The present invention relates to an apoptosis regulatory gene detected in irradiated thymic lymphoma cells, and more particularly to a method of detecting an apoptosis regulatory gene in low-dose-rate low-level-irradiated mouse thymic lymphoma cells.
Generally, high levels of radiation are known to damage DNA and cause cancer. In contrast, low-dose radiation is known to suppress cancer, but the genes involved and the mechanism of cancer suppression are unknown.
Low-dose radiation indicates radiation having low intensity, typically radiation of 100 mSv or less. A large amount of radiation may harm organisms, but low-dose radiation has beneficial effects, such as promoting the physiological activity of organisms to thus prolong the lifespan thereof, or promoting growth or reducing the incidence of tumors. These effects are referred to as radiation hormesis.
Low-dose radiation in the range of milligray (mGy), unlike high-dose radiation, has been reported to increase disease resistance and elicit adaptive protection effects (Sakai K., et al., Dose Response, 2006, 4(4):327-332; Sakai K., et al, Int. J. Low Radiat., 2003, 1(1):142-146; Scott B R., Dose Response, 2008 6(3):299-318), and experimental mice with diabetic genetic factors have been reported to show amelioration in disease after irradiation with a predetermined amount of low-dose radiation (Sakai K., et al., Dose Response, 2006, 4(4):327-332). Furthermore, there have been reports in which radiation of 0.25 Gy or less is effective at preventing DNA damage and restoring damaged DNA (Le X C., et al., Science, 1998, 280(5366):1066-1069), and also in which low-dose radiation is effective at immune enhancement (Nogami M., et al., Int. J. Radiat. Biol., 1993, 63(6):775-783; Nogami M., et al., Radiat. Res., 1994, 139(1):47-52).1. Low-dose radiation).
Accordingly, the present invention is intended to provide an apoptosis regulatory gene detected in irradiated thymic lymphoma cells, and more particularly a method of detecting an apoptosis regulatory gene in low-dose-rate low-level-irradiated thymic cancer cells.
In addition, the present invention is intended to provide a composition for the diagnosis of thymic lymphoma, comprising the base sequence of an apoptosis regulatory gene or a base sequence complementary thereto. In addition, the present invention is intended to provide a kit for the diagnosis of thymic lymphoma, comprising the above composition.
Therefore, the present invention provides a method of detecting an apoptosis regulatory gene in thymic lymphoma cells, comprising applying low-dose-rate low-level radiation to thymic lymphoma cells and detecting an apoptosis regulatory gene having altered gene expression in individual irradiated mouse thymic lymphoma cells.
The thymic lymphoma cells are preferably a mouse (Mus musculus) thymic lymphoma EL4 cell line. The low-dose-rate low-level radiation may be applied such that a final cumulative dose is 61.92 mGy at a dose rate of 2.58 mGy/hr, 139.2 mGy at a dose rate of 5.8 mGy/hr, and 557.28 mGy at a dose rate of 23.22 mGy/hr.
The detecting the apoptosis regulatory gene may include performing polymerase chain reaction and Western blotting.
In the detecting the apoptosis regulatory gene, the detected gene preferably includes Bik (Genebank accession No: NM_007546), Bmf (Genebank accession No: NM_138313), Ddit3 (Genebank accession No: NM_007837), Nod1 (Genebank accession No: NM_172729), and Tnfrsf19 (Genebank accession No: NM_013869).
In addition, the present invention provides a composition for the diagnosis of thymic lymphoma, comprising the base sequence of at least one apoptosis regulatory gene selected from the group consisting of apoptosis regulatory genes of thymic lymphoma, including Bik (Genebank accession No: NM_007546), Bmf (Genebank accession No: NM_138313), Ddit3 (Genebank accession No: NM_007837), Nod1 (Genebank accession No: NM_172729) and Tnfrsf19 (Genebank accession No: NM_013869) or the base sequence complementary thereto.
In addition, the present invention provides a diagnostic kit for the diagnosis of thymic lymphoma, comprising the above composition for the diagnosis of thymic lymphoma.
