KR20110060205A - Marker for diagnosis of inflammatory disease comprising cd93 or soluble fragment thereof - Google Patents

Abstract

PURPOSE: A marker containing CD93 or soluble fragment thereof for diagnosing inflammatory diseases is provided. CONSTITUTION: A marker for diagnosing inflammatory diseases contains CD93 or soluble fragment thereof as an active ingredient. The soluble fragment is ectodomain of CD93 which is released after cell culture. A kit for diagnosing inflammatory diseases contains an antibody which specifically binds to CD93 or soluble fragment thereof or aptamer. A method for detecting CD93 or the soluble fragment comprises: a step of coating a biological sample and proteins of a control group to a fixture; a step of adding the antibody for antigen-antibody binding reaction; a step of detecting the antigen-antibody binding reactant using a marker of secondary antibody conjugate and chromogenic substrate; and a step of comparing the biological sample and a control group.

Description

Marker for diagnosis of inflammatory disease comprising CD93 or soluble fragment according to CD93 or a soluble fragment thereof as an active ingredient

The present invention provides a diagnostic marker for inflammatory diseases containing CD93 or a soluble fragment thereof as an active ingredient, an inflammatory disease diagnostic kit including an antibody or aptamer specifically binding to CD93 or a soluble fragment thereof, and a marker for diagnosing inflammatory disease in a biological sample. The present invention relates to a method for detecting CD93 or a soluble fragment thereof through an antigen-antibody binding reaction using an antibody that specifically binds to an inCD93 or a soluble fragment thereof.

Human CD93 (C1qRp, AA4.1 (mouse)) is a type 1 transmembrane glycoprotein located on chromosome 20, p11.21 (Malhotra, R. et al., 1993. Immunology 78: 341-348; Nepomuceno, RR et al., 1997. Immunity 6: 119-129; Steinberger, P. et al., 2002. Biol . 71: 133-140). The protein has been reported to be involved in cell-cell interactions and phagocytosis in B cell development (Petrenko, O. et al., 1999. Immunity 10: 691-700; Norsworthy, PJ et al. , 2004. J. Immunol. 172: 3406-3414; Nepomuceno, RR et al., 1997. Immunity 6: 119-129; McGreal, EP et al., 2002. J. Immunol. 168: 5222-5232).

CD93 consists of a total of 652 amino acids and contains a leader sequence, a c-type carbohydrate-cognitive domain, five EGF-like domains, a mucin domain, a membrane transmembrane domain, and an intracellular domain of 47 amino acids. Nepomuceno RR et al., 1997. Immunity 6: 119-129; Park, M. et al., 2003. J. Cell Physiol . 196: 512-522; Nepomuceno, RR et al., J. Immunol. 162: 3583 ). CD93 is also expressed in myeloid lineage, hematopoietic stem cells, NK cells, platelets, microglia and endothelial cells (Nepomuceno, RR et al., 1997). Immunity 6: 119; Nepomuceno, RR et al., 1998. J. Immunol. 160: 1929; Danet GH et al., Proc Natl Acad Sci USA 2002; 99: 10441-5; Lovik G. et al., J. Immunol 2000; 30: 3355-62; Fonseca MI et al., J Leukoc Biol 2001; 70: 793-800; Webster, SD et al., 2000. J. Leukocyte Biol. 67: 109). Another study reported that CD93 is not expressed in tissue macrophage but is expressed in vascular endothelial cells (Petrenko, O. et al., 1999. Immunity 10: 691; Dean, YD et al., J. Immunol. 31: 1370; Fonseca, MI et al., 2001. J. Leukocyte Biol. 70: 793.). The potential of CD93 to be involved in C1q-mediated enhancement of phagocytosis in phagocytosis in the results with anti-CD93 antibodies has been raised as a potential receptor for C1q. However, in subsequent experiments it was reported in several groups that it did not directly bind C1q (Nepomuceno, RR et al. 1999. J. Immunol. 162: 3583; McGreal, EP et al., 2002. J. Immunol. 168: 5222 ..; Steinberger, P. et al , 2002. J. Leukocyte Biol 71: 133).

