KR20160101889A - Use of ODAM as a biomarker for periodontal disease - Google Patents

Abstract

Disclosed is a composition for diagnosing periodontal diseases, including an odontogenic ameloblast-associated protein (ODAM) detection reagent. Also, disclosed are a kit and a method. According to the present invention, the marker enables an early diagnosis of periodontitis in a simple way via a non-invasive method, and also allows a user to make a prediction on the degree and the way to treat the disease. Thus, the composition of the present invention can contribute to the reduction in the huge amount of medical expenses required for treating periodontal diseases and enables effective treatment for periodontal diseases.

Description

Use of ODAM as a periodontal disease biomarker {Use of ODAM as a biomarker for periodontal disease}

The present invention relates to a biomarker for periodontal disease diagnosis.

Periodontal disease is an inflammatory reaction of the gums and surrounding connective tissues caused by plaque accumulation or bacterial infection of the teeth. It is characterized by the sudden onset of progression while progressing and sudden symptoms. When left untouched, severe silence silent.

According to the National Oral Health Survey of 2012, the rate of tooth decay in Korea is decreasing year by year, but the periodontal disease is steadily increasing. According to the data of National Health Insurance Corporation in 2013, Second, gingivitis and periodontal disease account for a large proportion of medical expenses.

However, periodontal disease can also be stopped by early diagnosis and treatment, or the damaged tissue around the teeth can be recovered through tissue regeneration.

Currently, the diagnosis of periodontal disease depends on the patient's subjective symptoms, the depth of the periodontal gingiva using the periodontal probe using a periodontal probe, the degree of progression of periodontitis, or the degree of bacterial infection.

However, in order to effectively treat periodontal disease, development of periodontal disease diagnosis factors that can be used for early diagnosis and / or simple examination is required.

U.S. Publication No. 2008-0027146 discloses a biomarker for periodontal disease diagnosis including V-FOS FBJ murine osteosarcoma virus cancer homolog B, matrix metalloproteinase I or III, caspase 10, and the like.

US-A-2002-0012944 discloses the use of PAI-2 and T-PA as periodontal disease markers.

However, the development of more biomarkers is necessary for accurate diagnosis and early diagnosis of periodontal disease.

The present invention provides a biomarker capable of diagnosing the presence and / or the degree of periodontal disease in the periodontal tissue in a non-invasive manner, especially early.

In one embodiment, the present invention provides a composition for diagnosing periodontal disease in a specimen containing an ODAM marker detection reagent. The composition according to the present invention detects ODAM in specimens, particularly gingival crevicular fluid.

The compositions according to the present invention can be used for the diagnosis of periodontal diseases, for example gingivitis, periodontitis or periplasmic periarthritis.

The detection reagent contained in the composition according to the present invention is a reagent capable of detecting the marker according to the present invention at the protein level, for example, a protein detection reagent is Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, But are not limited to, reagents used in immuno-staining, immunoprecipitation assays, complement fixation assays, FACS, mass spectrometry, or protein microarrays. In one embodiment, a reagent for detecting a marker protein according to the present disclosure includes, but is not limited to, a monoclonal antibody, polyclonal antibody, substrate, aptamer, avimer, peptidomimetic, receptor, ligand or cofactor It is not.

In another aspect, the disclosure also provides a kit for the diagnosis of periodontal disease in a sample comprising an ODAM marker detection reagent.

The kit according to the present invention can be used for ELISA analysis, dip stick rapid kit analysis, microarray use, gene amplification, or immunoassay.

In another aspect, the present invention provides a method of detecting ODAM at a protein level from a sample derived from a subject, and detecting the level of the protein at the level of the protein, in order to provide information necessary for diagnosis or prognosis of periodontal disease or periplasmic environment, Associated with a diagnosis or prognosis of peripheral inflammation. In the method according to the present invention, the associating step is to compare the detection result of the determined marker with a threshold value for each marker determined in the comparative group.

The method according to the present invention uses a gingival crevicular fluid as a specimen.

In one embodiment of the method according to the present invention, the method is performed by a microarray, a gene amplification, an antigen-antibody reaction, or a mass spectrometry method, but is not limited thereto.

