KR102332653B1 - Mouse Model for Laryngopharyngeal Reflux Disease and Method for Preparing the Same - Google Patents

Abstract

The present invention relates to a pharyngeal reflux disease mouse model and a method for manufacturing the same, using the pharyngeal reflux disease mouse model of the present invention. It is useful for screening drugs for laryngeal reflux disease by evaluating the decrease in MMP-7 expression.

Description

Laryngopharyngeal Reflux Disease and Method for Preparing the Same

The present invention relates to a mouse model of laryngeal reflux disease and a method for manufacturing the same, and more particularly, to a mouse model for laryngeal reflux disease in which lesions were induced by ingesting an acidic solution in a mouse, a method for manufacturing the mouse model, and a biomarker composition for diagnosing laryngeal reflux disease it's about

Laryngopharyngeal reflux-related diseases account for 4-10% of all patients visiting an otolaryngology clinic. Regurgitation of the contents of the gastrointestinal tract has been suggested as a common cause of the disease.

The pathophysiology and objective diagnosis of laryngopharyngeal reflux disease are still unclear despite the rapid increase in the incidence of laryngopharyngeal reflux disease in recent years (Hicks DM et al., J Voice . 16, 564-579, 2002). The most commonly debated theory about the pathogenesis of pharyngeal reflux disease is that the symptoms are directly caused by exposure and cell malformation of the laryngeal mucosa due to acid reflux, and is considered a disease different from typical reflux or gastroesophageal reflux disease. Diagnosis of laryngeal reflux disease is made by surveying specific symptoms and monitoring the laryngeal or double probe pH. Ambulatory 24-hour dual probe (pharyngeal and esophageal) pH monitoring is highly sensitive and specific for the diagnosis of pharyngeal reflux disease. However, this method is not widely used in clinical practice because of its inconvenience and cost compared to the easily accessible videolaryngoscopic examination.

In addition, although the standard of care consists of high-dose proton pump inhibitor (PPI) therapy hypothesized for the pathophysiology of pharyngeal reflux disease, there is insufficient evidence to conclude that PPI treatment is superior to placebo in randomized clinical trials. . Also, it is relatively poor to have a response to these treatments with improvement of symptoms of laryngeal reflux disease. As such, the pathophysiology proposed so far does not adequately explain the inconsistency of the diagnosis or treatment response of pharyngeal reflux disease.

The pathophysiological mechanisms and other influencing factors for acid exposure may result from reflux biomarkers, including inflammatory cytokines, carbonic anhydrase and mucus, as well as adverse factors for gastric reflux, although none of these are currently clinically involved. nothing can be

Recently, since there is no record of the existence of pharyngeal acid reflux disease that occurs naturally in animals such as mice, it is necessary to create a model artificially, but there is a problem in that acid reflux does not occur to the pharynx and larynx. In addition, there are studies on a model of esophagitis caused by acid reflux disease, but there is no study on a model of pharyngitis caused by acid reflux disease.

Accordingly, the present inventors have made diligent efforts to solve the above problems. As a result, the concentration of soluble E-cadherin fragment in saliva increases and the concentration of soluble E-cadherin fragments in saliva increases in a mouse model of laryngeal reflux disease produced by ingesting an acidic solution in a mouse. It was confirmed that the decrease in the expression level of E-cadherin in the mucosal layer and the increase in the expression level of MMP-7 was reproduced, and the present invention was completed.

Hicks DM et al, J Voice. 16, 564-579, 2002

It is an object of the present invention to provide a mouse model for laryngeal reflux disease and a method for manufacturing the same.

Another object of the present invention is to provide a biomarker composition for diagnosing laryngeal reflux disease.

Another object of the present invention is to provide a screening method for a therapeutic agent for laryngeal reflux disease, which is characterized by using the mouse model.

In order to achieve the above object, the present invention provides a method for manufacturing a mouse model of laryngeal reflux disease, comprising the step of ingesting an acidic solution to the mouse.

The present invention also provides a mouse model of laryngeal reflux disease produced by the above method.

The present invention also provides a biomarker composition for diagnosing pharyngeal reflux disease, comprising matrix metalloproteinases-7 (MMP-7), whose expression is relatively increased in the pharyngeal mucosal layer of a patient with pharyngeal reflux disease than in the pharyngeal mucosal layer of a normal person.

The present invention also comprises the steps of (a) treating a candidate material in a mouse model of laryngeal reflux disease to determine the concentration of soluble E-cadherin fragment in saliva and the expression levels of E-cadherin and MMP-7 in the pharyngeal mucosal layer; And (b) when the soluble E-cadherin fragment concentration and the expression levels of E-cadherin and MMP-7 of the mouse model treated with the candidate material are similar to normal, selecting the candidate material as a treatment for pharyngeal reflux disease. A screening method for a therapeutic agent for reflux disease is provided.

The laryngeal reflux disease mouse model of the present invention can be usefully used for screening drugs for pharyngeal reflux disease by reducing the concentration of soluble E-cadherin fragment in saliva, increasing the expression of E-cadherin in the pharyngeal mucosal layer, and evaluating the decrease in MMP-7 expression in the pharyngeal mucosal layer. can

1 is a result confirming the expression of E-cadherin and MMP-7 in the pharyngeal tissue of an animal model of laryngeal reflux disease.
Figure 2 shows the change of E-cadherin according to acid exposure and incubation time.
Figure 3 shows the change with respect to the 80-kDa E-cadherin fragment (soluble E-cadherin) from the culture supernatant.
Figure 4 shows changes in E-cadherin and mRNA expression changes for MMP-2, 3, 7, and 9 after treatment with an MMP inhibitor.
Figure 5 shows changes in protein expression with respect to enzyme activity when exposed to acids and MMP inhibitors.
Figure 6 is the result of confirming the effect on the MMP inhibitor through the immunocytochemical staining method and the permeability of the change after exposure to the acid and the MMP inhibitor.
7 is a result confirming the expression of E-cadheirn and MMP-7 in the pharyngeal tissue of a patient with laryngeal reflux disease.
8 is a graph showing the results of MMP-7 protein expression in saliva.
9 is a graph showing the results of MMP-7 protein expression in saliva by RSI (Reflux Symptom Index).
10 shows the expression results of MMP-7 protein in saliva after taking Noltech, a treatment for laryngeal reflux disease.

