KR102177324B1 - A low-dose radiation-specific biomarker including a PPP1R15A gene and a PPP1R15A protein, a low-dose radiation exposure diagnostic method using the biomarker, and a diagnostic kit including the biomarker - Google Patents
A low-dose radiation-specific biomarker including a PPP1R15A gene and a PPP1R15A protein, a low-dose radiation exposure diagnostic method using the biomarker, and a diagnostic kit including the biomarker Download PDFInfo
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Abstract
본 발명은 저선량방사선 피폭에 의한 세포생물학적 초기 반응을 확인할 수 있는 바이오마커, 상기 바이오마커를 포함하는 진단키트 및 상기 바이오마커를 이용하는 저선량방사선 피폭 진단 방법에 관한 것으로서, PPP1R15A유전자 또는 PPP1R15A단백질을 포함한다.
상기와 같은 본 발명에 따르면, 100 mSv 이하의 저선량방사선 노출에 특이적으로 반응하는 새로운 유전자 (PPP1R15A, SMAD6)의 발현량 변화를 확인함으로써, 의료 및 방사선 작업종사자, 일반인의 저선량방사선 피폭을 짧은 시간 내에 확인 할 수 있는 효과가 있다.The present invention relates to a biomarker capable of confirming the initial cellular biological response due to low-dose radiation exposure, a diagnostic kit including the biomarker, and a method for diagnosing low-dose radiation exposure using the biomarker, comprising the PPP1R15A gene or the PPP1R15A protein. .
According to the present invention as described above, by confirming the change in the expression level of new genes (PPP1R15A, SMAD6) specifically responding to exposure to low-dose radiation of 100 mSv or less, the low-dose radiation exposure of medical and radiation workers and the general public is reduced for a short time There is an effect that can be checked within.
Description
본 발명은 저선량방사선에 특이적 바이오마커 및 상기 바이오마커를 이용한 저선량방사선 피폭 진단방법 및 상기 바이오마커를 포함하는 진단 키트에 관한 것으로서, 더욱 상세하게는 저선량방사선에 특이적인 유전자 및 단백질을 이용하는 것이다.The present invention relates to a biomarker specific for low-dose radiation, a method for diagnosing low-dose radiation using the biomarker, and a diagnostic kit comprising the biomarker, and more particularly, to a gene and protein specific for low-dose radiation.
후쿠시마 원전 폭발 사고, 라돈 침대 논란 등의 사회적 이슈로 인해 저선량방사선이 인체에 미치는 영향에 대한 관심이 고조되고 있다. 고선량방사선 노출에 의한 확정적 영향에 대해서는 잘 알려져 있으나, 100 mSv 이하의 저선량방사선이 인체에 미치는 영향에 대해서는 과학적 증명이 부족하다. 또한 저선량방사선 피폭은 그 증상이나 영향이 즉각적으로 나타나지 않기 때문에 잠재적인 영향에 대한 우려가 있다.Interest in the effects of low-dose radiation on the human body is increasing due to social issues such as the Fukushima nuclear power plant explosion and the radon bed controversy. Although the definitive effect of exposure to high-dose radiation is well known, scientific evidence is insufficient for the effect of low-dose radiation below 100 mSv on the human body. In addition, since the symptoms or effects of low-dose radiation exposure do not appear immediately, there is concern about the potential effects.
따라서 저선량방사선 피폭에 의한 인체 영향에 대해 과학적 근거를 마련해야 하며, 이를 위해 저선량에 특이적으로 반응하는 생체 지표를 발굴하고 이를 통한 생체 영향 기전을 규명하는 것이 필요하다. Therefore, it is necessary to prepare a scientific basis for the effects of low-dose radiation on the human body, and for this purpose, it is necessary to discover a biomarker that specifically responds to low-dose and to investigate the mechanism of bio-influence through this.
저선량방사선에 노출된 경우, 즉각적인 증상이나 영향이 나타나지 않기 때문에 저선량방사선으로 인한 인체 영향을 확인하기까지는 많은 시간이 소요되고 이를 검출하는 데에도 많은 한계가 있다. In the case of exposure to low-dose radiation, since no immediate symptoms or effects appear, it takes a lot of time to determine the effect of the human body due to low-dose radiation, and there are many limitations in detecting it.
이러한 이유로, 저선량방사선 피폭에 관련한 연구는 현상학적 효과를 관찰하는 수준에 머물러 있을 뿐 구체적 영향 기전이 밝혀지지 않았다. 따라서 저선량방사선의 인체 영향 기전을 명확하게 규명하여, 보다 효율적인 저선량방사선 검출 방법을 제공할 필요가 있다.For this reason, studies on low-dose radiation exposure remain at the level of observing phenomenological effects, but no specific mechanism of effect has been identified. Therefore, there is a need to provide a more efficient method for detecting low-dose radiation by clearly clarifying the mechanism of influence on the human body of low-dose radiation.
