KR102147928B1 - Biomarker for identifying exposure to air pollutants and method for identifying exposure to air pollutants using the same - Google Patents

Abstract

The present invention relates to a biomarker for confirming exposure to air pollutants, and specifically, Calreticulin, a biomarker according to the present invention, is expressed by exposure to diesel exhaust particles (DEP), which are air pollutants. As it increases, it can be usefully used as a biomarker for confirming exposure of diesel exhaust particles.

Description

Biomarker to check exposure of air pollutants and how to check exposure to air pollutants using it{BIOMARKER FOR IDENTIFYING EXPOSURE TO AIR POLLUTANTS AND METHOD FOR IDENTIFYING EXPOSURE TO AIR POLLUTANTS USING THE SAME}

The present invention relates to a biomarker for determining whether air pollutants are exposed, and more particularly, to a biomarker for determining whether an individual is exposed to diesel exhaust particles (DEP), which are air pollutants.

Diesel exhaust particles (DEP) refer to all particles contained in exhaust produced by the combustion of diesel fuel and discharged to the outside from the diesel engine exhaust pipe. It is not only a major component of air pollutants, but also causes respiratory diseases. (Riedl, Marc et al., Journal of Allergy and Clinical Immunology 115.2 (2005): 221-228).

Specifically, as a result of conducting a study on changes in lung function due to exposure to diesel exhaust particles in asthma patients in the UK, Effort Expiratory Volume (FEV) and Forced Expiratory Volume (FVC) were respectively weaker than the control group (park street exposure patients) It was found to decrease by 6.1% and 5.4%. In addition, as a result of a study conducted on adults in the United States, it has been reported that the FEV decreases in residents within 150m of the road than in residents other than 150m of the road, resulting in decreased lung function (Korean Patent Publication 10-2015-0116082).

As such, problems such as the onset of respiratory diseases and respiratory function decline due to exposure of diesel exhaust particles are becoming international issues, but the provision of information according to exposure of diesel exhaust particles in Korea is insignificant. Accordingly, there is a need to develop an indicator material such as a biomarker capable of confirming the exposure of diesel exhaust particles, and development of a method capable of providing information on the exposure of diesel exhaust particles is required.

Korean Patent Publication 10-2015-0116082 (2015.10.15)

Riedl, Marc, and David Diaz-Sanchez. "Biology of diesel exhaust effects on respiratory function." Journal of Allergy and Clinical Immunology 115.2 (2005): 221-228.

Accordingly, the present inventors studied how the exposure of diesel exhaust particles according to concentration and time affects protein expression in order to develop a biomarker for determining whether diesel exhaust particles, which are air pollutants, are exposed. As a result, it was confirmed that the expression of a specific protein was increased in the lungs of mice exposed to the diesel exhaust particles for a long time or periodically, and by discovering that the specific protein has an excellent effect in confirming the exposure of the diesel exhaust particles, the present invention was made. Completed.

Accordingly, an object of the present invention is to provide a biomarker for determining whether air pollutants are exposed, and a method for checking whether air pollutants are exposed using the same.

In order to achieve the above object, the present invention provides a biomarker for confirming exposure to air pollutants including calreticulin protein.

In addition, the present invention provides a kit for confirming exposure to air pollutants comprising a detection agent for a caleticulin protein or a gene encoding the same.

In addition, the present invention comprises the steps of: 1) measuring the expression level of caleticulin in a sample isolated from an individual suspected of exposure to air pollutants; 2) comparing the expression level and the expression level of the normal control sample; And 3) when the expression level is higher than that of the control group, it provides a method of providing information on exposure to air pollutants, including determining that the individual is exposed to air pollutants.

In addition, the present invention is a DNA microarray chip for confirming exposure of an air pollutant in which a gene of caleticulin or a nucleic acid complementary thereto is exposed, and an air pollutant in which a protein of caleticulin or an antibody specifically binding thereto It provides a chip for detection.

Since caleticulin, a biomarker according to the present invention, increases in expression by exposure of air pollutants, for example, diesel exhaust particles, it can be usefully used as a biomarker for confirming exposure of diesel exhaust particles.