In the present invention, in a mouse model of pharyngeal reflux disease prepared by ingesting an acidic solution in a mouse, the concentration of soluble E-cadherin fragment in saliva increases, and the expression level of E-cadherin in the pharyngeal mucosal layer decreases, as in the case of pharyngeal reflux disease in humans, and MMP- 7 It was confirmed that the increase in expression level was reproduced.

Accordingly, the present invention, in one aspect, relates to a method of manufacturing a mouse model of laryngeal reflux disease, comprising the step of ingesting an acidic solution to the mouse.

In the present invention, given that the luminal environment of the pharynx is pH-neutral at pH 7.0 and the stomach secretes acid at pH 1.5-2.0, acid reflux into the laryngopharyngeal epithelium can significantly reduce the pH of the pharynx. Damage to the pharynx can result from a decrease in pH or exposure to harmful components of the reflux, including pepsin, bile salts and pancreatic enzymes.

In the present invention, the acidic solution may be characterized in that pH 3 to pH 5, it may be characterized in that a mixture of acid or pepsin in drinking water.

In the present invention, it may be characterized in that the normal drinking water and the acidic solution are alternately supplied to the mice in a cycle of 1 to 2 days, and ingested for about 5 to 10 weeks in total.

In the present invention, the mouse is not supplied with drinking water per day, and then alternately supplied with normal drinking water and acidic solution on a cycle of 1 to 2 days, characterized in that it is ingested for about 5 to 10 weeks in total.

In the present invention, the mouse may be characterized as wild-type C57BL/6.

In another aspect, the present invention relates to a mouse model of laryngeal reflux disease produced by the above method.

In the present invention, the mouse animal model of laryngitis, (a) increase the concentration of soluble E-cadherin fragment in saliva; (b) decreased expression of E-cadherin in the pharyngeal mucosal layer; (c) it may be characterized as having one or more characteristics selected from characteristics consisting of increased expression of matrix metalloproteinases-7 (MMP-7) in the pharyngeal mucosal layer.

In the present invention, E-cadherin is a type-I transmembrane glycoprotein and is defined as an adhesive junction and a basolateral membrane in various epithelial cells. The structure of E-cadherin consists of 1) a large extracellular domain, 2) a transmembrane segment, and 3) a conserved cytoplasmic domain. The extracellular portion, E-cadherin, contains five extracellular cadherin domain repeats that bind calcium ions to form a modified linear molecule. The binding function of E-cadherin plays an important role in the physiology of epithelial cells. The fully formed cadherin-catenin complex with bound actin filaments forms the core of adherens junctions and binds two homologous plasma membranes with a cell gap of 25 nm. E-cadherin-mediated adherens junctions are also essential for the formation of apical tight junctions, an important epithelial tissue that forms a permeable barrier that prevents passive diffusion of most solutes between joints and fissures.

In the present invention, E-cadherin fragments can be generated at several points. MMP cleavage occurs on the extracellular surface of the plasma membrane to generate sE-cad (N-terminal 80-kDa fragment). In a previous study using the esophageal epithelium in gastroesophageal reflux disease (GERD), it was confirmed through protein expression analysis that a C-terminal fragment with a molecular weight of 35 kDa exists in the esophageal mucosa due to the cleavage of E-cadherin. In addition, serum levels of the 80 kDa N-terminal fragment were significantly higher in GERD patients. Similarly, when the pharyngeal mucosal cells were exposed to acid, MMP-7 was activated, and E-cadherin was degraded by this enzyme, resulting in a decrease in intact E-cadherin and an increase in the degradation product, sE-cad, in the culture supernatant.

In the present invention, expression means mRNA transcript or protein expression.

In one embodiment of the present invention, since the cleavage of E-cadherin and the weakening of the intercellular binding force of the larynx are induced by exposure to acidic contents, matrix metalloproteinases (MMPs) for e-cadherin expression change after acid exposure to pharyngeal epithelial cells to evaluate the role of For this purpose, human pharyngeal mucosal epithelial cells from healthy volunteers were cultured. E-cadherin was significantly decreased by the correlation between the acid exposure organ and the number of repetitions. When treated with actinonin, an overall MMP inhibitor, it was confirmed that the change was inhibited. When analyzed for various MMPs, the expression of MMP-7 mRNA was increased, and the cleavage activity of MMP-7 in the acid-treated cell culture supernatant was increased. Immunochemical staining and cell permeability results showed that the cell surface staining permeability of E-cadherin was decreased in the acid-exposed cells, which was significantly inhibited by the MMP inhibitor. As a result, it was confirmed that MMP-7 plays an important role in inducing the degradation of E-cadherin after exposure to relatively mild acidic conditions similar to the more physiological conditions that occur in patients with laryngeal reflux disease.

In another example, the present invention confirmed the change of E-cadherin in pharyngeal epithelial cells by acid exposure and pathophysiological mechanisms mediated by MMP-7. In addition, if the mucosa is exposed to acidic conditions for more than 10 minutes, epithelial cells are decomposed even in a single event. However, mucosal acid exposure for 1 to 5 minutes induced maximal degradation of E-cadherin in epithelial cells 24 hours after mucosal acid exposure. Moreover, 24 h after acid exposure, shorter acid exposures, such as 15 and 30 s, did not induce degradation of E-cadherin in a single exposure, but was significantly reduced by repeated acid exposure. In particular, similar to the physiological exposure seen in humans, repeated exposure to acid three times a day increased the degradation of E-cadherin in proportion to the increase in exposure time. Consequently, this phenomenon demonstrates that short-term acid exposure to the mucosa does not destroy E-cadherin in epithelial cells directly through chemical damage, but can induce degradation of E-cadherin by a mechanism that affects it over a 24-hour period. .

