KR101138953B1 - The biomarkers for identification of exposure to disruptors activating thyroid peroxidase and the identification method of disruptors activating thyroid peroxidase exposure using the same biomarkers - Google Patents

Abstract

The present invention provides a DNA microarray chip for confirming exposure to chemicals that enhance the activity of thyroid peroxidase and a method for confirming exposure to chemicals that enhance the activity of thyroid peroxidase using the microarray chip. Specifically, a biomarker for checking exposure to carbaryl or dibutyl phthalate, a chemical that activates thyroid peroxidase, which plays an important role in thyroid hormone synthesis, a DNA microarray chip integrated with the biomarker, and a method of using the same The biomarker of the present invention is useful for monitoring and determining the contamination of chemicals that activate thyroid peroxidase in environmental samples using ferritin-related genes selected through DNA microarrays as biomarkers. Can be used It can be used as a tool to identify mechanisms of toxic effects caused by chemicals that activate thyroid peroxidase.

Description

Increased thyroid peroxidase activity Biomarkers for identifying chemical exposures and methods of identifying using them {The biomarkers for identification of exposure to disruptors activating thyroid peroxidase and the identification method of disruptors activating thyroid peroxidase exposure using the same biomarkers}

The present invention relates to a biomarker for confirming exposure to chemicals activating thyroid peroxidase and a method of identifying the same.

Sodium iodide symporter, type 5 I-deiodinase, TH transport protein transthyretin, TH receptor, etc. It has been reported to be an important target for the study of the action that causes the disorder (Schmutzler C. et. al ., Endocrinology , 148 (6): 2835-2844, 2007). In addition, thyroid peroxidase (TPO) is used to target thyroid hormone production by the inhibition and activation of thyroid peroxidase (TPO) as a target.

Thyroid peroxidase is the iodine component of the was reported to play an important role in thyroid hormone biosynthesis of proteins, inhibition of thyroid hormone, thyroxine (T ₄₎ for controlling the body metabolism of the thyroid peroxidase located in the cell membrane of thyroid epithelial cells It has been reported to promote deficiency and goiter development, making it an important target for observing the thyroid gland's toxicity to substances that interfere with thyroid peroxidase activity (Schmutzler C. et. al ., Endocrinology , 148 (6): 2835-2844 2007). Thyroid peroxidase has been reported to promote the production of thyroxine by increasing the activity of thyroid peroxidase by acting on catalyzed diiotyrosine (DIT) (Turner, CD et. al ., Arch . Biochem. Biophys ., 222 (1): 245-258, 1983). Tiodothyronine (T ₃ ) -dependent Graves' disease has been reported to increase thyroid peroxidase activity among thyroid hormones (Takamatsu, J. et. al ., Rinsho Ketsueki . 29 (11): 2037-2041, 1988).

Among the endocrine disrupting chemicals, carbaryl and dibutyl phthalate have been reported to cause thyroid hormone disorders (Cheng S et. al ., Ther . Clin . Risk Manag . 2 (3): 297-301, 2006; Schmutzler C et al ., Endocrinology , 148 (6): 2835-2844, 2007). Cabaril is Clarias , a fish in the marble shark Bacterial dysfunction caused by changes in thyroid hormone circulation by a decrease in thyroxine (T ₄ ), an increase in triiodothyronine (T ₃ ), and an increase in T ₃ / T ₄ ratio (Cheng S et. al ., Ther . Clin . Risk Manag . 2 (3): 297-301, 2006). In addition, carbaryl has been reported to bind to thyroid hormone receptors to inhibit transactivation by thyroid hormones (Sun H et. al ., Toxicology, 249: 238-242, 2008). Dibutylphthalate induces hyperthyroidism in rat in vivo experiments and superoxide dismutase and glutathione peroxidase in testis of rats with hyperthyroidism. Increased activity of peroxidase has been reported to cause oxidative damage. In addition, PPAR-γ is known to increase the activity of peroxisome proliferator-activated receptor-γ. Hyperthyroidism is caused by oxidative damage to tissue by increased gene expression of PPAR-γ and metabolic activity of dibutyl phthalate. It has been reported (Lee E et al ., Environ Toxicol . 22 (3): 245-255, 2007).

