생쥐 발달과정 중 난포 내 c-Kit/SCF 및 Inhibin-α 단백질의 발현 변화

강재성 ( Kang Jae Seong ),이창주 ( Lee Chang Ju ),라중열 ( Rha Jung Yeol ),이종민 ( Lee Jong Min ),송강원 ( Song Gang Won ),박문향 ( Park Mun Hyang ) 대한산부인과학회 2003 Obstetrics & Gynecology Science Vol.46 No.7

목적 : 포유류 난포의 성장에 관여하는 기전은 밝혀져 있지 않으나, 줄기세포인자 (stem cell factor, SCF)는 난소의 성장과 발달에 관여하며, inhibin-α는 과립막세포의 분화에 관여하는 인자로 알려져 있다. 본 연구는 발달 단계에 따라 생쥐 난포 내 c-kit, 줄기세포인자 (stem cell factor, SCF)와 inhibin-α의 발현 변화를 면역조직 화학적 방법을 통해 조사하여 c-kit, SCF, inhibin-α 단백질이 난포성장의 어느 시기에 발현하는지를 알아보기 위하여 시행하였다. 연구 방법 : 임신 14일, 16일, 생후 2일, 7일, 그리고 21일된 생쥐 난소를 채취하여 4% paraformaldehyde에 고정한 후 c-kit/SCF, inhibin-α의 면역조직화학 염색을 실시하였다. 결과 : 태자의 경우 난모세포에서 c-kit/SCF의 발현을 확인하였으며, 신생자의 경우에는 원시난포 및 일차난포의 난자에서 SCF가 발현되었다. 미성숙 생쥐의 경우 난포의 발달에 따라 난자 내 c-kit/SCF의 발현이 감소 되었으며, SCF는 전동난포의 난자에서 강하게 발현되었다. Inhibin-α는 미성숙 생쥐의 전동난포 및 초기 동난포의 과립막세포에서 발현되었다. 결론 : c-kit/SCF의 발현은 난포의 성장이 개시된 이후에 감소되며, inhibin-α 의 발현이 증가한 난포에서 c-kit 및 SCF 단백질의 발현이 감소한다는 것을 알 수 있다. 따라서 c-kit/SCF 단백질은 난포가 성장을 개시하기 이전에 작용하는 것으로 생각한다. Objective : It is known that SCF stimulates cellular migration, prolifertation, differentiation and survival upon interaction with its receptor, c-kit. SCF is essential for a normal gonadal development. Inhibin-α subunit gene expression is important for granulosa cell (GC) differentiation. The mechanism concerning the development of the ovarian follicles is not clearly understood. In this study, an immunohistochemical study was performed to find out the follicular expressions of c-kit, SCF, and inhibin-α during different developmental stages in mouse ovary. Materials & Methods : Mouse ovaries obtained from 14 and 16 days post coitum and 2, 7, and 21 days post partum were fixed in paraformaldehyde. Immunohistochemistries for c-kit, SCF, inhibin-α were subsequently carried out. Results : Antigens for both c-kit and SCF were expressed constantly on oogonia regardless of their developmental stages. SCF was expressed on oocytes of primordial and primary follicles of neonate mouse ovaries. In immature mouse ovaries, there was a gradual decrease in expression of c-kit/SCF as the follicles develop. SCF was expressed strongly on the oocytes of preantral follicles, but weakly in granulosa and theca cells. Inhibin-α showed a large expression on granulosa cells of preantral and early entral follicles of the immature mouse ovaries. Conclusion : We concluded that the expression of c-kit/SCF is decreased after the follicle start to grow. The expression of inhibin-α is negatively correlated to the expressions of both c-kit/SCF in the ovarian follicles in mice.

