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기저 난포자극호르몬 농도에 따른 난포액 내 과립만 세포의 세포고사 , 증식율 , 생존율의 비교
강병문,김정훈,채희동,강은희,추형식,전용필,장윤석 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.6
목적 : 체외수정시술 도중 난자 채취시 얻어진 난포액 내의 과립막 세포에서 세포고사율, 증식율, 그리고 생존율을 측정하여 기저 혈중 난포자극호르몬의 농도에 따라 비교하여 보기 위함이다. 연구방법 : 1997년 3월부터 1998년 2월까지 울산대학교 의과대학 서울중앙병원 산부인과 불임크리닉을 방문한 총 64명의 환자들을 대상으로 하였다. 대상 환자들은 월경주기 제3일에 면역방사계측법으로 측정된 기저 혈중 난포 자극호르몬의 농도에 따라 저 FSH군[$lt;8.5 mIU/ml, n=43]과 고 FSH군[$gt;=8.5 mIU/ml, n=21]으로 분류되었다. 체외수정시술을 위한 과배란유도는 성선자극호르몬분비호르몬 작용제를 사용한 황체기 장기투여법을 사용하였다. 세포고사는 terminal deoxynucleotidy1 transferase[TdT]-mediated dUTP-digoxigenin nick end-labeling[TUNEL] 방법에 의한 in situ apoptosis detection kit를 사용하여 확인하였다. 통계학적 유의성은 Student`s t-검정, x square 검정, 그리고 Fisher의 직접확률법 등으로 검증하였다. 결과 : 체외수정시술의 임상 결과 비교에 있어 양 군간에 통계적으로 유의한 차이는 없었다. 세포고사율은 저 FSH군에 비하여 고 FSH군에서 다소 높게 나타났으나 통계학적으로 유의한 차이는 발견되지 않았고[3.4 -4.1% vs 4.1 -7.5%], 증식율에 있어서도 양 군 사이에 통계적인 의미가 발견되지 않았다. 그러나 생존율에 있어서는 저 FSH 군에서 77.9 -8.1%로 고 FSH군의 67.9 -7.2%에 비하여 유의하게 증가되어 있었다.[p$lt;0.01]. 결론 : 난포액 내의 과립막 세포의 생존율은 저 FSH군에서 고 FSH군에 비하여 유의하게 높게 나타났지만, 세포고사율에 있어서는 저 FSH군에서 고 FSH군에 비하여 다소 높은 것으로 나타났으나 통계적 유의성은 발견되지 않았다. Objective : To compare the apoptosis rate, proliferation ate, and viability of granulosa cells in follicular fluid of the patients undergoing in vitro fertilization and embryo transfer[IVF-ET] according to their basal follicle stimulating hormone [FSH] concentration. Materials and Methods : From March 1997 to februry 1998, total 64 patients were included in this study. Parients were allocated to the low basal FSH group [$lt;8.5 mIU/ml, n = 43] and the high basal FSH group[$gt;=8.5 mIU/ml, n=21]. The basal levels of FSH were measured in the 3rd day of menstrual cycle preceding ovearian stimulation cycle in total IVF cycles by immunoradiometric assay [IRMA]. The controlled ovarian hyperstimulation [COH] using luteal long protocol of gonadotropin releasing hormone agonist [GnRH-a] was used in all patients. Apoptosis was determined by terminal deoxynucleotidyl transferase[TdT]-mediated dUTP-digoxigenin nick end-labeling [TUNEL] mothod. Statistical analysis was performed using Student`s t-test, Fisher`s exact test, and X-square test as appropriate. Statistical significance was defined as p$lt;0.05. Results : There were no statistical significant differences in the clinical outcomes of the patients between the both groups. The apoptosis rate of hight FSH group seemed to be higher than the low FSH group, although there was no statistically significant difference [4.1 - 7.5% vs, 3.4 - 4.1%]. The proliferation rates were not differed between the both groups. However, the viability of low FSH group was significantly higher than high FSH group [77.9 -8.1% vs, 67.9 - 7.2%, p$lt;0.01]. Conclusion : It was true that the viability of the granulosa cells in low FSH group was significantly higher than high FSH group, however there was no significant differences in apoptosis rate of the granulosa cells between the two groups.
강병문,정병목,계명찬 한국발생생물학회 2002 발생과 생식 Vol.6 No.1
세포외기질(exrtracellular matrix, ECM)에 의한 생쥐 초기 배아의 발생 조절 현상의 기작 규명을 위한 연구의 일환으로 Engelbreth-Holm-Swarm(EHS) mouse sarcoma의 세포외기질로부터 추출한 ECM 복합체인 Matrigel의 성장인자 제거형(GFR-Matrigel)을 생쥐 포배에 처리한 후 mitogen activated protein kinase (MAPK, ERK1/2) 활성 의 변화를 조사하였다. Mat To elucidate the mechanism underlying the embryotropic effect of extracellular matrix(ECM) on the preimplantation development of mammalian embryos, the involvement of mitogen-activated protein kinase(MAPK) downstream the integrin signaling was examined in mouse blastocysts. Blastocysts were cultured in the presence of growth factor-reduced(GFR) Matrigel(0.5%, v/v). MAPK activity was measured by in vitro phosphorylation of myelin basic protein by the Erk1/2 antibody immunoprecipitates of embryonic extract following the Matrigei treatment. MAPK activity of the early blastocysts rapidly increased within 10 min fo1lowing the Matrigel treatment. When the embryos were cultured for 12 h in the presence of Matrigel, the MAPK activity was significantly higher than that ot the control embryos. PD098059, a MAPK kinase(MEK) inhibitor, attenuated the effect of Matrigel on the change in MAPK activity. Taken together, it suggested that the embryotropic effect of ECM proteins might be mediated by the activation of MAPK cascade.
생쥐 난자 및 초기배아에서 Leptin 수용체 발현 및 Leptin에 의한 Mitogen Activated protein Kinase 활성의 조절 및 난자의 성숙 조절
강병문,한현주,서혜영,홍석호,계명찬,Kang, Byung-Moon,Han, Hyun-Joo,Seo, Hye-Young,Hong, Suk-Ho,Gye, Myung-Chan 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.2
Objective: To verify the expression of leptin receptor (OB-R) in oocytes and preimplantation embryos, the involvement of mitogen activated protein kinase (MAPK or Erk1/2) in the leptin signaling, and effect of leptin on the oocyte maturation in mice. Method: RT-PCR analysis of OB-R was conducted in germinal vesicle (GV)-intact and MII stage oocytes, and 1, 2, 8-cell embryos and blastocysts. Germinal vesicle breakdown (GVB), polar body extrusion, monitored in the presence or absence of leptin ($1{\mu}M$). Following the leptin treatment, temporal changes in MAPK activity were verified by immunoprecipitation and in vitro kinase assay in MII oocytes. Results: The expression of OB-R mRNA was found in GV and MII oocyte but not in the embryos. MAPK activity of the MII oocytes was significantly increased by brief incubation in the HTF supplemented with leptin ($1{\mu}M$). Priming of PD098059, a MEK inhibitor to leptin treatment attenuated the activation of MAPK by leptin in MII oocytes. Following 24 hrs of culture of the GV oocytes, leptin significant increased the GVB and 1 st polar body extrusion. Conclusion: This result suggested that functional interaction between leptin and OB-R resulted in potentiation of MAPK (Erk1/2) activity in MII oocytes through MEK activation and that leptin might be a local regulator of meiotic maturation of the mouse oocytes.