KR102593991B1 - Biomarker composition for diagnosis of radiation exposure and method of providing information for diagnosis of radiation exposure using the same - Google Patents

Abstract

The present invention provides a biomarker for diagnosing radiation exposure, and relates to a method of providing information for diagnosing radiation exposure using the biomarker. More specifically, the present invention relates to a microarray by collecting blood from irradiated mice. As a result of the analysis, increased expression of the Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9 and CXCL10 gene groups was confirmed, and the genes were provided as biomarkers for diagnosing radiation exposure. By checking the level of increased expression of biomarkers, it is possible to easily and effectively diagnose whether a subject suspected of being exposed to radiation has been exposed to radiation.

Description

Biomarker composition for diagnosis of radiation exposure and method of providing information for diagnosis of radiation exposure using the same}

The present invention provides a biomarker for radiation exposure diagnosis and relates to a method of providing information for radiation exposure diagnosis using the biomarker.

Radiation is closely related to our lives, and its unique energy characteristics can be used to manufacture new materials and advanced parts for industries such as medicine, aerospace, and semiconductors, develop new varieties, produce safe food, and use biotechnology and high value-added radiation. It is used in many fields, including isotope development, radiation generator development, and accelerator development. The use of such radiation is increasing and the resulting human exposure is also increasing.

When a patient is exposed to radiation, it is essential to accurately determine the amount of radiation the patient received as soon as possible in determining the patient's treatment plan and prognosis. In addition, confirming the amount of radiation exposure will play an important role in predicting chronic effects of radiation that may occur in the future and in identifying the cause of diseases that may be caused by radiation exposure.

The dose range can be clinically estimated using the patient's symptoms and signs after radiation exposure, but a more objective and accurate method is the physical method of calculating the radiation dose by using a worn dosimeter or reconstructing the exposure situation, and the radiation Biological methods have been used to estimate radiation dose by measuring changes in the human body induced by radiation. However, there is a need to develop damage control factors or radiation exposure biomarkers that can diagnose radiation exposure more conveniently and accurately.

Recently, studies looking for radiation exposure biomarkers that can be found in normal tissues exposed to radiation have expanded, and these radiation exposure biomarkers can be used to determine the impact and prognosis of the human body due to radiation exposure from nuclear accidents, terrorism, or radiation therapy. You will be able to.

Republic of Korea Patent Publication No. 10-2020-0116744 (published on October 13, 2020)

The present invention provides a biomarker for diagnosing radiation exposure, and provides a method for providing information for diagnosing radiation exposure using the biomarker to quickly and accurately diagnose whether a subject suspected of being exposed to radiation has been exposed to radiation.

The present invention provides a biomarker for diagnosing radiation exposure containing one or more genes selected from the group consisting of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9, and CXCL10, or a protein encoded by the genes. A composition is provided.

In the present invention, one or two or more genes are selected from genes consisting of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9 and CXCL10, or proteins encoded by the genes, and the expression or activity level of the genes, Alternatively, a kit for diagnosing radiation exposure containing as an active ingredient an agent that detects the expression level of the protein encoded by the gene is provided.

In addition, the present invention relates to the expression or activity level of genes consisting of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9 and CXCL10 from samples isolated from the specimen, or the expression of the protein encoded by the genes. Provides a method of providing information for radiation exposure diagnosis including the step of confirming the level.

According to the present invention, blood from irradiated mice was collected and microarray analysis was performed, and the results showed increased expression of the Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9 and CXCL10 gene groups. As confirmed, the gene can be provided as a biomarker for diagnosing radiation exposure, and the increased expression level of the biomarker can be identified, making it possible to conveniently and effectively diagnose whether a subject suspected of being exposed to radiation has been exposed to radiation.

Figure 1 shows the results of confirming changes in lymphocyte cells and bone marrow cells after irradiating mice with radiation. Cell death was confirmed in the blood 1, 3, and 7 days after irradiating mice with radiation at doses of 1, 3, and 5 Gy. .
Figure 2 shows the results of microarray analysis that identified damage factors in lymphocyte cells due to radiation exposure.
Figure 3 shows the results of confirming the gene group identified by microarray through quantitative PCR analysis, and shows the results of performing microarray to select genes whose gene expression increased two-fold according to radiation dose.
Figure 4 shows the results of confirming genes selected by microarray by quantitative PCR analysis.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention believe that radiation is closely related to daily life, and as human exposure increases due to the increased use of radiation, diagnosing radiation exposure as soon as possible and proceeding with treatment of the patient can result in a high treatment effect. The present invention was completed to develop a method that can more conveniently and accurately diagnose radiation exposure.