Although some studies on thyroid hormone activity, hormone receptor binding, and gene expression changes of thyroid hormone obstructions have been reported, they are mostly limited to studies using mice. As mentioned above, there is not enough risk assessment data in the human body even in the possibility of human thyroid disorder due to increased activity of thyroid peroxidase, and the detection method for exposure is also GC-MS (Gas Chromatography-Mass Spectrometer). Or classical methods such as High Performance Liquid Chromatography (HPLC). When using the GC-MS or HPLC method, quantitative analysis is possible, but appropriate conditions for analysis must be set, and expensive equipment is required. Therefore, faster and easier screening methods, such as real-time reverse transcription polymerase chain reaction (RT-PCR) or DNA microarrays using primers, allow for rapid risk assessment in humans. It is an important task to identify and utilize molecular indicators that can explore the toxic effects of toxicants and to take appropriate measures and control against chemical exposure that increases thyroid peroxidase activity.

Several genome sequencing projects, including six mammals and 292 microbes, have been completed and reported to the National Center for Biotechnology Information (NCBI). Genome-wide expression studies are being conducted to study the function of genes based on the vast amount of data obtained. That is, DNA microarray analysis is performed to analyze the expression of thousands of genes in one experiment (Schena, M., et. al ., Proc . Natl . Acad . Sci . USA 93: 10614-10619, 1996).

Microarrays integrate a set of oligonucleotides of cDNA (complementary DNA) or 20-25 base pairs in length on glass. cDNA microarrays are produced by labs in schools or by companies such as Agilent and Genomic Solutions, either mechanically immobilizing the cDNA collection on a chip or by using ink jetting (Sellheyer K. et. al ., J. Am . Acad . Dermatol . 51: 681-692, 2004). Oligonucleotide microarrays are made by Affymetrix's method of direct synthesis on a chip using photolithography, and Agillent's, etc., are produced by immobilizing synthesized oligonucleotides (Sellheyer, K.). et al ., J. Am . Acad . Dermatol . 51: 681-692, 2004).

For analysis of gene expression, RNA is obtained from samples such as tissues and hybridized with oligonucleotides in a DNA microarray. The obtained RNA is labeled with fluorescence or isotope and converted to cDNA. Oligomicroarrays mainly label the control and experimental groups with two different fluorescences (eg Cy3 and Cy5), perform hybridization reactions simultaneously on the same chip, and then optically scan images to obtain fluorescence intensity and analyze the results. . Gene expression is determined by the ratio of the two fluorescence intensities (Somasundaram, K. et. al ., Genomics Proteomics I: 1-10, 2002).

In combination with recent research on toxic genomics (Toxicogenomics), an advanced technique using DNA microarray technology, high-throughput expression in specific tissues or cell lines by all chemicals including pharmaceuticals and new drug candidates, as well as representative environmental pollutants Analysis and quantitative analysis of the expression patterns of genes are now possible. Accordingly, by analyzing the frequency of expression of specific genes in specific cells, it is possible to discover genes related to adverse effects of drugs and harmful effects of environmental pollutants, and through this, molecules according to the harmful effects of environmental pollutants, drugs, and side effects. It will be possible to understand the mechanisms and further to search for substances that cause toxicity and side effects.

Therefore, the present inventors have developed a gene expression profile of carbaryl and dibutyl phthalate among chemicals that increase the activity of thyroid peroxidase by using oligomicroarray accumulated with 44,000 human genes in human thyroid vesicle cancer cells. By observing and analyzing the FTC-238 / hrTPO / RSK008 cell line, which is a recombinant cell line, it is possible to detect genes that are overexpressed by chemicals that increase the activity of thyroid peroxidase to detect chemicals that increase the activity of thyroid peroxidase. The present invention has been completed by establishing a biomarker and a method for confirming exposure using the same.