CD93 knock-out mouse experiments have been shown to cause problems with the elimination of dead cells due to the absence of CD93, and in vitro experiments have shown conflicting results in which such dysfunction is not seen (Guan, E. et al., 1994. J. Immunol. 152: 4005-4016; Norsworthy, PJ et al., 2004. J. Immunol. 172: 3406-3414; Norsworthy, PJ et al., 2004. J. Immunol. 172: 3406-3414) . This phagocytosis function plays an important role early in the immune response and plays an important role in the removal of apoptotic cells (Brown, EJ (1995) Phagocytosis. Bioessays 17, 109-117).

Recent studies on these functions have reported that CD93 has shedding of the extracellular domain (ectodomain) in the early pro-inflammatory states of TNF-α and LPS. (Park, M. et al., 2003. J. Cell Physiol. 196: 512-522 . ; Suzanne S. Bohlson et al., 2005. J. immunol . 175: 1239-1247). In addition, it has been reported that fragmentation occurs by phorbol-12-myristate-13-acetate (PMA) treatment in U937 cells, which is a lineage cell such as CD93, and is cut into soluble fragments (Ikewaki N., Tamauchi H., and Inoko H.). Decrease in CD93 expression in a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances.Microbiol.Imunmun., 51 (12): 1189-1200). In addition, WO 2008/082519 discloses that CD93-related polymorphs are useful for the diagnosis of type 1 diabetes and lupus erythematosus. However, no studies have used CD93 as a marker for diagnosing inflammatory diseases.

Therefore, there is a need for developing CD93 as a marker for diagnosing inflammatory diseases.

The inventors of the present invention are studying markers for diagnosing inflammatory diseases that can predict the diagnosis or prognosis of inflammatory diseases early, and confirm that the expression of CD93 is increased and the amount of CD93 soluble fragment is increased by the inflammatory inducer or in the inflammatory environment. The present invention has been completed.

The present invention is to provide a marker for diagnosing inflammatory diseases containing CD93 or a soluble fragment thereof as an active ingredient.

The present invention also provides an inflammatory disease diagnostic kit comprising an antibody or aptamer specifically binding to CD93 or a soluble fragment thereof.

The present invention also provides a method for detecting CD93 or a soluble fragment thereof through an antigen-antibody binding reaction using an antibody that specifically binds CD93 or a soluble fragment thereof as a diagnostic marker for inflammatory disease in a biological sample.

The present invention provides a marker for diagnosing inflammatory diseases containing CD93 or a soluble fragment thereof as an active ingredient.

The present invention also provides an inflammatory disease diagnostic kit comprising an antibody or aptamer specifically binding to CD93 or a soluble fragment thereof.

In addition,

1) coating the protein of the biological sample and the control to the fixture,

2) performing an antigen-antibody binding reaction by adding an antibody specifically binding to CD93 or a soluble fragment thereof, which is a marker for diagnosing an inflammatory disease, to the fixture;

3) detecting the antigen-antibody binding reactant generated through the antigen-antibody binding reaction using a label and a color substrate solution of the secondary antibody conjugate; And

4) A method for detecting a CD93 or a soluble fragment thereof, comprising comparing a detection result for a biological sample with a control.

Hereinafter, the present invention will be described in detail.

