KR20120072533A - Biomarker composition for diagnosing exposure to nano particle in air and the method for detecting using the same - Google Patents
Biomarker composition for diagnosing exposure to nano particle in air and the method for detecting using the same Download PDFInfo
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Abstract
Description
The present invention relates to a biomarker for diagnosing exposure of nanoparticles in air.
Extensive epidemiologic studies have been associated with the duration of exposure to airborne nanoparticles and the mortality rate of cardiopulmonary disease and lung cancer. However, the physical and chemical properties of airborne micronanoparticles and the mechanism of disease outbreaks are not well known. Recently, health risks caused by nanoparticles are known to be derived from oxidative stress initiated by the formation of reactive oxygen (ROS). ROS cause a change in the state of the redox reaction, resulting in chain reactions of inflammatory and apoptosis pathways. Oxidative stress and inflammation are associated with the destruction of DNA strands and the induction of oxidative DNA damage, and have been shown to be associated with carcinogenesis.
Recently, there are many concerns about ultra-fine particles or nano dust (less than 0.1 um) in the air, which can be more dangerous than large particles because they can settle deep into the lungs due to their small size and large surface area when they enter the human lung. . Recently, microparticles have been reported to cause local anemia / reperfusion, whereas large particles in the air are mainly toxic to the lungs. In other words, it is suggested that diseases caused by particle sizes may be different.
With the recent rapid increase in the production of manufactured nanoparticles, the potential toxicity of manufactured nanoparticles is considered to be an important issue.
Many people are routinely exposed to nanoparticles in the air. However, while exposure to nanoparticles is low in mass, they can be very large in terms of particle number, size, and surface area. In addition, the toxicity of nanoparticles in the air may differ from the particle size as well as the chemical composition of nanoparticles collected in different regions.
However, little research has been conducted on the potential toxicity of nanoparticles in air, and there are few data on the risk of nanoparticles in air. In addition, little is known about the analysis of whole proteins following exposure of nanoparticles to air. So there was a need to develop markers related to nanoparticle exposure.
The present invention to solve the above problems, to provide a biomarker composition for diagnosing exposure of nanoparticles in the air, a kit for diagnosing the exposure of nanoparticles in the air using the biomarker, and to provide a biomarker detection method for diagnosing the exposure of nanoparticles in the air. The purpose.
The present invention provides a biomarker composition for diagnosing exposure to nanoparticles in air containing at least one protein selected from the group consisting of: (1) ATP / GTP binding protein-like 5
The present invention also provides a kit for diagnosing exposure of nanoparticles in air including an antibody that specifically binds to at least one protein selected from the group.
In addition, the present invention comprises the steps of reacting a biological sample with an antibody that specifically binds to at least one protein selected from the group; And comparing the amount of the antigen-antibody complex formed by the reaction with a control group. The method provides a biomarker detection method for diagnosing exposure of nanoparticles in air.
The present invention is the first to reveal the difference in protein expression associated with nanoparticles or microparticles in the air, it can be used as a biomarker for detecting the toxicity of nanoparticles in the air by diagnosing the exposure of the nanoparticles in the air. .
Figure 1 shows the extraction process of nanoparticles in air.
Figure 2 shows cytotoxicity when treated to 20 ug / ml of nanoparticles in air to human bronchial epithelial cells.
3 and 4 shows that the differences seen in the 13 proteins the expression which appears after the control and the PM 0 .056 nanoparticles in air treatment.
5 is an enlarged view of the protein of FIG. 4.
6 is a result of reconfirming the increase of
Hereinafter, the present invention will be described in detail.
The present invention screens proteins that are specifically expressed in a biological sample of a subject exposed to nanoparticles in air compared to a subject not exposed, thereby securing the proteins described in Table 1 as biomarkers for diagnosing exposure of nanoparticles in air. It was.
The term "biomarker" refers to an organic biomolecular substance capable of diagnosing exposure by showing an increase or decrease in cells, tissues, etc. exposed to nanoparticles in air compared to normal cells, and in the present invention, the biomarker described in Table 1 By confirming their expression at the protein level, it was possible to diagnose the exposure. In this case, the number of proteins used for the analysis is not limited, and one or more proteins may be appropriately selected according to the purpose.
