KR101978407B1 - A biomarker for diagnosing asthma or pulmonary fibrosis comprising Claudin-7 and the uses thereof - Google Patents

Abstract

The present invention relates to a composition for diagnosing asthma or pulmonary fibrosis comprising an agent for measuring the level of mRNA of a protein of Claudin 7 (CLDN7) or its gene, and a kit for the diagnosis of asthma or pulmonary fibrosis comprising the composition, A method for providing information for diagnosing pulmonary fibrosis, and a method for screening for a therapeutic agent for asthma or pulmonary fibrosis.
The present invention provides a composition for the diagnosis of asthma or pulmonary fibrosis, which can be used for early diagnosis of asthma or pulmonary fibrosis by measuring and comparing the expression level of the protein of Claudin 7 whose expression changes in asthmatic or pulmonary fibrosis patients, And disease severity can be significantly predicted or understood.
In addition, the composition for diagnosing asthma or pulmonary fibrosis of the present invention is a simple and effective method for early diagnosis of severe asthma or pulmonary fibrosis due to non-invasive diagnosis.

Description

A biomarker for the diagnosis of asthma or pulmonary fibrosis including claudin 7 and its use,

The present invention provides a composition for diagnosing asthma or pulmonary fibrosis, a kit for diagnosing asthma or pulmonary fibrosis comprising the composition, and a method for providing information for diagnosing asthma or pulmonary fibrosis.

Asthma is a lung disease characterized by chronic airway inflammation and airway hyperresponsiveness. It is caused by air pollutants, dust, allergens, and the like. In particular, asthma is an inflammatory respiratory disease causing dyspnea, and about 3 million people in Korea have asthma. Asthma is a disease characterized by colorless asthma, dyspnea, sneezing, severe cyanosis due to lack of oxygen, and chest pain. Recently, it has been rapidly increasing due to exposure to air pollution and various chemical substances and westernization of diet. Especially, And it is increasing with the change of eating habit and westernization. However, in asthmatic patients, the cause and mechanism of the disease are not clear. In this pathogenesis, Th2 (T helper type 2) type of immune response is exaggerated, resulting in increased secretion of interleukin-4, 5, and 13 (Liu et al., 2006; Williams et al. 2012), many inflammatory cells including eosinophils migrate and infiltrate into lung tissue in association with this reaction (Zhou et al., 2011). In addition, inflammatory cells release a variety of proinflammatory and chemoattractant factors, further exacerbating the inflammatory response, increasing mucus secretion in intratracheal goblet cells, and causing airway hyperresponsiveness (Chibana et al., 2008). Because of this series of reactions, patients with asthma exhibit clinical symptoms such as dyspnea, cyanosis, and chest pain.

Currently, steroids, bronchodilators, and anti-inflammatory drugs are mainly used in the treatment of asthma. Steroids and antiinflammatory agents are used for the treatment of asthma through immunity and inhibition of inflammatory responses, and bronchodilators are used to counteract clinical manifestations such as dyspnea. The most widely used inhaled corticosteroids have excellent therapeutic effect, but when used for a long period of time, adrenal suppression, bone density reduction, growth disorder, eye and skin complications, collagen synthesis And the like. In addition, persistent beta-2 agonists, such as salmeterol and formeterol, have been shown to be protective against asthma attacks, . And long-term use has been found to have side effects, limited use as a treatment for asthma.

On the other hand, pulmonary fibrosis or idiopathic pulmonary fibrosis (IPF) is an unexplained disease characterized by chronic fibrosis of pulmonary epilepsy. The disease is mainly localized in the lungs and shows typical interstitial pneumonia (UIP). The prevalence of this disease varies from report to report, but it is known to be 2-29 out of 100,000 people, and it is designated as a rare incurable disease in Korea. The clinical course of IPF is variable, and it is a fatal disease that progresses to respiratory failure due to the progressive deterioration of pulmonary function in general and the average survival time after diagnosis is within 2-3 years. Because of this, the exact causes and etiologies of IPF have been identified and attempts have been made to develop therapeutic agents therefor, but there is no definite treatment yet.