Expression of CD93 according to the present invention is increased by LPS and TNF-α in THP-1 cells, which are human monocytes, and is not significantly increased by PMA treatment. However, the amount of CD93 soluble fragment is all increased by LPS, TNF-α, PMA treatment. In addition, after injection of PBS, thioglycolate, and LPS into mice, mouse CD93 expression in macrophages in peritoneal cells is most increased by LPS treatment, and similarly by thioglycolate treatment. In addition, the amount of CD93 soluble fragment in serum is not significantly changed by LPS or thioglycolate, but the amount of CD93 soluble fragment in peritoneal fluid is significantly increased by LPS or thioglycolate treatment. In addition, CD93 expression was significantly higher in synovial tissue from rheumatoid arthritis patients than in synovial tissue from patients with degenerative arthritis, and the amount of CD93 soluble fragment in synovial fluid from rheumatoid arthritis patients was significantly higher than that of degenerative arthritis patients. appear. In the present invention, the CD93 soluble fragment is an extracellular portion (ectodomain) released after cell culture of the CD93 protein, which is a transmembrane protein, and means that the fragment contains about 95 kDa.

As mentioned above, the CD93 or soluble fragments thereof according to the present invention increases CD93 expression and increases the amount of CD93 soluble fragments caused by or in an inflammatory environment. Therefore, the CD93 or soluble fragment thereof according to the present invention can be usefully used as a diagnostic marker of inflammatory disease.

The inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritability Bowel Syndrome, Inflammatory Pain, Migraine, Headache, Back Pain, Fibromyalgia, Fascia Disease, Viral Infection, Bacterial Infection, Fungal Infection, Burn, Surgical or Dental Surgery, Prostaglandin E Over Syndrome, Atherosclerosis Sclerosis, gout, degenerative arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iris, peritonitis, uveitis, dermatitis, eczema, multiple sclerosis, and the like.

In addition, an inflammatory disease diagnostic kit comprising an antibody or aptamer specifically binding to CD93 or a soluble fragment thereof of the present invention can be easily prepared by a manufacturing method commonly used in the art using the marker. have. The aptamer is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure in itself and is characterized by high affinity and specificity for binding to a target molecule.

The inflammatory disease diagnosis kit includes an antibody specifically binding to CD93 or a soluble fragment thereof, a secondary antibody conjugate conjugated with a label developed by reaction with a substrate, a solution of a colored substrate to react with the developed label, It may include a washing solution and a solution for stopping the enzyme reaction.

The label of the secondary antibody conjugate is preferably a conventional coloring agent that performs a color reaction, horseradish peroxidase (HRP), basic dephosphatase (alkaline phosphatase), colloidal gold (coloid gold), poly L-lysine-fluorescein (FITC) isothiocyanate, fluorescent materials such as rhodamine-B-isothiocyanate (RITC), dyes, and the like.

The chromogenic substrate solution is preferably used according to the label, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid )], OPD (o-phenylenediamine) and the like can be used. At this time, the color development substrate is more preferably provided in a dissolved state in a buffer solution (0.1M NaOAc, pH 5.5).

The wash preferably comprises phosphate buffer, NaCl and Tween 20, more preferably a buffer consisting of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20 (PBST). After washing the antigen-antibody-binding reaction, the washing solution is reacted with the secondary antibody to the antigen-antibody conjugate, and then washed three to six times by adding an appropriate amount to the fixture. As the reaction terminating solution, sulfuric acid solution may be used.

In addition, the present invention can detect CD93 or a soluble fragment thereof through an antigen-antibody binding reaction using an antibody that specifically binds CD93 or a soluble fragment thereof in a biological sample, thereby predicting the diagnosis or prognosis of an inflammatory disease early. . The biological sample includes, but is not limited to, tissue, cells, blood, serum, peritoneal fluid, synovial fluid, saliva, urine or feces. Specifically, CD93 or a soluble fragment thereof in a biological sample is subjected to electrophoresis on SDS-PAGE, fractionated and transferred to a fixture, followed by immobilization of an antibody that specifically binds to the immobilized CD93 or a soluble fragment thereof. Is performed and the expression level of CD93 or a soluble fragment thereof is measured. In other words, if the expression level of CD93 or a soluble fragment thereof is high in a biological sample, it is predicted to have an inflammatory disease or to predict the possibility of an inflammatory disease.