24 hours after the epithelial cells were exposed to acid, an increase in sE-cadherin in the supernatant was observed as opposed to a decrease in intracellular E-cadherin. This increase in sE-cadherin in the supernatant after acid exposure correlates with MMP activity. In particular, the acid exposure time correlated with an increase in MMP-7 mRNA expression and enzymatic activity along with an increase in epithelial cell permeability. As expected, treatment with an MMP inhibitor moderated the increase in permeability along with the denaturation of E-cadherin.

In the present invention, the monitoring of adventitious acidity of pharyngeal reflux disease is de facto controversial about defining a pH of less than 4 or 5 as a disease. This is because the acid exposure of the pharynx is shorter and shorter in duration compared to the peripheral esophagus, with dilution and neutralizing effects. For saliva and mucus, the pH of the pharynx usually does not drop below 4. Therefore, the pH of the acid exposure was determined to be 4 in the experiment, and the primary cultured pharyngeal mucosal cells were treated for up to 5 minutes, and 15 seconds or 30 seconds in repeated exposure.

Single acid exposure to pH 4 for pharyngeal reflux disease mucosal cells showed no significant change in mRNA but significantly decreased protein for 24 h after washing. These results may indicate an association with other mechanisms and may not be direct damage from acid exposure. Cleavage of E-cadherin is associated with an increase in MMP-7 expression and enzymatic activity after acid exposure, which may be why E-cadherin is cleaved after a delay after acid exposure.

Given the role of E-cadherin in maintaining intercellular binding, alteration of E-cadherin by acid exposure may increase the influx of various solutes into the intercellular gap and interstitial space, leading to the typical finding of mucosal damage. . Therefore, sE-cadherin by E-cadherin cleavage showed a positive correlation with the increase in mucosal permeability, so sE-cadherin can be a new diagnostic indicator for the presence and severity of pharyngeal reflux disease.

In the present invention, sE-cadherin from sputum or saliva was used as a diagnostic tool that can be evaluated. The severity of acid exposure can be assessed by evaluating sE-cadherin levels in sputum or saliva, which may be a useful diagnostic aid for laryngeal reflux disease. In addition, sE-cadherin levels may be useful in follow-up of patients with laryngeal reflux disease and may monitor the effectiveness of drug treatment.

sE-cadherin (sE-cad) may be a negative competitor for membrane-bound E-cadherin, interfering with hemophilic interactions of full-length E-cadherin homodimers between adjacent cells. In addition, sE-cad causes upregulation of major MMPs and affects cell-cell adhesion with E-cadherin. In the present invention, it was confirmed that acid exposure had a significant effect on pharyngeal epithelial cells in both single exposure and short repeated exposure. Considering that the effects of sE-cad and MMP are maintained for 24 hours after acid removal, single or repeated acid exposure, even for a short period of time, can induce symptoms and physical findings of pharyngeal reflux disease.

In the present invention, although the clinical criteria for diagnosing laryngeal reflux disease of dual probe pH monitoring are still controversial, it is believed that increasing the duration and repetition of acid exposure has a significant effect on increasing the cleavage and permeability of E-cadherin. Given this, the duration and repetition of acid exposure were included as part of the monitoring.

In the present invention, generation of sE-cad by mechanical cleavage by acid itself reduces the amount of full-length E-cadherin in the plasma membrane. By cleaving the E-cadherin, the existing adhesive junction is further disrupted, and thereafter, the expression of MMP-7 is increased to cleave the extracellular region, and thus E-cadherin is cleaved, Permeability increases ( FIG. 7E ).

In another aspect, the present invention provides a biomarker for diagnosing pharyngeal reflux disease, including matrix metalloproteinases-7 (MMP-7), whose expression is relatively increased in the pharyngeal mucosal layer of a patient with pharyngeal reflux disease than in the pharyngeal mucosal layer and/or saliva of a normal person. is about

In another aspect of the present invention, it relates to a composition for diagnosing laryngeal reflux disease, comprising an agent for measuring mRNA or protein levels of matrix metalloproteinases-7 (MMP-7) gene.

In another aspect, the present invention provides a kit for diagnosing laryngeal reflux disease comprising the composition.

In the present invention, the agent for measuring the mRNA level preferably includes a primer pair or probe that specifically binds to the MMP-7 gene, and the nucleic acid sequence of genes encoding MMP-7 is registered in the gene bank. Therefore, those skilled in the art can design primer pairs or probes for specifically amplifying a specific region of these genes based on the above sequence.

In the present invention, a "primer" is a nucleic acid sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template and is a starting point for copying the template. It refers to a short nucleic acid sequence that functions as Primers are capable of initiating 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, the sequence of the primer is not limited as long as the primer can be amplified by complementary binding to the MMP-7 gene.

In the present invention, "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to a gene or mRNA, and is labeled with a specific mRNA existence can be checked. 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, the sequence of the probe is not limited as long as the probe can complementarily bind to the MMP-7 gene.

The primers or probes of the present invention can be chemically synthesized using the phosphor amidite 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.).

In the present invention, the agent for measuring the protein level may preferably include an antibody specific for the MMP-7 protein.

In the present invention, "antibody" refers to a specific protein molecule directed against an antigenic site. Such an antibody can be prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the gene, and from the obtained protein by a conventional method. This also includes partial peptides that can be made from the protein, for example, the partial peptide contains at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and any part thereof is included in the antibody of the present invention as long as it has polyclonal antibody, monoclonal antibody or antigen-binding property, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody. Antibodies for use in the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.

