커로 활용 가능성이 제시되고 있다.On the other hand, exosomes are small extracellular vesicles with a diameter of about 200 nm or less that are formed inside the cell and secreted out of the cell through multivesicles. Exosomes, which contain protein and genetic information of parental cells, have recently been suggested to be useful as biomarkers for various diseases.
다만 엑소좀을 이용하여 아토피성 피부염 상관 스트레스를 진단하는 방법에 대한 기술은 아직 연구된 바 없어 이에 대한 심도 깊은 연구가 요구되고 있다.However, a technique for diagnosing atopic dermatitis-related stress using exosomes has not yet been studied, so an in-depth study is required.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Literature]
대한민국 공개특허공보 10-2016-0042703Korean Patent Publication No. 10-2016-0042703
본 발명의 목적은 엑소좀에서 유래한 특정 miRNA의 발현 수준을 측정하는 제제를 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단용 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for diagnosing stress induced by atopic dermatitis, comprising an agent for measuring the expression level of a specific miRNA derived from exosomes.
본 발명의 다른 목적은 상기 조성물을 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for diagnosing stress induced by atopic dermatitis, comprising the composition.
본 발명의 또 다른 목적은 개체의 엑소좀 유래 특정 miRNA의 발현 수준을 대조군의 엑소좀 유래 miRNA의 발현 수준과 비교하는 단계를 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단을 위한 정보제공 방법을 제공하는 것이다.Another object of the present invention is to provide an information providing method for diagnosing stress induced by atopic dermatitis, comprising comparing the expression level of a specific miRNA derived from an exosome of an individual with the expression level of an exosome-derived miRNA of a control group will provide
상기 본 발명의 목적을 달성하기 위해 본 발명은 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 엑소좀 유래 miRNA의 발현 수준을 측정하는 제제를 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단용 조성물을 제공한다.In order to achieve the object of the present invention, the present invention provides mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3) , mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu -miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu -miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p (SEQ ID NO: 15) , mmu-miR-185-5p (SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a-5p (SEQ ID NO: 18) 19), mmu-miR-29a-3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 21) 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) comprising an agent for measuring the expression level of one or more exosome-derived miRNAs selected from the group consisting of , To provide a composition for diagnosing stress induced by atopic dermatitis.
본 발명의 일 구현예로 상기 엑소좀은 신경 유래 엑소좀일 수 있다.In one embodiment of the present invention, the exosome may be a nerve-derived exo.
본 발명의 일 구현예로 상기 제제는 상기 하나 이상의 엑소좀 유래 miRNA에 특이적으로 결합하는 프라이머 또는 프로브를 포함할 수 있다.In one embodiment of the present invention, the agent may include a primer or probe that specifically binds to the one or more exosome-derived miRNAs.
또한 본 발명은 상기 조성물을 포함하는 아토피성 피부염으로 인해 유발된 스트레스 진단용 키트를 제공한다.The present invention also provides a kit for diagnosing stress induced by atopic dermatitis comprising the composition.
또한 본 발명은 (a) 개체의 생물학적 시료로부터 엑소좀을 분리하는 단계; 및The present invention also comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
(b) 상기 (a)단계에서 분리된 엑소좀에서 유래한 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준을 대조군 시료의 해당 엑소좀 유래 miRNA의 발현 수준과 비교하는 단계를 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단을 위한 정보제공 방법을 제공한다.(b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p ( SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a- 5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a- The expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) was measured for the corresponding exo of the control sample. It provides an information providing method for diagnosing stress induced by atopic dermatitis, comprising comparing the expression level of a moth-derived miRNA.
본 발명의 일 구현예로, 상기 생물학적 시료는 혈액일 수 있다.In one embodiment of the present invention, the biological sample may be blood.
본 발명의 일 구현예로, 상기 엑소좀은 신경 유래 엑소좀일 수 있다.In one embodiment of the present invention, the exosome may be a nerve-derived exo.
본 발명의 일 구현예로, 상기 (b)단계에서 개체의 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8) 및 mmu-miR-5128(서열번호 9)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준이 대조군에 비해 고발현인 경우 상기 개체는 스트레스 상태인 것으로 판정하는 단계를 더 포함할 수 있다.In one embodiment of the present invention, in step (b), mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), when the expression level of one or more miRNAs selected from the group consisting of mmu-miR-466i-5p (SEQ ID NO: 8) and mmu-miR-5128 (SEQ ID NO: 9) is higher than that of the control group, the subject is in a stress state It may further include the step of determining that it is.
본 발명의 일 구현예로 상기 (b)단계에서 개체의 mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준이 대조군에 비해 저발현인 경우 상기 개체는 스트레스 상태인 것으로 판정하는 단계를 더 포함할 수 있다.In one embodiment of the present invention, mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 11) of the subject in step (b) No. 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p (SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a-5p (SEQ ID NO: 19), mmu-miR-29a -3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o When the expression level of one or more miRNAs selected from the group consisting of -3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) is low compared to the control group, determining that the subject is in a stress state may further include.
또한, 본 발명은 (a) 개체의 생물학적 시료로부터 엑소좀을 분리하는 단계; 및In addition, the present invention comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
(b) 상기 (a)단계에서 분리된 엑소좀에서 유래한 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준을 대조군 시료의 해당 엑소좀 유래 miRNA의 발현 수준과 비교하는 단계를 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단 방법을 제공한다.(b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p ( SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a- 5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a- The expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) was measured for the corresponding exo of the control sample. It provides a method for diagnosing stress induced by atopic dermatitis, comprising comparing the expression level of a moth-derived miRNA.
본 발명은 아토피성 피부염으로 스트레스를 유발한 마우스의 엑소좀에서 유래한 miRNA를 분석한 다음, 스트레스 처리 전후에 발현양상이 달라지는 다수의 miRNA를 확인하여 이를 바이오마커로 활용 가능하다는 점을 도출하였는바, 피부염증과 스트레스의 연관성 및 스트레스 진단 사업 등 관련된 다양한 분야에 이용될 수 있다.The present invention analyzed miRNAs derived from exosomes of mice induced by stress due to atopic dermatitis, and then identified a number of miRNAs whose expression patterns change before and after stress treatment, and derived that they can be used as biomarkers. , it can be used in various fields related to the relationship between skin inflammation and stress and the stress diagnosis project.
도 1은 Balb/c 마우스에서 아토피성 피부염을 유발한 방법 및 각 군 설정에 대한 실험 설계 내용을 개략적으로 나타낸 것이다.1 schematically shows the experimental design for the method and each group setting for inducing atopic dermatitis in Balb/c mice.
도 2는 DNCB 처리 후 아토피성 피부염 유발 여부를 피부조직을 통해 확인한 것이다.Figure 2 shows whether atopic dermatitis is induced after DNCB treatment is confirmed through skin tissue.
도 3은 아토피성 피부염 유발 이후 2일, 6일, 14일에 뇌의 해마 (hippocampus) 부위에 특정 단백질 발현 양상 확인한 것이다.3 is a graph confirming the expression pattern of specific proteins in the hippocampus of the brain at 2 days, 6 days, and 14 days after induction of atopic dermatitis.
도 4는 아토피성 피부염 유발 동물 혈청 유래 엑소좀의 형태학적 확인(A), 엑소좀의 사이즈 분포 확인(B) 및 엑소좀 표면 특이 발현 단백질 확인 결과를 나타낸 것이다.Figure 4 shows the morphological confirmation of atopic dermatitis-induced animal serum-derived exosomes (A), the size distribution of the exosomes (B), and the results of confirming the exosome surface-specific expression protein.
