[구연19]돼지난소에서 난포폐쇄시 과립층세포의 세포자멸사와 큰포식세포와의 관계에 대한 형태학적 연구

한승로,김수일,김원식 대한체질인류학회 2004 대한체질인류학회 학술대회 연제 초록 Vol.47 No.-

[지상25]동맥에서 속막과다증식을 일으키는 세포에 관한 연구

한승로,김수일,이영호,김원식 대한체질인류학회 2002 대한체질인류학회 학술대회 연제 초록 Vol.45 No.-

말굽콩팥과 그 형성기전에 관한 고찰

한승로,조근자,김수일,김무강,김원식 대한체질인류학회 2002 대한체질인류학회지 Vol.15 No.2

TNFα and TNFR2 Immunohistochemistry During Ovarian Follicular Development and Atresia in the Rat

한승로(Seung Ro Han),손성경(Sung Kyong Son),양윤석(Yun Seok Yang),김원식(Won Sik Kim) 대한해부학회 2005 Anatomy & Cell Biology Vol.38 No.6

TNFα는 난소의 발생과 기능에 있어서 중요한 역할을 하는 사이토카인(cytokine)으로 알려져 있다. 그리고 TNFα 수용체의 확인은 TNFα의 역할을 입증하는데 기여하였다. TNFα는 TNF 수용체 1 (TNFR1) 또는 TNF 수용체 2 (TNFR2)와 결합함으로써 기능을 발휘하게 된다. TNFα가 TNFR2와 결합시에는 과립층세포의 증식을 초래하게 된다. 본 연구를 통해 흰쥐 난소에서, 건강한 난포에서는 과립층의 바깥층에서 TNFα와 TNFR2에 강한 반응이 관찰되었고 아울러 많은 증식세포를 관찰했으며 세포자멸사는 관찰되지 않았다. 반면 폐쇄난포에서는 TNFα와 TNFR2의 발현이 과립층에서 급격히 감소하였고 이곳에서 많은 세포자멸사를 볼 수 있었다. 이같은 결과로 볼 때 TNFα는 흰쥐 난소에서 난포폐쇄시 과립층세포의 생존요소로서 작용할 것으로 생각된다. Tumor necrosis factor alpha (TNFα) is an intraovarian cytokine that may play a role in ovarian development and function. Identification of ovarian TNFα receptors provides support for establishing a role of TNFα in ovarian development and function. TNFα exerts its effects by binding to either TNF receptor 1 or 2 (TNFR1 or TNFR2). When TNFα binds with TNFR2, expression of survival genes is up-regulated, resulting in proliferation of granulosa cells. In the present study, the authors identified the changes in localization of TNFα and the expression of TNFR2 in granulosa cells during follicular atresia in rat ovaries. In healthy follicles, intense signals for TNFα and TNFR2 were found in the outer surface of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased expression of TNFα and TNFR2 was observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNFα acts as a survival factor in granulosa cells during follicular atresia in rat ovaries.

방사선 조사에 의한 흰주 난포의 퇴축과 난포세포의 방사선 감수성

김수일,한승로,조근자,허대영,이영호,조문준,김무강,김원식 충남대학교 의과대학 의학연구소 2002 충남의대잡지 Vol.29 No.1

The aim of this study were to investigate radiation-induced atresia and radiation susceptibility in the rat ovary morphologically. Female rats (Sprague Dawley strain) of 4 weeks old were irradiated with dose of 4 Gy and 8 Gy, and sacrificed at hour 6, 12, and 24 after radiation. The H & E stain, the TUNEL method (ApopTag kit) and transmission electron microscopy (TEM) were used. In light microscopic observation, the number of atresia of ovarian follicles were increased significantly at 6h after irradiation (p<0.01). There was no significant difference between 4 Gy and 8 Gy irradiated groups. In TEM observation, granulosa cells in radio-sensitive follicles were characterized by several structural features including condensation of nuclear chromatin granules, nuclear fragmentation and apoptotic body formation. An apoptotic cell is observed to have been phagocytosed by a normal granulosa cell. Granulosa cells in radio-resistant follicles were characterized by several structural features including nuclear indentation, partial condensation of chromatin granules, mitochondrial swelling, dilatation of RER cisterns, accumulation of dense irregular masses, accumulation of lipid droplets, and increased lysosomal bodies. Number of gap junctions between granulosa cells were decreased, and intercellular space were widen than that of control animals. These findings were prominent at 6h after irradiation and were diminished at 12h and 24h after irradiation. With these results, it was concluded that radiation-induced follicular cell apoptosis and ovarian follicular atresia in rat ovary increased considerably at 6h after irradiation. Further studies are needed to reveal the more extensive differences between radiosensitive and radioresistant follicular granulosa cells.