As the fixture for the antigen-antibody coupling reaction, a nitrocellulose membrane, a polyvinylidene difluoride membrane (PVDF) membrane, a 96 well plate synthesized with polyvinyl resin or polystyrene resin, glass slide glass, or the like may be used.

The antigen-antibody binding reaction is conventional enzyme immunoassay (ELISA), radioimmunoassay (RIIA), sandwich ELISA method, Northern blot, Western blot, immunoprecipitation method, immunohistochemical staining method, fluorescence immunoassay, enzyme substrate coloration Method, antigen-antibody aggregation, surface plasmon resonance (SPR), biochips (DNA, RNA), electrophoresis, PCR, RT-PCR and the like.

Hereinafter, preferred embodiments of the present invention will be described in order 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 by the examples.

Example 1  : Determination of CD93 Expression and CD93 Soluble Fragment in THP-1 Cells, Human Monocytes

In order to examine the expression of CD93 and its fragmentation in various inflammatory inducers or in an inflammatory environment, CD93 expression in human monocytes, THP-1 cells, and the amount of CD93 soluble fragments in culture supernatants were measured.

1-1. Expression of CD93 in THP-1 Cells, Human Monocytes: FACS Experiment

THP-1 cells contain 10% fetal calf serum, penicillin G (100 IU / ml), streptomycin (100 μg / ml), L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate (1.0 mM). Cultured in a 37 ℃ cell culture in RPMI 1640 medium containing. Cultured THP-1 cells were treated with PBS, LPS (1.0 μg / ml), TNF-α (0.5 μg / ml) or PMA (0.1 μg / ml) for 24 hours and fixed with 4% paraformaldehyde for 20 minutes. It was. Washed once with PBS, blocked for 30 minutes using 3% NGS (normal goat serum) and then washed again three times. The goat anti-human CD93 antibody (R & D systems) was cooled and reacted for 1 hour. After washing three times with PBS again, and cold-cooled for 1 hour with anti-goat IgG-FITC antibody and stained. As a control group, each experimental group was separately stained with anti-CD14-FITC antibody. Each stained cell was analyzed using a flow cytometer (EPICS Elite (Coulter)).

The results are shown in FIG.

As shown in FIG. 1, the cell surface expression of CD93 in THP-1 cells was increased by LPS and TNF-α, but not significantly by PMA treatment. In addition, the expression of the cell surface of CD14 used as a control group was all reduced by LPS, TNF-α, and PMA treatment, which is thought to be due to fragmentation of CD14. Fragmentation of CD14 is known to be an important mechanism in down-modulation of monocyte-macrophage activation (Schutt, C. 1999. Cd14. Int. J. Biochem. Cell Biol. 31: 545. 23. Le ... -Barillec, K., M. Si-Tahar, V. Balloy, and M. Chignard 1999. Proteolysis of monocyte CD14 by human leukocyte elastase inhibits lipopolysaccharide-mediated cell activation J. Clin Invest 103:.. 1039 Bazil, V., and JL Strominger. 1991. Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes.J. Immunol. 147: 1567 ). Several studies to date have reported that collagenase, neutrophil elastase, and cathepsin G fragment CD14 molecules on the cell surface (Bryniarski, K., K. Maresz, M. Szczepanik, M. Ptak, and W. Ptak. 2003. Modulation of macrophage activity by proteolytic enzymes.Differential regulation of IL-6 and reactive oxygen intermediates (ROIs) synthesis as a possible homeostatic mechanism in the control of inflammation.Inflammation 27 :..... 333 Le -Barillec, K., M. Si-Tahar, V. Balloy, and M. Chignard 1999. Proteolysis of monocyte CD14 by human leukocyte elastase inhibits lipopolysaccharide-mediated cell activation J. Clin Invest 103: 1039. Coyne, CP, T. Howell III, H. Smodlaka, C. Willetto, BW Fenwick, and E. Chenney. 2002. Alterations in membrane-associated CD14 expression and the simultaneous liberation of soluble CD14 fragment in adherent macrophages mediated by a leukocyte carboxyl / aspartate protease.J. Endotoxin .Res. 8: 273. )