One embodiment of the invention (1) ATP / GTP binding protein-like 5 isoform 3 (ATP / GTP binding protein-like 5
In addition, one embodiment of the present invention (2) keratin 7 (Keratin 7, Genbank Accession No. NP — 005547); (3) Keratin 17 (Genbank Accession No. NP — 000413); And (8) Annexin A2, isoform CRA_c (Annexin A2, isoform CRA_c, Genbank Accession No. EAW77587); It provides a diagnostic biomarker composition for diagnosing exposure of nanoparticles in the air, which comprises essentially one or more proteins selected from the group consisting of.
Expression of keratin 7 (spot 2 and spot 10) in the biomarker for diagnosing exposure to airborne nanoparticles according to an embodiment of the present invention was reduced by the nanoparticles in the air. Furthermore, keratin 8 (spot 4), keratin 17 (Spot 3), keratin 18 (spot 6) and keratin 1 (spot 11) increased expression. Keratin is expressed in various forms in a tissue specific and differentiation dependent manner. Keratin intermediate filaments are the major cytoskeletal components of epithelial cells and they are composed as a mixed polymer of type I (KRT9-KRT20) and type II (KRT1-KRT8) intermediate filament proteins. The expression of keratin is widely used in the detection of various carcinogens because the expression of the cytoskeleton is mainly preserved during tumor formation.
Keratin 7 (spot 2 and spot 10) is originally described as 55 kDa type-II keratin expressed in mesothelial tissue.
Myosin XVIIIB (spot 5) in the biomarker for diagnosing exposure to airborne nanoparticles according to an embodiment of the present invention has increased expression by airborne nanoparticles. Myosin MVIIIB, which constitutes an actin-based cellular kinetic protein, uses ATP to play a fundamental role in eukaryotic motility such as cytoplasmic division, phagocytosis, and trafficking. The protein is also involved in determining the timing of the development and differentiation of unicellular and multicellular cells.
According to an embodiment of the present invention, the protein group including the Kelch motif in the biomarker for diagnosing exposure to the nanoparticles in the air, the Kelch protein (spot 7), has increased expression by the nanoparticles in the air. Kelch domains typically occur when 5-7 sets of quench are repeated to form a β-propeller tertiary structure. The function of the kelch protein is not clear but is known to be associated with the actin structure.
The annexin A2 protein (spot 8) in the biomarker for diagnosing exposure to airborne nanoparticles according to an embodiment of the present invention has increased expression by airborne nanoparticles. Annexin A2 is known to be involved in a variety of processes, including exocytics, motility of epithelial cells, the association of protein complexes with actinic membranes and actin cytoskeleton, fibrin degradation, and ion channel formation. In addition, annexin A2 play an important role in the regulation of cell growth and signaling pathways in Ca + 2-dependent protein attached to the phospholipid.
MBD1 (spot 9) in the biomarker for diagnosing exposure to airborne nanoparticles according to an embodiment of the present invention has been reduced expression by airborne nanoparticles. MBD1 is part of a protein with a domain attached to methylated CpG and is known to inhibit translation of methylated and unmethylated promoters. Methylated CpG moieties are recognized by protein factors with domains attached to methylated CpG. To date, five types of MBD, MeCP2, MBD1, MBD2, MBD3 and MBD4, have been known in mammals. Among them, MBD1 has been reported to play an important role in forming and maintaining local chromosomal states to regulate gene activity. In addition to regulatory regulation, MBD1 is involved in DNA repair through interaction with methylpurine DNA glycosylase, which removes purines damaged by methylation or by oxidative factors. In addition, MBD proteins are found in the same methylated promoter of human cancer cell lines.
Recently, environmental factors are known to be related to abnormal changes in epigenetic pathways. Although epigenetic changes are insignificant and progress very slowly, it is difficult to explain the definite relationship between environmental factors, epigenetic changes and disease, but epigenetic changes explain the correlation between disease and environmental factors. The fact that the expression of MBD1 decreased with exposure to air nanoparticles suggests that the epigenetic pathway has been altered by nanoparticles in air.