방사선조사가 흰주의 치아발생에 미치는 영향에 관한 형태학적 연구

이주환,한승로,김수일,조근자,조문준,김원식 대한체질인류학회 2004 대한체질인류학회지 Vol.17 No.4

돼지난소에서 난포폐쇄와 큰포식세포에 관한 형태학적 연구

김원식,한승로,김수일,박창식 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

난포폐쇄가 과립층세포의 세포자멸사에 의해 이루어지고 이 과정에 큰포식세포가 직? 간접적으로 관여한다는 사실은 널리 알려져 있다. 그러나 난포내에서 일어나는 세포자멸사가 과립층의 어디에서부터 개시되고 어떻게 파급되는지, 세포자멸사 산물과 퇴화된 난모세포의 제거 방법 그리고 난포폐쇄의 완성은 어떻게 이루어지는가에 대해서는 아직 확실히 밝혀져 있지 않다. 이에 저자들은 가임기 돼지(Yorkshire-breed)를 실험동물로 난소내 난포의 광학현미경적 및 투과전자현미경적 관찰과 돼지 대식세포 단크곤항체 4E9를 이용한 변역조직화학적 방법으로 본 연구를 실시하였다. 광학현비경적관찰 결과, 난포폐쇄는 과립층세포의 세포자멸사로부터 개시되고 이어 난모세포의 퇴화와 난포막 속층 세포들의 세포자멸사가 일어나면서 난포폐쇄가 완성되는 것으로 보인다. 과립층세포의 세포자멸사는 핵농축으로부터 시작되고 과립층의 깊이에 관계없이 짧은 시간 안에 모든 층으로 파급되고 최종적으로 난모세포의 투명대를 둘러싸고 있는 과립층세포의 세포자멸사포 이어지는 것으로 보인다. 투과전자현미경 관찰에서는 세포자멸사중에 있는 과립층세포들이 인접한 정상적인 과립층세포들과 큰포식세포들에 의해 포식되는 것을 확인할 수 있었고, 난포동내에 산재한 세포자멸사 소체들의 제거에도 큰포식세포들이 관여하고 있음을 알 수 있었다. 난포폐쇄와 큰포식세포와의 관계를 알아보기 위해 실시한 면역조직화학적 관찰에서는, 과립층세포의 세포자멸사 초기에는 큰포식세포가 과립층내에서만 발견되었으나, 세포자멸사의 진행과 함께 난포동과 난모세포를 둘러싸고 있는 투명대 주위에서 각각 순서대로 관찰되었고 마지막으로 난포막 속층에서도 발견되어, 이들이 과립층세포들의 세포자멸사 소체들을 제거하는 것은 물론 최종적으로는 난포막 속층 세포들의 세포자멸사 소체들을 제거하여 난소 실질화에 관여하는 것으로 보인다. 이러한 본 연구의 결과는 난포폐쇄와 관련된 큰포식세포의 구조, 기능적 연구에 유용한 자료로 생각된다. Apoptosis of granulosa cells leads follicular atresia and macrophages have an important role during the apoptotic process. However, the propagation of apoptosis within the follicle, the ways of elimination of apoptotic bodies and degenerated oocyte, and the completion of follicular atresia are still controversial and unidentified clearly. Using adult porcine (Yorkshire-breed) ovary, in this morphological study, transmission electron microscopic observation and immunohistochemical study with pig macrophage monoclonal antibody 4E9 were performed. In light microscopy, the follicular atresia initiated with apoptosis of granulosa cells, followed by degeneration of oocyte and apoptosis of theca interna cells. Apoptosis occured in random fashion among the granulosa cells and propagated multidirectionally, and finally to the granulosa cells surrounding zona pellucida of degenerating oocyte. Pyknosis of granulosa cells was the first sign of apoptosis. In immunohistochemistry, macrophages were found only in the granulosa layer at the stage of beginning of apoptosis. With progression of apoptosis, they were proliferated greatly in number enough to eliminate all the apoptotic bodies, and found within the follicular antrum. In advanced stage of atresia, macrophages surrounded the zona pellucida of degenerating oocyte, and found also in the theca interna. In transmission electron microscopy, phagocytic granulosa cells maintained characteristic gap junctions with neighboring granulosa cells and contained several apoptotic bodies and lipid droplets within their cytoplasm. Macrophages kept many apoptotic bodies, vacuoles and autophagosomes in their cytoplasm. Apoptotic granulosa cells were ingested by intact granulosa cells and macrophages initially, but lately, all the apoptotic granulosa cells and degenerated oocyte were eliminated by macrophages. Ovarian follicular atresia completed with phagocytosis of apoptotic theca interna cells by macrophages, and the remnants of the atretic follicle became ovarian stroma. It is well known that macrophages may play an important role during follicular atresia, such as elimination of apoptotic granulosa cells, theca intema cells and degenerated oocytes, but, the valid action mechanisms of macrophages on the initiation of granulosa cell apoptosis and on the completion of atresia through the secretion of paracrine factors and autocrine factors still unclear.