In one embodiment, the present invention may relate to a kit for diagnosing laryngeal reflux disease comprising the antibody specifically binding to MMP-7 as an active ingredient.

The kit of the present invention can be used to diagnose laryngeal reflux disease by checking the mRNA expression level or protein expression level of the MMP-7 gene corresponding to the marker. The kit of the present invention may include primers, probes or antibodies for diagnosing laryngeal reflux disease, as well as one or more other component compositions, solutions, or devices suitable for the assay method.

As a specific example, the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR.

The RT-PCR kit includes, in addition to a primer pair specific for a marker gene, a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- It may include water (DEPC-water), sterile water, and the like.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing a microarray chip. The microarray chip kit includes a substrate to which cDNA corresponding to a gene or fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative control gene or fragment thereof, and the marker of the present invention is used. Therefore, it can be easily manufactured by a manufacturing method commonly used in this technical field. In order to manufacture a microarray chip, a micropipetting method or a pin type using a piezoelectric method to immobilize the detected marker on a substrate of a DNA chip using the searched marker as a probe DNA molecule. It is preferable to use a method using a spotter of , but is not limited thereto. The substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto. . In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.

In addition, the kit of the present invention may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. In the above, as the substrate, a nitrocellulose membrane, a plate synthesized from polyvinyl resin, a plate synthesized from polystyrene resin, glass slide glass, etc. may be used, and the chromogenic enzyme is peroxidase, alkaline phosphatase (alkaline). phosphatase), etc. may be used, the fluorescent material may be FITC, RITC, etc., and the color substrate solution is ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

In another aspect of the present invention, i) measuring the mRNA or protein expression level of the MMP-7 (matrix metalloproteinases-7) gene in saliva or pharyngeal mucosa, which is a biological sample; and ii) comparing the mRNA or protein expression level of the MMP-7 gene with a control sample.

In one embodiment of the present invention, the saliva may be saliva isolated from the individual after waking up in the morning.

When it is confirmed that the expression level of the MMP-7 gene or its protein is higher than that of the control group, it can be diagnosed that the subject providing the biological sample has laryngeal reflux disease.

Measuring the mRNA level in step i) is a process of confirming the presence and expression level of marker genes in a biological sample. Polymerase reaction (PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction RT-PCR), real-time reverse transcription polymerase reaction (Realtime RT-PCR), RNase protection assay (RPA), Northern blotting, or DNA microarray assay may be used, but are not limited thereto. .

The protein level measurement in step i) is a process of confirming the presence and expression level of a protein expressed from a marker gene in a biological sample, such as western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay ( Radioimmunoassay, Radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, flow cytometry An analysis method (Fluorescence Activated Cell Sorter, FACS) or a protein chip analysis method may be used, but is not limited thereto.

In another aspect, the present invention comprises the steps of: (a) confirming the concentration of soluble E-cadherin fragment in saliva and the expression levels of E-cadherin and MMP-7 in the pharyngeal mucosal layer by treating a candidate material in a pharyngeal reflux disease mouse model; And (b) when the soluble E-cadherin fragment concentration and the expression levels of E-cadherin and MMP-7 of the mouse model treated with the candidate material are similar to normal, selecting the candidate material as a treatment for pharyngeal reflux disease. It relates to a screening method for the treatment of reflux disease.

In the present invention, in (b), when the concentration of soluble E-cadherin fragment in saliva decreases, the expression level of E-cadherin in the pharyngeal mucosal layer increases, and the MMP-7 expression level in the pharyngeal mucosal layer decreases, the candidate substance It may be characterized in that it is selected as a therapeutic agent for laryngeal reflux disease.

In another aspect, the present invention may relate to a composition for treating pharyngeal reflux disease containing the drug screened by the above method as an active ingredient.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Establishment of a mouse model of laryngeal reflux disease

8-week-old male C57BL/6 mice of 28-30 g were purchased from Orient Bio (Seongnam, Korea). Mice were bred in an environment where 25±2°C and 50±10% humidity were maintained, and food and water were provided freely in a pathogen-free facility at Korea University College of Medicine.

In order to establish a pharyngeal reflux disease mouse model, after abstaining from water (no drinking water) the day before the experiment, 1) acidified water (pH 4) was continuously supplied, or 2) water and acidified water were alternately supplied on a daily basis, 3) The mice were supplied in three ways by alternately supplying normal drinking water and acidified water on a daily basis. Among them, the mouse produced by the method of 3) was selected. That is, the mice were divided into two groups: 6 mice in group A and 9 mice in group B exposed to acid.

Changes in water intake and body weight were observed in mice fed only normal drinking water and mice fed acidic and normal drinking water alternately. As a result, over time, it was confirmed that the group supplied with acidic drinking water (Group B) lost weight from the 5th week compared to the group of mice supplied with only normal drinking water (Group A) (FIG. 1B). As a result of measuring the daily water intake of the group supplied with acidic drinking water (Group B) and the group supplied with only general drinking water (Group A), there was no difference in intake (Fig. It was confirmed that it did not reach.

Thereafter, the mice were fed the same diet as above for 5 or 9 weeks, thereby preparing an animal model of laryngeal reflux disease.

At the 9th week, compared to when only general drinking water was supplied, it was confirmed that about 20% of body weight was reduced after measuring the daily intake and body weight when normal drinking water and acidic drinking water were alternately supplied.

Experimental methods and experimental design parameters were reviewed and approved by the Korea University Hospital Clinical Trial Committee (IRB No. KOREA-2018-0026). All experiments were performed according to the guidelines and regulations.