In an embodiment of the method according to the present application, the associating step further comprises the step of obtaining non-marker clinical information of the subject, for example a result obtained by a method such as depth measurement of a subject's gingiva, bleeding or X- Can be used.

The composition, kit and method for periodontal disease diagnosis including the reagent for ODAM detection according to the present invention can easily diagnose periodontitis by a noninvasive method and can predict the direction and degree of the treatment so that effective treatment of periodontal disease It also contributes to a reduction in the cost of treatment of periodontal disease, which is currently being spent enormously.

In particular, with reference to the Examples herein, the markers according to the present invention may be more effectively used for the diagnosis of gingivitis, periodontitis or implant periitis accompanied by damage to the junctional epithelium.

FIG. 1 shows immunostaining results showing the timing and location of ODAM and RhoA expression in teeth and gingiva (gums) according to one embodiment of the present invention.
FIG. 2 is a result of immuno-staining showing that the expression of ODAM is reduced in inflammatory gingival cells and the expression of ODAM is observed in the gingival crevicular fluid.
FIGS. 3A to 3C are graphs showing the relationship between ODAM concentration and depth of gingival crevicular fluid in a periodontal disease patient, wherein the probe pocket depth, the periodontal pouch (FIG. 3A) (correlation coefficient t = 0.167 *, Sig. ) = 0.0127), clinical attachment level (CAL) (Fig. 3b) (correlation coefficient = 0.226 **, Sig. (2-tailed) = 0.0007), pocket bleeding 3c). The ODAM concentration (ng / ml) of the gingival crevicular fluid collected from each patient was measured by ELISA. *: Reliability 0.05 (2-tailed); **: Reliability 0.01 (2-tailed).
Figures 4A-4B are measurements of the ODAM concentration in a peri-implantitis patient showing inflammation around the implant after implantation.

This study is based on the results of identifying the presence of ODAM in periodontal disease patients. In particular, the present study demonstrated that ODAM is strongly expressed in the conjoined epithelium of the tooth development process and in the oral epithelium attached to the teeth after surgical periodontal treatment, but ODAM is not expressed in the damaged tissue found in gingivitis.

Accordingly, in one aspect, the present invention relates to a composition for diagnosing periodontal disease or periimplantitis of a specimen containing an ODAM marker detection reagent.

The odontogenic ameloblast-associated protein (ODAM) has never been reported as a protein in osteoblast and dental epithelial oocyte, but also as a protein in several cancers such as glands, teeth, cervix, or periodontitis.

According to the present invention, the higher the intracellular expression of ODAM, the greater the cell adhesion and the decreased degree of metastasis. In addition, ODAM is expressed in actin rings of cells that induce cell adhesion by forming actin rings such as macrophages, and the expression of ODAM is decreased in areas where lesions such as infection are present. However, ODAM protein was present in the gingival crevicular fluid of patients with inflammatory periodontal disease, and that the greater the depth of inflammation, the more ODAM protein was present in the gingival crevicular fluid.

Currently, periodontal disease is progressive and can be treated when it is painful or visible to the eye during treatment. However, as the periodontal disease progresses, the initial treatment is important because the periodontal pockets become deeper and the gums are gradually contracted, causing damage to the teeth as well as loss of bone tissue around the teeth. Therefore, if periodontal disease can be identified early through periodontal disease markers, various effects such as the shape and height of the gums, the health condition of the teeth, and prevention of the loss of surrounding bone tissue can be expected. In this respect, ODAM protein is released to the gingival fissure if inflammation is induced in the tooth and the junction of the epithelium and tooth is lost. Therefore, identification of the ODAM protein enables accurate diagnosis of the early progression of periodontal disease. The disappearance of the junction of the epithelium and the teeth indicates that the underlying tissue supporting the epithelium is damaged, so that the initial periodontal status is progressing. If the gingivitis progresses and the tissue destruction including bones becomes worse, the epithelium will descend downward and the gingiva deepens. At this time, although the epithelium can not attach to the teeth, it is physiologically trying to stick to it, As a result, the progression of periodontal disease and the detection of ODAM protein are correlated. Therefore, ODAM is useful as a diagnostic marker for early periodontal disease.

Herein, periodontal disease or periodontal disease refers to inflammation and / or degeneration of the gums and peripheral connective tissues and bones, including, but not limited to, gingivitis, periodontitis, and periplasmic periplasm.

