아크리딘 오렌지 형광염색법을 이용한 저선량 감마선 유도 말초혈액 B와 T-림프구 미소핵 분석 Acridine Orange Stained Micronucleus Assay in Human B and T-lymphocytes after Low Dose ${\\gamma}-irradiation$원문보기
방사선에 의해 유도되는 사람 말초혈액 림프구 미소핵 관찰빈도를 높이면서 생물학적 선량평가법으로서 활용 가능성을 확인하고자 본 연구를 수행하였다. 우선 5명의 건강한 사람으로부터 혈액을 제공 받아 선량영역을 0에서 800cGy로 하여 감마선$(^{137}Cs)$을 조사 한 후 김사와 아크리딘 오렌지 형광 염색하고 미소핵 출현빈도를 비교하였다. 아크리딘 오렌지 염색법을 이용하여 미소핵을 관찰하였을 때, 김사 염색법에 비교하여 적갈색의 비특이성 과립과 녹황색의 DNA가 붉은색의 세포질을 배경으로 명확히 구별되었을 뿐만 아니라 선량이 증가하면서 검출율도 높았다. 아울러, 말초혈액 T와 B-림프구에 대하여 선량영역을 0에서 50cGy로 하여 방사선을 조사한 후 미소핵 출현빈도를 아크리딘 오렌지 염색하여 김사염색과 비교한 바, B-림프구에서 선량이 증가하면서 적어도 2배 이상 높게 관찰되었다. 본 실험 결과, 사람 말초 혈액 B-림프구를 대상으로 한 아크리딘 오렌지 형광염색 미소핵 분석법은 저선량 방사선 인체영향 평가나 과피폭 선량추정시 활용이 가능 할 것으로 생각된다.
방사선에 의해 유도되는 사람 말초혈액 림프구 미소핵 관찰빈도를 높이면서 생물학적 선량평가법으로서 활용 가능성을 확인하고자 본 연구를 수행하였다. 우선 5명의 건강한 사람으로부터 혈액을 제공 받아 선량영역을 0에서 800cGy로 하여 감마선$(^{137}Cs)$을 조사 한 후 김사와 아크리딘 오렌지 형광 염색하고 미소핵 출현빈도를 비교하였다. 아크리딘 오렌지 염색법을 이용하여 미소핵을 관찰하였을 때, 김사 염색법에 비교하여 적갈색의 비특이성 과립과 녹황색의 DNA가 붉은색의 세포질을 배경으로 명확히 구별되었을 뿐만 아니라 선량이 증가하면서 검출율도 높았다. 아울러, 말초혈액 T와 B-림프구에 대하여 선량영역을 0에서 50cGy로 하여 방사선을 조사한 후 미소핵 출현빈도를 아크리딘 오렌지 염색하여 김사염색과 비교한 바, B-림프구에서 선량이 증가하면서 적어도 2배 이상 높게 관찰되었다. 본 실험 결과, 사람 말초 혈액 B-림프구를 대상으로 한 아크리딘 오렌지 형광염색 미소핵 분석법은 저선량 방사선 인체영향 평가나 과피폭 선량추정시 활용이 가능 할 것으로 생각된다.
Firstly, we compared the two staining techniques, Giemsa and Acridine orange, to determine micronuclei on samples of cultures of five healthy human peripheral blood lymphocytes after ${\gamma}-irradiation\;(^{137}Cs)$ in dose ranges of 0 to 800cGy. It was found that the Acridine orange st...
Firstly, we compared the two staining techniques, Giemsa and Acridine orange, to determine micronuclei on samples of cultures of five healthy human peripheral blood lymphocytes after ${\gamma}-irradiation\;(^{137}Cs)$ in dose ranges of 0 to 800cGy. It was found that the Acridine orange staining method gives more reliable results than the usual Giemsa staining method in micronucleus tests. Moreover, the frequency of micronuclei in cytokinesis-blocked human B-lymphocytes was studied after in vitro irradiation in dose ranges of 0 to 50cGy. After setting and separating the B-lymphocytes, the frequency of radiation-induced micronuclei were observed as the end-point markers for the low-dose radiation dosimetry after staining with Giemsa and Acridine orange dyes. The micronuclei frequency in B-lymphocytes was significantly elevated from 10 to 30cGy ${\gamma}-irradiation$. The determination of micronuclei in B-lymphocytes after staining with Acridine orange was higher than that of Giemsa. The frequency of micronuclei in B-lymphocytes was observed to be at least two times higher than those of T-lymphocytes Giemsa in dose increasing. Therefore, the determination of low-dose radiation-induced micronuclei in B-lymphocytes after staining with Acridine orange is likely to have the greatest potential in the estimation of low dose radiation exposure.