Example 2: Correlation between the onset of laryngeal reflux disease and E-cadherin and MMP-7

2.1 Cell Culture

The pharyngeal mucosa of the patient who underwent tonsillectomy was collected. This method and experimental design parameters were reviewed and approved by Korea University Hospital Hospital Evaluation Committee (IRB No. ED15303). informed consent was obtained from all participants, and all methods were performed in accordance with relevant guidelines and regulations. The human pharyngeal mucosa was stored in a 1 mg/ml dispase solution, and then incubated for 1 hour at _{37 °C and 5% CO 2 .} After that, only the pharyngeal epithelial cells were scraped, collected in a 15ml conical tube, and centrifuged. After washing the cells 3 times using Dulbecco's modified Eagle's medium/F12 (DMEM/F12), bovine pituitary extract, insulin, hydrocortisone, gentamicin, amphotericin B, retinoic acid, transferrin, It was cultured using a culture medium (BEBM, Lonza, Walkersville, MD, USA) containing triodothyronine, epinephrine, epidermal growth factor, penicillin and streptomycin. Then, when the growth reached about 70%, the cells were removed with 0.25% trypsin EDTA, washed with DMEM/F12, and then cultured in a 12-well culture plate (SPL, Seoul, Korea) for acid treatment. Typically, the culture was maintained in an incubator of 37 ˚C, 5% CO ₂ , and the culture medium was replaced every 3 days. Cell morphology was observed with an Olympus CKX41-A32PHP (Olympus, Tokyo, Japan) microscope.

2.2 Acid Exposure and MMP Inhibitor Treatment Conditions

To investigate the effect of acid on the pharyngeal epithelial cells, hydrochloric acid was mixed with the BEBM culture medium to adjust the acidity (pH 4), and then the epithelial cells were treated for 30 seconds, 1 minute, and 5 minutes. Control cells were treated only with BEBM culture. After each acid treatment, the acidic medium was washed, and the cells were cultured _{for 1, 4, 8 and 24 hours at 37 ˚C, 5% CO 2} only with BEBM culture medium, washed twice with saline, and then used for the experiment. did.

Thereafter, in order to investigate the effect of repeated acid exposure, a solution adjusted to acidity 4 (pH 4) by adding hydrochloric acid to the culture medium of epithelial cells was repeated 3 times a day for 15 seconds or 30 seconds at 8 hour intervals for 3 days. did. As a result, cells on the 1st, 2nd and 3rd days were used for the experiment.

In addition, in order to investigate the effect of exposure of acid/MMP inhibitor on pharyngeal epithelial cells, culture was performed using 10 uM MMP inhibitor (actinonin, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for about 1 hour before acid treatment. . Then, the culture medium having an acidity of 4 was treated for 30 seconds, 1 minute, and 5 minutes, respectively, and only the MMP inhibitor was treated as a control. Thereafter, each treatment group was cultured for 1, 4, 8, and 24 hours, and then used for the experiment.

2.3 Observation of E-cadherin and MMP-7 Expression Changes in Pharyngeal Mucosal Cells after Acid Exposure Using RT-PCR

Gene expression in epithelial cells was measured by a quantitative real-time gene amplification method. After preparing the synthesized cDNA, SYBR green master mix (Qiagen, Germantown, MD, USA) was used. The primers used were GAPDH, forward 5'-GAG TCA ACG GAT TTG GTC GT-3' (SEQ ID NO: 1), reverse 5'-TTG ATT TTG GAG GGA TCT CG-3' (SEQ ID NO: 2); MMP-2, forward 5'-TCT CCT GAC ATT GAC CTT GGC-3' (SEQ ID NO: 3) and reverse 5'-CAA GGT GCT GGC TGA GTA GAT C-3' (SEQ ID NO: 4); MMP-3, forward 5'-ATT CCA TGG AGC CAG GCT TTC-3' (SEQ ID NO: 5) and reverse 5'-CAT TTG GGT CAA ACT CCA ACT GTG-3' (SEQ ID NO: 6); MMP-7, forward 5'-TGA GCT ACA GTG GGA ACA GG-3' (SEQ ID NO: 7) and reverse 5'-TCA TCG AAG TGA GCA TCT CC-3' (SEQ ID NO: 8); MMP-9, forward 5'-TTG ACA GCG ACA AGA AGT GG-3' (SEQ ID NO: 9) and reverse 5'-GCC ATT CAC GTC CTT AT-3' (SEQ ID NO: 10); and E-cadherin, forward 5'-TGC TCT TGC TGT TTC GG-3' (SEQ ID NO: 11) and reverse 5'-TGC CCC ATT CGT TCA AGT AG-3' (SEQ ID NO: 12). The condition was a real-time thermocycling system (TP800/TP860, Takara, TP800/TP860, Takara, Kusatsu, Shinga, Japan) was used. Result analysis was performed using the ΔCt method.

In order to analyze changes in E-cadherin protein expression after acid exposure to pharyngeal mucosal cells, fresh culture medium was replaced after exposure to acid.

As a result, when the pharyngeal mucosal cells were exposed to acid for 10 minutes, the expression of E-cadherin decreased, and almost no immune response was observed. This means that the epithelial cells are decomposed.

After 30 seconds, 1, 5 minutes of short exposure to acid, or 10 minutes and 20 minutes of long exposure, washing was performed and then incubated for 1 hour. As a result, no change in expression of E-cadherin was observed (Fig. 2A). .

After culturing for 8 hours or 24 hours under the same conditions as above, it was observed that E-cadherin decreased (FIG. 2A). It was confirmed that the expression of E-cadherin was more markedly decreased in the culture conditions for 24 hours after 1 minute or 5 minute acid exposure ( FIG. 2B ). However, it was confirmed that the level of mRNA expression was slightly decreased ( FIG. 2C ).