An object of the present invention is to expose a DNA microarray chip incorporating a chemical specific biomarker that increases the activity of thyroid peroxidase and exposure to a chemical that increases the activity of thyroid peroxidase using the DNA microarray chip of the present invention. It is to provide a way to check whether.

In order to achieve the above object, the present invention provides an oligonucleotide comprising all or part of the nucleic acid sequence of Genebank NM_000146 (ferritin, light polypeptide) or Genebank NM_002032 (ferritin, heavy polypeptide 1). Or it provides a DNA microarray chip for confirming the exposure to chemicals that increase the activity of thyroid peroxidase, its complementary oligonucleotides are integrated.

In addition, the present invention provides an RNA sample isolated from an individual (experimental group) and a control group suspected of exposure to a chemical that increases the activity of thyroid peroxidase.

1) labeling the experimental and control groups with different fluorescent materials while synthesizing with cDNA;

2) hybridizing cDNA labeled with different fluorescent materials of step 1) with the DNA microarray chip;

3) analyzing the reacted DNA microarray chip of step 2); And,

4) Whether the exposure to the chemicals that increase the activity of the thyroid peroxidase comprising the step of confirming the expression level of the gene integrated in the DNA microarray chip of the present invention in the analyzed data of step 3) compared to the control group Provided are gene detection methods for identification.

In addition, the present invention provides an RNA sample isolated from an individual (experimental group) and a control group suspected of being exposed to a chemical that increases the activity of thyroid peroxidase.

1) Real-time reverse transcript polymerase chain reaction (RT-PCR) is performed using a pair of primers complementary to the gene integrated in the DNA microarray chip of the present invention and amplifying the gene. step; And,

2) provides a gene detection method for identifying the exposure to a chemical that increases the activity of the thyroid peroxidase comprising the step of checking the expression level of the gene product of step 1) compared to the control.

In addition, the present invention provides a kit for confirming exposure to chemicals that increase the activity of thyroid peroxidase including the microarray chip.

In addition, the present invention includes a primer pair that is complementary to the gene of Genebank NM_000146 (ferritin, light polypeptide) or Genebank NM_002032 (ferritin, heavy polypeptide 1), and can amplify the gene. To provide a kit for confirming exposure to chemicals that increase the activity of thyroid peroxidase.

A biomarker for confirming exposure to a chemical that increases the activity of thyroid peroxidase of the present invention and a method of identifying the same using bioactive markers selected through DNA microarray to increase the activity of thyroid peroxidase It is useful for monitoring and risk assessment of chemicals and as a tool to identify the mechanism of toxic effects caused by chemicals that increase the activity of thyroid peroxidase.

Figure 1 is a graph of the thyroid peroxidase activity increased concentration of human thyroid vesicle cancer thyroid peroxidase gene recombinant cell line of a chemical (carbaryl) that increases the activity of thyroid peroxidase.
FIG. 2 is a graph showing an increase in thyroid peroxidase activity in human thyroid vesicle cancer thyroid peroxidase gene recombinant cell line of a chemical (dibutyl phthalate) that increases the activity of thyroid peroxidase.
Figure 3 is a diagram showing the results of analyzing the gene expression patterns in human thyroid vesicle cancer thyroid peroxidase gene recombinant cell line treated with a chemical that increases the activity of thyroid peroxidase using a microarray.

Hereinafter, the present invention will be described in detail.

The present invention is a biomarker for confirming exposure to a chemical that increases the activity of the thyroid peroxidase, characterized in that the expression changes by the exposure of a chemical that increases the activity of the thyroid peroxidase [Genebank] NM_000146 (ferritin, light polypeptide) or genebank (Genebank) NM_002032 (ferritin, heavy polypeptide 1)].