Although the exact etiology of IPF has not yet been fully elucidated, it is believed that pulmonary fibrosis progresses as an abnormal healing process for sustained damage to the alveolar epithelial cells. As a result of various stimuli, lung epithelial cells are damaged and can not be easily regenerated. One of the healing processes of these injuries is abnormally activated myofibroblasts, which leads to excessive extracellular matrix formation and fibrosis of lung epilepsy Respectively.

The current treatments for asthma or pulmonary fibrosis have many side effects. Early detection and treatment of asthma and pulmonary fibrosis are important. Therefore, it is urgent to find a biomarker capable of early diagnosis of asthma and pulmonary fibrosis.

Korean Patent Publication No. 10-2016-0141075

It is an object of the present invention to provide a composition for diagnosing asthma or pulmonary fibrosis comprising an agent for measuring the level of mRNA of a protein of Claudin-7 (CLDN7) or its gene.

Another object of the present invention is to provide a kit for the diagnosis of asthma or pulmonary fibrosis comprising the above composition.

It is still another object of the present invention to provide a method for providing information for diagnosis of asthma or pulmonary fibrosis.

It is still another object of the present invention to provide a method for screening for asthma or a therapeutic agent for pulmonary fibrosis.

In order to achieve the above object, the present invention provides, in one aspect, a composition for diagnosing asthma or pulmonary fibrosis comprising an agent for measuring the level of mRNA of a protein of Claudin 7 (CLDN7) or its gene.

In the present invention, the composition for diagnosing asthma or pulmonary fibrosis may be a composition for diagnosing asthma or pulmonary fibrosis comprising an agent for measuring the level of mRNA of a protein of Claudin 7 (CLDN7) or its gene.

The term " asthma " in the present invention is a comprehensive disease name collectively referred to as various diseases characterized by inflammation of the airways leading to organs, bronchi, bronchioles, and alveoli, , Clin Exp Allergy 2005; 35: 1254-62). In particular, asthma is a condition in which the bronchus in the lung is very sensitive. Sometimes the bronchus narrows and the breathing, choking, and breathing sounds can be heard, causing severe coughing. Allergy caused by bronchial allergy inflammation Disease. The most common symptoms of asthma include dyspnea, cough, wheezing (wheezy breathing), a symptom reliever (bronchodilator) that alleviates the narrowed bronchus in a short time, or a disease modifier that prevents asthma attacks by inhibiting bronchial allergic inflammation Anti-inflammatory agents, and leukotriene modulators). In the present invention, the asthma may be, but not limited to, bronchial asthma, allergic asthma, atopic asthma, non-atopic asthma, exercise induced asthma, aspirin asthma, cardiogenic asthma or alveolar asthma.

The term " pulmonary fibrosis " as used herein refers to a type of chronic interstitial lung disease, also called idiopathic pulmonary fibrosis, in which pulmonary tissue cells are transformed into fibroblasts, resulting in dyspnea, cough, , Clubbing, and the like. A honeycomb or atypical fibrous cell cluster is observed during histological examination. Up to now, steroid therapeutic agents, interferon gamma, acetylcysteine, pyrennidone, and bosetan have been used as therapeutic agents, but no agents showing a specific therapeutic effect have been reported.

The term " diagnosis " as used herein means to identify the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the onset of asthma or pulmonary fibrosis, and it is possible to confirm early on whether asthma or pulmonary fibrosis develops.

The term " claudin 7 " as used herein can be denoted as Claudin-7 or CLDN7, and the specific nucleotide sequence and protein information of the gene encoding the claudin 7 is known from NCBI (Genebank: NM_001193619.1. NM_016887.6.). Claudin is a structural molecule of tight junctions. In the present invention, we first elucidated the relationship between CLUD7 (CLDN7) and asthma or pulmonary fibrosis.

In one embodiment of the invention, a human bronchial epithelial cell line (Human Bronchial Epithelial Cells, NHBE) dust mites by treating (Dermatophagoides pteronissinus, Derp1) asthma vitro model, dust mite treatment, and the titanium dioxide to (Derp1) a (TiO ₂₎ The lung fibrosis test tube model was prepared by exposing the severe asthma test tube model and TiO ₂ nanoparticles, which were exacerbated by exposure of the nanoparticles. The dust mites (Derp1) an asthma vitro model manufactured by processing, dust mites (Derp1) of the process and the manufactured deteriorated by exposing the nano-TiO ₂ particles of severe asthma in vitro model, the pulmonary fibrosis is prepared by exposing the TiO ₂ nanoparticles In the test tube model, trans-epithelial electrical resistance (TEER) was increased compared with the control group.