도 5는 혈청 유래 엑소좀 및 신경 유래 엑소좀에서 신경 특이적 마커 발현 여부를 확인한 것이다.Figure 5 confirms the expression of nerve-specific markers in some serum-derived exosomes and nerve-derived exosomes.
도 6은 정상군 대비 아토피성 피부염 유발군에서 신경 유래 엑소좀의 miRNA 발현 양상을 NGS를 통하여 비교 분석한 결과를 나타낸 것이다.6 shows the results of comparative analysis through NGS of the miRNA expression pattern of the nerve-derived exosomes in the atopic dermatitis-induced group compared to the normal group.
도 7은 아토피성 피부염 유발군에서 발현이 상향되는 miRNA를 나타낸 것이다.7 shows miRNAs whose expression is increased in the atopic dermatitis-induced group.
도 8은 아토피성 피부염 유발군에서 발현이 하향되는 miRNA를 나타낸 것이다.8 shows miRNAs whose expression is down-regulated in atopic dermatitis-induced group.
본 발명은 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 엑소좀 유래 miRNA의 발현 수준을 측정하는 제제를 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단용 조성물을 제공한다.The present invention provides mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3), mmu-let-7e-5p ( SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu-miR-466i-5p (SEQ ID NO: 7) 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 11) 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p (SEQ ID NO: 15), mmu-miR-185-5p ( SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a-5p (SEQ ID NO: 19), mmu-miR-29a- 3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o- 3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) Stress induced due to atopic dermatitis comprising an agent for measuring the expression level of one or more exosome-derived miRNAs selected from the group consisting of A diagnostic composition is provided.
본 발명에서 "miRNA(microRNA)"는, mRNA의 3' 비번역 영역(UTR) 부위와의 염기결합을 통해 전사후 억제자 역할을 하는 대략 22 nt의 비번역 RNA를 의미한다.In the present invention, "miRNA (microRNA)" refers to an approximately 22 nt untranslated RNA that acts as a post-transcriptional repressor through base binding with the 3' untranslated region (UTR) region of mRNA.
엑소좀 miRNAs의 검출은 통상적으로는 시료부터 엑소좀을 추출하여, 추출물 중의 엑소좀 miRNAs을 검출함으로써 실시할 수 있다. 이러한 엑소좀 miRNAs의 검출은 하이브리드화 반응 및 증폭반응에 의해 측정될 수 있으나, 이에 제한되지 않고 당업계에 공지된 다양한 기술을 이용하여 용이하게 실시될 수 있다.Detection of exosome miRNAs can be carried out by extracting exosomes from a sample and detecting exosome miRNAs in the extract. Detection of these exosomes miRNAs can be measured by hybridization and amplification reactions, but is not limited thereto and can be easily carried out using various techniques known in the art.
본 발명에서 상기 엑소좀은 신경 유래 엑소좀 일 수 있다.In the present invention, the exosome may be a nerve-derived exosome.
본 발명의 엑소좀 miRNA의 발현 수준을 측정하는 제제는 아토피성 피부염으로 인해 유발된 스트레스 상태에서 생물학적 시료의 검체에서 발현 수준이 감소되는 마커인 엑소좀 miRNA 및 발현 수준이 증가되는 마커인 엑소좀 miRNA의 발현 수준을 확인함으로써 마커의 검출에 사용될 수 있는 분자를 의미한다.The agent for measuring the expression level of exosomal miRNA of the present invention is exosomal miRNA, a marker whose expression level is decreased in a sample of a biological sample, and exosome miRNA, which is a marker whose expression level is increased, in a stress state induced by atopic dermatitis. It refers to a molecule that can be used for detection of a marker by checking the expression level of
본 발명에서 상기 제제는 상기 하나 이상의 엑소좀 유래 miRNA에 특이적으로 결합하는 프라이머 또는 프로브를 포함하는 것일 수 있다.In the present invention, the agent may include a primer or probe that specifically binds to the one or more exosome-derived miRNAs.
즉, 핵산의 검출은 핵산 분자 또는 상기 핵산 분자의 상보물에 하이브리드화되는 하나 이상의 올리고뉴클레오타이드 프라이머를 사용하는 증폭반응에 의해 수행될 수 있다. 예컨대, 프라이머를 이용한 엑소좀 miRNAs의 검출은 PCR과 같은 증폭 방법을 사용하여 유전자 서열을 증폭한 다음 당 분야에 공지된 방법으로 유전자의 증폭 여부를 확인함으로써 수행될 수 있다.That is, the detection of a nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers hybridized to a nucleic acid molecule or a complement of the nucleic acid molecule. For example, detection of exosome miRNAs using primers can be performed by amplifying the gene sequence using an amplification method such as PCR and then confirming whether the gene is amplified by a method known in the art.
프라이머는 짧은 자유 3말단 수산화기(free 3' hydroxyl group)를 갖는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍(base pair)을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 폴리머레이즈 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성이 개시될 수 있다. 본 발명에서는 상기 하나 이상의 miRNA에 특이적으로 결합하는 센스 및 안티센스 프라이머를 이용하여 PCR 증폭을 실시하여 발현 수준을 확인함으로써 스트레스 여부를 진단할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 것을 기초로 변형할 수 있다.A primer is a nucleic acid sequence having a short free 3' hydroxyl group. A short nucleic acid sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand copying. means Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. In the present invention, stress can be diagnosed by performing PCR amplification using the sense and antisense primers specifically binding to the one or more miRNAs to check the expression level. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.
프로브는 짧게는 수 염기 내지 길게는 수십 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링되어 있다. 프로브는 올리고뉴클로타이드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 본 발명에서는 위 하나 이상의 miRNA와 상보적인 프로브를 이용하여 혼성화를 실시하여 발현 수준을 확인함으로써 스트레스 여부를 진단할 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 당업계에 공지된 것을 기초로 변형할 수 있다.The probe refers to a nucleic acid fragment, such as RNA or DNA, corresponding to several bases to several tens of bases as long and is labeled. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, stress can be diagnosed by performing hybridization using one or more of the above miRNAs and complementary probes and checking the expression level. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.
이러한 프라이머 또는 프로브는 공지된 서열을 바탕으로 당업자가 적절히 디자인할 수 있다.Such primers or probes can be appropriately designed by those skilled in the art based on known sequences.
예컨대, 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡화, 천연 뉴클레오타이드 하나 이상의 동족체로의 치환, 및 뉴클레오타이드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.For example, primers or probes can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoros. amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
miRNA의 발현 수준은 당해 분야에서 통상 사용되는 방법에 따라 측정될 수 있는데, 예컨대 역전사효소 중합효소반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(Competitive RT-PCR), 실시간 역전사효소 중합효소반응 (Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting) 또는 유전자 칩 등이 포함되며 이들로 제한되는 것은 아니다.The expression level of miRNA can be measured according to a method commonly used in the art, for example, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting), or gene chip, and the like are included, but are not limited thereto.
본 발명의 다른 양태로서 본 발명은 상기 조성물을 포함하는 아토피성 피부염으로 인해 유발된 스트레스 진단용 키트를 제공한다.As another aspect of the present invention, the present invention provides a kit for diagnosing stress induced by atopic dermatitis comprising the composition.
상기 키트에는 상기 엑소좀 miRNA의 발현 수준을 측정하는 제제뿐만 아니라, 스트레스 진단 키트로 사용되기에 적합하도록 사용되는 당 분야에서 일반적으로 사용되는 도구, 시약 등이 포함될 수 있다.The kit may include not only an agent for measuring the expression level of the exosome miRNA, but also tools, reagents, etc. commonly used in the art that are used to be suitable for use as a stress diagnosis kit.
