KR101885596B1 - Composition for diagnosing radiation exposure and method for diagnosing radiation exposure using the same - Google Patents

Abstract

The present invention relates to a composition for the diagnosis of radiation exposure and a method of diagnosing radiation exposure using the same. More particularly, the present invention relates to a composition for diagnosing radiation exposure, which comprises CXCL3, CXCL5, IL-1β, IL-15, IL- Using NOTCH2, GADD45, BCL2 and GATA3, you can quickly and accurately diagnose radiation exposure from your blood. In addition, the present invention can provide a radiation exposure diagnostic kit comprising a DNA chip for radiation exposure diagnosis comprising the gene as a biomarker and a primer or a probe specifically binding to the gene. Furthermore, by developing a drug that enhances radiation sensitivity using the gene as a target, it can improve the radiation treatment effect and can be utilized for predicting or diagnosing the degree of radiation exposure from radiation-related workers and natural radiation exposure.

Description

TECHNICAL FIELD The present invention relates to a composition for diagnosing radiation exposure, and a diagnostic method using the same.

The present invention relates to a radiation exposure diagnostic composition for diagnosing radiation exposure in blood and a radiation exposure diagnostic method using the same.

Ionizing radiation (IR) was first used by Helsper in 1967 for cancer treatment purposes. At present, radiation and radiation isotopes are used in various fields such as diagnosis and treatment, aerospace, semiconductor, biotechnology and food production as well as various industrial uses, and are closely related to our lives. Particularly, in Korea, a large part of electric power production is dependent on nuclear power generation, and small-sized radiation irradiation devices are used in the industrial field for diagnosis of non-destructive structure of buildings. Accordingly, . This increase in the use of radiation, which is closely related to life, is also increasing human exposure.

In addition, there are increasing incidents of radiation exposure unintentionally, such as the accident of heavy water leakage at Wolsong nuclear power plant in 1999 or the accident at a uranium factory in Tokai-mura, Japan. Radiation exposure causes DNA repair, cell cycle abnormality, cell death leading to apoptosis and chromosomal aberrations in peripheral blood lymphocytes (PBL), tissue and organ damage, malformations and cancer It is known. However, there has not yet been developed a method for monitoring the human body effect caused by radiation exposure or for quickly estimating the radiation obstacle in case of unexpected accident such as nuclear accident.

If patients are exposed to radiation, accurate and accurate identification of the radiation dose received by the patient is essential to determine the patient's treatment plan and to determine the prognosis. In addition, confirming the radiation dose will play an important role in predicting future chronic effects of radiation and in identifying the cause of the disease that may be caused by radiation exposure.

Although the dose range can be clinically estimated using the patient's symptoms and signs after radiation exposure, the more objective and accurate method is to use a physiological method to calculate the radiation dose by using a losing dosimeter or reconstructing the exposure situation, A biological method has been used to estimate the radiation dose by measuring changes in the body induced by the human body. However, it is necessary to develop a damage regulator or exposure biomarker that can diagnose radiation exposure more conveniently and accurately.

In recent years, research has been expanded to search for an exposure biomarker that can be found in normal tissue exposed to radiation. Such an exposure biomarker is important for the impact of the human body due to radiation exposure caused by a nuclear accident, terrorism or radiation treatment, It is expected to be used.

Methods for measuring gene expression include differential display PCR (DD-PCR), serial analysis gene expression (SAGE), expressed sequence tags (EST), and DNA chip methods. Among them, DNA chip can be used in various application fields such as diagnosis of diseases, development of new drugs, as well as basic research of molecular biology because it enables to observe expression pattern of thousands or tens of thousands of genes in place of individual genes.

The susceptibility to radiation differs according to the gene expression pattern, and the difference in the reactivity of the tumor cells to the radiation and efforts to measure the difference are underway, but efforts to measure the degree of radiation exposure of normal persons have hardly been done.

Genes known to be involved in radiation sensitivity include H-Ras, c-Myc, thymidine kinase, basic fibroblast growth factor, insulin-like growth factor, p21, Bcl-2, p53, and acid sphingomyelinase are known, and when p21 ^WAF1 and GADD45 genes react between 2 and 50 cGy in myeloid cancer cells, .

On the other hand, as a method of measuring radiation exposure, lymphocytes of blood are separated to detect chromosomal abnormality, measurement of micronucleus formation, measurement of mutation of glycoporin A in erythrocytes, degree of DNA cleavage and enamel of teeth, (Electron spin resonance) method using EST (electron spin resonance). However, it has a disadvantage in that it is difficult to measure low-dose radiation with a different reactivity depending on an individual and takes a long time to measure. Also, it is necessary to develop a method to increase the accuracy because it is dangerous to measure the degree of exposure by a single method in order to measure biological radiation exposure.

