KR20190028221A - A biomarker for diagnosing asthma or pulmonary fibrosis comprising Annexin-A5 and the uses thereof - Google Patents

Abstract

The present invention provides a composition for diagnosing asthma or pulmonary fibrosis, comprising an agent measuring the level of a protein of Annexin-A5 or mRNA of a gene thereof; a kit for diagnosing asthma or pulmonary fibrosis, comprising the composition; a method of providing information for the diagnosis of asthma or pulmonary fibrosis; and a screening method for a therapeutic agent for asthma or pulmonary fibrosis. The present invention provides the composition for diagnosing asthma or pulmonary fibrosis to measure and compare the expression levels of proteins or genes of the proteins whose expression levels vary in a patient with asthma or pulmonary fibrosis, thereby enabling an early diagnosis of asthma or pulmonary fibrosis or significantly prognosticating a disease progression thereof. Furthermore, the composition for diagnosing asthma or pulmonary fibrosis according to the present invention enables a non-invasive diagnosis and thus enables a simple and effective method of making an early diagnosis of asthma, worsened severeasthma, or pulmonary fibrosis by a blood test, a urine test, or the like.

Description

A biomarker for the diagnosis of asthma or pulmonary fibrosis including annexin-A5, and a use thereof,

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 antibiotics are mainly used as medicines used for the treatment of asthma. Steroids and antibiotics are used in the treatment of asthma through immune responses 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. 10-2012-0104690

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 Annexin-A5 (Annexin-A5) or a gene thereof.

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, as one embodiment, a composition for diagnosing asthma or pulmonary fibrosis comprising an agent for measuring the level of mRNA of a protein of annexin-A5 (Annexin-A5) do.

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 annexin-A5 (Annexin-A5) or a gene thereof.

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.

In the present invention, Annexin-A5 may be represented by ANXA5. In the present invention, Annexin-A5 and ANXA5 are both used in English names of Annexin-A5. It is known that Annexin-A5 has a biological activity based on the activity and regulation of phosphatidylserine C and phospholipase A2, phosphatidylserine binding. Annexin A5 is a cell death marker and is mainly used for the detection of apoptotic cells by its ability to bind to phosphatidylserine. In the present invention, the relationship between annexin-A5 (ANXA5) and asthma or pulmonary fibrosis was first described.

In one embodiment of the present invention, asthma animal models were prepared by sensitizing mice with OVA, mice were sensitized with OVA and exposed to TiO ₂ NP to produce aggravated severe asthmatic animal models, mice were injected with TiO ₂ NP was exposed to produce an animal model of pulmonary fibrosis. The OVA, OVA + TiO ₂ NP or TiO ₂ NP-exposed mice had increased airway hyperresponsiveness (AHR) and bronchoalveolar lavage fluid (BALF) compared to the control group.

In addition, it was confirmed that the expression of annexin A5 (ANXA5) was decreased in the lung tissue of OVA-sensitized asthma model mouse (OVA-OVA), and OVA + TiO ₂ We observed that the expression of annexin A5 (ANXA5) was increased in the lung tissue of aggravated severe asthma model mice exposed to NP and in the lung tissue of lung fibrosarcoma model mice exposed to TiO ₂ NP compared to the control.

Therefore, it is possible to diagnose asthma when the expression of annexin A5 (ANXA5) decreases, and it is possible to diagnose severe asthma or pulmonary fibrosis when the expression of annexin A5 (ANXA5) increases .

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 level of mRNA may comprise a primer pair, probe or antisense oligonucleotide that specifically binds to the gene of the protein of Annexin-A5 have.

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 Annexin-A5.

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 Annexin-A5, 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 of the present invention comprises the steps of measuring the level of expression of a protein of Annexin-A5 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 Annexin-A5 has a characteristic that the gene expression level or the expression level of the protein changes in an individual having asthma, severe asthma or pulmonary fibrosis, as compared with the normal control group, If the level changes, it can be diagnosed as asthma or pulmonary fibrosis.

In addition, when the expression level or the expression level of the gene of the isolated sample of the suspected individual of asthma or pulmonary fibrosis is lower than the expression level of the protein of the normal control sample or the expression level of the gene, ii) If the expression level of the protein of the normal control sample or the expression level of the gene is higher than that of the normal control group, it can be judged that the severe asthma or pulmonary fibrosis is exacerbated.

