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대한생식의학회지 제27권 제1호 2010년
공배양의 작용기전에 관한 연구
부산대학교 의과대학 산부인과학교실1, 문화병원 불임클리닉2, 부산대학교 자연과학대학 분자생물학과3
김미경1,3, 주보선2,3, 김미선3, 문화숙2, 이규섭1, 김한도3,
Mechanism for the Action of Co-culture
Mi Kyoung Kim 1,3, Bo Sun Joo2,3, Mi Sun Kim3, Hwa Sook Moon2, Kyu Sup Lee1, Han Do Kim3
1Department of obstetrics and Gynecology, Pusan National University School of Medicine, 2The center of Reproductive Medicine and Infertility, Moon Hwa Hospital, 3Department of Molecular Biology, Pusan National University School of Natural Science
Objective: A number of studies to improve in vitro culture conditions have been tried over past ten years by using co-culture system with helper somatic cells. However, the mechanism of coculture is poorly understood. This study was designed to understand the mechanism for the mode of actual action of co-culture using co-culture system of ICR strain’s 1-cell embryos with human oviduct epithelial cells by examining the effect of conditioned medium and contactless coculture using a cell culture insert on the embryo development and by measuring the level of superoxide anion from conditioned medium after co-culture. Methods: ICR strain’s zygote embryos were cultured in medium alone (control), coculture, conditioned medium, or contactless coculture system for 6 days. Conditioned media (CM) were prepared as following 5 groups. All CM were collected after culturing oviduct cells for 2 days. CM-1 was stored at -20˚C until use, and CM-2 was prepared just before the use as a culture medium. CM-3 was cocultured with embryos and retrieved just before use. CM-4 and CM-5 were derived from the microfilteration of CM-2 and CM-3, respectively, using Miscrocon-10 (10 kDa molecular weight cut-off). The percentage of the embryos developed to hatched blastocyst stage and the level of superoxide anion in supernatant from medium alone culture (control), coculture, and contactless coculture were measured. Results: The rates of embryo development to the hatched blastocyst stage were significantly higher in coculture (43%) than in control (0%) (p<0.05). The CM-1 group had no embryo development since 2-cell embryonic stage, whereas the CM-2, CM-3, CM-4 and CM-5 groups had the improved development to 4 or 8 cell embryo stage, but the similar rate of development to hatched blastocyst compared to control. The effect of coculture on embryo development was disappeared in the contactless coculture group. The level of superoxide anion was significantly reduced in coculture group compared to control. Conclusion: It is concluded that the present coculture system overcomes the 2-cell block in vitro and improves the embryo development. This beneficial effect may be due to the direct dell-cell contact between embryo and helper cells or the removal of deleterious components from medium rather than the embryotrophic factor.
키워드 : Coculture, Embryo development, Conditioned media, Cell to cell contact, Superoxide anion
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