Exposure to nanoparticles in the air according to an embodiment of the present invention TPI (spot 12), a trisaccharide phosphate isomerase in the biomarker for diagnosis, increased expression by the nanoparticles in the air. Trisaccharide phosphate isomerase is a glycolygic enzyme that catalyzes the interconversion of 3-phosphate D-glyceraldehyde and dihydroxyacetone phosphate. Characteristics of TPI dysfunction cause chronic hemolytic anemia, neurological disorders, and rapid killing of homozygotes and heterozygotes.
Bimentin (spot 13) in the diagnostic biomarker exposure to nanoparticles in the air according to an embodiment of the present invention increased the expression by the nanoparticles in the air. Bimentin, a cytoplasmic intermediate fiber protein, is a marker of mesenchymal cells that are not expressed in epithelial cells. Recently, however, the expression of VIM in epithelial cancer cells has been shown to be associated with local invasion and metastatic cancer through mesenchymal transition. The association between abnormal VIM overexpression and cancer metastasis has been reported in melanoma, breast cancer, uterine cancer, liver cancer and prostate cancer. Non-mentin overexpression was also increased in lung cancer as well as many cancers of epithelial origin. In this study, bimentin was weighted by nanoparticles in air.
ATP / GTP-binding protein-like 5 isoform 3 (spot 1) in the biomarker for diagnosing exposure to nanoparticles in the air according to an embodiment of the present invention was reduced by the nanoparticles in the air. Binding protein-like 5 is a cytosolic carboxypeptidase-like protein 5, an enzyme that hydrolyzes the C-terminus of protein peptide bonds and plays an important role in the activation of proteins through hydrolysis. .
Biomarker according to an embodiment of the present invention is a variety of modified forms by replacing, deleting and / or adding the amino acid residue of the natural amino acid sequence to another residue as long as it can be used to detect the exposure of nanoparticles in the air Can be. It may also be in a form in which modifications such as phosphorylation, saccharification, methylation, and pansylation have occurred.
In addition, an embodiment of the present invention is a composition for detecting a biomarker for diagnosing exposure of nanoparticles in the air including an antibody specifically binding to one or more proteins, specifically 1 to 12 proteins of the following Table 1 To provide. The biomarker detection composition may include an agent for measuring the protein level, specifically, an antibody. The antibody includes all polyclonal antibodies, monoclonal antibodies and recombinant antibodies that specifically bind to the protein. Also included are functional fragments of antibody molecules, as well as complete forms of antibodies.
Polyclonal antibodies can be produced by methods well known in the art for injecting the biomarker protein antigens described above into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.
Monoclonal antibodies can be made by fusion methods well known in the art. One of the two cell populations that are fused to make a "hybridoma" secreting monoclonal antibodies utilizes cells from an immunologically suitable host animal, such as a mouse injected with a marker protein antigen, and into the other population. Uses cancer or myeloma cell lines. These two populations of cells are fused by methods well known in the art, such as polyethylene glycol, and then antibody-producing cells are propagated by standard tissue culture methods. After obtaining a homogeneous cell population by subcloning by the limited dilution technique, hybridomas capable of producing antibodies specific for the marker protein are mass-produced in vitro or in vivo according to standard techniques. Incubate. The monoclonal antibodies produced by the hybridomas may be used without purification, but in order to obtain the best results, the monoclonal antibodies are preferably purified and used according to methods well known in the art. Monoclonal antibodies can be identified and separated by using a marker protein to search for a recombinant combinatorial immunoglobulin library (eg, antibody phage display library) and then isolate members of the immunoglobulin library that bind to the marker protein. Phage display library preparation and screening kits are commercially available.
In addition, an embodiment of the present invention provides a kit for diagnosing exposure of nanoparticles in air including an antibody that specifically binds one or more proteins, specifically, 1 to 12 proteins of Table 1 below. The diagnostic kit may be a kit for diagnosing lung toxicity by exposure to nanoparticles in air.