이에 본 발명은 저선량방사선에 특이적인 바이오마커를 제시함으로써 보다 효율적인 저선량방사선의 피폭을 확인할 수 있도록 하고자 한다. Accordingly, the present invention proposes a biomarker specific to low-dose radiation, so that more efficient low-dose radiation exposure can be confirmed.
본 발명의 목적은, 저선량방사선 피폭에 의한 세포생물학적 초기 반응을 확인할 수 있는 바이오마커를 개발함으로써, 상기 바이오마커를 포함하는 진단키트 및 상기 바이오마커를 이용하는 저선량방사선 피폭 진단 방법 등을 제공함에 있다.An object of the present invention is to provide a diagnostic kit including the biomarker and a method for diagnosing low-dose radiation using the biomarker by developing a biomarker capable of confirming the initial cellular biological response due to low-dose radiation exposure.
상기 목적을 달성하기 위하여, 본 발명은 PPP1R15A 유전자 또는 PPP1R15A 단백질을 포함하는 저선량방사선 특이적 바이오마커, 상기 바이오마커를 이용한 저선량방사선 피폭 진단방법 및 상기 바이오마커를 포함하는 저선량방사선 피폭 진단 키트를 제공한다.In order to achieve the above object, the present invention provides a low-dose radiation-specific biomarker comprising the PPP1R15A gene or PPP1R15A protein, a low-dose radiation exposure diagnostic method using the biomarker, and a low-dose radiation exposure diagnostic kit comprising the biomarker. .
상기와 같은 본 발명에 따르면, 100 mSv 이하의 저선량방사선 노출에 특이적으로 반응하는 새로운 유전자 (PPP1R15A의 발현량 변화를 확인함으로써, 의료 및 방사선 작업종사자, 일반인의 저선량방사선 피폭을 짧은 시간 내에 확인 할 수 있는 효과가 있다.According to the present invention as described above, by confirming the change in the expression level of a new gene (PPP1R15A) that specifically responds to exposure to low-dose radiation of 100 mSv or less, it is possible to confirm low-dose radiation exposure of medical and radiation workers and the general public within a short time. It can have an effect.
도 1은 사람의 B림프구 세포주인 Ramos 세포주를 이용하여 저선량방사선에 의한 PPP1R15A의 발현 변화를 확인한 결과를 도시하였다.
도 2는 사람의 B림프구 세포주인 IM9 세포주에서 저선량방사선에 의한 PPP1R15A의 단백질 발현 변화를 확인한 결과를 도시하였다.
도 3은 BALB/c 마우스의 비장세포(A)와 골수세포(B)에서 저선량방사선에 의한 PPP1R15A 발현의 변화를 확인한 결과를 도시하였다. 1 shows the results of confirming the change in the expression of PPP1R15A by low-dose radiation using the Ramos cell line, which is a human B lymphocyte cell line.
Figure 2 shows the results of confirming the protein expression change of PPP1R15A by low-dose radiation in the human B lymphocyte cell line IM9 cell line.
3 shows the results of confirming the change in PPP1R15A expression by low-dose radiation in the splenocytes (A) and bone marrow cells (B) of BALB/c mice.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 형태에 따라 PPP1R15A 유전자 또는 PPP1R15A 유전자를 기반으로 합성된 PPP1R15A 단백질을 포함하는 저선량방사선 특이적 바이오마커를 제공한다. According to one aspect of the present invention, a low-dose radiation-specific biomarker comprising a PPP1R15A protein synthesized based on the PPP1R15A gene or the PPP1R15A gene is provided.
상기 PPP1R15A 유전자는 저선량방사선 노출시 저선량방사선 비노출에 비해 발현이 증가할 수 있다. The expression of the PPP1R15A gene may be increased when exposed to low-dose radiation compared to non-exposed to low-dose radiation.
상기 PPP1R15A 단백질는 저선량방사선 노출시 저선량방사선 비노출에 비해 합성이 증가할 수 있다. The PPP1R15A protein may increase in synthesis when exposed to low-dose radiation compared to non-exposed to low-dose radiation.
상기 저선량방사선은 10 내지 100 mSv의 선량일 수 있다. The low-dose radiation may be a dose of 10 to 100 mSv.
본 발명의 다른 형태에 따라 저선량방사선 피폭 진단방법을 제공한다. According to another aspect of the present invention, a method for diagnosing low-dose radiation exposure is provided.
