BUdR-표지에 의한 자매염색분체 교환에 관한 실험적 연구

Abstract

[한글]

체세포 분열에서 자처 염색분체 교환(sister chromatid exchange; SCE)에 관한 최초의 관찰을 Taylor등(1977)이 자가-방사기록법을 이용하여 성공한 후에, 자가-방사기록법이 세포유전학 연구에 큰 공헌을 한 것은 사실이나, 수기의 번잡함과 제한된 이용도 및 tritium 자체애 의한 SCE의 유발, 관찰의 곤란등으로 새로운 방법의 개발이 기대되어 왔다. 이에 Latt(1973)과 Perry 및 Wolff(1974)에 의하여 개발된 BUdR-표지에 의한 SCE의 관찰은 획기적인 새로운 방법으르 인정되나, 아직까지는 확립된 방법이라고 할 수 없다.

따라서 본 연구에서는 BUdR-표지에 의한 SCE의 관찰에서 염색체의 제반 배양조건, BUdR의 제품등을 각각 달리하면서, BUdR의 투여방법 및 최변이제인 Mitomycin C의 투여등의 제반여건을 달리하였을 때에 나타나는 SCE 변화를 관찰하여 다음과 같은 결과를 얻었다.

1. BUdR의 제품과 배지의 종류에 따라서 SCE의 출현율은 심하게 변하였다.

2. BUdR의 농도를 증가시킴에 따라서 SCE의 빈도 변화는 적었으나, 분별염색세포의 출현율은 감소하였다.

3. BUdR의 투여방법의 차이에 따라서 SCE 빈도 변화가 없었으나 분별염색세포의 출현율에는 차이가 컸다.

4. 최변이제인 Mitomycin C 0.02 ㎍/ml에서 1.0 ㎍/ml로 증가시킴에 따라서 세포당 SCE가 12.38에서 37.57로 증가하였다.

5. SCE표본의 핵형도 작성이 가능하였다.

이상의 결과로 보아 BUdR-표지에 의한 SCE의 관찰은 매 실험마다 철저한 대조군의 관찰이 필요하고, 가능한 한 모든 조건을 동일하게 하여야 하며, 그 평가는 항상 상대적으로 비교해야 하고, 핵형도의 작성은 임상에서의 응용가능성이 있다고 생각되었다.

[영문]

The first unequivocal demonstration of sister chromatid exchanges(SCEs) in mitotic chromosomes of higher organisms was made about two decades ago by Taylor and his colleagues (1957) with the aid of autoradiographic techniques. This autoradiographic techniques have made a great contribution to cytogenetic studies but there are several long-lasting cytogenetic controversies because of the limitations of the conventional autoradiographic method and SCEs induced by

tritium.

Recently, cytogeneticists have shown considerable interest over the development of a new method that detects SCEs using a largely improved resolution but without the use of tritium labelling. The technique utilizes a halogenated base analog of DNA, 5-bromo-2'-deoxyuridine(BUdR) to label chromosomes, hence it is called the

BUdR-labelling method. Now, the use of autoradiography for detecting SCEs has been replaced by these new methods, in which the sister chromatids are made to stain differentially. Subsequently, because of the ease of visualization and scoring of

SCEs and their dramatic effects, this method have contributed to basic studios on chromosome structure and the mechanisms involved in exchange formation. The method also had been approved as a new assay system for hazardous effects of various environmental mutagens and carcinogens.

But the BUdR-labelling method still has problems due to several technical difficulties and limitations. There are many factors affecting the incidence of SCEs ; BUdR concentration, visib1e light, cell types, viruses and culture conditions. Therefore there are many discrepancies between the reports about the occurance of SCEs by the BUdR-labelling method.

This report is attempting to resolve the above discrepancies and to improve the BUdR-labelling method. This study will also he purposed on experiments, using human Iymphocytes in vitro, which demonstrate the discrepancies in the case wheal BUdR concentration, BUdR administration method, culture media and other culture conditions are different.

The results of this study nay be summarized as follows;

1) The frequency of SCEs is different when the brands of BUdR and culture medium are different.

2) There is no discrepancy in the incidence of SCEs despite the fact that the concentration of BUdR differs considerable)F. But the rate of appearance of differentially stained cells decrease when the concentration of BUdR is increased.

3) There is no discrepancy in the frequency of SCEs even when the administration-method of BUdR is different, but the rate of differentially stained cells is in creased.

4) An increase in Mitomycin C concentration from 0.02 ㎍/ml to 1.O ㎍/ml results in at increase of SCEs per cell from 12.38 to 37.57.

5) It is possible to karyotype the chromosomes with the preparation of SCEs by the BUdR-labelling method.

Therefore, the protocol of the BUdR-labelling method requires a control culture due to many factors that affect the incidence of SCEs.

In conclusion, this method, which demonstrates and controls the discrepancies of SCE incidence, might be the groundwork for a clinical method to detect SCEs, although there are still several technical difficulties at the present.