A "kit" is intended to be any preparation (eg, package or container) that includes one or more reagents such as antibodies, nucleic acid probes, and the like for specifically detecting a marker of the present invention. The kit may be formulated, dispensed, or sold as a unit for carrying out the method of the present invention. The diagnostic kit further comprises other components or devices widely used in the art for the biomarker detection composition. Applications, including can be produced. And a kit for ELISA comprising antibodies specific for the protein and additionally a reagent capable of detecting the bound antibody, such as labeled secondary antibodies, chromophores, enzymes and substrates thereof. have. As another example, the kit may be manufactured as a kit for a protein chip containing essential elements necessary for carrying out the protein chip.
In addition, the present invention comprises the steps of reacting a biological sample with an antibody that specifically binds to one or more of the proteins listed in Table 1; And comparing the amount of the antigen-antibody complex formed by the reaction with a control group. The method provides a biomarker detection method for diagnosing exposure of nanoparticles in air. The biomarker detection method may be a biomarker detection method for diagnosing lung toxicity by exposure to nanoparticles in air.
Specific analysis method of the step of comparing the amount of the antigen-antibody complex with a control group, Western blot, ELISA, radioimmunoassay, radioimmunoproliferation method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunosuppression Precipitation assays, complement fixation assays, FACS, protein chips, and the like, but are not limited thereto. ELISA is a direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support, followed by reaction with another antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support. Various ELISA methods, such as indirect sandwich ELISA using the labeled secondary antibody which recognizes this antibody, are included.
In the biomarker detection method according to an embodiment of the present invention, the amount of antigen-antibody complex formed can be quantitatively measured through the magnitude of a signal of a detection label. Such detection labels may be selected from enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but are not limited thereto. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapase, glucose oxidase, hexokinase, GDPase, RNase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate decarboxylase, β -Latamase and the like, but is not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include, but are not limited to, ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, and the like. To detect antigen-antibody complex formation, colorimetric method (electrometric method), electrochemical method (electrochemical method), fluorescence method (fluorimetric method), luminescence (luminometry), particle counting method (particle counting method), visual measurement (visual A method selected from assessment and scintillation counting method may be used, but is not limited thereto.
The biological sample refers to a cell, tissue or bodily fluid sample capable of detecting a biomarker for diagnosing exposure to nanoparticles in the air, and specifically, blood, urine, lymph, female fluid, biopsy, sweat, feces, tears, or sputum. Can be mentioned.
In the detection method, the biomarker is (3) keratin 17 (
In the detection method, the biomarker includes (1) ATP / GTP binding protein-like 5 isoform 3 (ATP / GTP binding protein-like 5
Differences in the amount of expression between the biological sample of the detection subject and the control group may be compared with absolute or relative differences.
Hereinafter, the present invention will be described in detail by way of examples.
However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
Example
1. Cell line culture and airborne nanoparticle treatment
In the present invention, human lung cells, ie bronchial epithelial cells, BEAS-2B (Human Bronchial Epithelial cell line) were used. BEAS-2B cells were purchased from ATCC with ATCC number CRL-9609 ™ and frozen and stored in liquid nitrogen at -190 ° C. Medium and cells were placed in a 150 mm culture dish and cultured in a 5% CO 2 , 37 ° C incubator. Cell culture status and cell number were measured using an inverted microscope and hematocytometer. In order to supply nutrients to the cells, a medium in which 1% antibiotics (penicillin-streptomycin) was added to BEGM (Bronchial Epithelial Growth Media, Lonza) medium was prepared and used as cell culture medium.
Human bronchial epithelial cells (BEAS-2B) were placed in a 150 mm cell culture flask with 3 × 10 6 cells in a medium with 5% CO 2 , 37 ° C incubator for 24 hours. After incubation, the cells were treated with 355 ug of air nanoparticles extracted from nanofilters collected from Gwangyang and Asan, respectively, for 24 hours. The remaining control group used a solution extracted with empty soda with DMSO.
