KR101927351B1 - Estrogen receptor and aromatase marker for diagnosing nasolacrimal duct obstruction and the uses thereof - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing nasal mucous membrane related diseases, in particular, a nasal mucosa stenosis disease or a tear crease, and a biomarker for its use. According to the composition, kit and method according to one aspect, It is possible to diagnose the nail-related diseases, and it is not a method of diagnosing by administering the existing contrast agent, but a method of confirming the expression of the marker in the tissue.

Description

[0001] Estrogen receptor and aromatase markers for the diagnosis of nasolacrimal duct obstruction disease and uses thereof. [0002]

And more particularly, to a biomarker for diagnosis of nasolacrimal duct obstruction disease or tear leak and its use.

Nasolacrimal duct obstruction refers to a nasolacrimal duct obstruction that is narrowed or obstructed by nasolacrimal ducts and is a major symptom of excessive development of tears called epiphora.

The method of diagnosing nasolacrimal stenosis is to check whether perfusion is obtained by injecting physiological saline into the nasolacrimal duct mechanically or by imaging the contrast agent in the nasolacrimal duct. However, the method of confirming in the nail tube or the tear bag in which the lesion progresses substantially can only identify the inflammation of the tissue, so that there is a limitation in discrimination after the disease progresses.

Accordingly, the present inventors have completed the present invention by detecting a protein or gene overexpressed in nasal eye tube-related diseases in order to overcome the problem of the conventional method of diagnosing the nasal eye tube-related disease.

One aspect is to provide a composition for the diagnosis of a nasolacrimal duct-related disease, which comprises an agent for measuring the level of mRNA of an estrogen receptor (ER), MUCIN5AC, or an aromatase protein or a gene thereof .

Another aspect is to provide a kit for the diagnosis of a nociceptive-related disorder comprising the composition.

Yet another aspect is a method for detecting the expression level of an estrogen receptor (ER), MUCIN5AC, or aromatase protein or a gene thereof from an isolated biological sample; And comparing the expression level of the measured protein or the expression level of the gene with a normal control sample to provide information for diagnosing the nasal eye-related disease.

Yet another aspect provides a method of identifying a test substance, comprising contacting the test substance with an estrogen receptor (ER), MUCIN5AC, or aromatase protein; And a step of screening the test substance which has reduced the expression level of the estrogen receptor (ER) or aromatase protein as a therapeutic agent for a nasal eye tube-related disease as compared to a nontreated control group A method of screening a therapeutic agent.

One aspect provides a composition for the diagnosis of a nasolacrimal duct-related disease comprising an agent for measuring the level of mRNA of an estrogen receptor (ER), MUCIN5AC, or aromatase protein or a gene thereof.

Another aspect provides a kit for the diagnosis of a nociceptive-related disorder comprising the composition.

Yet another aspect is a method for detecting the expression level of an estrogen receptor (ER), MUCIN5AC, or aromatase protein or a gene thereof from an isolated biological sample; And comparing the expression level of the measured protein or the expression level of the gene with a normal control sample, to thereby provide information for diagnosing the nasal eye-related disease.

The term "diagnosis" as used herein means to identify the presence or characteristic of a pathological condition. For the purposes of the present invention, diagnosis may refer to ascertaining the onset of nasal eye-related diseases.

As used herein, the term "marker" refers to a substance that can be diagnosed by distinguishing a normal group individual from an individual having a nociceptor-related disease, and includes polypeptides, proteins, Nucleic acid, gene, lipid, glycolipid, glycoprotein or sugar, and the like. In particular, the present invention is not limited to the protein or gene that is changed in the lacrimal sac sample separated from the subject having the nasal eye tube-related disease of the present invention.

As used herein, the term "estrogen receptor" may refer to a receptor that is activated by estrogen (17 [beta] -estradiol). The estrogen receptor may be ER [alpha] or ER [beta].