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, immunohistochemistry, Western blotting, and enzyme linked immunosorbent assay (ELISA) were used to measure and compare the protein expression level of Annexin-A5 itself.

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 Annexin-A5 protein in the candidate substance treatment group; And

When the expression level of the protein of Annexin-A5 is higher than that of the control without treatment of the candidate substance, the candidate substance is judged to be an asthma treatment agent, or the measured Annexin-A5 (Annexin-A5) ) Of the protein of the present invention is lower than that of the control not treated with the candidate substance, it is selected as a treatment for severe asthma or pulmonary fibrosis which is exacerbated.

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 level of expression of Annexin-A5 protein.

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 Annexin-A5 is involved in asthma or pulmonary fibrosis, thereby inducing expression of Annexin-A5 With a control cell that has not been treated with a candidate therapeutic agent.

A5 (Annexin-A5) 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, The level of Annexin-A5 protein in the presence of the candidate substance is measured, and the level of Annexin-A5 protein in the presence of the candidate substance is compared with that of the candidate substance A substance which increases the level under the absence of the substance can be predicted by a preparation for the prevention or treatment of asthma, or if the expression level of the protein of Annexin-A5 is lower than that of the control substance not treated with the candidate substance, May be predicted as a prophylactic or therapeutic agent for worsening severe asthma or pulmonary fibrosis.

The isolated cells may be primary cells isolated from the human body, and established cell lines such as human bronchial epithelial cell line and human lung fibroblast cell line may be used.

As described above, the present invention provides a composition for the diagnosis of asthma or pulmonary fibrosis, thereby measuring and comparing the expression level of a protein or its gene whose expression is changed in a patient suffering from asthma or pulmonary fibrosis, Early diagnosis of illness and disease severity can be predicted or understood significantly.

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.

The left side of FIG. 1 shows airway hyperresponsiveness (AHR) induced in mice exposed to saline (control), OVA, OVA + TiO ₂ NP or TiO ₂ NP. 1 shows the number of inflammatory cells increased in bronchoalveolar lavage fluid (BALF) of mice exposed to saline (control), OVA, OVA + TiO ₂ NP or TiO ₂ NP.
FIG. 2 is an analysis of ANXA5 expression in mouse lung tissues sensitized with saline (control), OVA, OVA + TiO ₂ NP, and TiO ₂ NP by immunohistochemistry. Figure 2A shows immunohistochemical staining with hematoxylin and eosin (H & E) as anti-ANXA5 antibodies using mouse lungs following OVA-OVA sensitization, OVA + TiO ₂ exposure, and TiO ₂ exposure will be. Figure 2B is a histogram of% area data (mean ± SE) of Figure 1A result. * P <0.05 compared to control.
FIG. 3A shows OVA (OVA-OVA) sensitization, TiO ₂ exposure, and OVA + TiO ₂ Protein levels of ANXA5 were determined by Western blot in the lungs of the exposed mouse group and standardized with beta-actin. Figure 3B is a histogram of the data of Figure 3A as densitometric data (mean ± SE). * P <0.05 compared to control.
Figure 4A is an evaluation of the clagen deposition in mice by Maison trichrome staining. The blue part represents collagen associated with fibrosis. Figure 4B is a bar graph of the densitometric data (mean ± SE) of Figure 4A. * P <0.05 compared to control.
5 is prepared by the process and exposing the TiO ₂ NP human bronchial epithelial cells (NHBE) and human lung fibroblast cells (MRC-5) dust asthma prepared by treating a mite (Derp1) in vitro model, a dust mite (Derp1) Western blot analysis of the expression of annexin A5 (ANXA5) in vitro was performed using a pulmonary fibrosis test tube model prepared by exposing the deteriorated asthma test tube model and TiO ₂ NP.
FIG. 6 shows the levels of plasma Annexin-A5 (ANXA5) expression in asthmatic patients and controls. * P <0.05 compared with healthy controls.

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. Plasma Annexin-A5 (ANXA5) concentrations were measured in steady state and deteriorated state. Eight BALB / c mice were exposed to saline (control), OVA, OVA + TiO ₂ NP (nano particles) or TiO ₂ NP (200 μg / m 3) (FIG. Specifically, mice were sensitized with OVA to produce an asthmatic animal model. OVA was sensitized to mice and exposed to TiO ₂ NP to produce a severe asthmatic animal model that was exacerbated, and mice were exposed to TiO ₂ NP to induce pulmonary fibrosis Model. Expression of ANXA5 in lung tissue was measured by immunohistochemistry and collagen of lung tissue was analyzed by Masson trichrome staining.