2. Cell count measurement
The cell number was measured using a hemocytometer and trypan-blue. After treatment with Trypsin-EDTA solution, 10 ul of the isolated cell culture and trypan-blue solution [0.4% (w / v) trypan-blue, 0.8% (w / v) NaCl, 0.06% (w / v) KH 2 PO 4 , 0.05% (w / v) methylene-p-hydroxybenzonate (pH 7.2)] 10 ul were mixed well and 10 ul of the mixture was taken on a hemocytometer and the cell number was measured using a reversed phase microscope. Four sections of 0.1 mm 3 of the hemocytometer were determined to measure the number of living cells without staining blue.
3. Extraction of Nanoparticles
15 nanotubes of <56 nm in the air collected by the nanoparticle measuring instrument were placed in 15 tubes, and then mixed with a vortexer for 10 minutes, and then mixed with a vortexer for 10 minutes. The effect was observed. Figure 1 shows the extraction process of nanoparticles in air.
4. Cell harvesting and protein quantification
Human bronchial epithelial cells (BEAS-2B) were placed in medium to 3 × 10 6 cells and incubated for 24 hours in 150 mm Jano, and then treated with empty pores and 355 ug of airborne nanoparticles for 24 hours. Cells were washed with 1 × PBS, 1/2 PBS, 1/10 PBS and cells were harvested using scraper and centrifuged for 15 minutes at 4, 15,000 rpm. The separated cells were placed in a rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, and 100 mM DTT, and the cell mixture was crushed using an ultrasonic grinder and centrifuged at 4, 15,000 rpm for 30 minutes. Only got. Protein quantification of the obtained supernatant was performed using BSA (bovine serum albumin) as a standard protein and reacting Bio-rad's protein assay 200 with BSA at a concentration of 0-60 mg / ml. After absorbance was measured to prepare a standard curve, the samples were measured under the same conditions to determine protein concentration.
5. SDS-PAGE Electrophoresis
Each sample contains 0.06 M Tris-HCl (pH 6.8), 2% SDS, 14.4 mM β-mercaptoethanol, 25% glycerol, 0.1% bromophenol blue after protein quantification After mixing well with the sample buffer (sample buffer) and heated in water for 5 minutes, the protein was completely denatured. The electrophoresis was performed by injecting a sample and a standard protein sample into a flat gel containing a 5% acrylamide fast gel, a stacking gel and a 10% separating gel. 25 mM Tris-HCl buffer (pH 8.3) containing 192 mM glycine and 10% SDS (w / v) was used.
6. Analysis by Western Blot
Cells were lysed by adding 1 ml of lysis buffer [50 mM Tris, 1% PMSF, 1% Triton X-100] to the nanoparticle treated cells. The dissolved solution was centrifuged to remove cell by-products, supernatant was taken and protein quantified. Each protein sample in the same amount was then sample buffer [0.05 M Tris-Cl buffer (pH 6.8). 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.001% bromophenol blue] and then heated at 100 to 5 minutes to completely denature the protein. Samples and standard proteins were separated on stacking gels of 5% acrylamide and running gels of 10% acrylamide. The separated protein was transferred to 250 mA for 1 hour with PVDF membrane with the blot kit filled with transfer buffer (192 mM glycine, 25 mM Tris, 20% methanol). In 5% degreasing solution, the mixture was reacted at room temperature for 2 hours, washed with TBST, and then reacted with
7. Two-dimensional electrophoresis
7-1. Isoelectric point electrophoresis
The amount of protein in the cells treated with nanoparticles was 300, 250 rehydration solutions (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% ampholytes, 100 mM DTT) , 0.01% bromophenol blue) was used to dissolve the protein. Proteins of each condition dissolved in 250 rehydration solutions were injected into the IPG holder, and a 13 cm immobilized dry strip (pH 3-) made to move according to the pH of the protein for isoelectric electrophoresis. 10 NL, Amer sham Bioscience) was used to rehydrate for 12 hours. At 200 V / 200 Vh, 500 V / 500 Vh, 1,000 V / 1,000 Vh, 8,000 V / 13,500 Vh, and 8,000 V / 100,000 Vh conditions, the protein was shifted in the electric field according to the pH of the protein.