As used herein, the term " aromatase "is used interchangeably with" estrogen synthetase "or" CYP19A1 "and refers to any of the monooxygenase C450 superfamilies that catalyze various reactions involved in steroidogenesis It can mean one.

As used herein, the term "MUCIN5AC" may refer to a protein encoded by the MUC5AC gene.

In one embodiment, the nasolacrimal duct-related disorder may be a nasolacrimal duct obstruction or epiphora. More particularly, the nasolacrimal duct disease may be a chronic or acute nasal mucosa stenosis disease.

As used herein, the term " mRNA level measurement "refers to the measurement of the amount of mRNA in a biological sample to identify the presence or absence of mRNA and the degree of expression of the gene in a biological sample. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

Since the agent for measuring the mRNA level of the gene is a primer pair or a probe and the nucleic acid information of the genes is known in GeneBank and the like, a person skilled in the art will design a primer or a probe that specifically amplifies a specific region of these genes based on the sequence . Therefore, the agent for measuring the mRNA level of the gene may include a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene.

As used herein, the term "primer pair" is a primer pair that contains all combinations of primer pairs consisting of forward and reverse primers that recognize the target gene sequence, and specifically provides analysis results with specificity and sensitivity. The nucleic acid sequence of the primer is inconsistent with the non-target sequence present in the sample and can be given high specificity when it is a primer that amplifies only the target gene sequence containing a complementary primer binding site and does not induce nonspecific amplification .

As used herein, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in the sample through the binding do. The probe molecule may be a peptide nucleic acid (PNA), a peptide nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA or a DNA, although it is not limited as a substance commonly used in the art. More specifically, the probe may be a biomolecule derived from or derived from an organism, or prepared in vitro, such as an enzyme, a protein, an antibody, a microorganism, an animal or plant cell and an organ, a nerve cell, DNA, and RNA DNA includes cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.

As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or derivatives thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, which binds to a complementary sequence in the mRNA and inhibits translation of the mRNA into a protein . An antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to and capable of binding to the mRNAs of the genes. This can interfere with translation of the gene mRNA, translocation into the cytoplasm, maturation, or essential activity for all other overall biological functions. The length of the antisense oligonucleotide may be 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases. The antisense oligonucleotides may be synthesized in vitro by conventional methods and administered in vivo or may be used to synthesize antisense oligonucleotides in vivo. One example for the synthesis of antisense oligonucleotides in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is a multi-cloning site (MCS) in the opposite direction. It is preferred that the antisense RNA is such that translation stop codon is present in the sequence so that it is not translated into the peptide sequence.

As used herein, the term " protein level measurement "is a process for confirming the presence and expression level of a marker protein for diagnosis of nasal eye tube-related diseases in a biological sample in order to diagnose a nasal eye tube-related disease. The amount of the protein can be confirmed using an antibody that specifically binds to the marker protein, and the protein expression level itself can be measured, for example, without using an antibody.

As the protein level measurement or comparative assay, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Flock Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheronin immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis, liquid chromatography- Mass Spectrometry, LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbent assay (ELISA). Thus, the agent that measures the protein level may comprise an antibody that specifically binds to the estrogen receptor, MUCIN5AC, or aromatase protein.

The term "antibody ", as used herein, may refer to a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to the estrogen receptor, MUCIN5AC, or aromatase protein, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The production of the antibody can be easily carried out using techniques well known in the art. The antibodies herein also include functional fragments of antibody molecules as well as complete forms with 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 ') 2, F (ab') 2 and Fv.

In other embodiments, the kit may be a reverse transcription polymerase chain reaction kit (RT-PCR), a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, ) Kit.