1 shows the total protein of human bronchial epithelial cells exposed to diesel exhaust particles on SDS-PAGE.
2 shows that the expression of caleticulin was increased in human bronchial epithelial cells exposed to diesel exhaust particles.
3 shows that airway resistance was increased in a mouse model exposed to diesel exhaust particles.
Figure 4 shows that the inflammatory cells were increased in the bronchoalveolar lavage fluid of the mouse model exposed to the diesel exhaust particles.
FIG. 5 shows that the expression of caleticulin was increased in lung tissue of a mouse model exposed to diesel exhaust particles.

An aspect of the present invention provides a biomarker for confirming exposure to air pollutants including calreticulin protein.

The term "caleticulin" as used herein refers to a major calcium-binding protein with a molecular weight of 55 kDa that binds to a high amount of calcium and is present in the lumen of the endoplasmic reticulum. The sequence of the protein may refer to AAB51176.1 (NCBI), but is not limited thereto.

In one embodiment of the present invention, the air pollutant may be diesel exhaust particles. Specifically, diesel exhaust particles collectively refer to all particles contained in exhaust that is generated during combustion of diesel fuel and discharged to the outside from the diesel engine exhaust pipe. Diesel exhaust particles are complex mixtures of gases, vapors, liquid aerosols and particulate matter, which are produced by combustion. The main chemical components of diesel exhaust particles include carbon monoxide, carbon dioxide, nitrogen oxides, sulfur oxides, alcohols, aldehydes, ketones, hydrocarbons, and polycyclic aromatic hydrocarbons. On June 12, 2012, the International Cancer Research Institute (IARC) under the World Health Organization (WHO) upgraded diesel exhaust particles from class 2A (estimated carcinogen) to class 1 carcinogen, and airway hypersensitivity due to exposure of diesel exhaust particles. (airway hyperresponsiveness), airway inflammation, asthma, lung cancer, chronic obstructive pulmonary disease, etc. can cause various respiratory diseases.

In one embodiment of the present invention, the expression of caleticulin is increased by exposure to air pollutants for a long time or periodically, and through this, it can be used as a biomarker for confirming exposure to air pollutants, specifically diesel exhaust particles. Confirmed.

In addition, an aspect of the present invention provides a kit for confirming exposure to air pollutants including a detection agent for a caleticulin protein or a gene encoding the same.

In one embodiment of the present invention, the detection agent may be a primer or a probe capable of complementarily binding to a gene encoding the caleticulin protein.

The term "primer" as used herein is a nucleic acid sequence having a free 3'hydroxyl group, which can form a complementary template and a base pair, and is for template strand copying. It refers to a short nucleic acid sequence that functions as a starting point. Primers can initiate DNA synthesis in the presence of reagents for polymerization and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. At this time, the reagent for the polymerization reaction may be a DNA polymerase or reverse transcriptase.

The term "probe" as used herein refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to a few tens of bases capable of specific binding to a gene or mRNA. These probes may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc., and may be labeled for easier detection. It is not limited thereto.

The primer or probe can be chemically synthesized using a phosphoramidite solid support method or other well known method. Such nucleic acids can also be modified using a number of means known in the art. Examples of such modifications may be methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides.

In one embodiment of the present invention, the detection agent may be an antibody that specifically binds to the caleticulin protein, and may include a polyclonal antibody, a monoclonal antibody, or a recombinant antibody.

Antibodies that bind to the protein can be easily prepared using techniques well known in the art. For example, the monoclonal antibody is a hybridoma method well known in the art (see Kohler et al., European Jounral of Immunology, 6:511-519, 1976), or a phage antibody library ( Clackson et al., Nature, 352:624-628, 1991; see Marks et al., J. Mol. Biol, 222:581-597, 1991) technology. The antibody prepared by the above method can be separated and purified using a method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.

As an analysis method for confirming the amount of the target protein bound to it using the antibody, Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, and octeroni ( Ouchterlony) immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein chip ), but is not limited thereto.

The kit can be used to check whether diesel exhaust particles are exposed by measuring the expression level of mRNA or protein of caleticulin from an individual in order to confirm exposure to air pollutants, specifically diesel exhaust particles. In addition, primers or probes and antibodies for measuring the mRNA or protein level of the caleticulin, as well as one or more other components, compositions, solutions or devices suitable for the analysis method may be included.