7-2. Equilibration
Strips subjected to isoelectric point electrophoresis were equilibrated buffer [6 M urea, 0.375 M Tris-HCl (pH 8.8), 2% SDS, 20% glycerol, 5 mM tributylphosphine, 2.5% acrylamide, 0.01% bromophenol Blue] strip and equilibrate twice for 20 minutes.
7-3. 2-Dimensional SDS-Polyacrylamide Gel Electrophoresis
After the 12.5% acrylamide gel was prepared, the strips on which the isoelectric point electrophoresis was completed were placed on the gel, fixed with 1% agarose gel, and then subjected to two-dimensional electrophoresis. The current was first developed with 25 mA per gel for 15 minutes and then the protein was shifted according to molecular weight with 50 mA per gel for 5 hours.
7-4. Gel Dyeing and Bleaching
The developed gel was subjected to Coomassie blue G-250 staining solution (34% methanol, 0.1% Coomassie G-250, 17% ammonium sulfate, 3% phosphoric acid) to identify proteins isolated from the gels. After staining for more than a hour, it was decolorized using 5% acetic acid.
7-5. Gel image analysis
Image Master TM after scanning gels stained with UMAX powerLoook 1100 for gel image analysis Image analysis was performed with (2D software, Amersham). Three gels were analyzed per condition for accurate analysis.
8. MALDI-TOF Analysis
Protein mass spectrometry was used with Ettan MALDI-TOF (Amersham Bioscience). Peptide mass values analyzed using ProFound (Webpro Found.exe) manufactured by Rockfeller University were investigated through a database and proteins were identified based on the results. Select the spot changed by GSNO, cut the spot from the gel for analysis, put it in a tube and mix 1: 1 with 30 mM potassium ferricyanide and 100 mM sodium thiosulfate. It was added to the tube containing the spot. The dye was left until the dye disappeared and washed with distilled water until the dye was completely removed. After reacting in 200 mM ammonium bicarbonate for 20 minutes, the mixture was washed with distilled water. After dehydration with acetonitrile until the gel pieces became opaque white, the tube was placed in a vacuum centrifuge and dried for 30 minutes. 5-10 ng / ml of trypsin and ammonium bicarbonate were added and left at 37 ° C. for at least 12 hours. Thereafter, 10-20 ml of a H 2 O: acetonitrile (50:50) solution containing 5% TCA was added, and the peptide fragments were extracted three times. The extract was placed in a vacuum centrifuge and dried for 2 hours and analyzed using MALDI-TOF.
9. Results
With the development of MOUDI and nano-MOUDI, nanoparticles having a size of less than 0.056 um (56 nm) and ultra-fine particles having a size of less than 0.1 um (100 nm) can be separated from each other. Thus ultrafine particle in air is ultrafine (PM 0 .1) not only can be split back into the nanoparticles (PM 0 .056). In the present invention, were with a MOUDI sampler collect PM 0 .056 nanoparticles having a diameter of less than 0.056um in the atmosphere. To understand the toxicity of nanoparticles in the air, we analyzed proteins that were differentially expressed by airborne nanoparticle treatment in bronchial epithelial cells by PM 0.056 . In PM 0 .056 was detected in a variety of heavy metals such as Mn, Zn, Ni, Cu, Pb, Cr.
2 shows that the survival rate of the cells is lowered by the nanoparticle treatment in air. When the nanoparticles in the
FIG. 3 shows the control group and FIG. 4 shows the expression difference of 13 proteins after treatment with PM 0.056 , which is a nanoparticle in air. 5 shows an enlarged view of a protein showing differential expression changes.
The following table shows the proteins showing differential expression changes by treatment with air nanoparticles.