상기 도구 또는 시약의 일 예로, 적합한 담체, 검출 가능한 신호를 생성할 수 있는 표지 물질, 발색단(chromophores), 용해제, 세정제, 완충제, 안정화제 등이 포함되나 이에 제한되지 않는다. 표지물질이 효소인 경우에는 효소 활성을 측정할 수 있는 기질 및 반응 정지제를 포함할 수 있다. 담체는 가용성 담체, 불용성 담체가 있고, 가용성 담체의 일 예로 당 분야에서 공지된 생리학적으로 허용되는 완충액, 예를 들어 PBS가 있고, 불용성 담체의 일 예로 폴리스틸렌, 폴리에틸렌, 폴리프로필렌, 폴리에스테르, 폴리아크릴로니트릴, 불소 수지, 가교 덱스트란, 폴리사카라이드, 라텍스에 금속을 도금한 자성 미립자와 같은 고분자, 기타 종이, 유리, 금속, 아가로오스 및 이들의 조합일 수 있다.Examples of the tool or reagent include, but are not limited to, a suitable carrier, a labeling material capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling material is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. The carrier includes a soluble carrier and an insoluble carrier, and an example of the soluble carrier is a physiologically acceptable buffer known in the art, for example, PBS, and an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly It may be acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.
본 발명의 키트는 상술한 조성물을 구성으로 포함하므로, 중복된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the kit of the present invention includes the above-described composition as a component, redundant descriptions are omitted in order to avoid excessive complexity of the present specification.
본 발명의 또 다른 양태에 따르면, (a) 개체의 생물학적 시료로부터 엑소좀을 분리하는 단계; 및According to another aspect of the present invention, (a) separating the exosomes from the biological sample of the individual; and
(b) 상기 (a)단계에서 분리된 엑소좀에서 유래한 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준을 대조군 시료의 해당 엑소좀 유래 miRNA의 발현 수준과 비교하는 단계를 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단을 위한 정보제공 방법을 제공한다.(b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p ( SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a- 5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a- The expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) was measured for the corresponding exo of the control sample. It provides an information providing method for diagnosing stress induced by atopic dermatitis, comprising comparing the expression level of a moth-derived miRNA.
상기 “대조군 시료의 해당 엑소좀 유래 miRNA 발현 수준과 비교” 한다는 것은 개체와 대조군의 비교 대상이 동일한 종류의 엑소좀에서 유래한 동일한 종류의 miRNA 라는 것을 의미한다.The “comparison with the expression level of the corresponding exosome-derived miRNA in the control sample” means that the comparison target between the subject and the control group is the same type of miRNA derived from the same type of exosome.
본 발명의 상기 (b)단계에서 개체의 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준이 대조군에 비해 고발현인 경우 상기 개체는 스트레스 상태인 것으로 판정하는 단계를 더 포함할 수 있다.In the step (b) of the present invention, mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu- When the expression level of one or more miRNAs selected from the group consisting of miR-466i-5p (SEQ ID NO: 8) and mmu-miR-5128 (SEQ ID NO: 9) is higher than that of the control group, determining that the subject is in a stress state may further include.
본 발명의 상기 (b)단계에서 개체의 mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준이 대조군에 비해 저발현인 경우 상기 개체는 스트레스 상태인 것으로 판정하는 단계를 더 포함할 수 있다.In the step (b) of the present invention, mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p (SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16) ), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a-5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 19) No. 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 23) No. 24) and mmu-miR-93-5p (SEQ ID NO: 25) when the expression level of at least one miRNA selected from the group consisting of is low compared to the control, determining that the subject is in a stress state. can
본 발명에서 상기 엑소좀 miRNA의 발현 수준을 언급하면서 사용되는 용어 "저발현"은 바이오 마커(본 발명에서는 엑소좀 유래 miRNA)가 비정상 프로세스, 질환 혹은 개체 내 기타 병태를 나타내거나 이의 징후인 경우, 건강하거나 정상인 개체 또는 비교 대상인 개체로부터 획득된 생물학적 시료에서 검출되는 바이오 마커의 값 혹은 수준 범위 보다 낮은 생물학적 시료 내의 바이오 마커의 값 혹은 수준을 지칭한다.In the present invention, the term "low expression" used while referring to the expression level of the exosome miRNA is a biomarker (exosome-derived miRNA in the present invention) indicating an abnormal process, disease, or other condition in an individual or a symptom thereof. Refers to a value or level of a biomarker in a biological sample that is lower than a value or level range of a biomarker detected in a biological sample obtained from a healthy or normal individual or a comparison subject.
본 명세서에서 상기 엑소좀 miRNA의 발현 수준을 언급하면서 사용되는 용어 "고발현"은 바이오 마커가 비정상 프로세스, 질환 혹은 개체 내 기타 병태를 나타내거나 이의 징후인 경우, 건강하거나 정상인 개체 또는 비교 대상인 개체로부터 획득된 생물학적 시료에서 검출되는 바이오 마커의 값 혹은 수준 범위 보다 높은 생물학적 시료 내의 바이오 마커의 값 혹은 수준을 지칭한다.The term "high expression" used while referring to the expression level of the exosomal miRNA in the present specification is when the biomarker indicates or is a symptom of an abnormal process, disease, or other condition within the subject, from a healthy or normal subject or a subject for comparison Refers to a value or level of a biomarker in a biological sample that is higher than a value or level range of a biomarker detected in the obtained biological sample.
본 발명에서 "생물학적 시료"란 본 발명의 miRNA 발현이 검출될 수 있는 개체로부터 얻어지는 모든 시료를 의미한다.In the present invention, "biological sample" refers to any sample obtained from an individual in which the expression of miRNA of the present invention can be detected.
본 발명에서 “개체”는 아토피성 피부염으로 스트레스 유도시 본 발명의 아토피 유도군과 miRNA 발현이 유사한 양상을 보이는 대상이면 제한없이 해당할 수 있으며, 바람직하게는 포유동물 일 수 있다.In the present invention, the "individual" may correspond without limitation as long as it is a subject showing a similar pattern to the atopic induced group of the present invention when stress is induced by atopic dermatitis, and may preferably be a mammal.
본 발명의 바람직한 구현예에 따르면, 상기 생물학적 시료는 혈액, 타액(saliva), 생검(biopsy), 피부 조직, 액체 배양물, 분변 및 소변으로 이루어진 군에서 선택된 어느 하나이며, 특별히 이에 제한되지 않으나 바람직하게는 혈액이며, 본 발명의 기술분야에서 통상적으로 사용되는 방법으로 처리하여 준비될 수 있다.According to a preferred embodiment of the present invention, the biological sample is any one selected from the group consisting of blood, saliva, biopsy, skin tissue, liquid culture, feces and urine, but is not particularly limited thereto. Preferably, it is blood, and it can be prepared by processing by a method commonly used in the art.
본 발명에서 상기 엑소좀은 신경 유래 엑소좀 일 수 있다.In the present invention, the exosome may be a nerve-derived exosome.