간흡충: 충체 및 대사성 항원의 특성분석

양용석(Yong-Suk Ryang),류장근(Jang-Keun Ryu),주난영(Nan-Young Ju),송강원(Kang-Won Song) 대한의생명과학회 1996 Biomedical Science Letters Vol.2 No.2

저자들은 마우스를 실험모델로 하여 간흡충의 항원을 투여 했을 때 비장조직에 대한 CD3, CD4 및 CD8 모노클로날 항체의 반응 여부를 알아보고자 하였다. 즉, 간흡충에 대한 세포면역학적인 특성을 규명고자 하였으며 특히 비장 조직에 대한 phenotype을 관찰한 결과 다음과 같은 결과를 얻었다. 간흡충의 조항원을 면역증강제와 함께 복강 투여한 다음 일정 기간 후에 비장조직을 Avidin-biotin complex 면역조직염색을 실시한 결과 CD3에서 강한 양성 반응을 나타냈고 CD4와 CD8에서는 약한 반응을 나타냈다. 조직부위를 보면 피막, 혈관, 임파관, 백수부위와 림프구 및 대식세포의 세포막에서 양성반응을 보였다. The authors inquired into what reactions comprise the response of mice(as a model) CD3, CD4 and CD8 monoclonal antibodies in spleen tissue when injected intraperitoneally by antigens of Clonorchis sinensis. The author is objective was focused on investigating the property of cellular immunity for liver fluke. In particular, the results of having examined the phenotype of the tissue of spleen were revealed as follows: a certain length of time after having been intraperitoneally injected with antigens of Clonorchis sinensis and Freund's adjuvant, the tissue of spleen was embedded and immunohistochemically stained by the avidin-biotin complex method. A strong reaction in response to CD3, while a feeble reaction resulted from CD4 and CD8. The tissue region showed a positive reaction to all antibodies, especially from capsules, vascular areas, white pulps and membrane of blood cells.

류마티스관절염 환자의 활막세포에서 Stat 단백의 발현에 관한 연구

심승철 ( Seung Cheol Shim ),이인홍 ( In Hong Lee ),정자헌 ( Ja Hun Jung ),김태환 ( Tae Hwan Kim ),전재범 ( Jae Bum Jun ),송강원 ( Kang Won Song ),배상철 ( Sang Cheol Bae ),유대현 ( Dae Hyun Yoo ),김성윤 ( Seong Yoon Kim ) 대한류마티스학회 2000 대한류마티스학회지 Vol.7 No.1

Objective: To characterize Stat activity in synovial tissue in rheumatoid arthritis (RA) in order to see if Stat molecule contributes to the pathogenesis of RA by regulatory expression of genes that play an important role in inflammation and tissue destruction. Methods: Synovial tissue were obtained immediately after operative excision. Immuno-histochemistry was done with the antibodies for Stat 3 and Stat 5. Cells were stimulated with interleukin 6 (IL-6) and soluble interleukin 6 receptor (sIL-6R) or steroid using chambered slide. In supershift experiment, cell extracts were incubated with 0.5ng of 32P-labelled double-stranded oligonucleotide probe. Samples were resolved on 4.5% polyacrylamide gels, which was transferred to polyvinylidene fluoride membranes. Anti-phosphotyrosine Stat 3 antibody was used for Western blotting. Results: Stat 3 was not shown on the synovial tissue section done by immuno-histochemistry. However, activated Stat 3 was expressed on cultured synovial cell stimulated with IL-6 and sIL-6R, and also with IL-6 and dexamethasone using chambered slide. In contrast to Stat 3, activated Stat 5 was expressed on the synovial tissue section, especially around blood vessel. Conclusion: Stat is activated in cultured synovial cells as shown in other immune associated cells, and IL-6 is the strong activator of Stat 3. Further analysis of the regulation of Stats in synovitis and the role of Stats in driving synovial inflammation will yield insight into the pathogenesis of RA and the development of novel therapeutic modality.