In addition, dust mites (Derp1) an asthma vitro model prepared by treating the dust treated mites (Derp1) and produced by the exposure to the deterioration of severe asthma in vitro model, TiO ₂ nanoparticles prepared by exposing the nano-TiO ₂ particles lung In the fibrosis test tube model, the protein expression of CLDN7 was decreased compared to the control group, and the expression of CLDN7 protein was decreased in the plasma of asthmatic patients compared with the normal control group.

Therefore, it was confirmed that when the expression of claudin 7 (CLDN7) decreases, it can be diagnosed as asthma or aggravated severe asthma or pulmonary fibrosis.

The term " marker " in the present invention refers to a substance which can be distinguished from a normal group individual and individuals having asthma or pulmonary fibrosis, and is characterized in that it has an increase or decrease in individuals having asthma or pulmonary fibrosis of the present invention Polypeptides, proteins or nucleic acids, genes, lipids, glycolipids, glycoproteins or sugars, and the like. In particular, the present invention is not limited to the protein of the present invention which is changed in an individual having asthma or pulmonary fibrosis.

As used herein, the term " mRNA level measurement " is used to determine the presence and expression level of mRNA for genes for diagnosis of asthma or pulmonary fibrosis in a biological sample in order to diagnose asthma or pulmonary fibrosis. 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.

The agent for measuring the mRNA level of the gene is preferably a primer pair or a probe. Since the nucleic acid information of the genes is known in GeneBank and the like, those skilled in the art will be able to use primers or probes that specifically amplify specific regions of these genes Can be designed.

The agent for measuring the mRNA level of the gene may comprise a primer pair, a probe or an antisense nucleotide that specifically binds to the gene.

Preferably for the diagnosis of asthma or pulmonary fibrosis, the agent for measuring the mRNA level may comprise a primer pair, a probe or an antisense oligonucleotide that specifically binds to the gene of the protein of claudin 7 (CLDN7).

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, but preferably provides specific and sensitive assay results . 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, a polypeptide, a protein, an RNA, or a DNA, and may be a peptide nucleic acid It is preferably PNA. 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 a derivative 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 determining the presence and expression level of a marker protein for the diagnosis of asthma or pulmonary fibrosis in a biological sample for diagnosis of asthma or pulmonary fibrosis. The amount of the protein can be determined using an antibody that specifically binds to the marker protein. Preferably, the protein expression level itself is measured 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 / Immunohistochemistry analysis, immunocytochemistry analysis, complement fixation assay, two-dimensional electrophoresis, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, Analysis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA) But is not limited to.

For the diagnosis of asthma or pulmonary fibrosis, the agent that preferably measures the protein level may comprise an antibody that specifically binds to the protein of claudin 7 (CLDN7).

The term " antibody " as used herein refers 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 protein of Claudin 7 (CLDN7), 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 of the present invention 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 another aspect, the present invention provides a kit for the diagnosis of asthma or pulmonary fibrosis comprising the composition for diagnosing asthma or pulmonary fibrosis.

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

In addition, preferably, the kit for diagnosing asthma or pulmonary fibrosis may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

Preferably, the diagnostic kit comprises a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The RT-PCR kit (RT-PCR) contains each pair of primers 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.

Preferably a diagnostic kit comprising essential elements necessary for carrying out a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.

Also preferably, it may be a diagnostic kit characterized by comprising essential elements necessary for performing ELISA. 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.

In addition, it may preferably be a rapid kit, which includes essential elements necessary for carrying out a rapid test in which an analysis result can be obtained within 5 minutes. 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.

In another aspect, the present invention provides a method for providing information for diagnosis of asthma or pulmonary fibrosis.

Preferably, the method comprises measuring the expression level of a protein of Claudin 7 (CLDN7) or a gene thereof from a biological sample; And

And comparing the expression level of the protein measured in the step or the expression level of the gene with a normal control sample, may be a method for providing information for diagnosing asthma or pulmonary fibrosis.