Implant periitis refers to an inflammatory reaction around implants, including peripheral bone loss and soft tissue infection, after implantation.

In this study, the concentration of ODAM was increased according to the depth of the periodontal pouch. Therefore, we can diagnose the progression of periodontal disease including gingivitis, periodontitis, and implant periostitis. Gingivitis can be divided into early, middle, and advanced periodontitis according to the progress of the disease. In the early stages, periodontal hemorrhage, 3 to 4 mm gingivalis, local receding, attachment loss (3-4 mm) Bone loss, and Class I root fractures. In the mid-term, there was no evidence of hemorrhage at the probe, 4 to 6 mm periodontal ligament, attachment loss (4-6 mm), bone loss (horizontal and / or vertical), and Class I or II root openings and Class I tooth swing or crown- 1: 1. ≪ / RTI > End-stage hemorrhage, gingiva greater than 6 mm, adhesion loss (> 6 mm), Class II and / or III root openings and Class II and / or III tooth wobble, bone loss (horizontal and / or vertical) Root ratio of 2: 1 or more.

Periodontitis can also be categorized into sub-diseases such as adult periodontitis (eg, plaque-related periodontitis), early-onset periodontitis (pediatric periodontitis, rapid onset periodontitis), periodontitis associated with systemic diseases, ulcerative periodontitis, do.

Gingivitis presents symptoms such as inflammation of the gum tissue, gum bleeding, and pseudopockets. Gingivitis can be subdivided into plaque-associated gingivitis, chronic gingivitis, acute gingivitis, gingivitis necrosis, systemic disease or drug-related gingivitis (eg gingival inflammation due to hormones, gingivitis due to drugs, gum erythema) , Incorporated herein by reference.

In this study, ODAM protein was expressed in normal gingiva but not in inflammatory tissue, but ODAM was detected in gingival crevicular fluid. This is not to be construed as limiting to this theory, but it is thought that the ODAM protein, which was present in damaged cells, was released to the outside with the damage of the cells as the epithelium disappeared from the teeth.

The term diagnosis herein refers to determining susceptibility to a disease of a subject for a particular disease or disorder, determining whether a particular disease or disorder is presently present, the prognosis of a subject afflicted with the particular disease or disorder determining the prognosis (e. g., determining the stage or progression of a disease or responsiveness to treatment), or determining therametrics (e. g., monitoring the status of an object to provide information about the therapeutic efficacy) .

As used herein, the term biomarker or diagnosis marker is a substance that can diagnose a diseased tissue or cell by distinguishing it from a normal sample or an improved tissue or cell after receiving proper periodontal disease treatment. Is a protein, nucleic acid, or derivative thereof that shows an increase in the specimen derived from an object.

As used herein, a biological sample or a sample refers to a substance or mixture of substances, including one or more components capable of detecting biomarkers, including, but not limited to, cells, tissues, or body fluids derived from an organism, particularly a human. It also includes cells or tissues cultured in Invitro as well as those directly derived from the organism. In one embodiment according to the present application, a gingival crevicular fluid of the periodontal ligament was used.

Detection herein includes quantitative and / or qualitative analysis, including detection of presence or absence, detection of an amount present, and such methods are well known in the art and are well known to those skilled in the art, You will be able to choose a method.

The detection reagent herein is a substance capable of specifically detecting ODAM at a protein or nucleic acid level.

The reagents for protein detection herein may be used, for example, in Western blot, enzyme linked immunoabsorbent assay, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, or protein microarrays But are not limited to, a < / RTI > Also, a system based on labeled beads, binding with antibodies labeled in a solution / suspension, detection with a flow cytometer, or a mass spectrometer can be used. Such methods are well known, for example, chip-based capillary electrophoresis: Colyer et al. 1997. J Chromatogr A. 781 (1-2): 271-6; mass spectroscopy: Petricoin et al. 2002. Lancet 359: 572-77; eTag systems: Chan-Hui et al. 2004. Clinical Immunology 111: 162-174; microparticle-enhanced nephelometric immunoassay: Montagne et al. 1992. Eur J Clin Chem Clin Biochem. 30: 217-22.