number
change
8.87
5.40
4.97
5.52
5.27
5.78
5.93
7.96
5.40
8.16
6.51
In Table 1, PM0.056 treatment resulted in increased expression of
6 is a result of reconfirming the increase of
The results of this study is the first proteomic studies revealed a difference in protein expression according to the exposure of the PM 0 .056. The 12 proteins, represented by the 13 spots identified in this study, could be used as biomarker candidates for the potential toxicity of nanoparticles in air.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (6)
(1) ATP / GTP binding protein-like 5 isoform 3 (ATP / GTP binding protein-like 5 isoform 3, Genbank Accession No. NP — 001030584);
(2) keratin 7 (Keratin 7, Genbank Accession No. NP — 005547);
(3) Keratin 17 (Genbank Accession No. NP — 000413);
(4) Keratin 8 (Genbank Accession No. AAA35763);
(5) Myosin XVIIIB, Isoform CRA_d (Myosin XVIIIB, isoform CRA_d, Genbank Accession No. EAW59706);
(6) Keratin 18 (Genbank Accession No. CAA31377);
(7) Kelch protein-like protein; Similar to Kelch proteins (similar to BAA77027, Genbank Accession No. AAF03529);
(8) Annexin A2, isoform CRA_c (Annexin A2, isoform CRA_c, Genbank Accession No. EAW77587);
(9) MBD1-containing chromatin binding factor 2 (MBD1-containing chromatin associated factor 2, Genbank Accession No. AAT66299);
(10) Keratin 1 (Genbank Accession No. AF237621_1);
(11) Triosephosphate isomerase related human genetic disorders (Genbank Accession No. 1HTI_A); And
(12) Vimentin (Genbank Accession No. AAA61279).
The composition comprises (2) Keratin 7, Genbank Accession No. NP — 005547;
(3) Keratin 17 (Genbank Accession No. NP — 000413); And
(8) biomarker for diagnosing exposure to nanoparticles in the air, comprising at least one protein selected from the group consisting of annexin A2 and isoform CRA_c (Annexin A2, isoform CRA_c, Genbank accession number EAW77587); Composition.
(1) ATP / GTP binding protein-like 5 isoform 3 (ATP / GTP binding protein-like 5 isoform 3, Genbank Accession No. NP — 001030584);
(2) keratin 7 (Keratin 7, Genbank Accession No. NP — 005547);
(3) Keratin 17 (Genbank Accession No. NP — 000413);
(4) Keratin 8 (Genbank Accession No. AAA35763);
(5) Myosin XVIIIB, Isoform CRA_d (Myosin XVIIIB, isoform CRA_d, Genbank Accession No. EAW59706);
(6) Keratin 18 (Genbank Accession No. CAA31377);
(7) Kelch protein-like protein; Similar to Kelch proteins (similar to BAA77027, Genbank Accession No. AAF03529);
(8) Annexin A2, isoform CRA_c (Annexin A2, isoform CRA_c, Genbank Accession No. EAW77587);
(9) MBD1-containing chromatin binding factor 2 (MBD1-containing chromatin associated factor 2, Genbank Accession No. AAT66299);
(10) Keratin 1 (Genbank Accession No. AF237621_1);
(11) Triosephosphate isomerase related human genetic disorders (Genbank Accession No. 1HTI_A); And
(12) Vimentin (Genbank Accession No. AAA61279).
Comparing the amount of the antigen-antibody complex formed by the reaction with a control; Biomarker detection method for diagnosing exposure of nanoparticles in air, comprising:
(1) ATP / GTP binding protein-like 5 isoform 3 (ATP / GTP binding protein-like 5 isoform 3, Genbank Accession No. NP — 001030584);
(2) keratin 7 (Keratin 7, Genbank Accession No. NP — 005547);
(3) Keratin 17 (Genbank Accession No. NP — 000413);
(4) Keratin 8 (Genbank Accession No. AAA35763);
(5) Myosin XVIIIB, Isoform CRA_d (Myosin XVIIIB, isoform CRA_d, Genbank Accession No. EAW59706);
(6) Keratin 18 (Genbank Accession No. CAA31377);
(7) Kelch protein-like protein; Similar to Kelch proteins (similar to BAA77027, Genbank Accession No. AAF03529);
(8) Annexin A2, isoform CRA_c (Annexin A2, isoform CRA_c, Genbank Accession No. EAW77587);
(9) MBD1-containing chromatin binding factor 2 (MBD1-containing chromatin associated factor 2, Genbank Accession No. AAT66299);
(10) Keratin 1 (Genbank Accession No. AF237621_1);
(11) Triosephosphate isomerase related human genetic disorders (Genbank Accession No. 1HTI_A); And
(12) Vimentin (Genbank Accession No. AAA61279).
