KR100614980B1 - Reagent for Detectihg Radioactivity Using Cytogenetic Property and Detecting Method thereby - Google Patents
Reagent for Detectihg Radioactivity Using Cytogenetic Property and Detecting Method thereby Download PDFInfo
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Abstract
본 발명은 핵단편의 확인을 위해 아크리딘 오렌지를 염색물질로 포함하는 방사선 선량측정용 염색액 조성물을 개시한다.The present invention discloses a radiation dosimetry dye composition comprising acridine orange as a dyeing material for identification of nuclear fragments.
또한 본 발명은 아크리딘 오렌지 염색액을 측정대상 세포에 염색처리하는 단계; 상기 염색된 세포를 형광처리하여 이로부터 발하는 빛을 통해 핵단편의 출현빈도를 확인하는 단계를 포함하는 생물학적 방사선 선량측정방법을 개시한다.In another aspect, the present invention comprises the steps of staining the acridine orange staining solution cells; Disclosed is a biological radiation dosimetry method comprising fluorescence of the stained cells to confirm the frequency of appearance of nuclear fragments through light emitted therefrom.
상기 구성에 의하면 방사선에 대한 세포유전학적 효과를 신속하게 검출할 수 있으며, 세포내 핵단편물을 비특이성 과립 등과 명확하게 구분할 수 있어 20cGy 이하의 저선량 방사선에 대해서도 실험오차를 줄일 수 있다. 또한 짧은 시간에 대량의 시료를 처리할 수 있고, 절차가 간단해 비전문가라도 용이하게 검출할 수 있는 장점이 있다. According to the above configuration, the cytogenetic effect on radiation can be detected quickly, and the intracellular nucleus fragment can be clearly distinguished from non-specific granules and the like, thereby reducing the experimental error even for low dose radiation of 20 cGy or less. In addition, a large amount of sample can be processed in a short time, the procedure is simple, even non-expert can be easily detected.
Description
도 1은 본 발명에 의한 아크리딘 오렌지 염색액에 의해 사람 B-림프구내 핵 단편을 관찰한 사진1 is a photograph of a nuclear fragment in human B-lymphocytes by the acridine orange stain according to the present invention
도 2는 종래 김사염색법에 의해 사람 B-림프구내 핵 단편을 관찰한 사진Figure 2 is a photograph of observing nuclear fragments in human B-lymphocytes by conventional gimsa staining method
본 발명은 세포유전학적 특성을 이용한 방사선 선량측정용 시약조성물 및 이를 이용한 방사선 선량측정방법에 관한 것으로, 보다 상세하게는 염색물질로 아크리딘 오렌지를 이용한 시약조성물 및 이를 이용한 방사선 선량측정방법에 관한 것이다.The present invention relates to a reagent composition for measuring radiation dose using cytogenetic properties and a method for measuring radiation dose using the same, and more particularly, to a reagent composition using acridine orange as a dyeing agent and a method for measuring radiation dose using the same. will be.
방사선의 인체에 대한 영향을 판단하는데 있어서 혈액이나 조직세포의 형태ㆍ조직학적 변화를 관찰하는 방법이 이용되고 있다. 이를 위해 검출 예민도가 높은 염색법이 활용되고 있으며, 염색체의 이상을 관찰하여 보다 간단하고, 짧은 시간에 대량의 시료를 분석하고자 하는 노력들이 행해져 왔다.In determining the effect of radiation on the human body, a method of observing the morphological and histological changes of blood or tissue cells is used. For this purpose, a high sensitivity detection method is utilized, and efforts have been made to analyze a large amount of samples in a simpler and shorter time by observing abnormalities in chromosomes.
하지만 종래 방사선의 선량과 이로 인한 인체의 영향 등을 세포유전학적 방법을 이용해 관찰하고자 하는 경우 전문적인 지식, 고가의 장비 및 많은 시간이 투입되는 문제가 지적된다. 또한 현재 널리 활용되고 있는 김사(Giemsa)염색법은 세포내에 잔존하는 염기성 과립 등을 포함한 비특이적 인자를 핵단편물과 구별하지 못하여 실험오차를 좁힐 수 없는 한계가 있다.However, when it is desired to observe the dose of radiation and the effects of the human body by means of cytogenetic methods, it is pointed out that a problem in which expert knowledge, expensive equipment, and a large amount of time is used. In addition, the currently used Kimsa (Giemsa) staining method has a limit that can not narrow the experimental error because it can not distinguish the non-specific factors, such as basic granules remaining in the cell with the nuclear fragment.
본 발명은 상기한 종래 기술에 대한 문제를 해결하기 위해 안출된 것으로 방사선에 대한 세포유전학적 효과를 신속하게 검출할 수 있으며, 세포내 핵단편물을 비특이성 과립 등과 명확하게 구분할 수 있어 실험오차를 줄일 수 있는 새로운 측정용 조성물을 제공함에 목적이 있다.The present invention has been made to solve the above problems of the prior art, it is possible to quickly detect the cytogenetic effect on radiation, and to clearly distinguish the intracellular nucleus fragments from non-specific granules, etc. It is an object to provide a new measuring composition that can be reduced.