In one embodiment according to the present invention, a sandwich immunoassay such as an ELISA (Enzyme Linked Immuno Sorbent Assay) or an RIA (Radio Immuno Assay) may be used. This method can be applied to a first antibody conjugated to a bead, membrane, slide or microtiter plate made of a solid substrate such as glass, plastic (e.g. polystyrene), polysaccharide, nylon or nitrocellulose, And then labeled or labeled with a labeling substance capable of direct or indirect detection, for example, a radioactive substance such as ³ H or ¹²⁵ I, a fluorescent substance, a chemiluminescent substance, a hapten, a biotin, a digoxigenin, Protein can be detected qualitatively or quantitatively through the conjugation of an enzyme conjugated with an enzyme such as horseradish peroxidase, alkaline phosphatase, or malate dehydrogenase capable of coloring or luminescence.

Other immune response based methods may be used and in other embodiments, immunization such as Ouchterlony plates, Western blot, Crossed IE, Rocket IE, Fused Rocket IE, Affinity IE, which can simply detect the marker through antigen- Immunoelectrophoresis may be used.

Methods of immunoassay or immunostaining are described, for example, in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984. By analyzing the intensity of the final signal by the above-described immunoassay, that is, by performing signal contrast with a sample of a comparative group, it can be associated with diagnosis of a disease and the like.

Reagents used in the above methods include, but are not limited to, for example, monoclonal antibodies, polyclonal antibodies, substrates, aptamers, avimers, peptidomimetics, receptors, ligands or cofactors.

In one embodiment, the detection reagent is an antibody that specifically binds to one or more markers according to the present invention and is capable of quantitatively and / or qualitatively assessing the protein in the sample.

In another embodiment, such an antibody may be provided attached to a substrate, for example a well of a multiwell plate, a surface of a glass slide, or a nitrocellulose.

In other embodiments, the detection reagent may be provided in the form of an array or chip comprising a microarray. Detection reagents can be attached to the surface of substrates such as glass or nitrocellulose, and array fabrication techniques are described, for example, in Schena et al., 1996, Proc Natl Acad Sci USA. 93 (20): 10614-9; Schena et al., 1995, Science 270 (5235): 467-70; And U.S. Pat. Pat. Nos. 5,599,695, 5,556,752, or 5,631,734. Detecting reagents that can be attached to the array include, for example, antibodies, antibody fragments, aptamers, avidity multimers, or peptidomimetics capable of specific binding to a protein.

Detection reagents may be labeled indirectly, either directly or in sandwich form for detection. In the case of direct labeling, the sample used in the array is labeled with a fluorescent label such as Cy3 or Cy5. In the case of sandwiches, the unlabeled sample is first reacted and bound to the array with the detection reagent attached, and then the target protein is detected by binding with the labeled detection antibody. In the case of the sandwich method, sensitivity and specificity can be increased, and detection up to pg / mL level is possible. Other radioactive materials, coloring materials, magnetic particles, and high-density electron particles can be used as labeling materials. Fluorescence intensity can be measured using a scanning confocal microscope, for example Affymetrix, Inc. Or from Agilent Technologies, Inc., and the like.

In another aspect, the present invention also relates to a kit for detecting periodontal disease or periplasmic periplasm, comprising the above-described composition or a reagent for ODAM detection.

The kit of the present invention, which may include various detection reagents, may be provided for ELISA analysis, dip stick rapid kit analysis, microarray use, gene amplification, or immunoassay according to the analysis mode , And appropriate detection reagents can be selected according to the analysis mode.

In one embodiment, an ELISA or dipstick rapid kit is used, wherein an antibody recognizing the marker according to the invention can be attached to the surface of a substrate, for example a well of a multiwell plate or the surface of a glass slide or a nitrocellulose have. In the case of dip sticks, a technique widely used in the field of POCT (Point of Care Treatment), in which one or more antibodies recognizing the biomarker according to the present invention is bound to a substrate such as nitrocellulose, For example, when the tip of a dip stick is brought into contact with a sample of a gingival tear solution, the sample is moved by capillary phenomenon, and the marker is detected in such a manner that it binds to the antibody in the substrate and develops color.

The kits herein may further comprise one or more additional components necessary for the analysis and may further comprise, for example, buffers necessary for detection, reagents necessary for sample preparation, sampling tools or a negative and / or positive control have. In other embodiments, a guide may be further included on how to use the biomarkers herein.

