간흡충감염 마우스에 있어 담관상피세포의 병리조직학적 변화에 대한 aflatoxin의 영향

Abstract

간흡충감염으로 인하여 변화를 일으킨 담관상피세포의 악성화에 관한 올바른 기전은 아직까지 밝혀지지 않았지만 간흡충증이 농후하게 만연되고 있는 우리나라를 비롯하여 동남아제국에 있어 간흡충감염과 원발성 간암 특히 담관암의 합병례가 드물지 않다는 역학적 및 병리조직학적 연구 결과를 근거로 할 때 이들 사이에는 불과분의 상관관계가 개재하고 있음이 분명하다. 그러나 간흡충감염 단독만으로는 담관암의 유발은 불가능하며 간흡충은 오직 담관상피세포 악성화의 전단계적 변화로 간주되고 있는 선종성 증식, 점액세포화생 및 배세포화생을 유발하는 선행적 요인으로서 작용할 뿐, 모종의 발암성 물질의 관여가 반드시 개재되어야만 담관암이 발생한다고 추정되고 있다. Aflatoxin B_1은 범세계적으로 음식물에서 검출되고, 우리나라에 있어서도 메주나 된장을 비롯한 발효성 식품과 대두나 옥수수 등 가축의 사료에서 검출 가능하며 간독성이 강하고 간세포성 암을 유발하는 것으로 알려져 있다. BALB/c 마우스를 3개군으로 구분하여 제I군에는 1.0ppm aflatoxin B_1 수용액만을 12주간 경구 투여하였으며, 제II군에는 50개의 간흡충 피낭유충만을 투여, 감염시켰고, 제III군에는 50개의 간흡충 피낭유충의 투여와 함께 1.0ppm aflatoxin B_1 수용액을 12주간 투여하였다. 실험시작후 제4주부터 4주 간격으로 제32주까지 각군으로부터 3마리씩의 마우스를 무작위로 취하여 희생시켜 광학 및 전자현미경적 검색소견을 기간별로 관찰 비교하였다. 광학현미경적 검색에 있어 제I군의 경우에는 초기의 경미한 소담관의 증식과 염증세포의 침윤이 주로 문맥부에서 관찰되었으며 후기에는 정상수준으로 회복되었고, 제II군에서는 간흡충감염시 필발되는 담관의 선종성 증식, 상피세포의 점액세포화생 및 배세포화생과 섬유화 등 주요 병리조직학적 공통소견이 예외없이 나타났다. 그러나 전관찰기간을 통하여 발암증거는 찾을 수 없었다. 한편 제III군에 있어서의 제반 변화소견은 제II군의 것들과 유사하였으나 그 정도가 보다 증가되었으며, 제28주와 제32주에 있어 각각 1례씩 점액분비성의 잘 분화된 선암이 관찰되었다. 전자현미경적 검사에 있어서는 광학현미경적 관찰 소견과 잘 일치되는 변화들이 관찰되었다. 1.0ppm aflatoxin B_1의 단독투여나 간흡충 단독투여의 경우에 있어서는 담관암이 유발되지 않았으나 동일한 조건하에서 이들 양자를 겸용했을 때에는 분명히 담관암이 유발되었던 바, 이와같은 현상은 aflatoxin B_1이 간흡충감염으로 변화된 세포에 효과적으로 작용하여 암세포로 전환시킨 것으로 해석되었다. ; The present study was carried out to examine the effect of a carcinogen, aflatoxin B_1 on the pathogenesis of the bile duct changes in mice experimentally infected with Clonorchis sinensis metacercariae. A total of 75 male albino mice of the BALB/c strain was used, and divided into 3 groups; group I, treated with 1.0ppm aflatoxin B_1 in drinking water alone for 12 weeks, group II, given 50 Clonorchis sinensis metacercariae alone and group III, given 50 metacercariae at the beginning of the 12 week treatment with 1.0ppm aflatoxin B_1 in drinking water. Three mice were served as untreated-uninfected normal control. All mice were fed the standard protein diet which was synthesized and supplied by the Laboratory of Sam-lip Food Co., Korea. Since the 4th week three mice from each group were removed and sacrificed at 4 week interval up to the 32nd week. The hepatobiliary tissues were extracted and subsequently prepared for light microscopic and electron microscopic examinations. For light microscopy the tissue specimens were stained with hematoxylin and eosin. Light Microscopic Findings; Mice in group I showed no significant changes except the lower degrees of bile ductular proliferation and inflammatory cell infiltration at the portal regions at an earlier stage, and regression at later stage. In groups II and III there were remarkable adenomatous tissue proliferation, mucinous cell metaplasia and goblet cell metaplasia together with extensive acute and chronic inflammatory cell infiltrations, cellular atypia and periductal fibrosis from an earlier stage. However, in group III the histopathological changes observed were more prominent than those in group II throughout the observation. As the time elapsed, they showed gradual regression with some fluctuations in the degrees. No any evidences of malignant changes of epithelial cells were noted in group II, but in group III the first tumor was found at the 28th week and the second one was at the 32nd week, respectively. Histologically the tumors observed were well differentiated adenocarcinoma of the bile duct. It was appeared that the tumors were of peripheral origin with a tendency of multifocal growth. They also showed an infiltrative growth pattern, increased mucin droplets, increased nuclear-cytoplasmic ratio and nuclear hyperchromatism. No hepatocellular carcinoma was noticed throughout the observation. Electron Microscopic Findings; The most prominent ultrastructural changes observed in group I were remarkable enlargement in nuclear size, diminution in nuclear size, dispersed chromatin granules in nuclei and increased dense granules along the inner membrane of nuclei. In the cytoplasm there were slight proliferation of mitochondria and endoplasmic reticulum at an earlier stage. At the 12th week separation of the fibrillar and granular components of the nucleolus was the characteristic finding. Nuclear abnormalities persisted. As the time elapsed, epithelial cells showed flattened-cuboidal in form and a tendency of atrophy. Accordingly, most of nuclei were elongated and polygonal in shape. In group II the appearance of elaborate interwoven folds of lateral cytoplasm, forming a labyrinth of interconnected intercellular space and variety in nuclear shape were the prominent findings observed at an earlier stage. Cytoplasm showed slight proliferation and dilatation of mitochondria and endoplasmic reticulum, and a small number of mucin droplets. In the basement membrane scanty fibrous cells were seen. With time, variety in nuclear shape, marked proliferation and dilatation of rough endoplasmic reticulum and some collagen fibrils were demonstrated. Other figures of intracellular organelles and mucin droplets persisted. In group III cuboidal epithelial cells showed remarkably enlarged and irregular nuclei, increased chromatin granules in nuclei and along the inner membrane of nuclei, separated nucleoli, proliferated and dilatated rough endoplasmic reticulum. With time, sequestered mitochondria showed bleb-like evaginations which lacked cristae and dense matrix, and were limited by a single membrane. At the 32 nd week, when the second tumor was found, microvilli were relatively scanty and poorly developed. The cytoplasm of the elongated polygonal tumor cell is poor in organelles and inclusions. Nuclei were varying in shape. The most prominent changes observed at this stage were separation of nuclei from cytoplasm, and numerous, irregularly angled and highly electron dense granules in nuclei. In conclusion, it is suggested that Clonorchis sinensis infection promotes carcinogenic action of aflatoxin B_1, a carcinogenic agent.