Human lung fibroblast (MRC-5) and human bronchial epithelial cell line (NHBE) were treated with titanium dioxide nanoparticles (TiO ₂ ) and dust mites (Dermatophagoides pteronyssinus, Derp1). Specifically, asthma test tube models were prepared by treating human bronchial epithelial cell line (NHBE) and human lung fibroblast (MRC-5) with dust mites (Derp1). Human bronchial epithelial cell line (NHBE) and human lung fibroblast -5) dust mite handle (Derp1) and were prepared in vitro model of asthma exacerbated by exposing the TiO ₂ NP, human bronchial epithelial cells (NHBE) and human lung fibroblast cells (exposed to the TiO ₂ NP on MRC-5) in To produce a pulmonary fibrosis test tube model. 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.

< Example  2> Cell culture

MRC-5 (human fetal lung fibroblasts) were grown in T75 flasks (37 ° C, 5%) using MEM (minimum essential medium) (cat #: SH30024.01, Hyclone) containing EBSS, L- CO ₂ ), human bronchial epithelial cells (NHBE, cat # CC-2540, Lonza, Basel, Switzerland; inoculation density, 3000 / cm ² ) were purchased from BEGM BulletKit ™ (bronchial epithelial cell growth medium, cat # : CC-3170, Lonza) in T75 flask (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 before the experiment, the medium was replaced with MEM or BEBM base medium and treated with 100 μM / ml titanium dioxide nanoparticles (TiO ₂ ) or 10 μg / ml dust mite (Derp 1) for 4 hours.

< Example  3> airway hyperresponsiveness AHR , airway hyperresponsiveness ), Bronchoalveolar lavage (Bronchoalveolar lavage  fluid, BALF ), Morphology analysis

Mice were anesthetized with 2.5 mg / kg tiletamine and xylazine (Zolethyl and lumpum; Bayer Korea Co., Seoul, Korea), and treated with either 0, 5, 20 or 100 mg / Sigma-Aldrich) and then evaluated for airway hyperresponsiveness (AHR). On the next day, bronchoalveolar lavage fluid (BALF) was collected, centrifuged and the supernatant was stored (-20 ° C). The cell pellet was resuspended for cell count and cytospin slides were prepared (500 cells / mouse, Diff-Quick staining) for cell differential analysis. A portion of the lung was fixed in 4% phosphoric acid buffer paraformaldehyde, embedded in paraffin, fractionated (4 μm), and stained (H & E, IHC staining).

< Example  4> Immunohistochemical analysis

The paraffin of the mouse lung slice was removed and rehydrated with an ethanol gradient series. The sections were treated with methanol containing 1.4% hydrogen peroxide (H2O2) for 30 minutes to inhibit the intrinsic peroxidase, treated with 1.5% horse serum for nonspecific binding and incubated with anti-goat Annexin A5 (1:50, Santa Cruz Biotechnology, Inc). The next day, sections were incubated with ABC kit (Vector Laboratories, Burlingame, CA, USA). The color reaction was induced by staining with a liquid DAB + substrate kit (Golden Bridge International Inc., Mukilteo, WA, USA). After immunohistochemical staining, slides were stained for comparison with Harris hematoxylin for 1 minute. Images were analyzed with the ImageJ program (National Institutes of Health, Bethesda, Md.).

< Example  5> Western Blot

Mouse lung tissue was suspended in distilled water in a solution containing 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 And homogenized in a protein lysis solution containing methylsulfonyl 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 goat anti-Annexin A5 (ANXA5) (1: 200, Santa Cruz Biotechnology, Inc) And incubated overnight at 4 ° C. 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 normalized to the level of anti-β-actin monoclonal antibody (1: 5000, Sigma-Aldrich).

< Example  6> ELISA

ANXA5 was measured in the blood of patients with asthma 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 And 14.1 pg / ml for ANXA5.

