쥐의 과립막 세포 자연사에 EGF, TGF-α가 미치는 영향
Regulation of the apoptosis on the rat granulosa cell: effects of EGF, TGF-α
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목적 : 쥐의 과립막 세포 배양시 세포 자연사 조절 기전에 EGF와 TGF-α가 어떠한 세포내 기전을 가지고 영향을 주는지 알아보기 위해, 이들이 세포내 cAMP의 작용과 cAMP로 인한 세포 자연사에 미치는 영향에 대해 연구를 해보고자 한다. 연구 방법 : 암컷 Sprague Dawley 쥐에게 DES (2 mg/day)를 이틀간 근주하고 이틀 뒤 난소를 제거하여 과립막 세포를 분리하였다. 세포 자연사를 유도하기 위해서 서로 다른 기전으로 세포 자연사를 유도하는 것으로 알려진 난포자극호르몬 (25 ng/mL)와 환상 아데노신인산 (1 mM)를 각각 배양액에 추가한 뒤 8시간, 16시간, 40시간 동안 배양한 뒤 세포를 채취하여 자연사 정도를 확인하였다. 추가로 배양액에 EGF (10 ng/mL), TGF-α (150 units/mL)를 각각 배양 초기에 투여하여 배양하여 세포의 자연사 정도를 확인하였다. 세포 염색, DNA ladder 확인, flow cytometry로 세포의 자연사 정도를 확인하였다. 모든 실험은 3회 되풀이하여 mean results (± SEM)으로 표시하였다. 실험의 각종 처치에 따른 유의성 확인은 p<0.05일 때 유의하다고 확인하였다. 실험 데이터의 통계처리는 SPSS를 사용하였다. 결과 : cAMP (1 mM)는 과립막 세포에서 8시간 배양시 50%, 40시간 배양시 100%의 세포 자연사율을 나타내었는데, 이러한 세포 자연사율의 증가는 배양액에 EGF나 TGF-α를 첨가하여도 큰 변화를 나타내지 않았다. 같은 실험을 난포자극호르몬 (25 ng/mL)을 이용하여 실시하였을 때 EGF와 TGF-α는 모두 난포자극호르몬으로 인해 유도되는 세포 자연사율의 증가에 억제 효과를 나타내었다. 또한 이 두 가지 인자가 세포 자연사율을 감소시키는 기전에 자연사와 관련된 세포내 유전자의 발현 정도가 중요한 기능을 하는 것으로 나타났다. 결론 : EGF와 TGF-α는 난포자극호르몬의 세포내 기전에 영향을 줄 뿐만 아니라 anti/pro apoptotic gene의 발현을 조절하여 세포의 자연사에 영향을 준다.
Abstract
Objective : To evaluate the effect of the epidermal growth factor (EGF) and the transforming growth factor-α (TGF-α) on the apoptosis of the rat granulosa cells. Methods : For this study, granulosa cells were isolated from female rats, and granulosa cells were cultured in the presence or absence of different autocrine/paracrine factors such as EGF and TGF-α. In order to investigate the apoptotic process, the number of apoptotic cells were counted using Annexin V staining via flow cytometry. In addition, the expression levels of the Bcl-2, ICE and BAX genes were checked. Results : c-AMP induced apoptosis was inhibited neither by EGF nor by TGF-α, but FSH induced apoptosis was inhibited by EGF and TGF-α. Both EGF and TGF-α decreased the rate of apoptosis in the serum free cultured media in a time dependent manner by inducing survival gene expression such as Bcl-2 and suppressing death gene expression such as ICE and BAX. Conclusion : EGF and TGF-α can be used to decrease the apoptotic rate of the rat granulosa cells. It is hoped to conduct a similar study on the function of these growth factors in the human granulosa cells, and to use EGF and TGF-α for reducing the apoptosis rate of the human granulosa cells in IVF.
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