다른 양태로서, 본 발명은 (a) 개체의 생물학적 시료로부터 엑소좀을 분리하는 단계; 및In another aspect, the present invention comprises the steps of (a) isolating the exosomes from the biological sample of the individual; and
(b) 상기 (a)단계에서 분리된 엑소좀에서 유래한 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준을 대조군 시료의 해당 엑소좀 유래 miRNA의 발현 수준과 비교하는 단계를 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단 방법을 제공한다.(b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p ( SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a- 5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a- The expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) was measured for the corresponding exo of the control sample. It provides a method for diagnosing stress induced by atopic dermatitis, comprising comparing the expression level of a moth-derived miRNA.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
<실시예 1: 재료 및 방법><Example 1: Materials and Methods>
실시예 1-1. 실험동물Example 1-1. laboratory animal
실험은 8 주령 수컷 balb/c 마우스로 수행되었다. 상기 마우스는 온도, 습도 및 제어된 실내 조명 (24 ± 2 °C, 50 ± 20 % 상대 습도, 07:00에 조명을 켜고 12/12 시간 명/암주기)조건에 수용되었다. 또한 마우스는 음식과 물에 제한없이 접근 가능하도록 하였다. 상기 마우스 및 본 발명에서 사용되는 시약 등은 상업적으로 구매 가능하다.Experiments were performed with 8-week-old male balb/c mice. The mice were housed in conditions of temperature, humidity, and controlled room lighting (24 ± 2 °C, 50 ± 20 % relative humidity, 12/12 h light/dark cycle with lights on at 07:00). In addition, mice were given unrestricted access to food and water. The mice and reagents used in the present invention are commercially available.
실시예 1-2. DNCB에 의해 유발된 아토피 피부염 모델Example 1-2. Atopic dermatitis model induced by DNCB
1-Chloro-2,4-dinitrobenzene(DNCB)(Sigma)을 사용하여 아토피 피부염 모델을 유도하였다. 각 그룹에서 아토피 피부염을 유도하기 위한 프로토콜은 도 1에 나타난 바와 같다. 먼저 마우스를 대조군, DNCB 2일, DNCB 6일 및 DNCB 14일로 나눈 다음 등쪽 털을 제모하였다. DNCB 2일 그룹 마우스는 200 μL의 1% DNCB 희석된 아세톤 및 올리브 오일 (4 : 1)로 1 회 처리되었고 24 시간 후에 희생되었다. DNCB 6일 그룹에서는, 마우스를 0 일 및 3 일에 200μL의 1% DNCB로 2회 처리한 후 6일째 희생시켰다. 마지막으로, DNCB 14 일 그룹의 마우스는 0 일 및 3 일에 200 μL의 1% DNCB로 감작시킨 다음 6 일부터 13 일까지 매일 2% DNCB로 처리하고 14 일째 희생시켰다.Atopic dermatitis model was induced using 1-Chloro-2,4-dinitrobenzene (DNCB) (Sigma). The protocol for inducing atopic dermatitis in each group is shown in FIG. 1 . First, mice were divided into control groups, DNCB 2 days, DNCB 6 days and DNCB 14 days, and then the dorsal hair was removed. DNCB day 2 group mice were treated once with 200 μL of 1% DNCB diluted acetone and olive oil (4:1) and sacrificed 24 hours later. In the DNCB 6-day group, mice were treated twice with 200 μL of 1% DNCB on days 0 and 3, followed by sacrifice on day 6. Finally, mice in the DNCB 14-day group were sensitized with 200 μL of 1% DNCB on days 0 and 3, then treated with 2% DNCB daily from days 6 to 13, and sacrificed on day 14.
실시예 1-3. 해마의 신경염증 확인을 위한 조직병리검사Examples 1-3. Histopathological examination to confirm hippocampal neuroinflammation
뇌를 절개한 다음 10% 중성 완충 포르말린에 고정하였다. 그 다음 조직을 처리하고 파라핀에 넣었다. 뇌 파라핀 블록 절편은 해마 레벨에서 4μm 두께로 절단되었다. Brains were dissected and fixed in 10% neutral buffered formalin. The tissue was then processed and embedded in paraffin. Brain paraffin block sections were cut to a thickness of 4 μm at the level of the hippocampus.
헤마톡실린 및 에오신(H&E) 염색은 DAKO CoverStainer (Agilent, Santa Clara, CA, USA)를 사용하여 수행되었다. 신경염증 마커 단백질의 발현을 확인하기 위해 면역 조직 화학(Immunohistochemistry, IHC)을 수행하였다. 뇌 절편은 anti-BDNF (희석 1 : 500; Abcam, Abcam, Cambridge, UK), COX2 (희석 1 : 250; Abcam, Abcam, Cambridge, UK), GFAP (희석 1 : 500; Abcam, Cambridge, UK) 1차 항체로 염색되었다. 다음으로, 라벨이 붙은 폴리머 DAKO EnVisionTM + System-HRP (Agilent, Santa Clara, CA, USA)를 제조업체의 지침에 따라 면역조직화학적으로 절편하였다. 염색 후, Pannoramic SCAN II (3DHISTECH Kft., Budapest, Hungary)를 사용하여 뇌 절편을 스캔하였다. 현미경 사진은 CaseViewer (3DHISTECH Kft., Budapest, Hungary)로 촬영되었다. 마지막으로, 해마의 신경염증 마커는 Imaged J 소프트웨어 (NIH, Bethesda, MD, USA)로 정량화되었다.Hematoxylin and eosin (H&E) staining was performed using a DAKO CoverStainer (Agilent, Santa Clara, CA, USA). Immunohistochemistry (IHC) was performed to confirm the expression of the neuroinflammatory marker protein. Brain sections were prepared for anti-BDNF (dilution 1:500; Abcam, Abcam, Cambridge, UK), COX2 (dilution 1:250; Abcam, Abcam, Cambridge, UK), GFAP (dilution 1:500; Abcam, Cambridge, UK). Stained with primary antibody. Next, the labeled polymer DAKO EnVisionTM + System-HRP (Agilent, Santa Clara, CA, USA) was sectioned immunohistochemically according to the manufacturer's instructions. After staining, brain sections were scanned using a Pannoramic SCAN II (3DHISTECH Kft., Budapest, Hungary). Micrographs were taken with a CaseViewer (3DHISTECH Kft., Budapest, Hungary). Finally, hippocampal neuroinflammatory markers were quantified with Imaged J software (NIH, Bethesda, MD, USA).
실시예 1-4. 혈청에서 엑소좀 분리Examples 1-4. Isolation of exosomes from serum
ExoQuick 솔루션 (System Biosciences, Palo Alto, CA, USA)을 사용하여 제조업체의 지침을 수정하여 혈청에서 엑소좀을 분리하였다. 먼저, 3 mL 혈청을 3,500 × g에서 15 분간 4 ℃에서 원심 분리를 통해 펠렛화하여 세포 파편을 제거하였다. 다음으로, 3 mL의 데브리가 제거된 혈청(debris-deleted serum)과 885 μL ExoQuick 용액을 혼합하였다. 혼합물을 3,500 × g에서 10 분 동안 원심 분리한 후, 펠렛을 200 μl PBS로 재현탁시켰다.ExoQuick solution (System Biosciences, Palo Alto, CA, USA) was used to isolate exosomes from serum by modifying the manufacturer's instructions. First, 3 mL serum was pelleted through centrifugation at 3,500 × g at 4 °C for 15 min to remove cell debris. Next, 3 mL of debris-deleted serum and 885 μL of ExoQuick solution were mixed. After centrifugation of the mixture at 3,500 × g for 10 min, the pellet was resuspended in 200 μl PBS.