< Example  7> Mason Trichrome  analysis( Masson trichrome  assay)

Lung tissue slides were placed at 60 ° C for 30 minutes. Thereafter, the sample was immersed in xylene for 15 minutes and immersed in 100%, 95%, 90%, 80%, and 70% ethanol for 10 minutes, respectively. Slides were stained according to the manual of the Maeson tri chromium assay kit (Sigma-Aldrich, St. Louis MO, USA). In short, the Bouin solution was used as a mordant to enhance the color response. Nuclei and cytoplasm were stained with hematoxylin and scarlet acid. The positive charge was changed to negative charge using a solution of tungstic acid / phosphomolybdic acid. After staining with aniline blue, the sample was treated with 1% acetic acid. Prior to observation, the sample was mounted with a cover slip using mounting media (Amresco, OH, USA). Positive trichrome stained sections were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The value is expressed as a percentage of the positive area in the whole sample.

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> Asthma model, Worse  Increased AHR and inflammatory cells in severe asthma model, pulmonary fibrosis model

First, the manufacturing and sensitization to OVA in mouse asthma in animal models, sensitization to OVA in mice and in lung fibrosis in animal models manufactured by exposing the TiO ₂ NP on the deteriorating with severe asthma animal model, mice produced by exposing the TiO ₂ NP Bronchial hyperresponsiveness and inflammatory cells were evaluated.

The left side of FIG. 1 shows airway hyperresponsiveness (AHR) induced in mice exposed to saline (control), OVA, OVA + TiO ₂ NP or TiO ₂ NP. 1 shows the number of inflammatory cells increased in bronchoalveolar lavage fluid (BALF) of mice exposed to saline (control), OVA, OVA + TiO ₂ NP or TiO ₂ NP.

As shown in Fig. 1, mice exposed to OVA, OVA + TiO ₂ NP or TiO ₂ NP increased airway hyperresponsiveness (AHR) (Fig. 1 left) in saline-treated sham mice. In addition, OVA / OVA mice sensitized with OVA showed increased inflammatory cells such as eosinophils and macrophages of Bronchoalveolar lavage fluid (BALF) compared to sham mice. Overall, mice exposed to OVA, OVA + TiO ₂ NP or TiO ₂ NP increased the total number of inflammatory cells compared to control mice (FIG. 1 right).

< Experimental Example  2> Asthma model, Worse  Severe asthma model, lung fibrosis model Annexin  Immunohistochemical analysis of A5 (ANXA5)

Produced by sensitization to OVA in mouse asthma in animal models, sensitization to OVA in mice and in lung fibrosis in animal models manufactured by exposing the TiO ₂ NP on the deteriorating with severe asthma animal model, mice produced by exposing the TiO ₂ NP lung tissue The expression level of annexin A5 (ANXA5) was analyzed.

2 is an analysis of ANXA5 expression in mouse lung tissues sensitized with saline (control), OVA, OVA + TiO ₂ NP, and TiO ₂ NP by immunohistochemical staining. FIG. 2A shows immunohistochemical staining with hematoxylin and eosin (H & E) as anti-ANXA5 antibodies using mouse lungs after OVA-OVA sensitization, OVA + TiO2 exposure, and TiO2 exposure. Figure 2B is a histogram of% area data (mean ± SE) of Figure 2A result.

As shown in FIG. 2, it was confirmed that the expression of annexin A5 (ANXA5) was reduced in the lung tissue of OVA-sensitized asthma model mouse (OVA-OVA). On the other hand, the expression of annexin A5 (ANXA5) was increased in the lung tissue of aggravated severe asthma model mice exposed to OVA + TiO ₂ NP and in lung tissues of lung fibrosarcoma model mice exposed to TiO ₂ NP compared to the control Respectively.

Therefore, a decrease in the expression of annexin A5 (ANXA5) can be diagnosed as asthma, and when the expression of annexin A5 (ANXA5) is increased, it can be diagnosed as aggravated severe asthma or pulmonary fibrosis.

< Experimental Example  3> Asthma model, Worse  Severe asthma model, lung fibrosis model Annexin  Western blot of A5 (ANXA5)

Next, Western blot was used to analyze the expression of annexin A5 (ANXA5) in lung tissue. 3A is confirmed ANXA5 protein levels by Western blot analysis in the lungs of OVA (OVA-OVA) sensitized, OVA + TiO _2, TiO ₂ mice exposed group, beta-actin is one standardized. Figure 3B is a histogram of the densitometric data (mean ± SE) of Figure 3A.

