Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
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Effects of 3-dimensional Co-culture of Human Endometrial Cells Decidualized with Progesterone and TGF-${\beta}1$ on the Development of Mouse 2-cell Embryos In Vitro

Progesterone과 TGF-${\beta}1$에 의해 탈락막화가 유도된 인간 자궁내막세포의 삼차원 공배양이 2-세포기 생쥐배아의 체외발달에 미치는 영향

  • Kwon, Wook-Hyun (Department of Obstetrics and Gynecology, School of Medicine, Pusan National University) ;
  • Kim, Hwi-Gon (Department of Obstetrics and Gynecology, School of Medicine, Pusan National University) ;
  • Lee, Dong-Hyung (Department of Obstetrics and Gynecology, School of Medicine, Pusan National University) ;
  • Ko, Kyung-Rae (Infertility Clinic, Pusan National University Hospital) ;
  • Lee, Kyu-Sup (Department of Obstetrics and Gynecology, School of Medicine, Pusan National University)
  • 권욱현 (부산대학교 의학전문대학원 산부인과학교실) ;
  • 김휘곤 (부산대학교 의학전문대학원 산부인과학교실) ;
  • 이동형 (부산대학교 의학전문대학원 산부인과학교실) ;
  • 고경래 (부산대학교병원 불임클리닉) ;
  • 이규섭 (부산대학교 의학전문대학원 산부인과학교실)
  • Published : 2008.03.30
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Abstract

Objective: This study was carried out to investigate the effects of 3-dimensional co-culture of human endometrial cells decidualized with progesterone and TGF-${\beta}1$ on the development of 2-cell mouse embryos. Methods: Stromal and epithelial cells isolated from human endometrial tissue were immunostained for cytokeratin and vimentin. Expression of TGF-${\beta}1$, its receptor-1, -2, integrin-${\beta}3$ and prolactin in mono or co-culture according to three different hormone conditions was investigated by RT-PCR. Differential staining was used to investigate the number of ICM and trophectoderm of hatched mouse blastocysts in different three conditions. Results: The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. In co-culture, TGF-${\beta}1$, its receptor-1, integrin-${\beta}3$ and prolactin except TGF-${\beta}1$-r2 were expressed in progesterone dominant condition. The hatching and attaching rate were higher in the co-culture with decidualized cells (p<0.05). However, we observed that lots of the incomplete hatched blactocysts attached on non-decidualized cells. The ICM number of hatched mouse blastocysts was higher in co-culture with decidualized and non decidualized cells than media only culture (p<0.05). The trophectoderm number of hatched blastocyst was higher in the co-culture with decidualized cells than non-decidualized cells or media only culture (p<0.05). Conclusion: The administration of progesterone, estrogen and TGF-$\beta$ could induce decidualization of stromal and epithelial cells isolated from human endometrial tissue using 3-dimensional co-culture, and the decidualization of human endometrial cells could increase the hatching and attaching rate of 2-cell mouse embryos.

목 적: 자궁내막조직에서 분리한 상피세포와 기질세포를 삼차원 공배양을 통한 탈락막화 유도에서 성호르몬과 TGF-${\beta}1$의 역할을 알아보고 2-세포기 생쥐배아와 탈락막화가 유도된 자궁내막세포와의 공배양을 통하여 포배형성율, 부화율, 포배기배아의 내세포괴와 영양막세포수 및 부착율을 알아보기 위해 시행되었다. 연구방법: 인간 자궁내막조직에서 분리된 기질세포와 상피세포의 표지인자인 cytokeratin과 vimentin에 대한 면역조직 화학염색을 실시하여 분리를 확인하였으며, 성호르몬 우세환경 (progesterone, estrogen)에서 분리된 세포를 단일배양 혹은 3차원 공배양을 통하여 RT-PCR법으로 TGF-${\beta}1$, 수용체-1, -2, integrin-${\beta}3$, prolactin의 발현을 조사하였다. 배양액군을 대조군으로 하여 2-세포기 생쥐배아와 탈락막화 유도와 유도하지 않은 인간 자궁내막세포와의 공배양을 통하여 포배형성율, 부화율, 부착율과 부화된 포배의 영양막세포와 내세포괴수를 비교하였다. 결 과: 상피세포 표지인자인 cytokeratin과 기질세포 표지인자인 vimentin을 이용하여 면역조직화학염색을 한 결과 각각 95% 이상에서 양성반응을 나타내어 자궁내막조직으로부터 상피세포와 기질세포가 성공적으로 분리되었음을 확인하였다. 분리된 상피세포와 기질세포를 단일배양에서는 성호르몬의 조건에 관계없이 TGF-${\beta}1$과 수용체 type-1, type-2, integrin-${\beta}3$, prolactin mRNA가 발현되지 않았다. 공배양에서는 progesterone 우세환경일 경우 TGF-${\beta}1$ 수용체 type-2를 제외한 모든 mRNA가 발현하였으나 estrogen 우세환경에서는 TGF-${\beta}1$ 수용체 type-2와 prolactin이 발현되지 않았다. 2-세포기 생쥐배아를 배양액군, 비탈락막군 및 탈락막군으로 나누어 공배양하였을 때 포배기 발달율은 차이가 없었으나 부화율 (92%)과 부착율 (82%)은 탈락막군이 유의하게 높았으며 (p<0.05), 비탈락막군의 공배양에서 다수의 영양막세포가 투명대를 완전히 빠져나오지 않은 상태로 부착한 비정상형태를 보였다. 부화된 생쥐 포배기배아의 내세포괴수는 탈락막화에 관계없이 공배양한 포배의 내세포괴수가 유의하게 많았으며 (p<0.05), 영양막세포수는 탈락막군에서 배양액군과 비탈락막군보다 유의하게 많았다 (p<0.05). 결 론: 자궁내막조직에서 상피세포와 기질세포를 분리하여 다시 삼차원적 공배양을 통하여 progesterone (100 nM), estrogen (1 nM)과 TGF-${\beta}1$ (10 ng/ml)을 첨가하면 체외에서 탈락막화를 유도할 수 있으며, 탈락막화를 유도한 자궁내막 세포와 2-세포기 생쥐배아를 공배양하였을 때 탈락막화가 부화율, 부착율 및 영양막세포수에 유효한 영향을 미치는 것을 알 수 있었다.

Keywords

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