실시예 1-5. 혈청에서 분리된 총 엑소좀에서 뉴런 엑소좀 분리Examples 1-5. Isolation of neuronal exosomes from total exosomes isolated from serum
종래의 문헌(Plasma extracellular vesicles enriched for neuronal origin: A potential window into brain pathologic processes. Front Neurosci. 2017 May 22;11:278.)을 참조 및 수정하여 신경 엑소좀을 분리하였다. 200 μL PBS를 포함하는 총 엑소좀을 로테이팅 믹서(rotating mixer)에서 4 ℃에서 1 시간 동안 anti-CD171 항체 (Bioss Antibodies, Beijing, China) 4 μL와 함께 배양하였다. 15μL PierceTM streptavidin + UltralinkTM 수지 (Thermo Fisher Scientific)와 25μL PBS를 첨가한 후, 혼합물을 로테이팅 믹서에서 4 ℃로 1 시간 동안 배양한 다음, 4 ℃에서 10 분 동안 500 x g로 원심 분리하였다. 샘플에서 상청액을 제거하고 펠렛을 200 μL 0.1M Glycine-HCl (Biosesang, Seongnam, Korea)에 재현탁시켰다. 10초 동안 혼합하고 30초 동안 볼텍싱(voltexing) 한 후 혼합물을 4,500 x g에서 10분 동안 4 ℃로 원심 분리하여 펠릿화 하였다. 상청액을 새로운 튜브로 옮긴 다음 15 μL Tris-HCl (Biosesang, Seongnam, Korea) 및 25 μL PBS를 첨가하였다.Neuronal exosomes were isolated by referring and modifying the conventional literature (Plasma extracellular vesicles enriched for neuronal origin: A potential window into brain pathologic processes. Front Neurosci. 2017 May 22;11:278.). Total exosomes containing 200 μL PBS were incubated with 4 μL of anti-CD171 antibody (Bioss Antibodies, Beijing, China) for 1 hour at 4° C. in a rotating mixer. After addition of 15 μL Pierce™ streptavidin + Ultralink™ resin (Thermo Fisher Scientific) and 25 μL PBS, the mixture was incubated in a rotating mixer at 4° C. for 1 hour and then centrifuged at 500×g at 4° C. for 10 minutes. The supernatant was removed from the sample and the pellet was resuspended in 200 μL 0.1M Glycine-HCl (Biosesang, Seongnam, Korea). After mixing for 10 seconds and vortexing for 30 seconds, the mixture was pelleted by centrifugation at 4,500 x g for 10 minutes at 4 °C. The supernatant was transferred to a new tube and then 15 μL Tris-HCl (Biosesang, Seongnam, Korea) and 25 μL PBS were added.
실시예 1-6. 투과 전자 현미경 (Transmission electron microscopy, TEM)Example 1-6. Transmission electron microscopy (TEM)
혈청에서 분리된 엑소좀을 차가운 증류수에 재현탁시켰다. 엑소좀 현탁액을 폼바 카본 코팅 그리드 (formvar carbon coated grid, Ted Pella Inc.)에 로딩한 후, 현탁액을 2% 파라포름알데히드에 10분 동안 고정하고 용액을 제거한 다음 샘플을 건조시켰다. 그리드는 bioTEM (Hitachi HT7700)을 사용하여 기록되었다.Exosomes isolated from serum were resuspended in cold distilled water. After loading the exosome suspension onto a formvar carbon coated grid (Ted Pella Inc.), the suspension was fixed in 2% paraformaldehyde for 10 minutes, the solution was removed, and the sample was dried. Grids were recorded using a bioTEM (Hitachi HT7700).
실시예 1-7. 나노 입자 추적 분석 (Nanoparticle tracking analysis, NTA)Example 1-7. Nanoparticle tracking analysis (NTA)
NTA는 제조업체의 지침에 따라 PMX120 (Particle Metrix) nanosight 기기를 사용하여 수행되었다.NTA was performed using a PMX120 (Particle Metrix) nanosight instrument according to the manufacturer's instructions.
실시예 1-8. 유세포 분석Examples 1-8. flow cytometry
50μl의 총 엑소좀을 10μl의 알데히드/설페이트 라텍스 비드(Invitrogen, Carlsbad, CA, USA)와 함께 15분 동안 상온에서 배양하였으며, PBS 500 μl 보충된 3% BSA가 첨가되었다. 샘플은 로테이팅 믹서에서 밤새 배양되었다. 비드-결합된 엑소좀을 3,000 x g에서 10 분 동안 원심분리하고 500 μl PBS로 세척하였다. 세척 후 샘플을 3,000 x g에서 10분 동안 원심분리 하였다. 샘플에서 상청액을 버리고 펠렛을 상온에서 1시간 동안 anti-CD9, CD81 항체 (BioLegend, San Diego, CA, USA)를 포함하는 PBS 50μl로 재현탁시켰다. 샘플을 3,000 x g에서 10분 동안 원심 분리하였으며, 펠렛을 200 μl의 PBS로 재현탁시켰다. 엑소좀 마커는 유세포 분석기 Galios (Beckman Coulter)를 사용하여 검출되었으며, Kaluza 소프트웨어로 분석을 수행하였다.50 μl of total exosomes were incubated with 10 μl of aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA, USA) at room temperature for 15 minutes, and 3% BSA supplemented with 500 μl of PBS was added. Samples were incubated overnight in a rotating mixer. Bead-bound exosomes were centrifuged at 3,000 x g for 10 min and washed with 500 μl PBS. After washing, the samples were centrifuged at 3,000 x g for 10 min. The supernatant was discarded from the sample and the pellet was resuspended in 50 μl of PBS containing anti-CD9 and CD81 antibodies (BioLegend, San Diego, CA, USA) at room temperature for 1 hour. Samples were centrifuged at 3,000 x g for 10 min, and the pellet was resuspended in 200 μl of PBS. Exosome markers were detected using a flow cytometer Galios (Beckman Coulter), and analysis was performed with Kaluza software.
실시예 1-9. 웨스턴 블롯Examples 1-9. western blot
혈청에서 분리된 총 엑소좀(tEV) 및 뉴런 엑소좀(nEV) 각각은 M-PER, HaltTM Protease 및 Phosphatase Inihhitor Cocktail (Thermo Scientific)을 사용하여 용해되었다. 엑소좀 용해물의 농도는 제조업체의 지침에 따라 PierceTM BCA 단백질 분석 키트 (Thermo Fisher Scientific)를 사용하여 측정되었다. 다음으로, 20 μg의 엑소좀 용해물을 BoltTM 4-12 % Bis-Tris Plus 겔 (Invitrogen, Carlsbad, CA, USA)에서 분리하고 폴리비닐리덴 플루오라이드 (polyvinylidene fluoride, PVDF) 막 (Invitrogen, Carlsbad, CA, USA)으로 옮겼다. 그 후, 실온에서 1 시간 동안 TBS-T가 보충된 5 % 탈지유로 막을 블로킹 하였다. 차단 후, TSG101 (NovusBio, USA), CD171(Santa Cruz Biotechnology, Santa Cruz, CA, USA), TUJ1 (Abcam, Cambridge, UK), NSE (Abcam, Cambridge, UK) 및 NeuN (Abcam, Cambridge, UK) 일차 항체(diluted 1:1000)와 함께 4 ℃에서 밤새 배양하였다. 세척 후, 막을 horseradish peroxidase(HRP) 접합된 이차 항체 (1 : 2000 희석)와 함께 상온에서 1시간 동안 배양하고 TBS-T로 세척하였다. 마지막으로 EzWestLumi plus (ATTO, Japan)로 밴드를 감지하고 Image QuantTM LAS 4000 (GE Healthcare, UK)을 사용하여 분석하였다.Total exosomes (tEV) and neuronal exosomes (nEV) isolated from serum were each lysed using M-PER, Halt™ Protease and Phosphatase Inihhitor Cocktail (Thermo Scientific). Concentrations of exosome lysates were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Next, 20 μg of exosome lysates were separated on a Bolt™ 4-12% Bis-Tris Plus gel (Invitrogen, Carlsbad, CA, USA) and polyvinylidene fluoride (PVDF) membrane (Invitrogen, Carlsbad, CA, USA). Then, the membrane was blocked with 5% skim milk supplemented with TBS-T for 1 h at room temperature. After blocking, TSG101 (NovusBio, USA), CD171 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TUJ1 (Abcam, Cambridge, UK), NSE (Abcam, Cambridge, UK) and NeuN (Abcam, Cambridge, UK) Incubated overnight at 4 °C with primary antibody (diluted 1:1000). After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution) at room temperature for 1 hour and washed with TBS-T. Finally, bands were detected with EzWestLumi plus (ATTO, Japan) and analyzed using Image Quant™ LAS 4000 (GE Healthcare, UK).