The term " biological sample " used in the present invention refers to a sample such as a tissue, cell, whole blood, serum, plasma, saliva, cerebrospinal fluid or urine which shows a difference in gene expression level or protein expression level due to the onset of asthma or pulmonary fibrosis But are not limited thereto.

Since all of the clodin7 (CLDN7) has a characteristic that the gene expression level or the expression level of the protein changes in an individual having asthma, worsened severe asthma or pulmonary fibrosis, as compared with the normal control group, Asthma or pulmonary fibrosis can be diagnosed.

In addition, when the expression level or protein expression level of said gene in a separate sample of asthmatic or pulmonary fibrosis suspect individuals is lower than the expression level of the protein of the normal control sample or the gene expression level, asthma, severe asthma or lung fibrosis It can be judged as a fire.

Preferably, the level of expression of the gene of the invention can be measured or compared to mRNA expression levels.

The measurement or comparison of the mRNA expression level may be performed using 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, but the present invention is not limited thereto . Through the above-described methods, mRNA expression level in a normal control group and mRNA expression level in a patient with asthma or pulmonary fibrosis can be confirmed, and the degree of expression of these mRNAs can be compared to diagnose or predict the onset of asthma or pulmonary fibrosis.

Preferably, the protein expression level of the present invention can be measured and compared using an antibody that specifically binds to the protein. The antibody and the protein in the biological sample are allowed to form an antigen-antibody complex, and a method of detecting the antibody-antibody complex is used.

As used herein, the term " antigen-antibody complex " refers to a conjugate of a corresponding protein antigen in a biological sample and an antibody recognizing it. The detection of the antigen-antibody complex can be detected using methods such as those known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance, chemical and other methods.

For the purpose of the present invention, protein level analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry Analysis, Radiation Immunoassay, Radiation Immunodiffusion, Oucheroton Immunodiffusion, Rocket Immunoelectrophoresis, Immunohistochemistry Analysis, Immunocytochemistry Analysis, Complement Liquid chromatography-mass spectrometry (LCMS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and ELISA (enzyme linked immunosorbent assay ), But are not limited thereto.

In one embodiment of the present invention, Western blotting and ELISA (enzyme linked immunosorbent assay) were used to measure and compare the protein expression level of claudin 7 (CLDN7) per se.

In another aspect, the present invention provides a method for screening for an agent for treating asthma or pulmonary fibrosis.

The screening method for the therapeutic agent for asthma or pulmonary fibrosis of the present invention

Treating an asthma or pulmonary fibrosis candidate candidate agent in an isolated cell, isolated tissue or an animal other than a human;

Measuring the expression level of the protein of claudin 7 (CLDN7) in the candidate substance treatment group; And

If the measured expression level of the protein of CLAD7 (CLDN7) is higher than that of the control not treated with the candidate substance, the candidate substance is selected as an asthma treatment agent, a severe asthma or a treatment for pulmonary fibrosis.

The term " agent for treating asthma or pulmonary fibrosis " of the present invention means a substance that is presumed to have the potential of treating asthma or pulmonary fibrosis according to a conventional selection method, or may be an individual nucleic acid, protein, , Or a compound or the like.

Expression level measurement in the present invention may be to determine the expression level of the protein of claudin 7 (CLDN7).

The term " control group " of the present invention means a separated cell belonging to a parallel relationship with a group treated with the candidate substance as an isolated cell, a separated tissue, or an animal other than a human who has not treated with a therapeutic agent for asthma or pulmonary fibrosis Or an animal other than human beings.

The expression " method for screening for a therapeutic agent for asthma or pulmonary fibrosis of the present invention " confirms that the expression of clad7 (CLDN7) is involved in asthma or pulmonary fibrosis, whereby the expression of clad7 (CLDN7) Lt; RTI ID = 0.0 > control cells. ≪ / RTI >

(CLDN7) protein in isolated cells, isolated tissues or animals other than humans in the absence of candidate substances capable of preventing or treating asthma or pulmonary fibrosis, and also determining the presence of the candidate substance (CLDN7) protein in the presence of the candidate substance, and comparing the amount of the protein with that of the candidate substance in the presence of the candidate substance, wherein the level of the protein of CLDN7 is higher than that of the candidate substance in the absence of the candidate substance, For the prevention or treatment of severe asthma or pulmonary fibrosis.