상기 저선량방사선 피폭 진단방법은 사람을 제외한 동물의 생체시료를 준비하는 단계, 상기 생체시료의 PPP1R15A유전자 또는 PPP1R15A단백질을 분석하는 단계 및 상기 분석된 값을 저선량방사선에 비노출된 대조군의 PPP1R15A유전자 또는 PPP1R15A단백질 분석값과 비교하는 단계를 포함한다. The method for diagnosing low-dose radiation exposure includes preparing a biological sample of animals other than humans, analyzing the PPP1R15A gene or PPP1R15A protein of the biological sample, and using the analyzed value as the PPP1R15A gene or PPP1R15A protein of a control group that was not exposed to low-dose radiation. And comparing the analyzed value.
상기 생체시료는 혈액, 비장 또는 골수 일 수 있다. The biological sample may be blood, spleen or bone marrow.
상기 유전자를 분석하는 방법은 실시간 중합효소연쇄반응(Real-time PCR) 일 수 있다. The method of analyzing the gene may be real-time PCR.
상기 단백질을 분석하는 방법은 웨스턴 블롯(western blot) 일 수 있다. The method of analyzing the protein may be a western blot.
상기 PPP1R15A유전자 또는 PPP1R15A단백질 분석값을 비교하는 단계는 PPP1R15A유전자 또는 PPP1R15A단백질이 대조군에 비해 특이적으로 발현 또는 합성이 증가되는 것을 확인할 수 있다. Comparing the analysis values of the PPP1R15A gene or PPP1R15A protein may confirm that the PPP1R15A gene or the PPP1R15A protein is specifically expressed or synthesized compared to the control.
본 발명의 또 다른 형태에 따라 PPP1R15A유전자 또는 PPP1R15A 유전자를 기반으로 합성된 PPP1R15A 단백질을 포함하는 저선량방사선 피폭 진단 키트를 제공한다. According to another aspect of the present invention, there is provided a diagnostic kit for low-dose radiation exposure comprising a PPP1R15A protein synthesized based on the PPP1R15A gene or the PPP1R15A gene.
상기 PPP1R15A유전자 또는 PPP1R15A단백질은 저선량방사선 노출시 저선량방사선 비노출에 비해 특이적으로 발현 또는 합성이 증가하는 것을 특징으로 할 수 있다. The PPP1R15A gene or PPP1R15A protein may be characterized by a specific increase in expression or synthesis when exposed to low-dose radiation compared to non-exposed to low-dose radiation.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.
B림프구 세포주에서 저선량방사선에 의한 PPP1R15A의 발현 변화 확인Confirmation of the expression change of PPP1R15A by low-dose radiation in B lymphocyte cell line
실시예 1. Example 1.
방사선조사기 (137Cs, 204 mGy/min, Grammacell 40 Exactor, Best Theratronics Ltd.)를 이용하여, 인간 B 림프구 세포주인 Ramos 5개의 실험군에 각각 10, 25, 50, 100, 1000 mGy의 이온화방사선을 조사하였다. Using a radiation irradiator (137Cs, 204 mGy/min, Grammacell 40 Exactor, Best Theratronics Ltd.), the human B lymphocyte cell line Ramos 5 experimental groups were irradiated with 10, 25, 50, 100, 1000 mGy of ionizing radiation, respectively. .
세포주 Ramos 5개의 실험군에 저선량방사선을 각각 10, 25, 50, 100, 1000 mGy로 조사하고 24시간 배양한 후, Ramos 세포를 취하여 total RNA를 추출하였다. 저선량방사선이 조사된 세포에서 total RNA를 추출하기 위하여 RNA isolation kit (RNeasy Mini Kit, Qiagen, Germany)를 사용하여 제조사가 제공한 방법에 따라 추출하였다. 그 후 One Step SYBR PrimeScriptTM PLUS RT-PCR Kit (Takara)를 사용하여 세포에서 추출한 RNA와 PPP1R15A 프라이머를 이용하여 실시간 중합효소연쇄반응(Real-time PCR)을 수행하여 분석하였다. Low-dose radiation was irradiated with 10, 25, 50, 100, and 1000 mGy, respectively, to the experimental groups of five cell lines Ramos, and cultured for 24 hours, and then Ramos cells were taken to extract total RNA. In order to extract total RNA from the cells irradiated with low-dose radiation, it was extracted according to the method provided by the manufacturer using an RNA isolation kit (RNeasy Mini Kit, Qiagen, Germany). After that, real-time PCR was performed using RNA extracted from cells and PPP1R15A primer using One Step SYBR PrimeScriptTM PLUS RT-PCR Kit (Takara) and analyzed.