In addition, it was confirmed that the expression of E-cadherin and MMP-7 decreased in the cell extract and increased in the cell culture medium when the acid was exposed for 15 seconds and 30 seconds three times a day and observed for 1 to 3 days. .

2.4 Changes in expression of E-cadherin and sE-cadherin in pharyngeal mucosal cell supernatant after acid exposure

In order to evaluate the fragment and cleavage site of E-cadherin, the expression of E-cadherin was confirmed using the culture supernatant after exposure to acid. sE-cad (soluble 80-kDa E-cadherin) in the culture supernatant was the predominant fragment and was also associated with the discovery of the full-length E-cadherin in the cell composition (Fig. 3A). It was confirmed that the cleavage of E-cadherin was prevented when the MMP inhibitor actinonin was used. Therefore, it was confirmed that cleavage of E-cadheirn is related to MMP.

Since sE-cadherin is released when the extracellular portion of E-cadherin is cleaved, acid exposure in the pharyngeal mucosa may affect E-cadherin damage. Therefore, the increase in the expression of sE-cadherin and the decrease in the expression of E-cadherin when exposed to acid is a result of MMP activity (Fig. 3B).

In Fig. 3B, CTF means C-terminal fragment, CD means cytoplasmic domain, EC means extracellular cadherin domain, and T means transmembrane domain.

After exposure to acid and culturing for 24 hours after exposure to cells, E-cadherin was observed to decrease, and it was confirmed that the cells were improved by MMP inhibitor ( FIGS. 4A and 4B ).

2.5 Confirmation of MMP subset change after acid exposure

As a result of performing RT-PCR after treating the pharyngeal mucosa with acid, the expression of MMP-9 was significantly reduced while the expression of MMP-7 was increased ( FIG. 4C ). And although the expression level of MMP-3 was not statistically significant, it showed a tendency to increase when exposed to acidic conditions (FIG. 4C).

In order to examine the effect of the transcription inhibitor, pharyngeal epithelial cells were pretreated with Actinomycin D (Sigma) 5ug/ml at 37 ˚C, 5% CO ₂ conditions for 30 minutes. Then, after exposing the cells to a culture medium of acidity 4 (pH 4) for 30 seconds, 1 minute, and 5 minutes, respectively, the culture medium was changed to a culture medium containing only Actinomycin D and cultured for 24 hours, then mRNA for MMP-7 factor The expression level was confirmed.

As a result, it was confirmed that the expression of MMP-7 in the mRNA increased when the cells were treated with actinomycin D, a transcription inhibitor (FIG. 4D).

For quantitative protein detection analysis of MMP subset, pharyngeal epithelial cells treated with acid or acid/MMP inhibitor were extracted with 5% Laemmli buffer and 5% β-mercaptoethanol, and protein binding was disrupted for 10 minutes. The extracted protein was separated on an 8% SDS-polyacryl amide gel, and a transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS and 20% methanol, acidity 8.3) was transferred to a nitrocellulose membrane at 350 mA for 30 min. Blocked with 5% skim milk for 90 min at room temperature to prevent non-specific binding on the membrane. Then, E-cadherin, MMP-3 (Santa Cruz), MMP-7 (Sigma-Aldrich, St. Louis, MO, USA), and b-actin were used as protein expression primary antibodies. Next, rabbit and mouse antibodies were used as secondary antibodies for each antibody. Next, expression was amplified using a chemiluminescence kit (Santa Cruz), and then expression was confirmed using ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA).

As a result, at the protein level, the expression of MMP-7 in the cells decreased as the time for exposing the cells to the acid increased, and correspondingly, the expression of MMP-7 in the supernatant increased ( FIG. 5A ). These changes tended to be restored again in the cells treated with the MMP inhibitor. However, there was no change in the expression of MMP-3 under the same culture conditions (FIG. 5B).

Then, in order to evaluate the protein function itself, the enzymatic activity of MMP-7 and MMP-3 was analyzed. As the acid exposure time increased, the activity of MMP-7 significantly increased, whereas the activity of MMP-3 was almost the same. In addition, when the MMP inhibitor was treated, the activity of MMP-7 was significantly reduced compared to that of MMP-3 ( FIGS. 5C and 5D ). Therefore, it can be seen that the cleavage of E-cadherin after acid exposure is mainly due to MMP-7.

2.6 Analysis of MMP-3 and MMP-7 Enzyme Substrates

In order to measure the activity of matrix metalloproteinase MMP-3 and MMP-7, culture supernatant of pharyngeal epithelial cells treated with acid or acid/MMP inhibitor was transferred to Centricon spin tube (3kDa cutoff, Merck Millipore, Billerica, MA, USA). It was concentrated by centrifugation at ˚C and 3000 g for 40 minutes. For analysis, the concentrate was incubated with 10 uM APMA (4-aminophenylmeric acetate) at 37 °C for 1 hour. Then, the MMP-3 substrate (5-FAM/QXLTM520FRETpeptide) or the MMP-7 substrate (5-FAM/QXLTM520FRETpeptide) was diluted 1:100 and reacted for 1 hour, and then the excitation wavelength was 490 nm using a SpectraMax M2eplatereader (MolecularDevices). and emission wavelength 520 nm (SOFTMAX PRO V5 software, Molecular Devices, Sunnyvale, CA, USA).

As a result, the enzyme activity of MMP-7 significantly increased as the acid exposure time increased, whereas the activity of MMP-3 was almost the same. In addition, when the MMP inhibitor was treated, the activity of MMP-7 was significantly reduced. Therefore, it is presumed that MMP-7 is involved in the mechanism by which E-cadherin is cleaved after acid exposure and soluble E-cadherin is increased.