Specifically, the kit for confirming exposure to air pollutants of the present invention may include essential elements necessary for performing RT-PCR. The RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, in addition to each primer pair specific for the caleticulin gene. RNAse inhibitors, sterile water, etc. may be included. In addition, a primer pair specific to a gene used as a quantitative control may be included.

In another aspect of the present invention, 1) measuring the expression level of caleticulin in a sample isolated from an individual suspected of exposure to air pollutants; 2) comparing the expression level and the expression level of the normal control sample; And 3) when the expression level is higher than that of the control group, it provides a method of providing information on exposure to air pollutants, including determining that the individual is exposed to air pollutants.

In one embodiment of the present invention, the sample may be an individual's bronchoalveolar lavage fluid or lung cells, specifically lung epithelial cells.

In one embodiment of the present invention, the step of measuring the expression level of caleticulin may be performed through measurement of mRNA of caleticulin or protein. Specifically, the mRNA measurement may be performed using a probe or primer that specifically binds to the caleticulin gene, and the protein measurement may be performed using an antibody that specifically binds to the caleticulin protein. I can.

The mRNA measurement is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RT-PCR, RNase protection method. ), northern blotting, or a gene chip, but is not limited thereto.

The protein measurement is measured using two-dimensional (2-D) Electrophoresis, LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry), Western blot, ELISA, tissue immunostaining, or immunoprecipitation assay. It can be, but is not limited thereto.

Another aspect of the present invention is a DNA microarray chip for confirming exposure to air pollutants in which a gene of caleticulin or a nucleic acid complementary thereto is accumulated, and an air in which a protein of caleticulin or an antibody specifically binding thereto is accumulated. It provides a chip for detecting contaminants.

The DNA microarray chip is made of a conventional microarray, except that the gene of caleticulin or a nucleic acid complementary thereto is included. The DNA microarray chip may be attached with a probe corresponding to a gene or a fragment thereof.

A method of manufacturing the microarray chip is as follows. In order to immobilize on the substrate of the DNA microarray chip by using the searched biomarker as a probe DNA molecule, a micropipetting method using a piezoelectric method or a spotter in the form of a pin ), etc. The DNA microarray chip substrate may be coated with an active group such as amino-silane, poly-L-lysine, and aldehyde. In addition, the substrate may be a slide glass, plastic, metal, silicon, nylon film, or nitrocellulose film. In addition, the kit is composed of a buffer solution used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, cNTPs, rNTP (premixed or separately supplied type), a labeling reagent such as a chemical inducer of fluorescent dye, and a washing buffer. I can.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1. Confirmation of changes in caleticulin expression in human bronchial epithelial cells (NHBE) according to exposure of diesel exhaust particles

Example 1.1. Cell culture and treatment of diesel exhaust particles

Human bronchial epithelial cells (cat#. CC-2540; Lonza, Basel, Switzerland) (3000 cells/cm ² ) were placed in a T-flask BEGM™ BulletKit™ (Lonza, Basel, Switzerland) at 37°C, 5% CO ₂ Cultured. The medium was changed every 48 hours until cell saturation reached 90%. The cells used in the experiment were subcultured in a 6-well plate. 24 hours before the experiment, the medium was replaced with a bronchial epithelial cell growth basal medium (BEBM) added with 0.1% (v/v) FBS and incubated for 30 minutes, followed by a diesel exhaust at concentrations of 5 µg/ml and 10 µg/ml. The particles were treated for 8 hours and 24 hours for each concentration, and then incubated again.

Example 1.2. Two-dimensional gel electrophoresis and image analysis

Human bronchial epithelial cells were harvested by centrifuging the cell culture, and disrupted with lysis buffer containing 5 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% Triton X-100, and 2 mM PMSF. After centrifuging the cell lysate at 12,000× g for 30 minutes, a supernatant portion was obtained. Protein concentration was measured with a BCA assay kit (Pierce). Immobiline DryStrips (Amersham Biosciences) were used for isoelectric point electrophoresis (IEF) performed with 1 mg of protein extracted from the IPGphor system (Amersham Biosciences). After IEF separation, the proteins were separated on a two-dimensional gel by SDS-PAGE (Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis).