간흡충: 충체 및 대사성 항원의 특성분석 (1)항원투여 마우스 비장조직에 대한 면역조직화학적 연구

송강원,주난영,류장근,양용석 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.2

저자들은 마우스를 실험모델로 하여 간흡충의 항원을 투여 했을 때 비장조직에 대한 CD3, CD4 및 CD8 모노클로날 항체의 반응 여부를 알아보고자 하였다. 즉, 간흡충에 대한 세포면역학적인 특성을 규명고자 하였으며 특히 비장 조직에 대한 phenotype을 관찰한 결과 다음과 같은 결과를 얻었다. 간흡충의 조항원을 면역증강제와 함께 복강 투여한 다음 일정 기간 후에 비장조직을 Avidin-biotin complex 면역조직염색을 실시한 결과 CD3에서 강한 양성 반응을 나타냈고 CD4와 CD8에서는 약한 반응을 나타냈다. 조직부위를 보면 피막, 혈관, 임파관, 백수부위와 림프구 및 대식세포의 세포막에서 양성반응을 보였다. The authors inquired into what reactions comprise the response of mice(as a model) CD3, CD4 and CD8 monoclonal antibodies in spleen tissue when injected intraperitoneally by antigens of Clonorchis sinensis. The author is objective was focused on investigating the property of cellular immunity for liver fluke. In particular, the results of having examined the phenotype of the tissue of spleen were revealed as follows: a certain length of time after having been intraperitoneally injected with antigens of Clonorchis sinensis and Freund's adjuvant, the tissue of spleen was embedded and immunohistochemically stained by the avidin-biotin complex method. A strong reaction in response to CD3, while a feeble reaction resulted from CD4 and CD8. The tissue region showed a positive reaction to all antibodies, especially from capsules, vascular areas, white pulps and membrane of blood cells.

간흡충 피낭유충에서 Alkaline Phosphatase 및 Acid Phosphatase의 특성

양용식,송강원,주난영 연세대학교 보건과학연구소 1997 보건과학논집 Vol.7 No.-

Cell cycle evaluation of granulosa cells in the γ-irradiated mouse ovarian follicles

Song,Kang-Won,Lee,Young-Keun,Kim,Jin-Kyu,Lee,Chang-Joo,Yoon,Yong-Dal 대한방사선 방어학회 1999 방사선방어학회지 Vol.24 No.1

이온화방사선이 동물 생식세포에 미치는 생화학적 및 형태학적 영향을 알아보기 위해 본 연구를 시행하였다. 미성숙 생쥐 (ICR, 3 주령)에 γ선을 선량 LD으로 전신조사하였다. 방사선 조사 후 6시간, 12 시간, 1 일, 그리고 2 일 후에 난소를 적출하였다. 난소에서 추출한 과립세포의 세포주기를 DNA에 대한 유세포 분석으로 분석하였다. 세포자연사를 확인할 수 있는 A 세포주기는 이온화방사선이 조사된 실험군에서 대조군에 비해 현저히 높은 값을 보였다. 이온화방사선을 조사한 후 6 시간 군에서 TUNEL 면역조직화학 염색도를 나타낸 난포의 수가 대조군에 비해 현저히 증가 하였다. 따라서, 본 실험의 결과 방사선 조사에 의해 유발되는 난포의 퇴화는 6 시간 이내에 급성으로 진행되는 방사선에 의해 유발되는 세포자연사 기작의 이해와 퇴화난포의 정량화를 위한 수단이 될 수 있다. This study was carried out to evaluate the biochemical and morphological effects of ionizing radiation on mouse ovarian follicles. Immature mice (ICR, 3 week-old) were irradiated with a dose of LD at KAERI. The ovaries were collected after 6 hours, 12 hours, 1 day, and 2 days post irradiation. With the morphological basis of the histological staining with hematoxylin-eosin, immunohistochemical preparation using in situ 3'-end labeling was evaluated. Flowcytometric evaluation of DNA extracted from the whole ovary was performed. The percentage of A (subpopulation of cells with degraded DNA and with lower DNA fluorescence than G/G₁ cells), apoptotic, cells in the cell cycle was significantly higher in the irradiated group than in the control group. The number of in situ 3'-end labeled follicles increased at 6 hours post irradiation. All the analyses represented that the ionizing radiation-induced follicular atresia was taken place via an apoptotic degeneration. Such a degeneration underwent very fast and acutely. Therefore, it is concluded that the radiation-induced follicular degeneration is, like the spontaneous atresia, mediated by an acute appoptosis of follicular granulosa cells. Flowcytometric evaluation of cell cycles can make the role for quantifying the atretic follicles and understanding the mechanism of the radiation-induced cell death.