The isolated cells may be primary cells isolated from the human body and include human normal bronchial epithelial cells (NHBE), human fetal lung fibroblast cells (MRC-5), adenocarcinoma cells Bronchial Epithelial cells transformed with Ad12-SV40 2B cells were transfected with human umbilical vein endothelial cells (EA.hy926) or adenocarcinomic human alveolar basal epithelial cells (A549) Ad12-SV40 2B, BEAS-2B) may also be used.

As described above, the present invention provides a composition for diagnosing asthma or pulmonary fibrosis by measuring and comparing the expression level of a protein or gene of claudin 7 whose expression is changed in asthma or pulmonary fibrosis patients, Or early diagnosis of lung fibrosis and disease severity can be predicted or understood.

In addition, the diagnostic composition of the present invention makes non-invasive diagnosis possible and can provide an initial diagnosis of simple and effective, asthma, severe asthma or pulmonary fibrosis with blood, urine test or the like.

Figure 1 is a process of human bronchial epithelial cells (NHBE) an asthma vitro model prepared by treatment of dust mite (Derp1), a dust mite (Derp1) and titanium dioxide (TiO ₂₎ is manufactured by exposing the nanoparticles deterioration severe asthma The change in trans-epithelial electrical resistance (TEER) in vitro was confirmed using a pulmonary fibrosis test tube model prepared by exposing the TiO ₂ nanoparticles to a test tube model (compared to 0 hours) p < 0.05, # p < 0.05 compared to Derp1).
Figure 2 is a human bronchial epithelial cells (NHBE) dust asthma prepared by treating a mite (Derp1) in vitro model to dust processing mites (Derp1) and a test tube of the deterioration severe asthma prepared by exposing the nano-TiO ₂ particles model, TiO ₂ nano-particles were exposed for 4 hours, 8 hours, and 24 hours in vitro using a pulmonary fibrosis test tube model, and the degree of expression of clad7 was determined by Western blotting. (P <0.01 compared to the control group).
Figure 3 compares the expression of claudin 7 in plasma of healthy control and stabilized asthmatic patients (p < 0.05 compared to control).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

A. Experimental Method

Example 1 Experimental Design

Fifty asthmatic patients were recruited and observed for 6.6 ± 3.6 years. The plasma level of Claudin-7 (CLDN7) was measured in a stable state.

Human Bronchial Epithelial Cells (NHBE) were treated with titanium dioxide nanoparticles (TiO ₂ ) and dust mites ( Dermatophagoides pteronyssinus , Derp1). Specifically, by treatment of dust mite (Derp1) in human bronchial epithelial cells (NHBE) were prepared asthma vitro models, deterioration by treatment of dust mite (Derp1) and exposing the nano-TiO ₂ particles into the human bronchial epithelial cells (NHBE) , And a lung fibrosis test tube model was prepared by exposing TiO ₂ nanoparticles to human bronchial epithelial cell line (NHBE). The patient's study was approved by the Review Committee of Soonchunhyang University. This animal study was approved by the Animal Care and Use Committee of Soonchunhyang University.

&Lt; Example 2 > Cell culture

Human bronchial epithelial cells (. Human Bronchial Epithelial Cells, NHBE , cat # CC-2540, Lonza, Basel, Switzerland; inoculation density, 3000 / cm ²⁾ is BEGM BulletKit ™ (bronchial epithelial cell growth medium, cat #: CC-3170 , Lonza) in T75 flasks (37 ° C, 5% CO ₂ ). The medium was replaced every 48 hours (37 ° C, 5% CO ₂ ) until the cell number reached 90% confluence. Cells were then inoculated into 6-well plates. Twenty-four hours prior to the experiment, 100 μM TiO ₂ nanoparticles or 10 μg / ml dust mite (Derp1) were incubated for 4 hours, 8 hours, and 10 hours after replacing the medium with MEM or BEBM base medium. And treated for 24 hours.