Japanese Patent Laid-Open No. 2007-093341 (disclosed on Apr. 12, 2007)

An object of the present invention is to provide a radiation exposure diagnostic composition comprising a gene selected from the group consisting of CXCL3, CXCL5, IL-1 ?, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3, .

It is another object of the present invention to provide a method of screening for a disease or disorder in a sample isolated from a human subject suspected of having radiation exposure, comprising administering to the subject a therapeutically effective amount of one or more genes selected from the group consisting of CXCL3, CXCL5, IL-1 ?, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 Or measuring the level of protein expression thereof.

Another object of the present invention is to provide a method for diagnosing a radiation exposure diagnosis comprising the step of irradiating a subject with at least one gene selected from the group consisting of CXCL3, CXCL5, IL-1 ?, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 as a biomarker Chip. ≪ / RTI >

Another object of the present invention is to provide a primer or a probe that specifically binds to at least one gene selected from the group consisting of CXCL3, CXCL5, IL-1 ?, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 And to provide a kit for the diagnosis of radiation exposure.

In order to accomplish the above object, the present invention provides a method for diagnosing radiation exposure including a gene selected from the group consisting of CXCL3, CXCL5, IL-1 ?, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3, Lt; / RTI >

The present invention also relates to a method for screening for a disease or disorder in a sample isolated from a human suspected of radiation exposure, comprising the steps of: (a) And measuring the level of protein expression.

The present invention also provides a DNA chip for radiation exposure diagnosis comprising at least one gene selected from the group consisting of CXCL3, CXCL5, IL-1 ?, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 as a biomarker to provide.

The present invention also relates to a method for screening a compound that specifically binds to one or more genes selected from the group consisting of CXCL3, CXCL5, IL-1 ?, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 A radiation exposure diagnostic kit is provided.

The present invention relates to a composition for the diagnosis of radiation exposure comprising the genes CXCL3, CXCL5, IL-1β, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 or proteins thereof having high susceptibility to radiation. Can be diagnosed. In addition, it is possible to provide a radiation exposure diagnostic kit comprising a DNA chip for radiation exposure diagnosis comprising the gene as a biomarker and a primer or a probe specifically binding to the gene.

In addition, the present invention can diagnose radiation exposure quickly and accurately by using a sample as blood, and by developing a drug that enhances radiation sensitivity by targeting the gene, it is possible to enhance the effect of radiation therapy, Can be used to easily predict or diagnose the degree of radiation exposure from radiation exposure.

FIG. 1 is a schematic diagram of a study for identifying a gene group for diagnosis of radiation exposure.
FIG. 2 is a result of comparing the number of lymphocytes and bone marrow cells with that of a complete blood cell count (CBC) after irradiating the mouse with radiation.
FIG. 3 is a result of irradiating a mouse with radiation, separating lymphocytes from the blood, and performing a microarray.
FIG. 4A shows a gene group in which the expression is increased more than 2 times and a gene group in which the expression is decreased less than 2 times in comparison with the control in the microarray result.
FIG. 4B is a result of verifying the gene selected through the microarray using a polymerase chain reaction (PCR).

As used herein, the term " radiation irradiation " refers to exposure to the action of electromagnetic radiation or alpha or beta particles including, for example, X-rays, ultraviolet rays, alpha particles and gamma rays. Radiation can affect both cancer cells and normal cells, but using a variety of methods and techniques, cancer cells are destroyed by treating cancer with fewer effects on normal cells. It does not kill cancer cells instantly, but destroys the ability of cancer cells to divide and multiply, preventing new cancer cells from dividing and producing, and the cancer cells that are no longer dividing will die due to their life span. Each treatment kills more cells, dead cells are degraded and transported to the blood, drained out of the body, and the size of the tumor is reduced. Most of the normal cells are recovered, but some normal cells are not recovered, resulting in side effects of radiation therapy. Changes in cells and tissues that can be caused by side effects include changes in cell division timing, cell death and necrosis, tissue damage, and immune function.

As used herein, the term " Gray (Gy) " refers to a dose of radiation that can release one line (Joule) in 1 kg of biological tissue.

The term " primer " as used herein means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand replication . The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In addition, primers may incorporate additional features that do not alter the primer properties of the primers that serve as a starting point for DNA synthesis, as sense and antisense nucleic acids with 7 to 50 nucleotide sequences.