Effects of Follicle Stimulating Hormone on γ-Ray Irradiated Immature Mouse Ovarian Follicles

Kim, Jin-Kyu,Lee, Chang-Joo,Lee, Young-Keun,Song, Kang-Won,Yoon, Yong-Dal 대한방사선 방어학회 1998 방사선방어학회지 Vol.23 No.2

뇌하수체 분비호르몬인 FSH의 방사선방어 효능을 조사하고자 생후 3주된 미성숙 생죄 γ선을 전신조사하거나(R), 전신조사 후 FSH 5 IU를 복강주사(RF)하였다. 대조군은 동량의 식염수를 복강주사하거나(C) 혹은 FSH를 복강주사(F)하였다. 0시간, 6시간, 12시간 1일, 2일, 4일 그리고 8일 후에 각 군별로 5마리씩 경추파열로 도살한 후 난소를 채취하였다. 난소는 neutral buffered formalin에 고정한 후 통상적인 표본조직을 만들었다. HE 염색을 기본으로 면역조직화학염색을 비교하였으며 전체 DNA를 추출하여 AGAROSE GEL 전기영동을 실시하여 DNA fragmentation의 정도를 알아보았다. 방사선 조사군인 R군과 RF군에 있어서 6시간에서 12시간에 apoptosis를 반영하느 ㄴ면역조직화학적 염색률이 높게 나타났으며, 8일 경과시 분열세포의 존재비를 반영하는 염색률이 높게 나타났다. 또한 DNA fragmentation의 정도를 보면, 모든 실험군에서 185bp의 DNA ladder가 시간에 따라 증가하는 경향을 보였다. 370bp의 DNA는 R군의 경우 6시간에 나타났으며 1일 이후 감소하였다. RF군의 경우 12시간에 나타나 1일 이후 감소하였다. F군에서는 370bp가 보이지 않았다. 본 실험의 결과, 강소형성 난포가 피폭되는 경우 4일 이후 8일에 이르러 난소내에서 완전히 소멸되고, 8일에 새로운 원시난포가 성장하는 것이 확인되었다. FSH는 방사선에 피폭된 난포의 퇴화를 지연 혹은 억제시키는 방사선방어효과가 있었으며, 방사선에 의한 난포의 퇴화는 과립세포의 apoptosis를 매개로 하여 일어남을 알 수 있었다. To assess the radioprotective effects of follicle stimulating hormone (FSH) on ovarian follicles, 3 week-old female mice were irradiated with 8.33 Gy of γ-ray (group R) and followed by 5 IU ip-injection of FSH (group RF). For control groups, 5 IU of saline (group C) or 5 IU of FSH (group F) was ip-injected. Ovaries were collecte 0h, 6h, 12h, 1d, 2d, 4d, and 8d after irradiation or saline/FSH injection, and followed by fixation in neutral buffered formalin for rountine histochemistry. Immunohistochemistry was used to assess the status of follicles and DNA fragmentation was analyzed by agarose gel electrophoresis for total DNA. Staining specific for apoptotic follicles showed high intensity at 6h and 12h in group R and RF. On the other hand, staining specific for proliferating follicles showed noticeably high intensity at 8d in group R and RF. DNA fragmentation of 185bp increased with time in all experimental groups. Especially 370bp appeared at 6h in group R, then disappeared after 1d. in cease of group RF, it appeared at 12h and disappeared after 1d. From the above results, the irradiated antral follicles become completely disappeared from 4d to 8d, and then new follicles started to grow again at 8d. FSH had delaying or suppressing effects on follicular atresia after irradiation, it became clear that radiation-induced follicular atresia was mediated by granulose cell apoptosis.