As shown in FIG. 3, it was confirmed that the expression of annexin A5 (ANXA5) was reduced in the lung tissue of the asthma model mouse (OVA-OVA) sensitized with OVA. On the other hand, there was no significant difference in the expression of annexin A5 (ANXA5) compared with the control group in aggravated severe asthma model mice exposed to OVA + TiO ₂ , but in the lung fibrosis mice exposed to TiO ₂ NP, ANXA5) was increased compared with the control group.

Therefore, when the expression of annexin A5 (ANXA5) decreases, it can be diagnosed as asthma, and when the expression of annexin A5 (ANXA5) increases, it can be diagnosed as pulmonary fibrosis.

< Experimental Example  4> asthma model, Worse  Severe asthma model, evaluation of collagen accumulation in pulmonary fibrosis model

Next, pulmonary fibrosis of OVA (OVA-OVA) sensitization, OVA + TiO ₂ , and TiO ₂ exposed mouse group was confirmed by Maison trichrome staining. Figure 4A is an evaluation of the clagen deposition in mice by Maison trichrome staining. The blue part represents collagen associated with pulmonary fibrosis. Figure 4B is a bar graph of the densitometric data (mean ± SE) of Figure 4A.

As a result, collagen precipitation was observed in OVA (OVA-OVA) sensitization, OVA + TiO ₂ and TiO ₂ -expression mice as compared with the control group, and collagen precipitation was highest in the TiO _2- exposed mouse model of pulmonary fibrosis .

< Experimental Example  5> Asthma model, Worse  Severe asthma model, lung fibrosis model Annexin  A5 (ANXA5) expression evaluation

An asthma test tube model prepared by treating human bronchial epithelial cell line (NHBE) with dust mite (Derp1), an aggravated asthma test tube model prepared by treating human bronchial epithelial cell line (NHBE) with dust mite (Derp1) and exposing TiO2 NP , And the expression level of annexin A5 (ANXA5) in vitro was determined by Western blotting using a pulmonary fibrosis test tube model prepared by exposing TiO ₂ NP to human bronchial epithelial cell line (NHBE).

The same test tube model was also prepared using human lung fibroblast (MRC-5).

As a result, as shown in FIG. 5, it was observed that the expression of annexin A5 (ANXA5) was reduced in a human bronchial epithelial cell line (NHBE) treated with dust mite (Derp1), and the aggravated asthma model , And the expression of annexin A5 (ANXA5) was increased in the pulmonary fibrosis model.

In addition, it was observed that the expression of annexin A5 (ANXA5) was reduced in asthma model treated with dust mite (Derp1) in human lung fibroblast (MRC-5). In the asthmatic model and the lung fibrosis model It was confirmed that the expression of annexin A5 (ANXA5) was increased.

Therefore, as in the in vivo experiment using a mouse, when the expression of annexin A5 (ANXA5) decreases, asthma can be diagnosed. When the expression of annexin A5 (ANXA5) is increased, the severely aggravated asthma or lung fibroblast And it was confirmed that the diagnosis can be made by the symptom.

< Experimental Example  6> In the plasma of asthmatic patients Annexin  A5 ( ANXA5 ) Decreased expression

Next, we confirmed the decrease in the expression of annexin A5 (ANXA5) in the plasma of patients with asthma. FIG. 6 shows the level of plasma Annexin-A5 (ANXA5) expression in asthmatic patients and control group. FIG. 6 shows that the expression of annexin A5 (ANXA5) was significantly reduced in asthmatic patients compared to the control group, When the expression of A5 decreases, asthma can be diagnosed.

As described above, when the expression of annexin A5 is significantly reduced in asthmatic patients compared to the control group, the decrease in expression of annexin A5 can be diagnosed as asthma.

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 an Annexin-A5 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 level of expression of a protein of annexin-A5 (Annexin-A5) 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 (i) lower than the expression level of the protein of the normal control sample or the gene expression level, , And (ii) the expression level of the protein of the normal control sample or the expression level of the gene is judged to be severe asthma or pulmonary fibrosis which is deteriorated. Way. 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 6, 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; 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 Annexin-A5 protein in the candidate substance treatment group; And
When the expression level of the protein of Annexin-A5 is higher than that of the control without treatment of the candidate substance, the candidate substance is judged to be an asthma treatment agent, or the measured Annexin-A5 (Annexin-A5) ) Of the protein of the present invention is lower than that of the control not treated with the candidate substance, the method comprising the step of selecting asthma or pulmonary fibrosis as a therapeutic agent for aggravated severe asthma or pulmonary fibrosis.

Images