1-2. Western blot experiment

THP-1 cells contain 10% fetal calf serum, penicillin G (100 IU / ml), streptomycin (100 μg / ml), L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate (1.0 mM). Cultured in a 37 ℃ cell culture in RPMI 1640 medium containing. Cultured THP-1 cells were treated with PBS, LPS (1.0 μg / ml), TNF-α (0.5 μg / ml) or PMA (0.1 μg / ml) for 24 hours, followed by lysate and culture supernatant. Got. The sample was boiled for 5 minutes and then stored frozen. Samples were loaded onto 8% SDS-PAGE gels and then electrophoresed. The gel was transferred back to the nitrocellulose membrane and blocked with 4% skim milk. Thereafter, goat anti-CD93 antibody (R & D systems) was diluted to 0.5 µg / ml and reacted at room temperature for 1 hour. After washing three times with PBST, the anti-goat IgG-HRP antibody (Santa cruz) was 0.5 μg / ml and reacted at room temperature for 1 hour. Anti-β actin (Santa cruz) and anti-goat IgG-HRP (Santa cruz) were used as internal controls. The ECL kit (ECL Plus, Amersham, USA) was then processed according to the manufacturer's method.

The results are shown in Fig.

As shown in FIG. 2, self-expression of CD93 molecules was increased by LPS and TNF-α treatment, but not by PMA treatment, as in FACS results. However, the amount of CD93 soluble fragment was all increased by LPS, TNF-α, PMA treatment. In view of the above, in terms of increased expression in THP-1 cells and Jukat cells by an inflammatory environment or an inflammatory inducer, the expression of CD93 itself increases, especially considering that the amount of CD93 soluble fragment also increases. It is contemplated that CD93 or soluble fragments thereof may be utilized as diagnostic markers of inflammatory diseases.

1-3. RT-PCR Experiment

THP-1 cells were cultured in 6 well plates, and then treated with PBS, LPS (1.0 μg / ml, InvivoGen) and PMA (0.1 μg / ml, Sigma), respectively. RNA was identified 24 hours after treatment using the RNA Identification Kit (Qiagen). For reverse transcription, reverse transcriptase (Bio-Rad) and dNTP were mixed and reacted at 42 ° C. for 1 hour to obtain respective cDNAs. The CFX96 system (Bio-Rad) was used for real-time PCR analysis. Initial denaturation at 95 ° C. for 3 minutes, followed by annealing at 95 ° C. for 15 seconds, and elongation at 60 ° C. for 1 minute at 40-45 cycles was performed. Fluorescence measurements were recorded at each annealing step, and data was automatically stored at each step. Specifically, 2 μl of cDNA template and CD93 primer were mixed with 23 μl of iQ SYBR Supermix (Bio-Rad) to react. All reactions were repeated twice. In order to measure relative mRNA expression, the average Ct (average threshold cycle) value was used as a reference value. All Ct values were standardized using GAPDH. Primer sets were as follows [CD93: Forward CTC TGG GGC TAC TGG TCT ATC, Reverse TGT CGG ACT GTA CTG GTT CTC; GAPDH: Forward CAT GTT CGT CAT GGG TGT GAA, Reverse GGA CTG TGG TCA TGA GTC CTT].

The results are shown in FIG.

As shown in FIG. 3, CD93 expression in THP-1 cells was significantly increased by LPS and PMA treatment.

Example 2  : Measurement of CD93 Expression in Mouse Peripheral Blood Mononuclear Cells and Peritoneal Cells, and Measurement of CD93 Soluble Fragment in Serum and Peritoneal Fluid (in vivo)

CD93 expression in vitro experiments to determine whether the same expression pattern and fragmentation occurs in vivo experiments, the following experiment was performed. In other words, CD93 expression in mouse peripheral blood mononuclear cells (PBMC) and peritoneal cells after injection of peritonitis or an inflammation-inducing substance (thioglycollate or LPS) in mice Aspects and the amount of CD93 soluble fragment in serum and peritoneal lavage fluid (PLF) were measured.