In another aspect, the present invention provides a method of detecting ODAM at a nucleic acid or protein level from a specimen from a subject, and detecting the level of the nucleic acid or protein at a level of the periodontal disease or protein, in order to provide information necessary for the diagnosis or prognosis of periodontal disease or periplasmic environment, Disease, or periplasmic < RTI ID = 0.0 > inflammation, < / RTI >

The method further comprises comparing the nucleic acid or protein level detection results for the marker according to the present invention with the results corresponding to the markers of the normal control sample and, if there is a change in the nucleic acid or protein level of the subject sample, Related to the diagnosis or prognosis of periarthritis.

The associating in the method according to the present invention is to compare the detection result of the thus determined marker with the threshold value for each marker determined in the comparative group. The comparative group includes samples from normal subjects that did not develop periodontitis, or samples that were treated with appropriate treatment.

For example, a normal range cutoff of a biomarker in a comparative group is determined, and if the value of the marker of the suspect patient is increased by about 50% or more from the threshold value, the patient is diagnosed as a periodontal disease or periodontal disease . In particular, early differential diagnosis of patients with severe periodontal disease, in which the amount of biomarkers is increased more than twice the threshold value, is possible. However, the figures are not limited thereto.

In addition, patients with periodontal disease or periplasmic infection can be identified and followed up by checking whether the biomarker is recovered within the normal range after treatment. Diagnosis of a disease using the biomarker according to the present invention can be used alone or in combination with known methods.

The biological sample, the biomarker detection method and reagents used therein are as described above. The methods herein are directed to mammals, especially humans. Human subjects include those who are suspected of having periodontal disease or peri-implantitis, and those who are not suspected of needing periodontal disease or implant diagnosis.

The method according to the present invention can be used in conjunction with non-marker clinical information used in the diagnosis of periodontal disease or periplasmic periitis, including, but not limited to, periodontal depth, bleeding and / or X-ray examination.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Materials and Methods

Reagents and Antibodies: In the present invention, fibronectin fibronectin, laminin, was purchased from sigma and collagen was purchased from Roche. ODAM antibodies (amino acid residues 102-114 and 241-251) were synthesized in rabbits by synthesizing peptides. RhoA, ROCK, p-myosin, p-paxillin, paxillin and E-cadherin antibodies were detected in cell signaling and active RhoA (GTP-RhoA) antibodies were detected in Biosource, ARHGEF5, integrin av, integrin b6, cytokeratin, HA, E-cadherin Antibodies were purchased from Santa Cruz Biotechnology, Flag antibodies from Sigma-Aldrich, and F-actin antibodies from Invitrogen.

Tissue preparation and immunochemical tissue analysis: All experiments using animals were in accordance with a protocol (SNU-111013-2) approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. The rat teeth on day 16 (P16), 20 (P20) and 26 (P26) and 10 days after birth (P10) were demineralized in 10% EDTA (pH 7.4) Immunohistochemical analysis was performed. The teeth of patients with inflammatory periodontal disease were obtained from the Chosun University Dental College as teeth of the patients who were extracted for the implant. The primary antigen was an ABAM kit (Vector Labs, Burlingame, Calif.) Using rabbit anti-ODM, RhoA, active RhoA, F-actin, biotinylated goat anti- Rabbit IgG , RhoA, active RhoA, and F-actin, respectively.

Cell culture differentiation: Wet air containing 5% CO ₂ Two types of cells cultured at 37 ° C were used in the experiment. Mouse ameloblastoma was treated with Ameloblast-Lineage Cell (ALC, provided by Dr. T. Sugiyama, Akita Medical School, Japan) and ALC was performed on MEM medium supplemented with 5% heat-inactivated bovine serum (FBS) (EGF, 50 ng / ml, R & D systems, Minneapolis, Minn., USA) was added to the plate and incubated in a collagen-coated dish. HAT7 (apical bud cells, apical bud cells) The cells were cultured in DMEM / F12 medium supplemented with 10% heat inactivated bovine serum (FBS) and received from Harada H (Iwate Medical College School of Dentistry, Morioka, Japan). ALC and HAT7 cells were cultured on fibronectin, laminin, collage coated culture plates according to the experimental purpose. RAW 264.7 (osteoclasts) were cultured in DMEM supplemented with 10% heat-inactivated bovine serum (FBS). Bone marrow-derived macrophages were cultured in a-MEM medium supplemented with 10% heat-inactivated bovine serum (FBS).

