간세포 Apoptosis에 대한 형태학적 연구 = Morphologic Study on the Liver Cell apoptosis

국문 초록 (Abstract)

목적 : 본 연구는 간세포에 괴사를 주로 일으키는 물질로 알려진 사염화탄소와 apoptosis를 일으키는 것으로 알려진 cycloheximide로 간세포에 일어난 괴사와 apoptosis의 차이를 형태학적으로 알아보고 또 최근 개발된 in situ nick translation방법을 이용한 ApopTag염색을 통해 apoptosis를 쉽게 찾을 수 있는가를 알아보기 위해 시도하였다.
대상 및 방법 : 표준식으로 사육한 쥐에 cycloheximide를 체중 1㎏당 1.5㎎ 또는 50㎎이 되도록 정맥내에 주사하였고, 다른 동물은 사염화탄소를 체중 1㎏당 1㎎씩 복강내로 주사하였다. Cycloheximide 주사 후 30분에서 6시간 사이에, 사염화탄소 주사 후는 12시간에서 24시간 사이에 도살하여 H-E 및 ApopTag로 염색하에 apoptosis수를 계수하였고, 전자현미경으로도 관찰하였다.
결과 : Cycloheximide군은 H-E염색상 1.5㎎/㎏군에서는 30분후 100개의 간세포당 0.82개, 6시간후에 4.29개의 apoptosis가 나타났고 50㎎/㎏투여군에서는 처음 1.85개에서 6시간 후 9.86개가 나타나 용량의존성을 보였다. 사염화탄소 군은 H-E 염색에서 처음 0.46개에서 후에 1.88개로 증가하였다. ApopTag염색에서는 cycloheximide 투여군에서는 10-15%의 검출률이 더 높았으나 사염화탄소 투여군에서는 2-3배의 검출률을 보여 상반된 결과를 보였다. 결론 : 이상의 성적으로 보아 ApopTag는 apoptosis의 쉬운 검출을 위해 매우 유용한 것으로 생각되나 괴사 세포에 양성을 보일 수도 있으므로 결과해석에는 신중한 검토가 필요할 것으로 생각된다.

다국어 초록 (Multilingual Abstract)

It is well known that there are two ways of cell death; necrosis and apoptosis. Recently apoptosis is vigrously studied in concern with mechanism of hyperplasia and tumor promotion. This study was carried out to compare the morphologic differences between necrosis and apoptosis and to verify the usefulness of recently developed ApopTag for the detection of apoptotic cells. Necrosis was induced by carbon tetrachloride and apoptosis was induced by cycloheximide.
Rats were received intravenous single dose of 1.5㎎/㎏ or 50㎎/㎏ of cycloheximide and sacrificed 30 minutes, 1 hour, 3 hours, and 6 hours after injection, other rats were received intraperitoneal single does of 1㎖ of carbon tetrachloride and sacrificed 12, 15 and 24 hours after injection. Numbers of apoptotic cells were counted in hematoxylin and eosin and ApopTag stained tissue. Apoptosis was also observed under electron microscope.
Cycloheximide 1.5㎎/㎏ group showed 0.82 apoptotic cells among 100 liver cell nuclei after 30minutes; 2.19 after 1 hour; 2.64 after 3 hours; 4.29 after 6 hours. Cycloheximide 5㎎/㎏ group showed 1.85 apoptotic cells among 100 liver cell nuclei after 30 minutes; 5.42 after 1 hour; 9.69 after 3 hours; 9.86 after 6 hours respectively. Apoptosis appeared very early in 30 minutes and the number was increased with time pass and there was dose-dependency.
Carbon tetrachloride group also showed increased number of apoptotic cells; 0.46 apoptotic cells among 100 liver cell nuclei after 12 hours; 1.85 after 15 hours 1.88 after 24 hours.
ApopTag staining showed 10-15% increased number of apoptotic cells than H-E staining in cycloheximide groups, while 2-3 times increased number of apoptotic cells in carbon tetrachloride group. Some of necrotic cells in carbon tetrachloride group were also positive.
The results suggested that ApopTag staining is superior than H-E staining with easy detection of the apoptosis, but caution should be paid in interpreting the results because necrotic cell may show positive reaction.