As used herein, the term " probe " refers to an oligonucleotide that specifically binds to a target sequence, and includes, for example, a nucleic acid sequence that hybridizes to a target nucleic acid sequence in a sequence-specific manner with a complementary region of a specific nucleic acid sequence Lt; RTI ID = 0.0 > polynucleotides < / RTI >

The term " measurement " as used in the present invention is to confirm the degree of radiation exposure or irradiation in a cell. In one embodiment, the present invention can determine the level of mRNA expression of the gene or the expression level of the protein from the biological sample obtained from the subject, and compare the expression level of the gene with the expression level of the normal control sample to determine the degree of radiation exposure or irradiation.

The present invention relates to the use of a nucleic acid encoding a polypeptide selected from the group consisting of CXCL3 (GenBank accession no. NM_002090), CXCL5 (GenBank accession no. NM_002994), IL-1β (GenBank accession no NM_000576), IL- (GenBank accession number NM_00122295), NOTCH2 (GenBank accession number NM_024408), GADD45 (GenBank accession number NM_001924), BCL2 (GenBank accession number NM_000633), and GATA3 (GenBank accession number NM_001002295). A gene selected from the group or a protein thereof.

Preferably, the radiation exposure diagnostic composition further comprises CXCL2 (GenBank accession no. NM_002089) gene or a protein thereof.

The present invention relates to a pharmaceutical composition comprising at least one gene selected from the group consisting of CXCL3, CXCL3, CXCL5, IL-1β, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3, And measuring the level of expression of the radiation.

Preferably, the radiation exposure diagnostic method may further comprise a CXCL2 gene or a protein thereof.

Preferably, the sample may be but is not limited to blood.

Preferably, it is determined that the expression levels of CXCL3, CXCL5, IL-1β, IL-15, IL-19, CCR1, NOTCH2 and GADD45 were 1.5 times higher than the expression level of the control without radiation exposure .

Preferably, it can be determined that the expression level of BCL2 and GATA3 is less than 1.5 times the expression level of the control without radiation exposure.

Preferably, the expression is measured by real-time polymerase chain reaction (RT-PCR), competitive PCR, reverse transcription PCR (RT) (PCR), microarray, northern blotting, western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunoprecipitation immunoprecipitation, and flow cytometry (FACS), but are not limited thereto.

The present invention provides a DNA chip for radiation exposure diagnosis comprising at least one gene selected from the group consisting of CXCL3, CXCL5, IL-1β, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 as a biomarker .

Preferably, the DNA chip for radiation exposure diagnosis further comprises a CXCL2 gene.

The present invention provides a method for detecting a radiation dose comprising a primer or a probe specifically binding to at least one gene selected from the group consisting of CXCL3, CXCL5, IL-1 ?, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 Provide a diagnostic kit.

Preferably, the radiation exposure diagnostic kit further comprises a primer or a probe that specifically binds to the CXCL2 gene.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1: Radiation and cell count analysis of lymphocyte and bone marrow cells

Radiation doses (1 Gy, 3 Gy, 5 Gy) were measured using a gamma ray generator (Gamma cell-3000, Nordion International INC.) In BALB / c mice Mice were anesthetized with tilethylamine / Zolazepam, Zolethyl 50, Virbac Korea, and Xylazine (Rompun, Bayer Korea, Korea) on the 7th day. Blood was collected from the saphenous vein, stored in a heparin tube, and bone marrow cells were isolated from the femoral bone marrow and stored in a 30% fetal bovine serum (FBS) solution. Lymphocytes were isolated from the blood using Ficoll solution, and mononuclear cells were isolated from the bone marrow and analyzed for cell numbers. The results of cell count analysis were compared with the results of complete blood cell count (CBC).

As a result, referring to FIG. 2, it was confirmed that lymphocytes and bone marrow cells were decreased in the mice irradiated with 1 Gy radiation until day 3, and then recovered to the normal mouse level without irradiation at 7 days. However, lymphocyte cells in the mice irradiated with 3 Gy and 5 Gy radiation showed a significant decrease on days 3 and 7. Therefore, experiments were conducted under conditions of 1 Gy, 1 day, 2 days, 3 days, and 7 days, and 3 Gy and 5 Gy radiation for 1 day and 2 days, and a microarray sample was prepared Respectively.

Example  2 : Microarray  analysis

RNA was isolated from the lymphocytes of Example 1 using trizol in order to derive a damage regulator and an exposure diagnostic gene by irradiation. RNA was purified using a purification kit (Qiagen RNase-free DNase kit and RNeasy spin column) and quantified using a UV spectrophotometer.