When the cells reached 80-90% confluence, they were differentiated for 2 weeks using differentiation medium containing 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate.

Western analysis: To prepare total cell extracts, the cells were washed three times with PBS, scrapped in a 1.5 ml tube, and centrifuged at 12,000 rpm for 2 minutes at 4 ° C to pellet. After removing the supernatant, the pellet was resuspended in lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EDTA, pH 7.4) and incubated on ice for 15 minutes. Cell debris was removed by centrifugation. 30 micrograms of protein was separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. Membranes were blocked with 5% skimmed milk in PBS containing 0.1% Tween 20 (PBS-T) for 1 hour and incubated overnight at 4 ° C with primary antibody diluted in PBS-T (1: 1000). (Sc-2031), anti-rabbit (sc-2004), or anti-goat (sc-2768) IgG secondary antigen (1: 5000) conjugated with horseradish peroxidase All purchased from Santa Cruze Biotechnology) were incubated for 1 hour. Labeled protein bands were detected using an enhanced chemiluminescence system (GE Healthcare, UK), and the bend was measured by concentration analysis of the diluted self-emissive film.

Gene expression profiling: Gene profiling was performed using an mRNA microarray database with gingival epithelial cells in genital gingival fibrosis, Integrin b6 gene deficient mice, and oral pathogen infection. Microarray data was downloaded and analyzed from NCBI's GEO database (accession numbers GSE4250, GSE2255, and GSE9723).

Patient Sampling: All human-based experiments were conducted using the protocol approved by the IRB (Institutional Review Board for Human Subjects) of Korea University (IRB No. ED13162) and Seoul National University Bundang Hospital (IRB No. B-1410-271-003) I followed. Periodontal examination of 4 healthy subjects and 10 periodontal disease patients evaluated the degree of ODAM expression according to presence or absence of inflammation. Periodontal examination of 4 patients with periodontal disease was performed with 10 teeth or more per patient, 4 sites per tooth A total of 222 specimens were collected and assessed for plaque grade, plaque grade, probe pocket depth, loss of tooth - periodontal cell adhesion in pocket, and intra - pocket hemorrhage. After removing the dentate gingival margin of the periodontal patient with a hand curette and drying it with compressed air for about 10 seconds, the absorbent paper strips (Oraflow, USA) were used to remove the gingival crevicular fluid A sample was taken. Absorbent strips were placed in tubes containing 100 μl PBS and stored at -70 ° C for ELISA analysis.

ELISA: The ODAM concentration in the gingival crevicular fluid sample was measured using an ODAM ELISA kit (CUSABIO company) according to the user's method.

In this experiment, statistical treatment was carried out using Student t-test. All statistical analyzes were performed using SPSS software ver. 19.0. ≪ / RTI >

Example  One Ameloblast  And Junction epithelium (Junctional Epithelium, JE Analysis of ODAM, ARHGEF5 and RhoA expression during development and identification of markers

Immunochemical staining was used to analyze the degree of ODAM expression in the gingival ameloblast - tooth boundary during tooth development. The results are shown in Fig. As a result, ODAM expression was confined to the basal cells of the first molar OE (oral epithelium) of rats 16 days after birth. ODAM was observed in the nuclear portion of the reducing oral epithelial cell, and the label was diffused and distributed at the boundary. In the first molar of rat 20 days after birth, immunostaining for ODAM was strongly observed in cells located between the enamel organ and the junctional epithelium. ODAM was also expressed at the boundary of the teeth. In the case of rats 26 days after birth, in which three molars were all born and gum (JE) was well developed, it was expressed in junctional epithelial (JE) cells.