For image analysis, the gel was visualized using Coomassie Brilliant Blue G-250 according to the manufacturer's instructions. The 2-D gel was scanned in transmission mode of ImageScanner (Amersham Biosciences). Spot detection and matching were performed using ImageMaster 2D version 5.0 (Amersham Biosciences). Digitized images were analyzed and normalized using the ImageMaster program to measure the intensity of the 2-D spot while integrating the optical density as well as the area of the spot ("volume" of the spot). The intensity measurements were normalized and sent to SPSS 12.0 for statistical analysis. All spots identified on each gel were confined to a pH range of 3 to 10 with a molecular weight range of 10 to 200 kDa. The 2-D gel image was used as a master gel and a reference map.

As shown in FIG. 1, in the case of the cell extract treated with diesel exhaust particles, when the diesel exhaust particles were treated at a concentration of 5 μg/ml and 10 μg/ml for 8 hours, 16 protein spots were changed. Proteins have been shown to include calcium regulation, cytoskeleton, cell cycle regulation and signaling. As described above, 16 proteins changed in cells treated with diesel exhaust particles for 8 hours are shown in Table 1 below. In addition, when the diesel exhaust particles were treated at a concentration of 5 µg/ml and 10 µg/ml for 24 hours, 18 protein spots were changed, and the proteins were chloride channels, calcium regulation, glycolytic enzyme, cytoskeleton, and action. It has been shown to contain regulatory and antimicrobial peptides. As described above, 18 proteins changed in cells treated with diesel exhaust particles for 24 hours are shown in Table 2 below. The spots were cut out from the gel, incubated with trypsin to digest the protein in the gel, and then analyzed using LC-MS/MS.

Example 1.3. Western Blot

Protein lysis solution by adding 50 mM Tris-HCL (pH 7.4), 50 mM NaCl, 0.1% (v/v) SDS, 1% (v/v) TritonX-100, 0.5 mM EDTA, and 100 mM PMSF to distilled water. Was prepared. Human bronchial epithelial cells exposed to diesel exhaust particles and lung tissue of an animal model were homogenized by treatment with the protein lysis solution. Then, the sample was centrifuged at 14,000 rpm for 30 minutes at 4°C. The supernatant was collected from the centrifuged solution. Proteins in the supernatant were separated using SDS-PAGE and transferred to a PVDF membrane. To proceed with the blocking process, a TBS buffer to which 0.1% (v/v) Tween 20 and 5% (v/v) BSA were added to the membrane was treated and reacted at room temperature for 2 hours. Thereafter, the membrane was washed 3 times with TBS buffer to which 0.1% (v/v) Tween 20 was added.

The washed membrane was treated with a rabbit anti-caleticulin antibody (1:1000, Novus, St. Charles, MO, USA), and reacted at 4°C overnight. The next day, the membrane was washed 3 times with TBS buffer to which 0.1% (v/v) Tween 20 was added.

The washed membrane was treated with mustard radish peroxidase (HRP)-labeled goat anti-rabbit antibody (Santa Cruz) and reacted for 1 hour. Thereafter, the membrane was washed 3 times with TBS buffer to which 0.1% (v/v) Tween 20 was added. The washed membrane was treated with a mouse anti-goat (Santa Cruz) antibody and reacted for 1 hour. The measurement was performed using a WEST-ZOL plus western blot detection system (iNtRon, SungNam, Korea). The relative amount of protein was compared through β-actin (Sigma-aldrich, St Louis, USA).

As a result of confirming the change in the expression of caleticulin in human bronchial epithelial cells according to the exposure of diesel exhaust particles through western blot, it was found that the expression of caleticulin (CALR) was increased when exposed to diesel exhaust particles (FIG. 2 ).

Example 2. Confirmation of changes in caleticulin expression in mice according to exposure to diesel exhaust particles

Example 2.1. Preparation of mice and exposure to diesel exhaust particles

6-week-old BALB/c female mice were placed in the chamber, and diesel exhaust particles having a concentration of 100 ug/m ³ were exposed to the chamber for 4 weeks and 8 weeks for 5 days each week and inhaled. Control mice were exposed to ambient air. Thereafter, airway hyperresponsiveness (AHR) was measured, bronchoalveolar lavage fluid (BALF) was collected, and lung tissue was processed to measure protein expression.