The biomarker is a gene whose expression is increased by 1.5 times or more when a chemical that increases the activity of thyroid peroxidase in carbaryl human thyroid vesicle cancer cells is treated with thyroid peroxidase inhibitory concentration.

In order to identify biomarkers for exposure to chemicals that increase the activity of thyroid peroxidase, the present inventors have applied a chemical substance that increases the activity of thyroid peroxidase, carbaryl or dibutyl phthalate, to human thyroid vesicle cancer cell line. Peroxidase was treated in FTC-238 / hrTPO / RSK008, a recombinant cell line, to determine the activity of thyroid peroxidase, which is known to be important for cytotoxicity and thyroid hormone production. As a result, it was confirmed that carbaryl and dibutyl phthalate activate the action of thyroid peroxidase in FTC-238 / hrTPO / RSK008 (see Figs. 1 and 2), based on the above experiment without showing cytotoxicity The concentrations of carbaryl and dibutylphthalate that activate the enzyme were determined. Subsequently, after treating FTC-238 / hrTPO / RSK008 with carbaryl or dibutyl phthalate at the concentration determined above, mRNA was isolated to obtain a 4 × 44 k human genome microarray chip (Human whole genome microarray, Agilent, USA). The gene expression patterns were analyzed. In the analysis, when the Cy5 / Cy3 ratio was 1.5 times or more, the expression was classified as an increased gene. As a result, genes whose expression of Cy5 / Cy3 is overexpressed or underexpressed by more than 1.5-fold by carbaryl and dibutyl phthalate are 0.209% (92 out of 44,000 genes), which have a p-value of less than 0.01 Confirmed genes were 0.211% (93 of 44,000 genes) (see FIG. 3). In the above case, the ferritin-related genes, Genebank NM_000146 (ferritin, light polypeptide) and Genebank NM_002032 (ferritin, heavy polypeptide 1), were treated with both carbaryl and dibutylphthalate. Expression increased more than 1.5-fold (see Table 2). Ferritin is an important protein that stores iron in the human body and is known as a marker for the distribution of iron in the human body. It has been reported that ferritin gene expression is increased in hyperthyroidism (Hess SY et. al ., J. Nutr . 132 (7): 1951-1955, 2002). However, there is no report that the gene is associated with toxicity in human thyroid follicular cancer cells when treated with a chemical that increases the activity of thyroid peroxidase used in the present invention.

In addition, the present invention is a DNA microarray chip for confirming exposure to chemicals that enhance the activity of thyroid peroxidase, in which an oligonucleotide comprising all or a portion of the nucleic acid sequence of a ferritin associated gene or its complementary oligonucleotide is integrated. To provide.

The ferritin associated gene is Genebank NM_000146 (ferritin, light polypeptide) or Genebank NM_002032 (ferritin, heavy polypeptide 1).

The oligonucleotide or its complementary oligonucleotide comprises 18 to 30 nucleic acids of the gene integrated in the DNA microarray chip of the present invention, preferably 20 to 25 nucleic acids.

The chemical that enhances the activity of the thyroid peroxidase is preferably, but not limited to, carbaryl or dibutyl phthalate.

DNA microarray chip for chemical detection that increases the activity of the thyroid peroxidase of the present invention can be produced by methods known to those skilled in the art. The method of manufacturing the microarray chip is as follows. In order to immobilize the found gene as a probe DNA molecule on a substrate of a DNA chip, a micropipetting method using a piezoelectric method or a pin-shaped spotter is used. It is preferable to use a method and the like, but is not limited thereto. In a preferred embodiment of the present invention, a microarray, which is a pin-shaped spotter, is used. The substrate of the DNA microarray chip of the present invention is coated with one active group selected from the group consisting of amino-silane, poly-L-lysine and aldehyde. Preferred but not limited to. In addition, the substrate may be selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane, and nitrocellulose membrane, but is not limited thereto, and in the preferred embodiment of the present invention, amino-silane may be used. Coated slide glass was used.