2-1. Expression of CD93 in Mouse Peripheral Blood Mononuclear Cells and Peritoneal Cells: FACS Experiment

Six-week-old balb / C mice were injected with 1 ml of PBS, 4% thioglycolate or 0.1 mg / ml of LPS and maintained for 3 days. Three days later, blood was collected from mice using heparin-coated glass capillary tubes via retro-orbital vein and centrifuged to obtain serum and PBMC cells. Immediately, mice were sacrificed to obtain peritoneal fluid (PLF) and peritoneal cells (mostly intraperitoneal macrophages) from the intraperitoneal cavity. In order to remove red blood cells contained in each cell, 10 ml of red blood cell lysing buffer (Sigma) was reacted twice at 37 ° C. for 10 minutes to completely remove red blood cells. Thereafter, fluorescent dyeing was carried out in the same manner as described above in Example 1-1. However, in the case of antibodies, both anti-mouse CD93 antibodies (R & D systems) and anti-sheep IgG-FITC (Santa Cruz) were used. In addition, to determine the degree of macrophage differentiation, double staining was performed using a CD11b antibody (BD sciences), a differentiation marker.

The results are shown in FIG.

As shown in FIG. 4, mouse CD93 expression in macrophages among peritoneal cells was most increased by LPS treatment, and similarly increased by thioglycolate treatment. Therefore, it can be seen that macrophages present in PBMCs were transferred to the peritoneum by treatment with thioglycolate and LPS in mice.

2-2. Western blot experiment

After lysing the remaining half of the cells used in 2-1, Western blot was performed. Western blot experiments were performed in the same manner as in Example 1-2 above, except that both anti-mouse CD93 antibodies (R & D systems) and anti-sheep IgG-HRP (Santa Cruz) were used to detect mouse CD93. Was performed. In addition, to determine the degree of macrophage differentiation, double staining was performed using a CD11b antibody (BD sciences), a differentiation marker. Anti-β actin (Santa cruz) and anti-goat IgG-HRP (Santa cruz) were used as internal controls. The ECL kit (ECL Plus, Amersham, USA) was then processed according to the manufacturer's method.

The results are shown in FIG.

As shown in FIG. 5, the expression of CD93 molecules in peritoneal cells was significantly increased by treatment with thioglycolate and LPS in mice, as in the FACS results.

2-3. Determination of the amount of CD93 soluble fragments: sandwich ELISA experiment

After observing the increase in the amount of CD93 molecules themselves by FACS and Western blot experiments, to determine how the CD93 soluble fragments changed, the sandwich ELISA method was used to determine the CD93 soluble fragments in serum and peritoneal fluid (PLF). The amount was measured. First, 100 ng / well of rat anti-mouse CD93 antibody (R & D systems) were coated overnight in a 93-well Beccon Dickinson plate. After washing three times with PBST, it was blocked for 30 minutes at room temperature with 3% skim milk. After washing three times with PBST again, 100 μl of each mouse serum and peritoneal fluid collected were loaded and left for 2 hours. Mouse CD93 soluble fragment was used as a standard material for quantification. After three more washes, the cells were left for 1 hour with 1 µg / ml of anti-mouse CD93 antibody (R & D systems), followed by 1 µg / ml of anti-sheep IgG-HRP antibody (Santa cruz). Reacted. After washing three times with PBST again, the color was developed using OPD (Sigma), and the OD value was measured using a spectrophotometer (VERSAmax tunable microplate reader, Molecular Devices). The measured values were each quantified using a standard curve.

The results are shown in FIG.

As shown in FIG. 6, the amount of CD93 soluble fragment in serum was not significantly changed by LPS or thioglycolate. However, the amount of CD93 soluble fragment in peritoneal fluid was significantly increased by LPS or thioglycolate treatment. Therefore, it is thought that the CD93 soluble fragment may be utilized as a diagnostic marker for inflammatory diseases.