분석결과는 도 1에 도시하였다. The analysis results are shown in FIG. 1.
도1을 참조하면, Ramos 세포주에서 저선량방사선에 의해 PPP1R15A의 발현이 증가하는 것을 확인할 수 있었다.Referring to Figure 1, it was confirmed that the expression of PPP1R15A was increased by low-dose radiation in the Ramos cell line.
실시예 2. Example 2.
방사선조사기 (137Cs, 204 mGy/min, Grammacell 40 Exactor, Best Theratronics Ltd.)를 이용하여, 인간 B 림프구 세포주인 IM9 5개의 실험군에 10, 25, 50, 100, 1000 mGy의 이온화방사선을 조사하였다. Using an irradiator (137Cs, 204 mGy/min, Grammacell 40 Exactor, Best Theratronics Ltd.), Ionizing radiation of 10, 25, 50, 100, 1000 mGy was irradiated to the five experimental groups of IM9, a human B lymphocyte cell line.
IM9 세포주에 저선량방사선을 10, 25, 50, 100 mGy로 조사하고 24시간 배양한 후 세포를 취하여 단백질을 추출하였다. SDS lysis buffer를 이용하여 세포를 용해하고 10분동안 가열 후 원심분리하여 세포잔해를 제거하여 단백질을 얻었다. 그 후 pierce BCA protein assay kit (Thermo Fisher Scientific, USA)를 이용하여 추출한 단백질을 정량하였다. 단백질 정량 후 PPP1R15A 항체 (ab9869, abcam, England)와 β-actin 항체 (sc-47778, Santa Cruz, USA)를 이용하여 western blot analysis를 진행하여 단백질 발현량을 확인하였다. The IM9 cell line was irradiated with low-dose radiation at 10, 25, 50, 100 mGy, cultured for 24 hours, and then the cells were taken to extract proteins. Cells were lysed using SDS lysis buffer, heated for 10 minutes, and centrifuged to remove cell debris to obtain proteins. Then, the extracted protein was quantified using the pierce BCA protein assay kit (Thermo Fisher Scientific, USA). After protein quantification, western blot analysis was performed using PPP1R15A antibody (ab9869, abcam, England) and β-actin antibody (sc-47778, Santa Cruz, USA) to confirm the protein expression level.
단백질 발현량 결과는 도 2에 도시하였다. The protein expression level results are shown in FIG. 2.
도 2를 참조하면 IM9 세포주에서 저선량방사선에 의해 PPP1R15A의 단백질 양이 증가하는 것을 확인할 수 있었다.Referring to FIG. 2, it was confirmed that the amount of protein of PPP1R15A was increased by low-dose radiation in the IM9 cell line.
실험용 마우스 비장, 골수세포에서 저선량방사선에 의한 PPP1R15A의 발현 변화 확인Confirmation of the expression change of PPP1R15A by low-dose radiation in experimental mouse spleen and bone marrow cells
실시예 3. Example 3.
방사선조사기 (137Cs, 204 mGy/min, Grammacell 40 Exactor, Best Theratronics Ltd.)를 이용하여, BALB/c 마우스 5개 실험군의 전신에 각각 10, 25, 50, 100, 1000 mGy의 이온화방사선을 조사하였다. Using a radiation irradiator (137Cs, 204 mGy/min, Grammacell 40 Exactor, Best Theratronics Ltd.), the whole body of the five experimental groups of BALB/c mice was irradiated with 10, 25, 50, 100, 1000 mGy of ionizing radiation, respectively. .
BALB/c 마우스 5개 실험군에 각각 저선량방사선을 전신 조사하고 24시간 후 BALB/c 마우스우의 유전자 발현을 분석하기 위해 비장세포와 골수세포를 분리하여 total RNA를 분리하였다. 이때 40 um cell strainer를 이용하여 비장과 골수에서 세포를 분리한 후, RNA isolation kit (RNeasy Mini Kit, Qiagen, Germany)를 사용하여 제조사가 제공한 방법에 따라 total RNA를 추출하였다. 그 후 One Step SYBR PrimeScriptTM PLUS RT-PCR Kit (Takara)를 사용하여 분리한 RNA와 PPP1R15A 프라이머를 이용하여 실시간 중합효소연쇄반응(Real-time PCR)을 수행한 후 결과를 분석하였다.Each of the five experimental groups of BALB/c mice was irradiated with low-dose radiation throughout the body, and after 24 hours, splenocytes and bone marrow cells were separated and total RNA was isolated to analyze gene expression in BALB/c mice. At this time, cells were separated from the spleen and bone marrow using a 40 um cell strainer, and then total RNA was extracted using an RNA isolation kit (RNeasy Mini Kit, Qiagen, Germany) according to the method provided by the manufacturer. After that, real-time PCR was performed using RNA isolated using One Step SYBR PrimeScriptTM PLUS RT-PCR Kit (Takara) and PPP1R15A primer, and the results were analyzed.