Example 3 Measurement of trans-epithelial electrical resistance (TEER)

TEER was used as a measure of tight junction formation in NHBE, and NHBE was cultured at a cell concentration of 8 × 10 ³ cells / ml. They were treated Derp1 or TiO ₂ nanoparticles for 4, 8, 24 hours before the experiment 24 at 37 ℃, 5% CO _2. TEER was measured weekly using an EVOM meter (World Precision Instruments, FL).

&Lt; Example 4 >

Human bronchial epithelial cells (NHBE) were suspended in 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 0.5 mM ethylenediaminetetraacetic acid (EDTA) And 100 mM phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 4 ° C and 1,4000 rpm for 30 minutes, the soluble material was collected. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with Tris buffered saline (TBS) containing 0.1% Tween-20 containing 5% skim milk for 1 hour at room temperature and incubated with rabbit anti-Claudin-7 (CLDN7) (1: 200, abcam) Overnight. The cells were then incubated for 1 hour at room temperature using HRP-conjugated secondary antibody (1: 5000, Santa Cruz Biotechnology, Dallas, USA). (ECL) and Western blot detection system (ATTO, Tokyo, Japan) on X-ray film. Relative protein levels were determined by quantitative concentration method and standardized to the level of anti-beta-actin monoclonal antibody (1: 5000, Sigma-Aldrich).

Example 5 Enzyme-linked immunosorbent assay (ELISA)

Claudin 7 in the blood of asthmatic patients was measured by ELISA (USCN, Wuhan, China). To compare the results with the other plates, the optical density (OD) of the test sample was adjusted for the positive and negative control samples provided per kit. The average OD of the two wells was calculated. The exponential value of the test plasma is defined by the following equation: exponent = (OD of OD-negative control of test plasma) / (OD of negative control - OD of negative control) x 100. Low detection limits are determined according to manufacturer's recommendations In the case of Claudin-7, it was set at 15.6 pg / ml.

Statistical analysis

Data were expressed as mean ± standard error (SEM) and Mann-Whitney U test was used to determine significant differences between the two groups. p <0.05 was considered to mean statistical significance.

B. Experimental Results

EXPERIMENTAL EXAMPLE 1 Increase in trans-epithelial electrical resistance (TEER) in asthma, aggravated severe asthma, pulmonary fibrosis test tube model

1 is a dust mite (Dermatophagoides pteronissinus, Derp1) the processing by the processing of the prepared asthma vitro model, Derp1 and made worse by exposing the nano-TiO ₂ particles of severe asthma in vitro model of human bronchial epithelial cells (NHBE), TiO ₂ The change in trans-epithelial electrical resistance (TEER) is shown in vitro using a pulmonary fibrosis tube model prepared by exposing nanoparticles.

As it is shown in Fig. 1, and only the processing Derp1 treated NHBE, Der1 and significantly increased both as compared to the 0 hours TEER in NHBE was only exposed NHBE and TiO ₂ nanoparticles by exposing the nano-TiO ₂ particles. This suggests that the connection between cells and cells is weakened and the permeability changes in the bronchial epithelium and becomes sensitive to the stimulation of foreign substances.

EXPERIMENTAL EXAMPLE 2 Expression of Clad7 (CLDN7) Expression in Asthma Models, Severe Severe Asthma Models and Pulmonary Fibrosis Models

Next, Western blot was used to analyze the expression of CLD7 in human bronchial epithelial cells (NHBE). Figure 2 is a human bronchial epithelial cells (NHBE) dust mite (Derp1) an asthma vitro model prepared by treating the dust treated mites (Derp1) and a test tube of the deterioration severe asthma prepared by exposing the nano-TiO ₂ particles model to, TiO The expression level of claudin 7 after 4 hours, 8 hours, and 24 hours in vitro was confirmed by Western blotting using a pulmonary fibrosis test tube model prepared by exposing ₂ nanoparticles and standardized with beta-actin It is.

As shown in FIG. 2, the expression of CLDN7 was increased in the asthma test tube model treated with dust mite (Derp1) for 4 hours, but after 8 hours and 24 hours, the dust mite (Derp1) the handle asthma vitro model, dust mites (Derp1) and the keulrawoodin 7 expression in both the pulmonary fibrosis in vitro model produced by the manufactured deterioration severe asthma vitro model exposes the nano TiO ₂ particles, by exposing the TiO ₂ nanoparticles was reduced .