In addition, the kit for diagnosing nose-related diseases may further comprise one or more other component compositions, solutions or devices suitable for the assay method. For example, the diagnostic kit may be a diagnostic kit containing the necessary elements necessary for performing the reverse transcription reaction. The RT-PCR kit contains the respective primer pairs specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each of the above genes, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other reverse transcriptase reaction kits include enzymes such as test tubes or other appropriate containers, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC- Water (DEPC-water), sterile water, and the like. Further, for example, the DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof. Also, for example, the kit may be a diagnostic kit that includes the necessary elements necessary to perform LISA. The ELISA kit comprises an antibody specific for the protein. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like. Also, for example, the kit may be a rapid kit that includes the necessary elements necessary to perform a rapid test to know the analysis result. Rapid kits contain antibodies specific for proteins. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The rapid kit may also include antibodies specific for the control protein. Other rapid kits include reagents capable of detecting the bound antibody, such as a nitrocellulose membrane with a specific antibody and a secondary antibody immobilized thereon, a membrane bound to the bead conjugated with the antibody, Materials, and the like. Also, for example, the kit may be a multiple reaction monitoring (MRM) kit that is an MS / MS mode that includes the necessary elements to perform mass analysis. While SIM (Selected Ion Monitoring) is a method of using ions generated by collision with the source part of the mass spectrometer, MRM once again selects a specific ion among the broken ions to connect another consecutively connected MS source And then collide with another once more, and then use ions obtained from the collision. Through the above-described MRM (Multiple Reaction Monitoring) analysis methods, it is possible to compare the protein expression level in the normal control group or the same individual with the protein expression level in the nose-eye-tube-related disease and in the nose- , And it is possible to diagnose whether or not a nail-related disease-related disease has occurred.

The term "biological sample " as used herein refers to a sample such as a lacrimal sac sample, a nasal mucosa sample, a tissue, a cell, a whole blood, a serum, plasma, saliva, cerebrospinal fluid Urine, and the like. In detail, the biological sample may be a sample separated from the tear bag. The normal control group may be a sample separated from the same individual as the biological sample. For example, a sample separated from the tear bag may be used as the biological sample, and a sample separated from the nasal mucosa of the same individual as the normal control sample may be used.

The estrogen receptor (ER), MUCIN5AC, or aromatase may be administered to a normal control group (a subject not having a nociceptive-related disease or a sample not associated with a nociceptive-related disease in the same subject, ), The level of gene expression or the expression level of the protein is increased in an individual having a nociceptor-related disease, and thus, when the level is increased, it can be diagnosed as a nail-related disease. Therefore, a method according to one aspect may include judging a nose-eye-related disease if the expression level of the protein or the expression level of the gene is higher than the expression level of the protein of the normal control sample or the expression level of the gene.

Yet another aspect provides a method of identifying a test substance, comprising contacting the test substance with an estrogen receptor (ER), MUCIN5AC, or aromatase protein; And a step of screening the test substance which has decreased the expression or activity level of the estrogen receptor (ER) or aromatase protein as a therapeutic agent for a nasal eye tube-related disease as compared to the nontreated control group A method for screening a therapeutic agent for a disease.

The test substance, for example, the drug candidate substance, the test compound or the test composition may be a low molecular compound, an antibody, an antisense nucleotide, a short interfering RNA, a short hairpin RNA, a nucleic acid, a protein, Other extracts or natural products may be included.

The contacting may be carried out in vitro. For example, transforming a vector expressing the above proteins into a cell; And treating the transformed cells with a drug candidate substance.

The marker according to one embodiment of the present invention increases expression in a nociceptor-related disease, so that a candidate substance that decreases the expression or activity of the marker can be usefully used for the treatment of nasal eye tube-related diseases.

According to the composition, kit, and method according to one aspect, it is possible not only to diagnose the nose tear-related disease rapidly by collecting only a small amount of tissue but also to diagnose the expression of the marker in the tissue , There is an effect of less burden on the patient.

FIG. 1A is a graph showing protein expression levels in a lacrimal sac sample and a nasal mucosa sample (control sample) of a patient having acute nail tear duct stenosis.
FIG. 1B is a graph showing the amount of protein expression in a lacrimal sac sample and a nasal mucosa sample (control sample) of a patient having chronic nasal mucosal stenosis.
1C is a graph showing relative protein expression levels in a lacrimal sac sample and a nasal mucosa sample (control sample) of a patient having acute and chronic nail lacunar constriction disease.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example . Analysis of tissue samples from patients with nasolacrimal duct obstruction

Western blot analysis and immunofluorescence analysis were performed by separating tissue samples from the lacrimal sac and nasal mucosa of patients with nasolacrimal duct obstruction.