Example 3  : Determination of CD93 expression in synovial tissue and amount of CD93 soluble fragment in synovial fluid

3-1. CD93 Expression in Synovial Tissues: Immunohistochemistry

Synoviium tissue from three patients with rheumatoid-arthritis (RA) and two patients with osteo-arthritis (OA) was fixed overnight with 4% paraformaldehyde. Subsequently, cleavage was performed by dehydration with alcohol and embedding with paraffin. It was then reacted with methanolic H ₂ O ₂ to eliminate the intrinsic peroxidase activity. It was also blocked for 2 hours with 3% NGS (normal goat serum) to eliminate nonspecific reactions. The tissues were then reacted with goat anti-human CD93 antibody (R & D), biotinylated anti-chlorine IgG, streptavidin-peroxidase complex (Vector, Peterboredgh, UK) for 1 hour each. The stained tissues were counter stained with hematoxylin again and observed with an Olympus microscope (Tokyo, Japan).

The results are shown in FIG.

As shown in FIG. 7, CD93 expression was significantly higher in synovial tissue obtained from rheumatoid arthritis patients than synovial tissue obtained from degenerative arthritis patients. In particular, the expression of CD93 molecules was significantly higher in synovial lining, sublining, and perivascular region where lymphocyte infiltration occurs.

3-2. Determination of the amount of CD93 soluble fragment in synovial fluid: sandwich ELISA method

The amount of CD93 soluble fragment in synovial fluid obtained from 8 patients with rheumatoid arthritis and 8 patients with degenerative arthritis was measured using a sandwich ELISA method. First, 100 ng / well of rat anti-human CD93 antibody (R & D systems) was coated overnight in 93-well Bekcon plates (Becton Dickinson). After washing three times with PBST, it was blocked for 30 minutes at room temperature with 3% skim milk. After washing three times with PBST, 100 μl of synovial fluid (SF) was collected from each of the rheumatoid arthritis patients and eight degenerative arthritis patients, and left for 2 hours. Human soluble CD93 was used as standard material for quantification. After washing three more times, it was left for 1 hour with 1 µg / ml goat anti-human CD93 antibody (R & D systems), followed by 1 µg / ml anti-goat IgG-HRP antibody (Santa cruz) for another hour. Reacted. After washing three times with PBST again, the color was developed using OPD (Sigma), and the OD value was measured using a spectrophotometer. The measured values were each quantified using a standard curve.

The results are shown in FIG.

As shown in FIG. 8, the amount of CD93 soluble fragment in synovial fluid obtained from rheumatoid arthritis patients was significantly higher than that of the degenerative arthritis patients. Thus, it is contemplated that the CD93 molecule or soluble fragment thereof may be utilized as a diagnostic marker for inflammatory diseases.

CD93 or a soluble fragment thereof according to the present invention increases CD93 expression and increases the amount of CD93 soluble fragment by or in an inflammatory environment. Therefore, the CD93 or soluble fragment thereof according to the present invention can be usefully used as a diagnostic marker of inflammatory disease.

1 is a diagram showing the results of analyzing CD93 expression on the cell surface by FACS after treatment with LPS, TNF-α or PMA to THP-1 cells, which are human monocytes.

Figure 2 shows the results of observation of CD93 expression on the cell surface and soluble fragments thereof by Western blot after treatment of LPS, TNF-α or PMA to THP-1 cells.

3 is a diagram showing the results of quantitative measurement of CD93 mRNA expression on the cell surface after treatment with LPS or PMA to THP-1 cells by RT-PCR.

4 is a diagram showing the results of analysis of mouse CD93 expression by FACS in peritoneal macrophages after injection of PBS, thioglycolate, and LPS into mice.