본 발명의 다른 목적은 상기 측정용 시약을 이용해 짧은 시간에 대량의 시료를 처리할 수 있고, 절차가 간단해 비전문가라도 용이하게 검출할 수 있는 방사선 선량 측정 방법을 제공함에 있다.
Another object of the present invention is to provide a radiation dose measuring method which can process a large amount of samples in a short time by using the measuring reagent, and the procedure is simple and can be easily detected even by non-experts.
상기 목적을 달성하기 위한 본 발명은 핵단편의 확인을 위해 아크리딘 오렌지를 염색물질로 포함하는 방사선 선량측정용 염색액 조성물을 포함한다.The present invention for achieving the above object comprises a radiation dose measurement dye solution composition containing acridine orange as a dyeing material for the identification of nuclear fragments.
또한 본 발명은 아크리딘 오렌지 염색액을 측정대상 세포에 염색처리하는 단계; 상기 염색된 세포를 형광처리하여 이로부터 발하는 빛을 통해 핵단편의 출현빈도를 확인하는 단계를 포함하는 생물학적 방사선 선량측정방법을 포함한다.In another aspect, the present invention comprises the steps of staining the acridine orange staining solution cells; It includes a biological radiation dosimetry method comprising the step of identifying the frequency of appearance of the nuclear fragments by fluorescence treatment of the stained cells.
본 발명의 방사선 선량측정용 염색액 조성물은 염색물질로 아크리딘 오렌지가 사용된다. 아크리딘 오렌지 염색액은 시판되고 있는 제품(Waco사 제품, 일본)을 그대로 사용하거나 희석하여 사용할 수 있다. 희석액으로는 특별한 한정을 요하는 것은 아니며, 인산완충용액 등이 이용될 수 있다.In the dyeing composition for radiation dosimetry of the present invention, acridine orange is used as a dyeing material. Acridine orange stain can be used as it is or commercially available (available from Waco, Japan). The diluent is not particularly limited, and a phosphate buffer solution may be used.
아크리딘 오렌지를 함유하는 염색액을 염색대상인 세포에 적용하는 경우 세포의 핵은 진노란색의 형광을 발하며, 세포질이나 그 밖의 비특이성 과립들은 붉은 색과 진고동색깔을 띠게 되어 확연히 구분될 수 있다.When a dye containing acridine orange is applied to a cell to be stained, the nucleus of the cell emits a deep yellow fluorescence, and the cytoplasm and other nonspecific granules have a red color and an auburn color, which can be clearly distinguished. .
상기와 같이 본 발명에 의한 염색액 조성물 및 측정방법에 의하면 종래 김사염색법을 이용한 경우에 비해 핵단편에 대한 관찰예민도를 높일 수 있다.As described above, according to the dye solution composition and the measuring method according to the present invention, it is possible to increase the observed sensitivity to the nuclear fragment as compared to the case using the conventional gimsa staining method.
이하 본 발명의 내용을 실시예에 의해 보다 상세하게 설명하기로 한다. 다만 이들 실시예는 본 발명의 내용을 이해하기 위해 제시되는 것일 뿐 본 발명의 권리범위가 이들 실시예에 한정되어지는 것으로 해석되어져서는 아니된다.Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.
<실시예><Example>
선원을 Cs-137(0.8Gy/min)로 하는 감마선 발생장치를 이용해 건강한 사람 5명으로부터 말초혈액을 제공받은 후 조사선량에 따라 4개 그룹(0, 10, 20, 50cGy)으로 나누어 수행되었다.After receiving peripheral blood from five healthy persons using a gamma ray generator with a source of Cs-137 (0.8 Gy / min), it was divided into four groups (0, 10, 20, 50cGy) according to the irradiation dose.
방사선 조사 후 림파구를 히스토파크액을 이용해 원심분리한 후 ml당 2×06cell/ml가 되도록 최종 희석한 후 배양하였다. 림파구의 배양액은 RPMI 1640(Gibco, USA)에 FBS(15%), PWM(2.5㎕/ml) 및 PHA(20㎕/ml)를 첨가하여 조제하였고, 배양기(5% CO2, 37℃)에서 72시간 동안 세포배양을 수행하였다. After irradiation, lymphocytes were centrifuged with histopark solution, followed by final dilution to 2 × 0 6 cells / ml per ml, followed by incubation. Lymphocyte culture was prepared by adding FBS (15%), PWM (2.5 μl / ml) and PHA (20 μl / ml) to RPMI 1640 (Gibco, USA), and incubator (5% CO 2 , 37 ° C.). Cell cultures were performed for 72 hours.
배양이 끝난 림파구는 특정 항체(CD-19)와 미세철분으로 중복 처리한 후 자석장치가 부착된 분리기를 이용해 구분 채취하였다. 상기 과정을 통해 구분된 림파구는 통상적인 핵 단편물 구분법에 따라 처리하고 슬라이드로 제작하였다. 핵단편물은 관찰 시 정상핵과 비교해 3배 정도 크기가 작게 관찰되는 것으로 하였다.After culturing, lymphocytes were treated with a specific antibody (CD-19) and fine iron, and then separated using a separator attached to a magnetic device. Lymphocytes separated through the above process were processed according to the conventional nuclear fragment classification method and made into slides. Nuclear fragments were observed to be three times smaller in size than normal nuclei.