In addition,

RNA samples isolated from individuals (experimental group) and controls suspected of exposure to chemicals that enhance the activity of thyroid peroxidase

1) labeling the experimental and control groups with different fluorescent materials while synthesizing with cDNA;

2) hybridizing cDNA labeled with different fluorescent materials of step 1) with the DNA microarray chip of claim 1;

3) analyzing the reacted DNA microarray chip of step 2); And,

4) Gene detection method for identifying exposure to chemicals that enhance the activity of thyroid peroxidase by comparing the expression level of the gene integrated in the DNA microarray chip of step 1 with the control group in the analyzed data of step 3) to provide.

The RNA sample is an RNA sample derived from human thyroid, thyroid vesicles or thyroid vesicle cancer cells or tissues, preferably from FTC-238 / hrTPO / RSK008, a cell line recombining thyroid peroxidase into human thyroid vesicle cancer cell lines It is. At this time, the control group is derived from a healthy individual.

In addition, the fluorescent material of step 1) may be selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), and rhodamine (rhodamine), but is not limited thereto. The fluorescent substance known to those skilled in the art can be used.

The chemical that enhances the activity of the thyroid peroxidase is preferably, but not limited to, carbaryl or dibutyl phthalate.

In the above method, the hybridization of step 2) is preferably performed at 40 ° C. to 50 ° C. for 10 to 20 hours, but is not limited thereto.

In the above method, the DNA microarray chip of step 2) is a human genome of the present invention in the gene genome (Genebank) NM_000146 (ferritin, light polypeptide) or Genebank (Menebank) NM_002032 (ferritin, heavy polypeptide 1) It is preferable to use a mounted microarray chip, and it is preferable to use Whole Human Genome Oligo Microarray (Agilent, USA) and the like, but it is not limited thereto. .

In the above method, the analysis of step 3) is preferably performed by washing and staining the hybridized microarray chip, scanning, performing image processing, and then performing statistical analysis. The staining and washing is performed using Fluidics Station 450, the scan is GeneChip scanner  3000 is used, and image processing is performed using Affymetrix GeneChip Operating System (GCOS), and all procedures are preferably performed according to Affymetrix's protocol, but not limited thereto. The statistical analysis is preferably performed using GenPlex software version 2.3, but is not limited thereto. Analysis software known to those skilled in the art may be used.

In addition,

RNA samples isolated from individuals (experimental group) and controls suspected of exposure to chemicals that enhance the activity of thyroid peroxidase

1) performing a real-time reverse transcript polymerase chain reaction (RT-PCR) using primer pairs complementary to the gene integrated in the DNA microarray chip of claim 1 and amplifying the gene ; And,

2) comparing the gene product of step 1) with a control group to determine whether exposure to a chemical that enhances the activity of thyroid peroxidase, including determining whether exposure to a chemical that enhances the activity of thyroid peroxidase. It provides a gene detection method for.

The RNA sample is an RNA sample derived from human thyroid, thyroid vesicles or thyroid vesicle cancer cells or tissues, preferably from FTC-238 / hrTPO / RSK008, a cell line recombining thyroid peroxidase into human thyroid vesicle cancer cell lines It is. At this time, the control group is derived from a healthy individual.

The primer pair of step 1) is 15 to 50mers long, preferably 15 to 30mers long, more preferably designed so that the amplification product of the gene integrated in the DNA microarray chip of the present invention is 100 to 300 bp. Both forward and reverse primer pairs of 18 to 25 mer length are available.

The chemical that enhances the activity of the thyroid peroxidase is preferably, but not limited to, carbaryl or dibutyl phthalate.

In addition, the present invention provides a kit for confirming exposure to chemicals that increase the activity of thyroid peroxidase including the microarray chip.