실시예 1-10. 엑소좀 miRNA의 차등 발현 분석을 위한 차세대 시퀀싱(NGS)Examples 1-10. Next-generation sequencing (NGS) for differential expression analysis of exosomal miRNAs
혈청으로부터 분리된 뉴런 엑소좀을 모아 600 μL의 샘플을 제조하였다. 엑소좀의 농도는 제조업체의 지침에 따라 PierceTM BCA 단백질 분석 키트 (Thermo Fisher Scientific)를 사용하여 측정되었다. BCA 분석결과 각 샘플의 농도는 1.59 mg/mL (대조군), 2.34 mg/mL (DNCB 14 일)이었다. 엑소좀 smRNA 분리 및 라이브러리 준비는 제조업체의 지침에 따라 SMARTer smRNA-Seq Kit (Clontech Laboratories Inc., Mountain View, USA)를 사용하여 Macrogen (Seoul, Korea)에서 수행하였다. 그 다음 HiSeq 2500 시스템 사용 설명서 문서 # 15035786 v02 HCS 2.2.70에 따라 HiSeq 2500 시스템을 사용하여 miRNA 시퀀싱을 수행하였다.A 600 μL sample was prepared by collecting neuronal exosomes isolated from serum. The concentration of exosomes was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. As a result of BCA analysis, the concentrations of each sample were 1.59 mg/mL (control) and 2.34 mg/mL (DNCB 14 days). Exosome smRNA isolation and library preparation were performed in Macrogen (Seoul, Korea) using the SMARTer smRNA-Seq Kit (Clontech Laboratories Inc., Mountain View, USA) according to the manufacturer's instructions. MiRNA sequencing was then performed using a HiSeq 2500 system according to HiSeq 2500 System Instruction Manual Document # 15035786 v02 HCS 2.2.70.
실시예 1-11. 통계 분석Examples 1-11. statistical analysis
모든 통계 분석은 Student's t-test를 사용하여 수행되었으며, p<0.05는 유의한 차이로 간주되었다.All statistical analyzes were performed using Student's t-test, and p<0.05 was considered a significant difference.
<실시예 2: DNCB로 유도된 피부의 아토피성 피부염 확인><Example 2: Confirmation of atopic dermatitis of skin induced by DNCB>
피부의 병리학적 분석을 통해 DNCB에 의한 아토피성 피부염 유발 여부를 확인하였다. 먼저, 헤마톡실린 및 에오신 (H&E) 염색을 통해 아토피 피부염의 전형적 병변인 각질층과 표피 두께의 증가를 확인하였다(도 2의 A; control=12.05±2.51%; DNCB 2 days=80.6±31.46%, p=0.008; DNCB 6 days=108.54±43.89%, p=0.022; DNCD 14 days=125.74±19.25 %, p=0.00016). 다음으로 톨루이딘(toluidine) 블루 염색을 수행하여 진피의 비만 세포 침윤을 확인하였다. 그 결과, DNCB 처리가 반복됨에 따라 비만 세포가 증가하고 침윤이 증가하는 것이 관찰되었다(도 2의 B; control=6.75±1.5%, DNCB 2 days=12.75±2.87%, p=0.017; DNCB 6 days=13.75±1.89%, p=0.0014; DNCD 14 days=26.5±4.80%, p=0.0022).Through the pathological analysis of the skin, it was confirmed whether atopic dermatitis was caused by DNCB. First, an increase in the stratum corneum and epidermal thickness, which are typical lesions of atopic dermatitis, was confirmed through hematoxylin and eosin (H&E) staining (FIG. 2A; control=12.05±2.51%; DNCB 2 days=80.6±31.46%, p = 0.008; DNCB 6 days=108.54±43.89%, p = 0.022; DNCD 14 days=125.74±19.25 %, p =0.00016). Next, toluidine blue staining was performed to confirm mast cell infiltration of the dermis. As a result, as the DNCB treatment was repeated, it was observed that mast cells increased and invasion increased ( FIG. 2B; control=6.75±1.5%, DNCB 2 days=12.75±2.87%, p =0.017; DNCB 6 days) =13.75±1.89%, p =0.0014; DNCD 14 days=26.5±4.80%, p =0.0022).
<실시예 3: 아토피 피부염 마우스 모델의 해마 병리학적 분석><Example 3: Hippocampal pathological analysis of atopic dermatitis mouse model>
H&E 염색과 IHC를 수행하여 아토피성 피부염으로 유도된 스트레스로 인한 신경염증 표지자의 발현 정도를 분석하였다. 조직 병리학적 염색의 결과, 대조군과 비교하여 현미경사진에서 DNCB 2 일, DNCB 6 일 및 DNCB 14 일의 해마에서 유의한 차이가 발견되지 않았다 (도 3의 A). 그리고 IHC 염색을 수행하고 정량화 하였을 때 해마에서 BDNF 발현의 유의한 변화는 관찰되지 않았다(도 3의 B; control=0.55±0.11 %, DNCB 2 days=0.80±0.20 %, p=0.150; DNCB 6 days=0.70±0.077 %, p=0.130; DNCD 14 days=0.90±0.20 %, p=0.0706). 또한 COX2는 발현 수준에 큰 변화가 없었다(도 3의 C; control=0.71±0.084 %, DNCB 2 days=0.97±0.21 %, p=0.150; DNCB 6 days=0.90±0.35 %, p=0.448; DNCD 14 days=1.28±0.49 %, p=0.181). 그러나 GFAP 발현 수준은 DNCB 2 일, DNCB 6 일 및 DNCB 14 일에서 유의하게 변화하였다 (도 3의 D; 대조군 = 1.01 ± 0.0526 %, DNCB 2 일 = 1.10 ± 0.11 %, p = 0.327; DNCB 6 일 = 1.74 ± 0.016 %, p = 0.0102; DNCD 14 일 = 3.42 ± 0.077 %, p = 0.00000547).H&E staining and IHC were performed to analyze the expression level of neuroinflammatory markers due to stress induced by atopic dermatitis. As a result of histopathological staining, no significant difference was found in the hippocampus of DNCB 2 days, DNCB 6 days, and DNCB 14 days in the photomicrograph compared to the control group (FIG. 3A). And when IHC staining was performed and quantified, no significant change in BDNF expression was observed in the hippocampus (Fig. 3B; control=0.55±0.11%, DNCB 2 days=0.80±0.20%, p =0.150; DNCB 6 days) =0.70±0.077%, p =0.130; DNCD 14 days=0.90±0.20%, p =0.0706). Also, COX2 had no significant change in expression level (Fig. 3C; control=0.71±0.084%, DNCB 2 days=0.97±0.21%, p =0.150; DNCB 6 days=0.90±0.35%, p =0.448; DNCD 14 days=1.28±0.49%, p =0.181). However, GFAP expression levels were significantly changed at DNCB 2 days, DNCB 6 days and DNCB 14 days (Fig. 3D; control = 1.01 ± 0.0526%, DNCB 2 days = 1.10 ± 0.11%, p = 0.327; DNCB 6 days) = 1.74 ± 0.016%, p = 0.0102; DNCD 14 days = 3.42 ± 0.077%, p = 0.00000547).