According to the present invention, an apoptosis regulatory gene can be detected in irradiated thymic lymphoma cells, thereby revealing the function of the apoptosis regulatory gene by irradiation and providing a gene profile.
Also, the apoptosis regulatory gene detected in irradiated thymic lymphoma cells can be used to construct a gene profile that enables assessment of a dose-response relationship of industrial and healthcare workers in low-level radiation environments.
The apoptosis regulatory gene detected in irradiated thymic lymphoma cells can be used as a measure of cancer progression and treatment in patients with thymic lymphoma.
The method of detecting the apoptosis regulatory gene in irradiated thymic lymphoma cells can be used to manufacture a composition for the diagnosis of thymic lymphoma and a diagnostic kit.
Hereinafter, a detailed description will be given of the present invention.
An embodiment of the present invention pertains to a method of detecting an apoptosis regulatory gene in thymic lymphoma cells, comprising applying low-dose-rate low-level radiation, high-dose-rate low-level radiation and high-dose-rate high-level radiation to thymic lymphoma cells and detecting an apoptosis regulatory gene having altered gene expression in individual irradiated mouse thymic lymphoma cells.
The thymic lymphoma cells are preferably derived from mouse (Mus musculus) thymic lymphoma.
As the mouse thymic lymphoma cells, the EL4 cell line is a T-cell line established in lymphoma of C57BL/6, which is a pure mouse to which a chemical carcinogen is administered, and expresses Thy-1, 2 and H2b on the cell surface, and is reactive to phytohemagglutinin (PHA). This cell line produces interleukin-2 (IL-2) and is used for cell-mediated immunity experiments.
The low-dose-rate low-level radiation is preferably applied such that a final cumulative dose is 61.92 mGy at a dose rate of 2.58 mGy/hr, 139.2 mGy at a dose rate of 5.8 mGy/hr, and 557.28 mGy at a dose rate of 23.22 mGy/hr.
The detecting the apoptosis regulatory gene may be performed through polymerase chain reaction and Western blotting.
In the detecting the apoptosis regulatory gene, the detected gene may include Bik ((Genebank accession No: NM_007546), Bmf (Genebank accession No: NM_138313), Ddit3 (Genebank accession No: NM_007837), Nod1 (Genebank accession No: NM_172729), and Tnfrsf19 (Genebank accession No: NM_013869).
Another embodiment of the present invention pertains to a composition for the diagnosis of thymic lymphoma, comprising the base sequence of at least one apoptosis regulatory gene selected from the group consisting of apoptosis regulatory genes of thymic lymphoma, including Bik ((Genebank accession No: NM_007546), Bmf (Genebank accession No: NM_138313), Ddit3 (Genebank accession No: NM_007837), Nod1 (Genebank accession No: NM_172729) and Tnfrsf19 (Genebank accession No: NM_013869) or the base sequence complementary thereto.
A better understanding of the present invention will be given through the following examples, which are merely set forth to illustrate the present invention but are not to be construed as limiting the scope of the present invention, as will be apparent to those skilled in the art.
Mouse thymic lymphoma cells (EL4) (TIB-39, ATCC) were cultured using a cell incubator at 37° C. under 5% CO2 using an RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cultured cells were prepared at a concentration of 105 cells/ml.
For the low-dose-rate low-level-irradiated cell group, low-dose-rate low-level radiation (2.58 mGy/hr, 5.8 mGy/hr, 23.22 mGy/hr) was applied for 24 hr to the cells prepared as above such that the cumulative dose of the cells was 61.92 mGy, 139.2 mGy and 557.28 mGy.
For the high-dose-rate low-level-irradiated cell group, in comparison with the low-dose-rate low-level-irradiated cell group, high-dose-rate low-level radiation (0.8 Gy/min) was applied to the cells prepared as described above such that the final cumulative dose was 61.92 mGy, 139.2 mGy and 557.28 mGy.
For the high-dose-rate high-level-irradiated cell group, high-dose-rate high-level radiation (0.8 Gy/min) was applied such that the final cumulative dose was 2 Gy.
In order to screen the apoptosis regulatory gene of irradiated cells, a microarray was performed.