The present invention provides a biomarker for diagnosing radiation exposure containing one or more genes selected from the group consisting of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9, and CXCL10, or a protein encoded by the genes. A composition may be provided.

The expression of one or more genes selected from the group consisting of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9 and CXCL10 or the protein encoded by the genes may be increased upon radiation exposure. there is.

In the present invention, one or two or more genes are selected from genes consisting of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9 and CXCL10, or proteins encoded by the genes, and the expression or activity level of the genes, Alternatively, a kit for diagnosing radiation exposure containing as an active ingredient an agent that detects the expression level of the protein encoded by the gene may be provided.

The agent may be selected from the group consisting of primers, probes, antibodies, peptides, and aptamers.

In the present invention, one or two or more genes are selected from the genes consisting of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9, and CXCL10 from a sample isolated from the specimen, or the proteins encoded by the genes, and the genes A method of providing information for radiation exposure diagnosis including the step of confirming the expression or activity level of or the expression level of the protein encoded by the gene can be provided.

The sample may be selected from the group consisting of cells, tissues, blood, serum, urine, and saliva.

The expression level of one or two or more genes selected from the genes consisting of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9 and CXCL10, or the proteins encoded by the genes, or the proteins encoded by the genes If it is increased compared to the normal control group, it may be diagnosed as radiation exposure.

The expression level of the gene or the protein encoded by the gene is determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay. , immunohistochemistry, microarray, western blotting, and flow cytometry (FACS), but is not limited thereto.

The "biomarker" of the present invention is a substance that can diagnose the tissues or cells of a subject suspected of being exposed to radiation by distinguishing them from the tissues or cells of a normal control group, and is increased in the tissues or cells of a subject exposed to radiation compared to the normal control group. or organic biomolecules such as proteins or nucleic acids, lipids, glycolipids, glycoproteins, etc. that show a decrease pattern.

“Diagnosis” in the present invention broadly refers to judging the actual condition of a patient's disease in all aspects. The contents of the judgment include the name of the disease, etiology, type, severity, detailed conditions of the disease, and the presence or absence of complications.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Irradiation

The radiation irradiation experiment was conducted using Gamma cell-3000 (Nordion Inernational INC.) by irradiating 137Cs γ-radiation (3Gy/min) at each dose to male BALB/c mice.

2. Isolation of lymphocytes and bone marrow cells from experimental animals

Irradiated male BALB/c mice were anesthetized using Tiletamine/Zolazepam (Zoletil 50, Virbac Korea, Korea) and xylazine (Xylazine, Rompun, Bayer Korea, Korea), and the saphenous vein Blood was collected from and stored in a heparin tube. Additionally, bone marrow cells were isolated from the femoral bone marrow and stored in 30% FBS solution. Lymphocytes from blood and mononuclear cells from bone marrow were separated using Ficoll solution, and cell numbers were analyzed.

3. Real-time polymerase chain reaction (Real-time PCR)

To verify the microarray analysis results, the same RNA used in the microarray analysis was used in real-time PCR analysis. cDNA was synthesized using 1 μg of RNA, oligo (dT)20 primer, and Superscript-II reverse transcriptase (Invitrogen) (42°C for 50 minutes, 72°C for 15 minutes). The synthesized cDNA was diluted 10-fold, and then 2 μl was subjected to real-time PCR using power SYBR Green PCR Master Mix and 10 pmole primers. Pre-denaturation for PCR analysis; 10 minutes at 95°C, denaturation; 33 seconds at 95°C, annealing; 33 seconds at primer optimum temperature, extension; A program of 33 seconds at 72°C for a total of 40 reactions was used, and the reaction was checked on a 1.2% agarose gel. The 2-ΔΔCt method was used to calculate the c(t) value through real-time PCR, and the GAPDH gene value was used for normalization. Primers used in PCR were designed using Primer 3 software (http://frodo.wi.mit.edu) using NCBI base sequence information.

<Example 1> Confirmation of changes in lymphocyte cells and bone marrow cells after radiation irradiation

To develop a radiation exposure diagnostic gene group, mice were irradiated in vivo at doses of 1, 3, and 5 Gy, and blood was collected 1, 3, and 7 days later, and lymphocyte cells and bone marrow cells were separated to confirm apoptosis. , the results were compared with the CBC (complete blood count) results.