In addition, ODAM, ARHGEF5, and RhoA expression were analyzed by immunostaining in gum (JE) and developing molar embryo (germ). In the 20 day old rat, ODAM was strongly expressed in the junctional epithelium (JE), and ARHGEF5 and RhoA were also observed at the same site (Fig. 1B). In the case of 10-day old rats, ODAM was expressed in enamel cells, enamel matrix and enamel cell-tooth boundaries. Of particular interest, the expression of ODAM was consistent with the expression of RhoA (GTP-RhoA) activated at the enamel and ameloblast-tooth boundary, whereas ARHGEF5 was expressed only at the ameloblast-tooth boundary (Figure 1C) . Furthermore, F-actin was shown to be strongly expressed in the same region of p10 mice as compared to ODAM and RhoA (Fig. 1D).

Furthermore, Western blot analysis of HAT7 and ALC cells showed selective and time-dependent expression of ODAM, ARHGEF5, and RhoA during ameloblast differentiation. The expression of ODAM gradually increased during the incubation period (Fig. 1E) and similarly, ARHGEF5 and RhoA were also expressed continuously (Fig. 1E). These results indicate that ODAM, ARHGEF5, and RhoA are functionally related to the differentiation and maturation of ameloblasts and the formation of the junctional epithelium (JE). In particular, ODAM was strongly expressed in the conjoined epithelium of tooth development process and oral epithelium attached to tooth after surgical periodontal treatment. Based on this, ODAM was selected as a marker candidate for periodontitis.

Example  2 Damaged In the junctional epithelium (JE)  Expressed ODDAM  Intracellular location and expression analysis

The presence and distribution of ODAM and RhoA were investigated in the junctional epithelium (JE) injured by inflammation. Immunohistochemical staining was performed using mouse and human damaged tissues as described in the experimental materials and methods. As a result, ODAM was expressed in the normal junctional epithelium (JE) but not in the junctional epithelium (JE) of the mice induced by DDS or PG. The results are shown in FIG. FIG. 2 shows that the expression of ODAM is decreased in inflammatory junctional epithelial (JE) cells and the expression of ODAM is observed in the gingival crevicular fluid. In the mouse experiment, the conjunctival epithelium (JE) of the tongue area was severely damaged due to the oral administration of the inflammation-inducing drug, but the junctional epithelium (JE) (Fig. 2A). This indicates that ODAM is expressed in cells in the normal junctional epithelium (JE). When periodontal disease occurs, the junctional epithelium (JE) changes into invasive pockets of epithelial ridges and ulcer lesions surrounded by many inflammatory infiltrates. In order to observe the expression of ODAM in these invasive pockets, the teeth extracted for implants from patients with deep periodontal pockets around the teeth were analyzed. As shown in FIG. 2B, the joint epithelium (JE) ODAM and RhoA were not detected.

In order to further confirm ODAM expression in the inflamed junctional epithelium (JE), expression in the junctional epithelium (JE) cells infected with Porphyromonas gingivalis (PG) using the NCBI GEO microarray data set As a result, the expression of ODAM was inhibited as shown in Fig. 2C. Hereditary gingival fibromatosis (HGF) is a hereditary disease with junctional epithelium (JE), and when periodontal disease occurs, the tooth support site is seriously damaged. Analysis of ODAM expression of GEO data analyzed using the junctional epithelium (JE) tissue derived from HGF patients showed that ODAM expression was decreased in HGF patients compared to normal patients (FIG. 2D). However, the expression of ODAM in the gingival crevicular fluid of patients with inflammation in the junctional epithelium (JE) was analyzed and the expression was increased (Fig. 2E). These results suggest that ODAM is expressed in healthy joint epithelium (JE), and the expression of ODAM is decreased in the junctional epithelium (JE) when the periodontal disease is caused, while the expression of ODAM is increased in the gingival crevicular fluid, Can be used.

Example 3 Concentration of ODAM according to depth of gingival crevicular fluid in periodontal patients

In periodontal disease, bone is formed between the teeth and the junctional epithelium (JE), and the bone becomes deeper as the periodontal disease progresses. Thus, as the depth of gingival crevicular fluid becomes deeper, the periodontal disease progresses more. After the periodontal disease patients were evaluated, the ODAM concentration was measured according to the depth of the gingival crevicular fluid in the periodontal pouch, as described in the experimental method.