Specifically, tissue samples were collected from 5 patients with chronic nasal tear stenosis and 6 patients with acute nail stenotic lesions. Nasal mucosa was used as a normal control.

Thereafter, western blotting was carried out as follows. The tissue samples were dissolved in a pro-prep solution (Intron, Gyeonggi, Korea), quantitated using BCA technique, and then loaded with the same amount of protein to perform electrophoresis. After electrophoresis, the membranes were blocked with 5% skim milk at room temperature for 1 hour. Primary antibodies (SantaCruz Biotechnology, CA, USA) targeting ERα and aromatase (CYP19A1) (Tris-buffered saline-Tween 20) buffer (Intron) and reacted at 4 ° C overnight using a shaker. After the reaction was completed, the membrane was washed with TBS-Tween 20 buffer for 10 minutes three times. Then, a secondary antibody (SantaCruz Biotechnology) capable of detecting the primary antibody was diluted with TBS-Tween 20 (Intron) And reacted on a shaker at room temperature for 2 hours. The membranes were washed twice with TBST for 10 min and then HRP (horseradish peroxidase) activity was detected using ECL-solution (GenDEPOT, TX, USA), and ERα and aromatase (CYP19A1) Expression was analyzed, and the results are shown in Figures 1a (acute patient tissue) to 1b (chronic patient tissue) and relative protein expression is shown in Figure 1c.

FIG. 1A is a graph showing protein expression levels in a lacrimal sac sample and a nasal mucosa sample (control sample) of a patient having acute nail lacunar stenosis.

FIG. 1B is a graph showing the amount of protein expressed in a lacrimal sac sample and a nasal mucosa sample (control sample) of a patient having chronic nasal mucosal stenosis.

1C is a graph showing relative protein expression levels in a lacrimal sac sample and a nasal mucosa sample (control sample) of a patient having acute and chronic nail lacunar constriction disease.

As shown in FIG. 1, the expression of ERα or CYP19A1 according to one embodiment of the present invention is significantly increased in the lacrimal sac sample compared to the control sample.

Therefore, it can be seen that the ERa or CYP19A1 can be used to diagnose nasolacrimal duct obstruction.

Claims (11)

A composition for the diagnosis of nasolacrimal duct obstruction disease comprising an estrogen receptor alpha (ERα), and an agent for measuring the level of mRNA of an aromatase protein or a gene thereof. delete The diagnostic composition of claim 1, wherein the agent for measuring mRNA level of the gene is a primer pair, a probe, or an antisense oligonucleotide that specifically binds to the gene. The diagnostic composition of claim 1, wherein the agent for measuring the protein level comprises an antibody that specifically binds to the protein. Measuring the level of expression of estrogen receptor alpha (ERα), and an aromatase protein or a gene thereof from a separate lacrimal sac sample; And
And comparing the expression level of the measured protein or the expression level of the gene with a nasal mucosal sample. delete The method according to claim 5, wherein the determined level of expression of the protein or the expression level of the gene is higher than the expression level of the protein of the nasal mucosa sample or the expression level of the gene. 6. The method of claim 5, wherein the expression level of the gene is an mRNA expression level of the gene. The method according to claim 8, wherein the mRNA level is measured by a reverse transcription polymerase chain reaction, a competitive reverse transcription polymerase chain reaction, a real-time reverse transcription polymerase chain reaction, an RNase protection assay, a Northern blotting or a DNA chip. [7] The method according to claim 5, wherein the expression level of the protein is measured by white matter analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI- Time of Flight Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, 2-D electrophoresis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting, enzyme linked immunosorbent assay (ELISA) or a combination thereof.


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