As a result, as shown in Figure 1, it was confirmed that in mice irradiated with 1 Gy radiation, the lymphocyte cells and bone marrow cells decreased until 3 days, but recovered to the level of normal mice without radiation after 7 days, and microarray samples were prepared under the above conditions. Ready.

<Example 2> Identification of lymphocyte cell damage factors and identification of biomarkers for radiation exposure diagnosis

Under the previously confirmed conditions, mice were irradiated in vivo at doses of 1, 3, and 5 Gy, and blood was collected 1, 3, and 7 days later, lymphocyte cells were isolated, and microarray analysis was performed to determine damage control factors and radiation exposure biometrics. Factors with potential as markers were identified.

Mice were irradiated in vivo with 1, 3, and 5 Gy radiation, blood was collected on days 1, 2, 3, and 7, lymphocytes were isolated, RNA was isolated using trizol, and total RNA was purified using Qiagen RNase- It was purified using a free DNase kit and RNeasy spin column and quantified using a UV spectrophotometer. cDNA was synthesized from total RNA (cDNA synthesis kit), double-stranded DNA was purified (Purification of dsDNA, using Qiagen RNeasy Mini Kit), and then labeled with fluorescent dyes Cy3 and Cy5, dropped on a prepared microarray slide, incubated, and hybridized. . The Cy3 and Cy5 fluorescence intensities from each spot were captured and quantitatively recorded using a laser scanner. The intensity of the signal was quantitatively proportional to the actual mRNA expression level of the spot. Using an analysis program, genes whose gene expression increased more than two-fold as the radiation dose increased after irradiation compared to the control group were selected among the microarray analysis results as shown in Figure 3.

Additionally, the selected genes were confirmed using qPCR.

As a result, as shown in Figure 4, it was confirmed that the expression of Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9, and CXCL10 genes increased depending on the radiation dose.

No. gene NCBI accession number One Clu (clusterin) AY888165.1 2 csrnpl (cysteine and serine rich nuclear protein 1) KJ899465.1 3 Dap (death associated protein) AY266680.1 4 Egln3 (egl-9 family hypoxia inducible factor 3) KJ903617.1 5 Hcar2 (hydroxycarboxylic acid receptor 2) KU285440.1 6 RalB (RAS like proto-oncogene B) AF493911.1 7 ACOD1 NM_001258406.2 8 CXCR4 AY242129.1 9 CCL7 BC112258.1 10 CXCL9 BC095396.1 11 CXCL10 BC010954.1

From the above results, it was confirmed that the Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, ACOD1, CXCR4, CCL7, CXCL9, and CXCL10 genes are a useful gene group that can predict radiation exposure.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A biomarker composition for diagnosing radiation exposure comprising CXCL10 and ACOD1 genes or proteins encoded by the genes. The biomarker composition for diagnosing radiation exposure according to claim 1, further comprising the Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, CXCR4, CCL7 or CXCL9 genes or the proteins encoded by the genes. The biomarker composition for diagnosing radiation exposure according to claim 1, wherein expression of the CXCL10 and ACOD1 genes or the proteins encoded by the genes increases upon radiation exposure. A kit for diagnosing radiation exposure containing as an active ingredient an agent that detects the expression or activity level of the CXCL10 and ACOD1 genes, or the expression level of the protein encoded by the genes. The method of claim 4, wherein the kit for diagnosing radiation exposure is an agent that detects the expression or activity level of the Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, CXCR4, CCL7 or CXCL9 gene, or the expression level of the protein encoded by the gene. A kit for diagnosing radiation exposure, further comprising: The kit for diagnosing radiation exposure according to claim 5, wherein the agent is selected from the group consisting of primers, probes, antibodies, peptides, and aptamers. A method of providing information for diagnosing radiation exposure, comprising the step of confirming the expression or activity level of the CXCL10 and ACOD1 genes, or the expression level of the protein encoded by the genes, from a sample separated from the specimen. The method of claim 7, wherein the method of providing information for diagnosing radiation exposure includes the expression or activity level of the Clu, Csrnpl, Dap, Egln3, Hcar2, RalB, CXCR4, CCL7 or CXCL9 gene, or the expression of the protein encoded by the gene. A method of providing information for radiation exposure diagnosis, further comprising the step of checking the level. The method of claim 7, wherein when the expression level of the CXCL10 and ACOD1 genes or the protein encoded by the genes is increased compared to the normal control group, radiation exposure is diagnosed.