In addition to the identification kit provides a kit for confirming exposure to chemicals that increase the activity of thyroid peroxidase, including thyroid vesicle cancer cells. The thyroid follicular cancer cells preferably use the cell line FTC-238 / hrTPO / RSK008, which is a cell line recombined with thyroid peroxidase in human thyroid follicular cancer cell line, but is not limited thereto. Cells and tissues derived from human thyroid or thyroid cancer Any cell derived from can be used.

The chemical that enhances the activity of the thyroid peroxidase is preferably, but not limited to, carbaryl or dibutyl phthalate.

In addition to the kit may include a fluorescent material, the fluorescent material is a streptavidin-like phosphatease conjugate (streptavidin-like phosphatease conjugate), a chemifluorescent agent (chemifluorescent agent) and a chemiluminescent agent (chemiluminescent agent) It is preferably selected from the group consisting of, but is not limited thereto.

In addition, the kit may further include a reaction reagent, the reaction reagent used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (premixed or separated feed), fluorescent staining agent It may include, but is not limited to, labeling reagents such as chemical inducing agents and washing buffers, and may include all reaction reagents required for hybridization of DNA microarray chips known to those skilled in the art.

In addition, the present invention is complementary to the gene integrated in the DNA microarray chip of the present invention, including a primer pair capable of amplifying the gene, for confirming exposure to chemicals that increase the activity of thyroid peroxidase Provide the kit.

Primer pair of the identification kit is 15 to 50mers long, preferably 15 to 30mers long, more preferably designed so that the amplification product of the gene integrated in the DNA microarray chip of the present invention is 100 to 300 bp Preferably both forward and reverse primer pairs of 18 to 25 mer length are available.

The chemical that enhances the activity of the thyroid peroxidase is preferably, but not limited to, carbaryl or dibutyl phthalate.

In addition to the identification kit provides a kit for confirming exposure to chemicals that increase the activity of thyroid peroxidase, including thyroid vesicle cancer cells. The thyroid follicular cancer cells preferably use the cell line FTC-238 / hrTPO / RSK008, which is a cell line recombined with thyroid peroxidase in human thyroid follicular cancer cell line, but is not limited thereto. Cells and tissues derived from human thyroid or thyroid cancer Any cell derived from can be used.

In addition, the kit may further include a reaction reagent, which is a reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (premixed or separated feed), DNA polymerase, washing buffer, etc. It may be composed of, but not limited to, may include all the reaction reagents required for RT-PCR reaction known to those skilled in the art.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Cell culture and chemical treatment

<1-1> Cell Culture

FTC-238 / hrTPO / RSK008 cells, a human thyroid vesicle cancer thyroid peroxidase gene recombinant cell line, were prepared using DMEM / F12 medium (Gibro-BRL, USA) supplemented with 5% FBS, 2% penicillin / streptomycin, and 0.06% G418. Incubation was done in mm dishes until 80% growth. The present inventors have selected carbaryl and dibutyl phthalate, chemicals that increase the activity of thyroid peroxidase, and dissolved them in DMSO. Vehicle concentration was less than 0.1% in all experiments.

<1-2> Determination of Thyroid Peroxidase Activity and Determination of Chemical Substances

A guiacol assay was performed to determine the increased concentration of thyroid peroxidase activity using the FTC-238 / hrTPO / RSK008 cell line. After dispensing FTC-238 / hrTPO / RSK008 cells in a 100 mm dish at a concentration of 2 × 10 ⁶ cells / ml, hematin was treated with 1 μg / ml after 24 hours, and the cells were 1.5 after 48 hours. The membranes were collected in a ml tube and pulverized with a pulse of 0.6 seconds 10 seconds with a sonicator. The tube was then centrifuged at 14,000 rpm at 4 ° C. for 1 hour 30 minutes. After centrifugation, the supernatant was discarded, the precipitate was released well, and 500 μl of 1% digitonin was added, followed by incubation at 4 ° C. for 24 hours. After this, centrifuge at 14,000 rpm at 4 ° C. for another 1 hour and 30 minutes, and transfer the supernatant from the tube after centrifugation to a new tube. Supernatants were used in the following guiacol assay.