2.7 Observation of E-cadherin Expression Changes in Pharyngeal Mucosal Cells after Acid Exposure Using Immunocytochemical Analysis

nigshofen, Germany)에서 키운 다음 산 또는 산/MMP 억제제를 처리하였다. 면역 세포 화학 분석은 산 노출과 24시간 배양 후에 수행하였다. 세포를 4% glutaraldehyde에 30분 동안 고정시킨 후 염소 혈청(Vector Laboratories, Burlingame, CA, USA)을 이용하여 1시간 동안 비특이적인 결합을 차단시켰다. 그 다음 세포를 E-cadherin(1:50, Santa Cruz)항체로 4 ˚C에서 바매 항온 배양하였다. 다음날, 실온에서 60분 동안 토끼 2차 항체로 세포를 처리한 후 항원-항체 복합체인 아비딘-바이오틴 복합체 용액(Vectastain ABC Kit, Vector Laboratories)을 사용하여 발현을 증폭시켰다. Cytoslide는 DAB Substrate kit(Vector Laboratories)를 이용하여 발현 염색을 하였고, 세포의 핵은 mayer hematoxylin으로 염색한 후 Olympus BX51 현미경을 통해서 관찰하였다. 이미지는 Olympus DP72 및 DP2-BSW(Olympus) 프로그램을 이용하여 확인하였다. Cells were subjected to cytoslides (Marienfeld-Superior, Lauda-K) for immunocytochemical analysis.

nigshofen, Germany) and then treated with acids or acid/MMP inhibitors. Immunocytochemical analysis was performed after acid exposure and 24 h incubation. After fixing the cells in 4% glutaraldehyde for 30 minutes, non-specific binding was blocked for 1 hour using goat serum (Vector Laboratories, Burlingame, CA, USA). Then, the cells were incubated with E-cadherin (1:50, Santa Cruz) antibody at 4 ˚C. The next day, cells were treated with a rabbit secondary antibody for 60 minutes at room temperature, and then expression was amplified using an antigen-antibody complex avidin-biotin complex solution (Vectastain ABC Kit, Vector Laboratories). Cytoslide was stained for expression using DAB Substrate kit (Vector Laboratories), and cell nuclei were observed through an Olympus BX51 microscope after staining with mayer hematoxylin. Images were confirmed using Olympus DP72 and DP2-BSW (Olympus) programs.

As a result, it was found that the expression level of E-cadherin at the cell-cell binding junction was maintained when the acid and the MMP inhibitor were treated together, compared to the case of treatment with the acid ( FIG. 6A ) ( FIG. 6C ). In addition, when the acid was exposed for 5 minutes, the expression of E-cadherin in the cells was high. The increase in the cytoplasmic level of E-cadherin and the difference in the expression level of adhesion between cells indicate cleavage of the extracellular domain of E-cadherin.

2.8 Epithelial cell permeability analysis

Human pharyngeal epithelial cells were cultured at a density ^{of 1 x 10 5} cells/cm ^{2 in} a 0.4-μm polyester filter (SPL) 12 well plate. When 100% cells were grown, 50 ul of 100 uM rhodamine B isothiocyanate (RITC)-labeled Dextran 70S (Sigma) was added to the upper plate chamber after treatment with acid or acid/MMP inhibitor as mentioned above. After that, 50ul of culture solution samples were collected from the culture compartment at 30-minute intervals for 3 hours and analyzed with a SpectraMax M2eplatereader (MolecularDevices) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm (SOFTMAX PRO V5 software, Molecular Devices).

As a result of the permeability test of epithelial cells, the permeability gradually increased as the time increased when the acid was treated for 30 seconds, 1 minute, or 5 minutes (FIG. 6B). On the other hand, when the MMP inhibitor was treated, it was confirmed that the permeability was reduced (FIG. 6D). Thus, it enables cell-cell interactions by blocking further cleavage of E-cadherin by MMPs.

2.9 Organizational Analysis

Pharyngeal mucosa was collected from a total of 10 volunteers who underwent tonsillectomy for chronic tonsillitis. 1) hoarseness or problems with voice, 2) throat clearing, 3) excessive throat or water drops in eye drops, 4) difficulty swallowing food, liquids, or pills, 5) symptoms of diarrhea, 6) difficulty breathing or choking, 7) cough , 8) a feeling of something in the throat, 9) chest pain, indigestion, or stomach acid. According to the diagnostic criteria for laryngeal reflux disease, patients were divided into two groups: 1) patients with a reflux symptom index (RSI) less than 14 (n=5), and 2) patients with an RSI of 14 or more (n=5). is classified as The extracted mucosa was evaluated by immunohistochemical staining with E-cadherin and MMP-7 antibody, and quantitatively compared using the Image J program. The Institutional Review Board (IRB No. ED15303) of Korea University Anam Hospital approved all protocols and study designs, and written informed consent was obtained from all patients.

Patients who underwent tonsillectomy were classified into two groups according to the diagnostic criteria for laryngeal reflux disease. The expression of E-cadherin in the reflux symptom index (RSI) of 14 or higher ( FIG. 7B ) was significantly lower than that of the group with RSI less than 14 ( FIG. 7A ), and MMP in the pharyngeal mucosal layer of the group with RSI of 14 or higher The expression of -7 ( FIG. 7D ) was significantly higher than that of MMP-7 ( FIG. 7C ) in the pharyngeal mucosal layer of the group with an RSI less than 14 ( FIG. 7C ).

Example 3: Confirmation of the onset of pharyngeal reflux disease in a mouse model of laryngeal reflux disease

The experiment was conducted for a total of 9 weeks, and as a result of comparing the expression of E-cadherin and MMP-7 by extracting mouse pharyngeal tissues at 5 and 9 weeks from the mouse group (A and B) of Example 1, only normal drinking water was used. Compared to the supplied group (Group A), it was confirmed that the expression of E-cadherin was decreased while the expression of MMP-7 was increased in the group (Group B) supplied alternately with normal drinking water and acidic drinking water ( FIG. 1C ).