제작된 슬라이드에 아크리딘 오렌지 염색을 수행하고 김사염색 후 관찰된 핵 단편물의 출현빈도와 비교하였다. 아크리딘 오렌지 염색액(Wako, Japan)은 우선 40ug/ml 보존용액을 준비한 후, 소렌센 인산완충용액(pH 6.8)을 이용해 25배 희석한 것이 이용되었다.Acridine orange staining was performed on the prepared slides and compared with the frequency of appearance of the nuclear fragments observed after dyeing. Acridine orange stain (Wako, Japan) was prepared by first preparing a 40ug / ml preservation solution, and then diluted 25-fold with Sorensen phosphate buffer solution (pH 6.8).
제작된 슬라이드 상에 아크리딘 오렌지 희석액 30ul를 떨어뜨리고 커버글라스를 포배하여 형광현미경(BA-2필터장착)아래서 세포내 핵 단편물의 존재를 확인하 였다.30 μl of acridine orange dilution was dropped on the prepared slides and the cover glass was blasted to confirm the presence of intracellular nuclear fragments under a fluorescence microscope (BA-2 filter mounting).
도 1은 본 발명에 의한 염색액을 이용해 얻어진 결과사진으로, 염색된 핵은 진노랑색의 형광을 발하며, 세포질이나 그 밖의 비특이성 과립들은 붉은색과 진고동색깔로서 구별되어 관찰되고 있음을 확인할 수 있다.Figure 1 is a result photograph obtained using the dye solution according to the present invention, the stained nucleus emits a yellow fluorescence, the cytoplasm and other non-specific granules can be confirmed that the red and the true color is observed to be distinguished. have.
도 2는 종래 김사염색법에 의해 얻어진 결과사진으로, 김사염색 후 세포내에 존재하는 핵 단편물의 출현빈도를 확인하기 위해, 동일 시료를 적재한 슬라이드에 1/150 나트륨ㆍ칼륨 인산완충액(pH 6.8)으로 3∼4%희석한 김사액(Merck)을 염색하여 얻어진 것이다. 광학현미경으로 관찰한 결과 핵 단편물은 진청색으로 관찰되었다.Figure 2 is a result photograph obtained by conventional gimsa staining method, in order to confirm the frequency of appearance of nuclear fragments present in the cells after gimsa staining, with a 1/150 sodium potassium potassium phosphate buffer (pH 6.8) on a slide loaded with the same sample It is obtained by dyeing 3-4% of diluted Kimsa liquid (Merck). As a result of optical microscopy, the nuclear fragment was found in dark blue.
상기 실험결과는 하기 표 1에 정리된 바와 같으며, 동일 시료에 대해 처리한 결과 아크리딘 오렌지 염색 후의 관찰 결과에서 표준편차가 더 작게 관찰됨을 확인할 수 있다.The experimental results are summarized in Table 1 below, and as a result of treating the same sample, it can be confirmed that the standard deviation is smaller in the observation result after the acridine orange staining.
<표 1>TABLE 1
상기 표 1의 결과를 포함하여 종래의 김사염색법과 본 발명에 의한 아크리딘오렌지 염색법에 따른 구성 및 효과상의 차이점은 하기 표 2와 같다.
<표 2>Including the results of Table 1, the difference in construction and effect according to the conventional gimsa dyeing method and the acridine orange dyeing method according to the present invention is shown in Table 2.
TABLE 2
삭제delete
상기와 같이 김사염색법과 비교해 본 발명의 아크리딘 오렌지 염색법은 시료 관찰하는 전단계인 슬라이드 제작과정 중 건조, 염색, 세척, 건조 그리고 관찰에 필요한 시간을 적어도 26시간 정도 단축시킬 수 있을 뿐만 아니라 검출 예민도 역시 높일 수 있다.As described above, the acridine orange staining method of the present invention compared to the gimsa dyeing method can reduce the time required for drying, dyeing, washing, drying and observing at least 26 hours as well as detection sensitivity during the slide manufacturing process, which is a preliminary step of sample observation. You can increase it too.
본 발명에 의하면 방사선에 대한 세포유전학적 효과를 신속하게 검출할 수 있으며, 세포내 핵단편물을 비특이성 과립 등과 명확하게 구분할 수 있어 실험오차를 줄일 수 있다. 또한 짧은 시간에 대량의 시료를 처리할 수 있고, 절차가 간단해 비전문가라도 용이하게 검출할 수 있는 장점이 있다.According to the present invention, the cytogenetic effect on radiation can be detected quickly, and the intranuclear fragment can be clearly distinguished from non-specific granules and the like, thereby reducing the experimental error. In addition, a large amount of sample can be processed in a short time, the procedure is simple, even non-expert can be easily detected.
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