Proteins containing the extracted supernatant thyroid peroxidase were quantified using a BCA assay kit (pierce) and then transferred to each tube so that the concentration was 325 ㎍. A total volume of 600 μl increases thyroid peroxidase activity in a solution containing 325 μg of protein containing thyroid peroxidase, 100 mM potassium phosphate buffer, and 10 μM of hydrogen peroxide (H ₂ O ₂ ). The chemical carbaryl or dibutyl phthalate was treated at each concentration, incubated for 5 minutes, and 100 μl of this was transferred to a new tube. In addition, 50 mM potassium phosphate buffer solution, 40 mM guacol, and 220 μM hydrogen peroxide (H ₂ O ₂ ) were added and 100 μl of this was measured to measure absorbance at 470 nm to increase thyroid peroxidase activity. Was measured. The increase in thyroid peroxidase activity was 1 μM for carbaryl and 100 μM for dibutyl phthalate (see FIGS. 1 and 2). The concentration was determined according to the microarray experiment.

Microarray  Experiment

<2-1> RNA Isolation and Labeling of Fluorescent Materials

After dispensing FTC-238 / hrTPO / RSK008 cells in a 100 mm dish at a concentration of 2 × 10 ⁶ cells / ml, the concentration of carbaryl or dibutyl phthalate determined in Example 1-2 was treated for 48 hours. Subsequently, total RNA was isolated from the treated cells using a trizol reagent (Invitrogen life technologies, USA) according to the manufacturer's method, and purified using an RNeasy mini kit (Qiagen, USA). Genomic DNA was removed using RNase-free DNase set (Qiagen, USA) during RNA purification. The amount of each total RNA sample was measured by spectrophotometer, the concentration was confirmed by Xperion (Experion, Automated Electrophoresis station, Bio-Rad, USA).

<2-2> Labeled cDNA  Produce

For oligo microarray analysis, cDNA was prepared using total RNA from a chemical treatment group that increases the activity of thyroid peroxidase obtained in Example 2-1. Specifically, 30 μg of the obtained total RNA and 2 μg of oligo (dT) primer (1 μg / μl) were mixed and reacted at 65 ° C. for 10 minutes, and then annealed in ice immediately. Reagents were mixed as shown in Table 1 for Reverse Transcript reaction of the annealed RNA.

Configuration Volume (μl) 5X first strand buffer 6 dNTPs 0.6 0.1 M DDT 3 SuperScript II enzyme 3 Cy-3 or Cy-5 dUTP 2

Total RNA isolated from control FTC-238 / hrTPO / RSK008 cell line was labeled with Cy3-dUTP (green) and isolated from FTC-238 / hrTPO / RSK008 cell line treated with chemicals that increase the activity of thyroid peroxidase. RNA was labeled with Cy5-dUTP (red) respectively. The two samples were mixed and purified using a Microcon YM-30 column (Millipore, USA).

<2-3> Hybridization  reaction

Hybridization and washing process was performed according to the instructions of Gino 첵 Co., Ltd. (Korea). Hybridization was performed in a 62 ° C. oven for 12 hours. As a DNA microarray, a 44k human whole genome oligomicroarray (Agilent, USA) was used. After the washing process (washing in 2 × SSC / 0.1% SDS for 2 minutes, 1 × SSC for 3 minutes and 0.2 × SSC for 2 minutes), the slides were dried by centrifugation at 800 rpm for 3 minutes.

<2-4> Fluorescence Image Acquisition

Hybridization images on the slides were scanned with the Genepix 4000B (Axon Instruments, USA). Chips washed with unbound genes were obtained with a fluorescence image using a laser fluorescence scanner. In this case, the green fluorescence image in the control group, the red fluorescence image represents the activity level of the gene specifically expressed only in the experimental group, and the yellow fluorescence image is the complementary color of green and red, which means that there is no significant difference between the two groups. Scanned images were analyzed with GenePix 4.1 software (Axon Instruments, USA) to obtain gene expression rates. Marker genes for chemicals that increase the activity of thyroid peroxidase were selected from the data obtained by the method (see Table 2).