Example 2.2. Airway hyperresponsiveness measurement

In Example 2.1, the airway hyperresponsiveness of mice exposed to diesel exhaust particles was determined through airway resistance measurement. The degree of airway resistance was evaluated by measuring Penh (enhanced pause). Specifically, the mouse was placed in a chamber and the basal value was measured under normal breathing, and PBS was inhaled for 5 minutes using a nebulizer and then measured for 3 minutes. Then, methacholine was inhaled while gradually increasing to a concentration of 0 mg/ml, 5 mg/ml, 20 mg/ml or 100 mg/ml, and Pehn was measured. Pehn, used as an index of airway resistance, is proportional to the ratio of peak expiratory flow (PEF)/peak inspiratory flow (PIF) and the former expiratory volume for one cycle of breathing. It is expressed as a number of dimensions and has a good correlation with airway resistance and is known as an index of airway contraction.

Penh = (Te/RT-1)(PEF/PIF)

Te = expiratory time

RT = relaxation time

PEF = peak expiratory flow

PIF = peak inspiratory flow

As a result, it was confirmed that mice exposed to high concentrations of diesel exhaust particles exhibited higher airway resistance to methacholine and higher airway hyperresponsiveness (FIG. 3).

Example 2.3. Identification of inflammatory cells in bronchoalveolar lavage fluid

The next day after airway hyperresponsiveness measurement, bronchoalveolar lavage fluid was obtained from mice and stored at -20°C. Lung tissue was treated with 4% (v/v) buffered paraformaldehyde and fixed in paraffin. Lung tissue was cut into 4 μm thick slices. Thereafter, as a result of confirming the inflammatory cells in the bronchoalveolar lavage fluid, the larger the amount of inflammatory cells were found in mice exposed to a high concentration of diesel exhaust particles (FIG.

Example 2.4. Western Blot

Western blot was performed in substantially the same manner as in Example 1.3, except that the paraffinized lung tissue of Example 2.3 was used instead of human bronchial epithelial cells. As a result of confirming the change in expression of caleticulin in mouse lung tissues according to exposure to diesel exhaust particles through western blot, it was found that the expression of caleticulin increased as mice exposed to high concentrations of diesel exhaust particles (FIG. 5 ).

Claims (15)

As a biomarker for confirming exposure to air pollutants including calreticulin protein,
The air pollutant is diesel exhaust particles, a biomarker for checking whether air pollutants are exposed. delete The method of claim 1,
A biomarker for confirming whether the caleticulin is exposed to air pollutants, whose expression is increased by exposure to air pollutants. As a kit for confirming exposure to air pollutants comprising a detection agent for a caleticulin protein or a gene encoding the same,
The air pollutant is diesel exhaust particles, a kit for checking whether air pollutants are exposed. delete The method of claim 4,
The detection agent is a primer or probe capable of complementarily binding to a gene encoding the caleticulin protein, a kit for checking whether air pollutants are exposed. The method of claim 4,
The kit for checking whether air pollutants are exposed, wherein the detection agent is an antibody that specifically binds to the caleticulin protein. 1) measuring the expression level of caleticulin in a sample isolated from an individual suspected of exposure to air pollutants;
2) comparing the expression level and the expression level of the normal control sample; And
3) When the expression level is higher than that of the control group, as a method of providing information on exposure to air pollutants comprising determining that the individual is exposed to air pollutants,
The air pollutant is diesel exhaust particles, a method of providing information on exposure to air pollutants. delete The method of claim 8,
The sample is a bronchoalveolar lavage fluid or lung cells of an individual, a method of providing information on exposure to air pollutants. The method of claim 8,
A method of providing information on exposure to air pollutants, wherein the step of measuring the expression level of caleticulin is performed through measurement of mRNA or protein of caleticulin. The method of claim 11,
The mRNA measurement is performed using a probe or primer that specifically binds to the caleticulin gene, a method of providing information on exposure to air pollutants. The method of claim 11,
The protein measurement is performed using an antibody that specifically binds to the caleticulin protein, a method of providing information on exposure to air pollutants. As a DNA microarray chip for confirming exposure to air pollutants in which caleticulin genes or nucleic acids complementary thereto are accumulated,
The air pollutants are diesel exhaust particles, a DNA microarray chip for checking whether air pollutants are exposed. As a chip for detecting air pollutants in which a protein of caleticulin or an antibody specifically binding thereto is integrated,
The air pollutant is a diesel exhaust particle, an air pollutant detection chip.

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