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GnRH-agonist에 의한 인간 과립-황체화 세포의 세포사멸은 caspase-3와 -9 및 PARP의 활성화에 의해 유도됨

GnRH-agonist Induces Apoptosis of Human Granulosa-luteal Cells ViaCaspase-3 and -9 and PARP Cleavage

강병문|양현원|박원일|김평식|염윤희|박은주
대한산부인과학회 (Korean Soceity of Obstetrics and Gynecology)|2004.06|Obstetrics & Gynecology Science|제47권 제6호|pp. 1145-1153

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ApoptosisCaspase세포사멸PARP인간과립-황체화세포GnRH-agoinstHuman granulosa-luteal cellsGnRH-agonist
주제분류산부인과학

초록

목적 : 체외수정 및 배아이식을 위한 과배란 유도에 사용되고 있는 GnRH-agonist는 사람의 난소내 세포사멸에 직접적인 영향을 주는 것으로 알려져 있으나 그 기전은 명확히 밝혀져 있지 않다. 따라서 본 연구의 목적은 GnRH-agonist가 사람의 과립-황체화 세포의 세포사멸을 유도하는데 있어 caspase-3, caspase-9, PARP 등이 관여하는지를 알아보고자 하였다. 연구 방법 : 난자 채취 과정에서 획득된 과립-황체화 세포를 체외에서 배양하면서 10-6 M GnRH-agonist를 처리하였고 대조군으로는 생리식염수를 처리하였다. 배양된 세포에서 세포사멸을 관찰하고자 처리 후 24시간에 TUNEL 방법과 DNA 분절화 분석을 수행하였다. caspase-3와 caspase-9의 활성도는 형광 기질을 사용하여 조사하였으며, 또한 caspase-3, caspase-9, PARP 단백질의 활성화 정도는 Western blotting으로 분석하였다. 결과 : DNA 분절화는 GnRH-agonist를 10-6 M 이상 농도에서 명확히 증가하였다. TUNEL 분석 결과 생리식염수를 처리한 대조군에 비해 GnRH-agonist를 처리한 실험군에서 세포사멸이 유의하게 증가하는 것을 확인하였다. 또한 형광기질을 이용하여 caspase-3와 caspase-9의 활성도를 조사한 결과 세포사멸이 일어난 세포에서 활성도가 증가하였다. Western blotting 결과, 생리식염수를 처리한 대조군에 비해 GnRH-agonist를 처리한 실험군에서 활성화된 caspase-3, caspase-9, PARP 단백질이 증가하는 것을 알 수 있었다. 결론 : 본 실험 결과 GnRH-agonist에 의한 난소내 과립-황체화 세포의 세포사멸은 caspase-3, caspase-9, PARP 단백질의 활성화로 인하여 유도되는 것으로 사료된다.

Abstract

Objective : GnRH-agonist (GnRH-Ag) used in controlled ovarian hyperstimulation (COH) for IVF-ET has been known to affect directly on apoptosis of human ovarian cells, but its mechanism is not clearly understood. Therefore, the purpose of the present study was to investigate whether caspase-3 and -9 activation and poly-(ADP-ribose)-polymerase (PARP) cleavage are involved in the mechanism(s) by which GnRH-Ag induces apoptosis in human granulosa-luteal cells. Methods : Human granulosa-luteal cells collected from IVF-ET patients were cultured and treated with 10-6 M GnRH-Ag or saline as a control. To access apoptosis in the cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay and DNA fragmentation analysis were preformed 24 h after the treatment. Activity of caspase-3 and -9 in the cells was examined by using a fluorogenic substrate. Caspase-3 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage were analyzed by Western blotting. Results : DNA fragmentation in the cells increased in the higher concentration over 10-6 M GnRH-Ag. In the result of TUNEL assay, the rate of apoptotic cells in GnRH-Ag treatment increased significantly compared with that of saline treatment (p<0.05). The activity of caspase-3 and -9 investigated by using a fluorogenic substrate increased only in the apoptotic cells. In Western blot analysis, the cells treated with GnRH-Ag revealed an increase in active forms of caspase-3 and -9 compared with those of the saline treatment. In addition, cleavage of PARP also increased in the cells treated with GnRH-Ag. Conclusion : These results suggest that activation of caspase-3 and -9 and cleavage of PARP might be involved in apoptosis of human granulosa-luteal cells induced by GnRH-Ag.

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