Therefore, the expression of Claudin 7 was temporarily increased in the early dust mite stimulation of 4 hours, but the expression of Claudin 7 (CLDN7) was decreased in all three models as time elapsed. If it declines, it can be diagnosed as asthma, worsening severe asthma or papillomatosis.

Experimental Example 3: Expression of CLD7 in plasma of asthmatic patients

Next, it was confirmed whether the expression of clodin 7 (CLDN7) was reduced in the plasma of asthmatic patients. FIG. 3 is a graph showing the expression level of claudin 7 (CLDN7) in plasma in asthmatic patients and healthy controls. 3, the expression of CLDN7 was decreased in asthmatic patients compared to the control group. The asthmatic patient identified in Fig. 3 is a stabilized asthmatic patient and corresponds to a test tube model in which asthma is induced by stimulation with a dust mite for 8 hours or more as shown in Fig.

As shown above, the expression of claudin 7 is decreased in both the test tube model of asthma and asthmatic patients. If the expression of claudin 7 decreases, asthma can be diagnosed.

In summary, the dust mites (Derp1) and / or titanium dioxide (TiO ₂₎ was the CLDN7 protein expression in the processing of nanoparticles human bronchial epithelial cells (NHBE) decreases, and increase in the TEER. This indicates that the dust mite and / or titanium dioxide affects the bronchial epithelium tight junction, so that Claudine 7 is involved.

CLDN7 is one of the structural molecules of the tight junction. In the present invention, it was confirmed that CLDN7 was reduced in a human bronchial cell model of asthma and pulmonary fibrosis. In the case of asthma, (CLDN7), indicating that expression changes can play a role in the onset of asthma by being involved in tight junctions.

Therefore, when the expression of cladin-7 is decreased in human bronchial epithelial cells and asthmatic patients, the expression of cladin-7 is decreased compared to the control group. Thus, when the expression of cladin-7 decreases, asthma, severe asthma or pulmonary fibrosis can be diagnosed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (12)

A composition for diagnosing asthma or pulmonary fibrosis comprising an agent for measuring the level of mRNA of a Claudin-7 protein or a gene thereof. The composition for diagnosing asthma or pulmonary fibrosis according to claim 1, wherein the agent for measuring the mRNA level of the gene is a primer pair, a probe, or an antisense oligonucleotide that specifically binds to the gene. The composition for diagnosing asthma or pulmonary fibrosis according to claim 1, wherein the agent for measuring the protein level comprises an antibody that specifically binds to the protein. A kit for the diagnosis of asthma or pulmonary fibrosis comprising the composition according to claim 1. 5. The method of claim 4, wherein the kit is an RT-PCR (reverse transcription polymerase chain reaction) kit, a DNA chip kit, an ELISA (enzyme-linked immunosorbent assay) kit, a protein chip kit or a rapid kit. Kits for the diagnosis of pulmonary fibrosis. Measuring the expression level of a protein of Claudin-7 or a gene thereof from a biological sample; And
Comparing the expression level of the protein measured in the step or the expression level of the gene with that of a normal control sample, for the diagnosis of asthma or pulmonary fibrosis. 7. The method according to claim 6, wherein the expression level of the protein or the expression level of the gene in the isolated sample of asthmatic or pulmonary fibrosis suspect individuals is lower than the expression level of the protein of the normal control sample or the expression level of the gene, Characterized in that the disease is judged to be severe asthma or pulmonary fibrosis. 7. The method according to claim 6, wherein the expression level of the gene is an mRNA expression level of the gene, for the diagnosis of asthma or pulmonary fibrosis. The method according to claim 8, wherein the mRNA expression 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, / RTI &gt; 7. The method of claim 6, wherein the protein expression level measurement utilizes an antibody that specifically binds to the protein. 7. The method of claim 6, wherein the protein expression level measurement or comparison is performed using protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF), Sulfate Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheronian immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry analysis, immunocytochemistry analysis, complement fixation assay, 2-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blot, and enzyme linked immunosorbent assay (ELISA) Wherein the method is carried out using a compound selected from the group consisting of: &lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; delete

Images