<실시예 4: 혈청에서 분리된 총 엑소좀 확인><Example 4: Identification of total exosomes isolated from serum>
뉴런 엑소좀을 분리하기 전에 혈청에서 분리된 총 엑소좀의 특성을 확인하였다. 먼저 투과 전자 현미경 (TEM)을 이용하여 엑소좀의 모양과 크기를 확인하였다. TEM 이미지는 엑소좀이 구형 이중층 막이고 직경이 30-150 nm임을 보여주었다(도 4의 A). 다음으로, 나노 입자 추적 분석 (NTA)을 이용한 결과 엑소좀 크기가 30-150 nm 범위임을 확인하였다(도 4의 B). 마지막으로, CD9, CD81과 같은 엑소좀 마커의 존재를 확인하기 위해 면역 표지 방법을 사용한 유세포 분석을 수행하였다. 그 결과 혈청에서 분리된 전체 엑소좀에서 98.08 %의 CD9-양성 및 80.16 %의 CD81-양성 입자를 나타냈다(도 4의 C).Before isolating neuronal exosomes, the characteristics of total exosomes isolated from serum were confirmed. First, the shape and size of the exosomes were confirmed using a transmission electron microscope (TEM). TEM images showed that the exosomes were spherical bilayer membranes and had a diameter of 30-150 nm (Fig. 4A). Next, as a result of using nanoparticle tracking analysis (NTA), it was confirmed that the exosome size was in the range of 30-150 nm (FIG. 4B). Finally, to confirm the presence of exosome markers such as CD9 and CD81, flow cytometry using an immunolabeling method was performed. As a result, 98.08% of CD9-positive and 80.16% of CD81-positive particles were expressed in total exosomes isolated from serum ( FIG. 4C ).
<실시예 5: 분리된 뉴런 엑소좀의 특성화> <Example 5: Characterization of isolated neuronal exosomes >
혈청으로부터 총 엑소좀을 분리한 후, 면역 침강을 위해 anti-L1 세포 부착 분자(L1CAM; CD171) 비오틴화된 항체를 사용하여 뉴런 엑소좀의 분리를 수행하였다. 뉴런 엑소좀의 식별은 웨스턴 블롯팅을 사용하여 수행되었다. 그 결과 CD171이 총 엑소좀 (tEV)보다 뉴런 엑소좀 (nEV)에 더 많이 포함되어 있고 Tuj1, NSE 및 NeuN과 같은 뉴런 마커가 뉴런 엑소좀에 제시되었지만 총 엑소좀에는 제시되지 않았음을 확인하였다. 마지막으로, 엑소좀마커 TSG101은 총 엑소좀과 뉴런 엑소좀 모두에서 확인되었다 (도 5).After isolation of total exosomes from serum, isolation of neuronal exosomes was performed using an anti-L1 cell adhesion molecule (L1CAM; CD171) biotinylated antibody for immunoprecipitation. Identification of neuronal exosomes was performed using Western blotting. As a result, it was confirmed that CD171 was contained more in neuronal exosomes (nEV) than total exosomes (tEV), and neuronal markers such as Tuj1, NSE and NeuN were presented in neuronal exosomes but not total exosomes. . Finally, the exosome marker TSG101 was identified in both total exosomes and neuronal exosomes ( FIG. 5 ).
<실시예 6: 아토피성 피부염 유발 스트레스에 의해 변화된 exosomal miRNA 발현 확인><Example 6: Confirmation of exosomal miRNA expression changed by atopic dermatitis-induced stress>
NGS(Next-generation sequencing)를 사용하여 아토피 피부염에 의해 변화된 miRNA 발현 패턴을 확인하였다. 그 결과 대조군과 비교하여 14 일 동안 DNCB에서 9 개의 miRNA가 크게 상향 조절되었고 16 개의 miRNA가 크게 하향 조절되었음을 확인하였다(도 6). 대조군과 비교하여 14 일 동안 DNCB에서 상향 조절된 miRNA는 mmu-let-7a-5p, mmu-let-7b-5p, mmu-let-7c-5p, mmu-let-7e-5p, mmu-miR-126a-5p, mmu-miR-3473b, mmu-miR-3473e, mmu-miR-466i-5p 및 mmu-miR-5128 이었다(도 7). 하향 조절된 miRNA는 mmu-let-7i-5p, mmu-miR-130a-3p, mmu-miR-140-3p, mmu-miR-142a-3p, mmu-miR-16-5p, mmu-miR- 17-5p, mmu-miR-185-5p, mmu-miR-19b-3p, mmu-miR-24-3p, mmu-miR-27a-5p, mmu-miR-29a-3p, mmu-miR-301a- 3p, mmu-miR-451a, mmu-miR-669a-3p, mmu-miR-669o-3p 및 mmu-miR-93-5p이었다 (도 8).Next-generation sequencing (NGS) was used to confirm the miRNA expression pattern changed by atopic dermatitis. As a result, it was confirmed that 9 miRNAs were significantly up-regulated and 16 miRNAs were significantly down-regulated in DNCB for 14 days compared to the control group (FIG. 6). miRNAs upregulated in DNCB for 14 days compared to controls were mmu-let-7a-5p, mmu-let-7b-5p, mmu-let-7c-5p, mmu-let-7e-5p, mmu-miR- 126a-5p, mmu-miR-3473b, mmu-miR-3473e, mmu-miR-466i-5p and mmu-miR-5128 (FIG. 7). Down-regulated miRNAs are mmu-let-7i-5p, mmu-miR-130a-3p, mmu-miR-140-3p, mmu-miR-142a-3p, mmu-miR-16-5p, mmu-miR-17 -5p, mmu-miR-185-5p, mmu-miR-19b-3p, mmu-miR-24-3p, mmu-miR-27a-5p, mmu-miR-29a-3p, mmu-miR-301a-3p , mmu-miR-451a, mmu-miR-669a-3p, mmu-miR-669o-3p and mmu-miR-93-5p (Fig. 8).
상기 miRNA의 서열은 아래 표 1에 나와 있다. 그리고 이러한 miRNA 중 일부는 우울증 관련 연구에서 발현 변화를 보여주는 miRNA로 확인되었다. 아토피 피부염은 심리적 스트레스를 유발하고 스트레스는 우울증을 유발한다는 이전 연구에 따르면 NGS 데이터는 이러한 miRNA가 심리적 스트레스 바이오 마커로 사용될 수 있음을 의미한다.The sequence of the miRNA is shown in Table 1 below. And some of these miRNAs were identified as miRNAs showing expression changes in depression-related studies. According to previous studies that atopic dermatitis induces psychological stress and stress induces depression, NGS data suggest that these miRNAs can be used as psychological stress biomarkers.
Figure PCTKR2021005258-appb-T000001
Figure PCTKR2021005258-appb-T000001
본 발명은 아토피성 피부염으로 스트레스를 유발한 마우스의 엑소좀에서 유래한 miRNA를 분석한 다음, 스트레스 처리 전후에 발현양상이 달라지는 다수의 miRNA를 확인하여 이를 바이오마커로 활용 가능하다는 점을 도출하였는바, 피부염증과 스트레스의 연관성 및 스트레스 진단 사업 등 관련된 다양한 분야에 이용될 수 있다.The present invention analyzed miRNAs derived from exosomes of mice induced by stress due to atopic dermatitis, and then identified a number of miRNAs whose expression patterns change before and after stress treatment, and derived that they can be used as biomarkers. , it can be used in various fields related to the relationship between skin inflammation and stress and the stress diagnosis project.