The biomarker comprises (3) Keratin 17 (Genbank Accession No. NP — 000413);
(4) Keratin 8 (Genbank Accession No. AAA35763);
(5) Myosin XVIIIB, Isoform CRA_d (Myosin XVIIIB, isoform CRA_d, Genbank Accession No. EAW59706);
(6) Keratin 18 (Genbank Accession No. CAA31377);
(7) Kelch protein-like protein; Similar to Kelch proteins (similar to BAA77027, Genbank Accession No. AAF03529);
(8) Annexin A2, isoform CRA_c (Annexin A2, isoform CRA_c, Genbank Accession No. EAW77587);
(10) Keratin 1 (Genbank Accession No. AF237621_1);
(11) Triosephosphate isomerase related human genetic disorders (Genbank Accession No. 1HTI_A); And
(12) at least one protein selected from the group consisting of Vimentin (Genbank Accession No. AAA61279),
The method of detecting a biomarker for diagnosing exposure of nanoparticles in air, characterized in that it further comprises the step of determining that the protein expression in the biological sample to be detected is exposed to nanoparticles in the air when compared to the control.
The biomarker comprises (1) ATP / GTP binding protein-like 5 isoform 3 (Genbank Accession No. NP — 001030584);
(2) keratin 7 (Keratin 7, Genbank Accession No. NP — 005547); And
(9) at least one protein selected from the group consisting of MBD1-containing chromatin associated factor 2 (Genbank Accession No. AAT66299),
When the amount of expression of the protein in the biological sample to be detected is reduced compared to the control group, further comprising the step of determining that the exposure to nanoparticles in the air biomarker detection method for diagnosing whether the nanoparticles in the air.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MD4200C1 (en) * | 2012-07-05 | 2013-09-30 | Институт Микробиологии И Биотехнологии Академии Наук Молдовы | Method for assessing the toxicity of nanoparticles by means of red microalga Porphyridium cruentum |
| KR101381079B1 (en) * | 2012-07-23 | 2014-04-10 | 아주대학교산학협력단 | Evaluation Method of Toxicity of Nanoparticles |
| KR20160114716A (en) * | 2014-02-05 | 2016-10-05 | 후지레비오 디아그노스틱스 에이비 | Composition and method for detecting malignant neoplastic disease |
| KR20180077806A (en) * | 2016-12-29 | 2018-07-09 | 순천향대학교 산학협력단 | Biomarker for identification of exposure to air pollutants and method of identifying exposure to air pollutants using the same |
| CN116202918A (en) * | 2023-03-07 | 2023-06-02 | 中国疾病预防控制中心环境与健康相关产品安全所 | Method and system for identifying key toxic components of atmosphere particulate matter component mixed exposure |
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2010
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MD4200C1 (en) * | 2012-07-05 | 2013-09-30 | Институт Микробиологии И Биотехнологии Академии Наук Молдовы | Method for assessing the toxicity of nanoparticles by means of red microalga Porphyridium cruentum |
| KR101381079B1 (en) * | 2012-07-23 | 2014-04-10 | 아주대학교산학협력단 | Evaluation Method of Toxicity of Nanoparticles |
| KR20160114716A (en) * | 2014-02-05 | 2016-10-05 | 후지레비오 디아그노스틱스 에이비 | Composition and method for detecting malignant neoplastic disease |
| KR20180077806A (en) * | 2016-12-29 | 2018-07-09 | 순천향대학교 산학협력단 | Biomarker for identification of exposure to air pollutants and method of identifying exposure to air pollutants using the same |
| CN116202918A (en) * | 2023-03-07 | 2023-06-02 | 中国疾病预防控制中心环境与健康相关产品安全所 | Method and system for identifying key toxic components of atmosphere particulate matter component mixed exposure |
| CN116202918B (en) * | 2023-03-07 | 2023-10-13 | 中国疾病预防控制中心环境与健康相关产品安全所 | Method and system for identifying key toxic components of atmosphere particulate matter component mixed exposure |
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