Microarray analysis proceeded in three steps: labeling, hybridization, and washing. First, cDNA was synthesized from RNA using a cDNA synthesis kit, and double-stranded DNA was purified using a purification kit (Qiagen RNeasy Mini Kit), followed by fluorescence staining using Cy3 and Cy5 to label the cDNA . The labeled cDNA was dropped onto the prepared microarray slides and reacted to hybridize. The fluorescence intensities of Cy3 and Cy5 from each spot were captured using a laser scanner and quantitatively recorded. The intensity of the signal is quantitatively proportional to the amount of actual mRNA expression of the spot. Using the post - scan analysis program, we selected genes whose expression was increased more than 2 times or less than 2 times as much as that of the control when compared with the control. The results are shown in FIG.

Example  3: Analysis and verification of radiation reaction genes

The gene group derived from the microarray was analyzed by referring to the NEN web page (www.nen.com). After irradiation, genes with more than 2 times increase in expression or less than 2 times decrease in expression compared to the control were selected as genes responsive to radiation. 4, the gene expression of CXCL2, CXCL3, CXCL5, SNAP23, IL-19, IL-15, CD14, IL-1 ?, CCR1, NOTCH2 and GADD45 was more than 2 times higher than that of the control. The expression of JADE1, BCL2, IL-4, CD24A, CD2, BCL2, IL-7, GATA3, GPX1 and TRNP1 was reduced by 2-fold or less.

Example  4: Real-time polymerase chain reaction (Real-time PCR ) analysis

Real - time PCR was performed using the same RNA used for microarray analysis to verify selected genes as a result of microarray analysis. CDNA was synthesized using 1 ug of RNA, oligo (dT) 20 primer and Superscript-II reverse transcriptase (Invitrogen).

The synthesized cDNA was diluted 10-fold and then real-time PCR was performed using a reaction mixture (power SYBR Green PCR master mix, Applied Biosystems) and primers for each gene. The PCR conditions were as follows; Pre-denaturation; 95 ° C, 10 min, denaturation; 95 ° C, 33 sec, annealing; Primer optimum temperature, 33 seconds, extension; 72 < [deg.] ≫ C, 33 seconds. Thereafter, the reaction was confirmed on 1% agarose gel. The 2 ^{- ΔΔCt} method was used to calculate the c (t) value and the GAPDH gene value was used for the normalization. The primers used in PCR were designed using Primer 3 software (http://frodo.wi.mit.edu) using NCBI nucleotide sequence information.

As a result, referring to FIG. 4B, it was confirmed that the selected genes increased expression more than 2 times or less than 2 times as compared with the control in the same manner as the result of microarray analysis. Therefore, when the patient is diagnosed with radiation exposure, if the expression level of the selected gene group is 2 times or more than 2 times that of the control, the radiation exposure may be diagnosed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (12)

A composition for diagnosing gamma radiation exposure comprising CXCL3, CXCL5, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 genes or a protein thereof. The composition for diagnosing gamma radiation according to claim 1, wherein the composition further comprises one or more genes selected from the group consisting of CXCL2 and IL-1? Or a protein thereof. The present invention relates to a method for detecting gamma radiation exposure in a sample isolated from a human suspected of gamma radiation exposure, comprising the step of measuring levels of CXCL3, CXCL5, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 genes or protein expression thereof. How to provide information. 4. The method according to claim 3, wherein the method further comprises the step of measuring the level of expression of one or more genes selected from the group consisting of CXCL2 and IL-1? Or its protein . 4. The method according to claim 3, wherein the sample is blood. The method according to claim 3, wherein the expression levels of CXCL3, CXCL5, IL-15, IL-19, CCR1, NOTCH2 and GADD45 are determined to be exposed to gamma rays when the expression level is 1.5 times or more than the expression level of a control not exposed to gamma radiation To provide information necessary for gamma ray exposure diagnosis. 4. The method according to claim 3, wherein the expression level of BCL2 and GATA3 is determined to be exposed to gamma rays when the expression level of the BCL2 and GATA3 is 1.5 times or less as compared with the expression level of a control not exposed to gamma radiation. 4. The method according to claim 3, wherein the expression is measured using real-time polymerase chain reaction (RT-PCR), competitive PCR, reverse transcription polymerase chain reaction , RT-PCR), microarray, northern blotting, western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunoprecipitation Wherein the method is selected from the group consisting of immunoprecipitation and flow cytometry (FACS). A DNA chip for gamma-ray irradiation diagnosis comprising CXCL3, CXCL5, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 genes as biomarkers. The DNA chip according to claim 9, wherein the DNA chip further comprises one or more genes selected from the group consisting of CXCL2 and IL-1 ?. Wherein the primer or probe specifically binds to CXCL3, CXCL5, IL-15, IL-19, CCR1, NOTCH2, GADD45, BCL2 and GATA3 genes. [Claim 13] The kit according to claim 11, wherein the kit further comprises a primer or a probe specifically binding to any one or more genes selected from the group consisting of CXCL2 and IL-1 [beta].

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