5 is a diagram showing the results of observing the expression pattern of mouse CD93 by Western blot in PBMC and peritoneal macrophages after injecting PBS, thioglycolate, and LPS into mice.

6 is a diagram showing the results of measuring the amount of mouse CD93 soluble fragments after injection of mice with PBS, thioglycolate, and LPS by a sandwich ELISA method in serum and peritoneal fluid.

7 is a diagram showing the results of staining the synovial tissue obtained from rheumatoid arthritis patients (RA) and degenerative arthritis patients (OA) with a CD93 antibody and analyzed the expression pattern by immunohistochemistry.

8 is a diagram showing the results of measuring the amount of CD93 soluble fragments present in the synovial fluid from rheumatoid arthritis patients (RA) and degenerative arthritis patients (OA) by the sandwich ELISA method.

Claims (11)

Marker for diagnosing inflammatory diseases containing CD93 or a soluble fragment thereof as an active ingredient. The marker for diagnosing inflammatory disease according to claim 1, wherein the soluble fragment is 95 kDa as an extracellular portion (ectodomain) in which the CD93 protein is released after cell culture. The method of claim 1, wherein the inflammatory disease is asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary Disease, pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascia disease, viral infections, bacterial infections, fungal infections, burns, wounds from surgical or dental surgery, prostagladins E excess syndrome, atherosclerosis, gout, degenerative arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, peritonitis, uveitis, dermatitis, eczema and multiple sclerosis Marker for diagnosing inflammatory disease, characterized in that. An inflammatory disease diagnostic kit comprising an antibody or aptamer specifically binding to CD93 or a soluble fragment thereof. 1) coating the protein of the biological sample and the control to the fixture, 2) performing an antigen-antibody binding reaction by adding an antibody specifically binding to CD93 or a soluble fragment thereof, which is a marker for diagnosing an inflammatory disease, to the fixture; 3) detecting the antigen-antibody binding reactant generated through the antigen-antibody binding reaction using a label and a color substrate solution of the secondary antibody conjugate; And 4) A method of detecting CD93 or a soluble fragment thereof, comprising comparing the detection results for the biological sample and the control. The method of claim 5, wherein the biological sample of the CD93 or a soluble fragment thereof, characterized in that at least one selected from the group consisting of tissue, cells, blood, serum, peritoneal fluid, synovial fluid, saliva, urine and feces Detection method. 6. The fixed body of claim 5, wherein the fixture comprises at least one member selected from the group consisting of nitrocellulose membranes, polyvinylidene difluoride membranes, 96 well plates synthesized from polyvinyl resins or polystyrene resins, and glass slide glass. CD93 or a method for detecting soluble fragments thereof. The method of claim 5, wherein the antigen-antibody binding reaction is enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich ELISA, Northern blot, Western blot, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay CD93, characterized in that it comprises one or more selected from the group consisting of enzyme substrate coloration, antigen-antibody aggregation, surface plasmon resonance (SPR), biochip (DNA, RNA), electrophoresis, PCR and RT-PCR Method for detecting soluble fragments thereof. The method of claim 5, wherein the label of the secondary antibody conjugate is horseradish peroxidase (HRP), basic dephosphatase (alkaline phosphatase), colloidal gold (colloid gold), poly L-lysine-fluorescein isothiocyanate (FITC), RITC ( rhodamine-B-isothiocyanate), and a method for detecting a CD93 or a soluble fragment thereof, comprising at least one selected from the group consisting of dyes. The method of claim 5, wherein the chromogenic substrate solution is TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], and Cd93 or a soluble fragment thereof, characterized in that it comprises one or more selected from the group consisting of OPD (o-phenylenediamine). [Claim 6] The CD93 or its solubility according to claim 5, wherein in step 4), if the expression level of the CD93 or a soluble fragment thereof in the biological sample is higher than that of the control group, the CD93 or its solubility is predicted to be diagnosed as having an inflammatory disease or a possibility of an inflammatory disease. Method of Detection of Fragments.

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