Total RNA of the low-dose-rate low-level-irradiated cell group of Example 1 was extracted and labeled with Cyanine3. A microarray chip in which all the genes of total DNA were arranged was prepared, and the Cyanine3-labeled total RNA was allowed to react therein. The signal in response to the above reaction was analyzed and the extent of expression of each RNA was verified.
Furthermore, the high-dose-rate low-level-irradiated cell group and the high-dose-rate high-level-irradiated cell group were subjected to microarray in the same manner as above.
Based on the results of microarray, genes, the expression of which was increased two times or more in the low-dose-rate low-level-irradiated cell group compared to the non-irradiated cell group, were selected. The selected genes were Adm, Btg2, II12a, Mapt, Mnda, Bmf, Nod1, Bik, Ddit3, Prkcz, and Tnfrsf19.
2-2 Measurement of RNA-Level Expression of Apoptosis Regulatory Gene of Irradiated Mouse Thymic Lymphoma Cells
In order to verify RNA-level expression of 11 genes selected through microarray, polymerase chain reaction (PCR) was performed.
Using a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA), RNA of the apoptosis regulatory gene was subjected to reverse transcription. Then, PCR of RNA subjected to reverse transcription was carried out using primers shown in Table 1 below. The PCR results were normalized using the housekeeping gene GAPDH.
Furthermore, in the high-dose-rate low-level-irradiated cell group and the high-dose-rate high-level-irradiated cell group, the amount of apoptosis regulatory gene expression was measured in the same manner as above.
The results of measurement of RNA-level expression of apoptosis regulatory genes upon irradiation are shown in
Upon low-dose-rate low-level irradiation, among the apoptosis regulatory genes selected in Example 2-1, genes, the RNA-level expression of which was increased compared to the non-irradiated cell group, were detected. The detected genes were Adm, Btg2, II12a, Mnda, Bmf, Nod1, Bik, Ddit3, and Tnfrsf19.
In the high-dose-rate low-level-irradiated cell group and the high-dose-rate high-level-irradiated cell group, the apoptosis regulatory gene tended to decrease in RNA-level expression.
In order to measure protein expression of the apoptosis regulatory gene detected in Example 2-2, Western blotting was performed.
A cell lysis buffer was prepared by mixing 150 mM NaCl, 1% Triton X0199, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM Tris (pH 7.4) and 1 mM EDTA. Also, the prepared cell lysis buffer was added with a protease inhibitor (Thermo Scientific, USA) at a concentration of 1% based on the total concentration of the solution.
The low-dose-rate low-level-irradiated cell group of Example 1 was mixed with the cell lysis buffer, and thus the cells were lysed to give a protein sample. Western blotting was performed using the protein sample, and the amount of protein expression was measured. Upon Western blotting, an antibody (1:200, Abcam, Cambridge, Mass.) for detecting Ddit3 and Tnfrsf19 and an antibody (1:200, OriGene) for detecting Nod1, Bmf, and Bik were used.
Also, in the high-dose-rate low-level-irradiated cell group and the high-dose-rate high-level-irradiated cell group of Example 1, the amount of apoptosis regulatory protein expression was measured in the same manner as above.
The results of measurement of the amount of apoptosis regulatory protein expression upon irradiation are shown in
Based on the above results, apoptosis regulatory genes, the RNA-level expression and protein-level expression of which were increased when applying the low-dose-rate low-level radiation to mouse thymic lymphoma cells, were detected. The detected genes were Bmf, Nod1, Bik, Ddit3, and Tnfrsf19. The detected apoptosis regulatory genes were increased in RNA-level expression and protein-level expression when applying the low-dose-rate low-level radiation to mouse thymic lymphoma cells, thus promoting apoptosis to thereby suppress thymic lymphoma (
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that these embodiments are merely set forth to illustrate but are not to be construed to limit the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Number | Date | Country | Kind |
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10-2016-0081973 | Jun 2016 | KR | national |
Filing Document | Filing Date | Country | Kind |
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PCT/KR2016/010072 | 9/8/2016 | WO | 00 |