Firstly, we compared the two staining techniques, Giemsa and Acridine orange, to determine micronuclei on samples of cultures of five healthy human peripheral blood lymphocytes after ${\gamma}-irradiation\;(^{137}Cs)$ in dose ranges of 0 to 800cGy. It was found that the Acridine orange staining method gives more reliable results than the usual Giemsa staining method in micronucleus tests. Moreover, the frequency of micronuclei in cytokinesis-blocked human B-lymphocytes was studied after in vitro irradiation in dose ranges of 0 to 50cGy. After setting and separating the B-lymphocytes, the frequency of radiation-induced micronuclei were observed as the end-point markers for the low-dose radiation dosimetry after staining with Giemsa and Acridine orange dyes. The micronuclei frequency in B-lymphocytes was significantly elevated from 10 to 30cGy ${\gamma}-irradiation$. The determination of micronuclei in B-lymphocytes after staining with Acridine orange was higher than that of Giemsa. The frequency of micronuclei in B-lymphocytes was observed to be at least two times higher than those of T-lymphocytes Giemsa in dose increasing. Therefore, the determination of low-dose radiation-induced micronuclei in B-lymphocytes after staining with Acridine orange is likely to have the greatest potential in the estimation of low dose radiation exposure.
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가설 설정
These early data indicated the availability of B-lymphocytes as a biological radiation dosimeter [18, 19]. In this study, we investigated whether the validation of the micronucleus method by Acridine orange staining supports its use for human lymphocyte micronuclei for low-dose radiation biological dosimetry.
제안 방법
Dose-response data were obtained in our study by scoring micronuclei after staining with Giemsa and Acridine orange in cytokinesis- blocked cells of five donors. The micronucleus frequency after staining with both dyes in peripheral blood lymphocytes was significantly elevated from 50cGy T~irradiation.
This is further strengthened by an earlier report that described that micronuclei are observed to be higher in B-lymphocytes than T-lymphocytes for low-dose radiation under IGy [17, 18]. The results also support our findings, which relate to range of dose estimation. However, they scored micronuclei after Giemsa staining, which has some disadvantages because it shows considerable variation in frequency of micronuclei, dependant on both observers and laboratory conditions.
However, they scored micronuclei after Giemsa staining, which has some disadvantages because it shows considerable variation in frequency of micronuclei, dependant on both observers and laboratory conditions. Therefore, the present investigation is unique as a study, as it was done using low-dose radiation induced micronuclei in B-lymphocytes with Acridine orange stain, thus minimizing the artifact. Although similar trends in the frequency of micronuclei have been reported previously, in this study more reliable data were obtained with the utilization of Acridine orange, and the experimental errors by misscored micronuclei could be minimized.
데이터처리
A statistical analysis was carried out using the INST AT GRAPHPAD program (Version 3.0).
성능/효과
Therefore, the present investigation is unique as a study, as it was done using low-dose radiation induced micronuclei in B-lymphocytes with Acridine orange stain, thus minimizing the artifact. Although similar trends in the frequency of micronuclei have been reported previously, in this study more reliable data were obtained with the utilization of Acridine orange, and the experimental errors by misscored micronuclei could be minimized. However, these results are independent of the opinion that B-lymphocytes have a high probability to operate various mechanisms that relate to radiosensitivity.
Hence, the study of genetic phenomena between the frequency of micronuclei in B-lymphocytes and their repair mechanisms after low dose radiation needs deeper study. In summary, the results in this study suggest that the Acridine orange staining method gives more reliable results than the usual Giemsa staining method in micronucleus assay. The determination of radiation-induced micronuclei in T-lymphocytes as wellas B-lymphocytes after staining with Acridine orange is likely to have the greatest potential in the estimation of low-dose radiation exposure.
Since the frequency of micronuclei may be influenced by various conditions, for each subject duplicate slides were prepared and stained from each culture. The results obtained by the two staining methods are in close agreement with each other, even though the frequency of Giemsa stained micronuclei was shown to be a little higher than Acridine orange after irradiation (Fig. 2). It was interesting to observe that the frequency of micronuclei per cells with micronuclei was generally high after Acridine orange staining (Fig.
4). The results obtained from this experiment on stain methods, and the trend of determination of micronuclei after staining with Acridine orange, were slightly higherthan those of Giemsa stain.
96). The results obtained from this experiment show that there was a significant difference in micronucleus determination dependant on the stain method, and the trend of micronuclei per cell with micronuclei after staining with Acridine orange was slightly higher than that of Giemsa staining following irradiation (Fig. 3).
The results of this study show that the frequency of micronuclei in B-lymphocytes was about two times higher when compared to those in T-lymphocytes in dose ranges of 10 to 50cGy (Fig. 5). Moreover, 산le micronuclei frequencies of B-lymphocytes found in the study of Vral et al [17] for the applied doses of 10 (0.
Micronuclei were round in shape and exhibited a strong yellow-green fluorescence, compared to the dark blue of Giemsa. These results show that the staining characteristics of micronuclei marked with Acridine orange versus those marked with Giemsa showed a more precise counting of micronucleated lymphocytes.
후속연구
It has been reported that a mutual relationship for Ku86 exists between the mechanism of DNA repair and the sensitivity of B-lymphocytes to low-dose irradiation [22]. Therefore, the results of this study acknowledge the possibility of using B-lymphocytes as a biological parameter for estimating the low-dose radiation effects. It is a generally established theory that, when exposed to ionizing radiation during radiation therapy or accidents, the numbers of lymphocytes in peripheral blood decrease and change in the formation of lymphocytes that react to radiation [23].
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