As a result, among the approximately 44,000 genes present on the oligo chip, carbaryl and dibutyl phthalate, chemicals that increase the activity of thyroid peroxidase, have a common overexpression of Cy5 / Cy3 by 1.5 times or more. As for the genes, p-values of 0.01 or less were found to be 0.209% (92 out of 44,000 genes) and 0.211% (93 out of 44,000 genes) decreased in expression (see FIG. 3). Of these genes, ferritin-related genes that specifically express increased thyroid peroxidase activity were identified (see Table 2).

Gene whose expression is altered by chemicals that increase the activity of thyroid peroxidase Genebank Gene abbreviation Gene name Oligo Microarray Value Cabaril Dibutyl phthalate NM_000146 FTL ferritin, light polypeptide 2.78 1.59 NM_002032 FTH1 ferritin, heavy polypeptide 1 1.68 1.55

Claims (14)

Oligonucleotides or their complementary oligonucleotides comprising the nucleic acid sequence of the ferritin gene of Genebank NM_000146 (ferritin, light polypeptide) or Genebank NM_002032 (ferritin, heavy polypeptide 1) DNA microarray chip for confirming exposure to carbaryl or dibutyl phthalate to enhance the activity of thyroid peroxidase.
delete delete From RNA samples isolated from individuals suspected of exposure to carbaryl or dibutylphthalate (experimental group) and controls that enhance the activity of thyroid peroxidase
1) labeling the experimental and control groups with different fluorescent materials while synthesizing with cDNA;
2) hybridizing cDNA labeled with different fluorescent materials of step 1) with the DNA microarray chip of claim 1;
3) analyzing the reacted DNA microarray chip of step 2); And,
4) For carbaryl or dibutyl phthalate, which enhances the activity of thyroid peroxidase, comprising the step of checking the expression level of the gene integrated in the DNA microarray chip of claim 1 in the analyzed data of step 3). Gene detection method to confirm exposure.
The method of claim 4, wherein the fluorescent material of step 1) is Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (rhodamine-B-isothiocyanate, RITC) and rhodamine (rhodamine), characterized in that any one selected from the group consisting of.
From RNA samples isolated from individuals suspected of exposure to carbaryl or dibutylphthalate (experimental group) and controls that enhance the activity of thyroid peroxidase
1) performing a real-time reverse transcript polymerase chain reaction (RT-PCR) using primer pairs complementary to the gene integrated in the DNA microarray chip of claim 1 and amplifying the gene ; And,
2) Gene detection method for confirming the exposure to carbaryl or dibutyl phthalate to enhance the activity of the thyroid peroxidase comprising the step of checking the expression of the gene product of step 1) compared to the control.
The method of claim 6, wherein the primer pair of step 2) is a forward and reverse primer pair of 18 to 30mer length, capable of amplifying a gene integrated in the DNA microarray chip of claim 1.
The identification method according to claim 4 or 6, wherein the RNA sample is derived from human thyroid, thyroid vesicle, or thyroid vesicle cancer cells or tissues.
delete Claim 1 kit for detecting exposure to carbaryl or dibutyl phthalate to enhance the activity of the thyroid peroxidase comprising the microarray chip of claim 1.
Complementary to the ferritin gene of Genebank NM_000146 (ferritin, light polypeptide) or Genebank NM_002032 (ferritin, heavy polypeptide 1), and comprises a primer pair capable of amplifying the gene. , Kit for confirming exposure to carbaryl or dibutyl phthalate to enhance the activity of thyroid peroxidase.
12. The kit of claim 11, wherein the primer pairs are 18-30 mer long forward and reverse primer pairs designed to have an amplification product of 100-300 bp.
12. The kit of claim 10 or 11, further comprising thyroid follicular cancer cells.
delete

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