The results are shown in Figures 3a and 3c. FIG. 3 is a graph showing the relationship between ODAM concentration and the depth of gingival crevicular fluid in patients with periodontal disease. FIG. 3 is a graph showing the relationship between ODAM concentration and the depth of probe pocket depth, periodontal ligament (FIG. 3A), and clinical Attachment Level (CAL) The ODAM concentration (ng / ml) of the gingival perfusate collected from each patient was evaluated by ELISA method according to the depth of the pocket, It is written. The probe pocket depth was measured as the depth of the pocket compared to normal. The correlation coefficient between ODAM and probe depth, ie, periodontal pouch, was 0.167, Sig. (2-tailed) = 0.0127 (reliability 0.05), correlation coefficient with CAL and ODAM 0.226, Sig. (2-tailed) = 0.0007 , Which is close to 100% reliability. Therefore, according to the present invention, ODAM can be used not only for the initial periodontal disease, but also for the diagnosis of periodontal disease according to the progression stage or progress.

Example 4 Concentration of ODAM in Patients with Peri-implantitis

As a result of measuring the ODAM concentration as described in the experimental method after collecting the gingival peritoneal fluid at the site where the inflammation occurred and the site where the inflammation did not occur after the implant treatment, as shown in FIG. 4A, it was compared with the site of the implant showing no inflammation And the concentration of ODAM increased at the site of inflammation.

The results of measurement of the ODAM concentration of the normal periodontal control group and the periplasmic area without the implant treatment are shown in FIG. 4B. Compared with normal periodontal tissue, ODAM concentration was significantly increased in patients with peri-implantitis.

The results of the above example show that when only periodontal tissue such as periodontal ligament, cementum, and alveolar bone is destroyed without damaging the junctional epithelium in the entire periodontal disease including gingivitis and periodontal disease, Rather than periodontal disease, is more effective in the diagnosis of periodontal disease in which the junctional epithelium is damaged or destroyed.

While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (9)

Gingivitis, periodontitis or periimplantitis To provide information necessary for diagnosis or prognosis,
Detecting ODAM at the protein level from the oral specimen derived from the subject; And
Detecting gingivitis, periodontitis, or implant periosteum, or detecting a marker for prognosis, comprising the step of associating said subject-derived subject with gingivitis, periodontitis or implant periplasm when said ODAM protein level in said sample is increased compared to a normal control sample Way.
In order to provide information necessary for the diagnosis or prognosis of gingival diseases, periodontitis or periplasmic implants with damaged junctional epithelium,
Detecting ODAM at the protein level from the oral specimen derived from the subject; And
Wherein said ODAM protein level in said sample is increased relative to a normal control sample, said method comprising the step of associating said subject-derived subject with a gingival epithelium with impaired gingivitis, periodontitis or periplasmic implant,
Methods for detecting gingivitis, periodontitis, or periplasmic implants or markers for prognosis.
The method according to claim 1,
Wherein the oral specimen is a gut saliva, periodontitis, or periplasmic circumference irritation, or a method for detecting a marker for prognosis.
3. The method of claim 2,
Wherein the oral specimen is a gut saliva, periodontitis, or periplasmic circumference irritation, or a method for detecting a marker for prognosis.
The method according to claim 3 or 4,
Wherein the oral fluid is saliva or gingival crevicular fluid, gingivitis, periodontitis, or a method for detecting a marker for peripheral inflammation or prognosis.
In order to provide information necessary for the diagnosis or prognosis of gingival diseases, periodontitis or periplasmic implants with damaged junctional epithelium,
Detecting ODAM at the protein level from the gingival crevicular fluid derived from the subject; And
Comprising contacting said gingival tegmental-derived subject with a gingival epithelium damaged gingivitis, periodontitis, or implant periplasm when said ODAM protein level in said gingival crevice fluid is increased compared to a normal control sample.
Methods for detecting gingivitis, periodontitis, or periplasmic implants or markers for prognosis.
The method according to any one of claims 1 to 4 or 6,
Wherein the method is performed by a microarray, an antigen-antibody reaction, or a mass spectrometry method.
The method according to any one of claims 1 to 4 or 6,
Wherein the associating further uses non-marker clinical information of the subject. ≪ Desc / Clms Page number 21 >
9. The method of claim 8,
Wherein the non-marker clinical information is at least one of depth measurement of the subject's gingiva, bleeding or X-ray examination, gingivitis, periodontitis, or a marker for detecting a prophylactic or peripheral implant.

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