돼지난소에서 난포폐쇄시 과립층세포의 아포토시스와 대식세포에 관한 형태학적 연구

박창식,한승로,김수일,조근자,김원식 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

It is known widely that granulosa cell apoptosis leads follicu1ar atresia and macrophages exert their effects directly and/or indirectly from the initiation to the completion of follicular atresia by phagocytosis of apoptotic bodies and secretion of various cytokines. However, the site of initiation, propagation routes and the elimination methods of apoptotic bodies, and the time and methods of penetration of macrophages into the follicles are not known completely. Using pig(Yorkshire-breed) ovary, immunohistochemical studies with TUNEL far apoptotic bodies and pig macrophage monoclonal antibody 4E9 for macrophages, and light and transmission election microscopic observations were performed. In the pig, follicular atresia began with the granulosa cell apoptosis, and the apoptosis of theca intema cells occured at the same time. The apoptosis of granulosa cells initiated randomly within the granulosa cell layer and propagated rapidly into the whole layer. Ultrastructurally, apoptotic ranulosa cells showed characteristic pyknotic and deformed nucleus and intracytoplasmic vesicles. Apoptotic bodies were eliminated by intact granulosa cells and macrophages. Intact granulosa cells inserted apoptotic bodies transiently, soon after they fell into the apoptotic. Finally, apoptotic bodies and degenerated oocyte were phagocytosed by macrophages. Macmphages entered the ovarian follicle at the time of initiation of granulosa cell apoptosis, and migrated with the progession of apoptosis. By elimination of theca cells, macrophages contributed the completion of follicular atresia. These results will provide valuable informations on the study of the interrelation between mac개phage and ovarian follicular atresia.

위암환자에서 BrdU에 의하여 유도되는 Fragile sites의 발현

윤숙자,한승로,정옥,김수일,박경란,이영호,김원식 충남대학교 의과대학 지역사회의학연구소 2001 충남의대잡지 Vol.28 No.1

The expression frequency of BrdU-induced fragile sites was examined in stomach cancer patients to determine whether this parameter could be used as a marker of genetic susceptibility in at-risk individuals. G banding performed to detect fragile sites. No difference was found in expression frequency between the stomach cancer patients and a group of normal individuals(p=0.7). This indicates that the expression frequency of BrdU-induced fragile sites is not a suitable marker for assessing genetic susceptibility in stomach cancer patient.

돼지 황체에서 황체용해와 대식세포와의 관계

김원식,한승로,손성경,박창식,양윤석 한국동물자원과학회 2006 한국축산학회지 Vol.48 No.2