분석 결과는 도 3에 도시하였다. The analysis results are shown in FIG. 3.
도 3을 참조하면, BALB/c 마우스의 비장과 골수에서 저선량방사선에 의해 PPP1R15A의 발현이 증가하는 것을 확인할 수 있었다.3, it was confirmed that the expression of PPP1R15A was increased by low-dose radiation in the spleen and bone marrow of BALB/c mice.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다. As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.
<110> Korea Hydro & Nuclear Power Co. Ltd.
<120> A low-dose radiation-specific biomarker including a PPP1R15A gene
and a PPP1R15A protein, a low-dose radiation exposure diagnostic
method using the biomarker, and a diagnostic kit including the
biomarker
<130> P19-E054
<160> 2
<170> KoPatentIn 3.0
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<213> Mus musculus
<220>
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ggtgtatcgc ccaggagagg acacagagga agaaaacagc gatttggatt cagctgagga 1440
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aggagaggac acagaagatg acacagaaga ggaagaggac agtgagaatg tggccccagg 1680
tgactcagaa acagctgact caagccagag tccctgcctt cagccccagc gttgtctacc 1740
aggagagaag accaagggac gtggggaaga gccccctctc ttccaggtgg ccttctattt 1800
acccggagag aagccagaat caccttgggc tgcacctaag ctgccccttc gactgcagag 1860
gcggctcaga ttgttcaaag cccccacccg ggatcaggac cccgagattc ctctaaaagc 1920
tcggaaggta cacttcgctg agaaagtcac agtccatttc cttgctgtct gggcaggacc 1980
agcccaagct gcccgtcgag gtccctggga gcagtttgca cgagatcgaa gccgctttgc 2040
tcgacgcatt gcccaggcag aggagaagct gggtccctac cttacccctg attccagggc 2100
cagagcatgg gcacgcctta gaaacccatc tcttccacag tccgagcctc gctcttcctc 2160
tgaggccact cccttgaccc aagatgtgac cacaccctct ccccttccca gtgaaacccc 2220
ttcgcccagc ctgtacttgg gagggaggcg gggctaagcc tgagtagttt cctattattt 2280
atttatttat ttatttgaat aagaaataaa gccttttaat ttgtagtgat aaaaaaaaaa 2340
aaaaa 2345
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<110> Korea Hydro & Nuclear Power Co. Ltd.
<120> A low-dose radiation-specific biomarker including a PPP1R15A gene
and a PPP1R15A protein, a low-dose radiation exposure diagnostic
method using the biomarker, and a diagnostic kit including the
biomarker
<130> P19-E054
<160> 2
<170> KoPatentIn 3.0
<210> 1
<211> 2345
<212> RNA
<213> Mus musculus
<220>
<221> mRNA
<222> (1)..(2345)
<223> Mus
Claims (11)
A composition for detecting the biomarker specific for low-dose radiation of 25 to 50 mSv as a biomarker comprising a PPP1R15A protein synthesized based on the PPP1R15A gene or PPP1R15A gene of the sample using a biological sample of the spleen or bone marrow as a sample.
상기 PPP1R15A 유전자는 25 내지 50 mSv의 저선량방사선 노출시 25 내지 50 mSv의 저선량방사선 비노출에 비해 발현이 증가하는 것을 특징으로 하는 저선량 방사선 특이적 바이오마커를 검출하기 위한 조성물.
The method of claim 1,
The composition for detecting a low-dose radiation-specific biomarker, characterized in that the expression of the PPP1R15A gene is increased when exposed to low-dose radiation of 25 to 50 mSv compared to non-exposed to low-dose radiation of 25 to 50 mSv.
상기 PPP1R15A 단백질은 25 내지 50 mSv의 저선량방사선 노출시 25 내지 50 mSv의 저선량방사선 비노출에 비해 합성이 증가하는 것을 특징으로 하는 저선량 방사선 특이적 바이오마커를 검출하기 위한 조성물.
The method of claim 1,
The PPP1R15A protein is a composition for detecting low-dose radiation-specific biomarkers, characterized in that the synthesis increases when exposed to low-dose radiation of 25 to 50 mSv compared to non-exposed to low-dose radiation of 25 to 50 mSv.
Low-dose radiation exposure diagnostic kit comprising the composition according to claim 1.
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