Example 4: Confirmation of MMP-7 expression in saliva

The subjects were those with a score of 13 or higher through the reflux syptom index for patients with suspected laryngeal reflux disease. The total number of participants was 15. Saliva was collected 8 times a day (after waking up, before breakfast, after breakfast, before lunch, after lunch, before dinner, after dinner, before going to bed) for 5 days to check whether the MMP-7 protein was expressed in the saliva of the experiment participants. did. As for the collection method, 10 ml of distilled water was gargled for 30 seconds in the mouth, and then saliva was collected in a tube. Thereafter, the sample of the same protein concentration was confirmed through the BCA protein quantification method through the Western blot experimental technique and MMP-7 enzyme activity technique, which are special protein detection methods (FIG. 8). As shown in FIG. 8 , it was confirmed that the MMP-7 protein had a high activity of about 5 times in saliva after waking up.

Example 5: Confirmation of MMP-7 protein expression in saliva by RSI (Reflux Symptom Index)

A participant with a Reflux Symptom Index (RSI) score of 13 or higher as a suspected symptom of laryngeal reflux disease is diagnosed as having a high possibility of laryngeal reflux disease. In order to see the difference in the amount of saliva expression in proportion to the RSI score, the saliva after waking up of the test participant was collected in the same manner as in Example 4 for 3 days, and MMP-7 protein expression was performed through Western blot and MMP-7 enzyme activity test technique. was confirmed (FIG. 9).

As shown in FIG. 9 , it was confirmed that the protein expression level was repeatedly increased in saliva having an RSI score of 13 or more. Therefore, it can be seen from this result that it is possible to diagnose pharyngeal acid reflux disease by confirming the expression of MMP-7 protein in saliva.

Example 6: Confirmation of decrease in expression of MMP-7 protein in saliva after taking a treatment for laryngeal reflux disease

Patients with symptoms of RSI score of 13 or higher were instructed to take Noltec, a treatment for laryngeal reflux disease, for 3 weeks. The saliva was retaken for 3 weeks once a week during the ingestion, and the enzyme activity for MMP-7 was also confirmed through Western blot and MMP-7 experiments (FIG. 10). As shown in FIG. 10 , it was confirmed that the protein expression levels for E-cadherin and MMP-7 after taking the therapeutic agent gradually decreased in proportion to the number of weeks taken.

Therefore, the present invention shows that salivary MMP-7 protein can be a biomarker for the diagnosis of pharyngeal acid reflux disease.

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Mouse Model for Laryngopharyngeal Reflux Disease and Method for Preparing the Same <130> DHP18-227 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 1 gagtcaacgg atttggtcgt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 2 ttgattttgg agggatctcg 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MMP-2 forward primer <400> 3 tctcctgaca ttgaccttgg c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MMP-2 reverse primer <400> 4 caaggtgctg gctgagtaga tc 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MMP-3 forward primer <400> 5 attccatgga gccaggcttt c 21 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MMP-3 reverse primer <400> 6 catttgggtc aaactccaac tgtg 24 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-7 forward primer <400> 7 tgagctacag tgggaacagg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-7 reverse primer <400> 8 tcatcgaagt gagcatctcc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-9 forward primer <400> 9 ttgacagcga caagaagtgg 20 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> MMP-9 reverse primer <400> 10 gccattcacg tccttat 17 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin forward primer <400> 11 tgctcttgct gtttcgg 17 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin reverse primer <400> 12 tgccccattc gttcaagtag 20

Claims (14)

delete delete delete delete delete delete delete delete A screening method for a therapeutic agent for laryngeal reflux disease, comprising the steps of:
(a) confirming the concentration of soluble E-cadherin fragments in saliva and the expression levels of E-cadherin and MMP-7 in the pharyngeal mucosal layer by treating the candidate material in the pharyngeal reflux disease mouse model; and
(b) When the concentration of soluble E-cadherin fragment in saliva of the mouse model treated with the candidate substance decreases, the expression level of E-cadherin in the pharyngeal mucosal layer increases, and the expression level of MMP-7 in the pharyngeal mucosal layer decreases, the candidate substance selecting as a treatment for laryngeal reflux disease.
delete an agent for measuring the concentration of Soluble E-cadherin fragment;
an agent for measuring the mRNA or protein expression level of the E-cadherin gene; and
In a kit for diagnosing laryngeal reflux disease, comprising an agent for measuring the mRNA or protein expression level of MMP-7 (matrix metalloproteinases-7) gene,
The soluble E-cadherin frament concentration increases in saliva, the expression level of E-cadherin decreases in the pharyngeal mucosal layer, and the expression level of MMP-7 increases in the pharyngeal mucosal layer, A kit for diagnosing laryngeal reflux disease.
delete i) Measuring the soluble E-cadherin frament concentration of saliva or pharyngeal mucosa, which is a biological sample, the expression level of E-cadherin gene or its protein, and the mRNA or protein expression level of MMP-7 (matrix metalloproteinases-7) gene to do;
(b) comparing the soluble E-cadherin frament concentration, the E-cadherin gene mRNA or protein expression level, and the MMP-7 gene mRNA or protein expression level with a control sample; and
(c) the concentration of the soluble E-cadherin fragment in the saliva is higher than that of the control sample, the mRNA of the E-cadherin gene of the pharyngeal mucosa or the expression level of its protein is lower than that of the control sample, and the mRNA of the MMP-7 gene of the pharyngeal mucosa Or, when the expression level of its protein is higher than that of the control sample, determining that it has a high probability of suffering from oropharyngeal reflux disease;
A method of providing information for diagnosing laryngeal reflux disease comprising a.
14. The method of claim 13,
The method of providing information for diagnosing laryngeal reflux disease, wherein the saliva is saliva isolated from the individual after waking up in the morning.

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