Claims (10)

  1. mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 엑소좀 유래 miRNA의 발현 수준을 측정하는 제제를 포함하는, 아토피성 피부염으로 인해 유발된 개체의 스트레스 진단용 조성물.mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4) ), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p (SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16) ), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a-5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 19) No. 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 23) No. 24) and mmu-miR-93-5p (SEQ ID NO: 25) for diagnosing stress of an individual induced by atopic dermatitis, including an agent for measuring the expression level of one or more exosome-derived miRNAs selected from the group consisting of composition.
  2. 제1항에 있어서, 상기 엑소좀은 신경 유래 엑소좀인 것인, 조성물.The composition of claim 1, wherein the exosome is a neuron-derived exosome.
  3. 제1항에 있어서, 상기 제제는 상기 하나 이상의 엑소좀 유래 miRNA에 특이적으로 결합하는 프라이머 또는 프로브를 포함하는 것인, 조성물.The composition of claim 1, wherein the agent comprises a primer or probe that specifically binds to the one or more exosome-derived miRNAs.
  4. 제1항 내지 제4항 중 어느 한 항의 조성물을 포함하는, 아토피성 피부염으로 인해 유발된 스트레스 진단용 키트.A kit for diagnosing stress induced by atopic dermatitis, comprising the composition of any one of claims 1 to 4.
  5. (a) 개체의 생물학적 시료로부터 엑소좀을 분리하는 단계; 및(a) isolating the exosomes from the biological sample of the subject; and
    (b) 상기 (a)단계에서 분리된 엑소좀에서 유래한 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준을 대조군 시료의 해당 엑소좀 유래 miRNA의 발현 수준과 비교하는 단계를 포함하는, 아토피성 피부염으로 인해 유발된 개체의 스트레스 진단을 위한 정보제공 방법.(b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p ( SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a- 5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a- 3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) The expression level of one or more miRNAs selected from the group consisting of the exo of the control sample An information providing method for diagnosing stress in an individual induced by atopic dermatitis, comprising comparing the expression level of a moth-derived miRNA.
  6. 제5항에 있어서, 상기 생물학적 시료는 혈액인 것인, 정보제공 방법.The method of claim 5, wherein the biological sample is blood.
  7. 제5항에 있어서, 상기 엑소좀은 신경 유래 엑소좀인 것인, 정보제공 방법.The method of claim 5, wherein the exosome is a neuron-derived exosome.
  8. 제5항에 있어서, 상기 (b)단계에서 개체의 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8) 및 mmu-miR-5128(서열번호 9)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준이 대조군에 비해 고발현인 경우 상기 개체는 스트레스 상태인 것으로 판정하는 단계를 더 포함하는, 정보제공 방법.The method of claim 5, wherein in step (b), mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) of the individual 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 7) , mmu-miR-466i-5p (SEQ ID NO: 8) and mmu-miR-5128 (SEQ ID NO: 9) When the expression level of one or more miRNAs selected from the group consisting of is higher than that of the control group, the subject is considered to be in a stress state. Further comprising the step of determining, the information providing method.
  9. 제5항에 있어서, 상기 (b)단계에서 개체의 mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준이 대조군에 비해 저발현인 경우 상기 개체는 스트레스 상태인 것으로 판정하는 단계를 더 포함하는, 정보제공 방법.According to claim 5, wherein in step (b), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 11), mmu-miR-140-3p (SEQ ID NO: 11) of the individual 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p (SEQ ID NO: 15), mmu-miR-185-5p ( SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a-5p (SEQ ID NO: 19), mmu-miR-29a- 3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a-3p (SEQ ID NO: 23), mmu-miR-669o- When the expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) is low compared to the control group, determining that the subject is in a stress state Further comprising, a method of providing information.
  10. (a) 개체의 생물학적 시료로부터 엑소좀을 분리하는 단계; 및(a) isolating the exosomes from the biological sample of the subject; and
    (b) 상기 (a)단계에서 분리된 엑소좀에서 유래한 mmu-let-7a-5p(서열번호 1), mmu-let-7b-5p(서열번호 2), mmu-let-7c-5p(서열번호 3), mmu-let-7e-5p(서열번호 4), mmu-miR-126a-5p(서열번호 5), mmu-miR-3473b(서열번호 6), mmu-miR-3473e(서열번호 7), mmu-miR-466i-5p(서열번호 8), mmu-miR-5128(서열번호 9), mmu-let-7i-5p(서열번호 10), mmu-miR-130a-3p(서열번호 11), mmu-miR-140-3p(서열번호 12), mmu-miR-142a-3p(서열번호 13), mmu-miR-16-5p(서열번호 14), mmu-miR-17-5p(서열번호 15), mmu-miR-185-5p(서열번호 16), mmu-miR-19b-3p(서열번호 17), mmu-miR-24-3p(서열번호 18), mmu-miR-27a-5p(서열번호 19), mmu-miR-29a-3p(서열번호 20), mmu-miR-301a-3p(서열번호 21), mmu-miR-451a(서열번호 22), mmu-miR-669a-3p(서열번호 23), mmu-miR-669o-3p(서열번호 24) 및 mmu-miR-93-5p(서열번호 25)로 이루어지는 군으로부터 선택된 1종 이상의 miRNA의 발현 수준을 대조군 시료의 해당 엑소좀 유래 miRNA의 발현 수준과 비교하는 단계를 포함하는, 아토피성 피부염으로 인해 유발된 개체의 스트레스 진단 방법.(b) mmu-let-7a-5p (SEQ ID NO: 1), mmu-let-7b-5p (SEQ ID NO: 2), mmu-let-7c-5p (SEQ ID NO: 2) derived from the exosome isolated in step (a) ( SEQ ID NO: 3), mmu-let-7e-5p (SEQ ID NO: 4), mmu-miR-126a-5p (SEQ ID NO: 5), mmu-miR-3473b (SEQ ID NO: 6), mmu-miR-3473e (SEQ ID NO: 6) 7), mmu-miR-466i-5p (SEQ ID NO: 8), mmu-miR-5128 (SEQ ID NO: 9), mmu-let-7i-5p (SEQ ID NO: 10), mmu-miR-130a-3p (SEQ ID NO: 9) 11), mmu-miR-140-3p (SEQ ID NO: 12), mmu-miR-142a-3p (SEQ ID NO: 13), mmu-miR-16-5p (SEQ ID NO: 14), mmu-miR-17-5p ( SEQ ID NO: 15), mmu-miR-185-5p (SEQ ID NO: 16), mmu-miR-19b-3p (SEQ ID NO: 17), mmu-miR-24-3p (SEQ ID NO: 18), mmu-miR-27a- 5p (SEQ ID NO: 19), mmu-miR-29a-3p (SEQ ID NO: 20), mmu-miR-301a-3p (SEQ ID NO: 21), mmu-miR-451a (SEQ ID NO: 22), mmu-miR-669a- The expression level of one or more miRNAs selected from the group consisting of 3p (SEQ ID NO: 23), mmu-miR-669o-3p (SEQ ID NO: 24) and mmu-miR-93-5p (SEQ ID NO: 25) was measured for the corresponding exo of the control sample. A method for diagnosing stress in an individual induced by atopic dermatitis, comprising comparing the expression level of a moth-derived miRNA.
PCT/KR2021/005258 2020-11-13 2021-04-26 Technique for diagnosing stress associated with atopic dermatitis using exosome-derived mirnas WO2022102885A1 (en)

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