보고서 정보
주관연구기관 |
서울여자대학교 Seoul Womans University |
연구책임자 |
김종일
|
참여연구자 |
김해권
,
이기원
,
이희진
,
김소라
,
김자영
,
정현주
,
박상희
|
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2000-11 |
주관부처 |
과학기술부 |
사업 관리 기관 |
서울여자대학교 Seoul Womans University |
등록번호 |
TRKO200200022934 |
DB 구축일자 |
2013-04-18
|
초록
▼
본 연구의 목표는 사람을 포함한 포유류의 배발생 과정에서 MT-MMP가 최초로 발현되는 시기를 알아보고 나아가서 배아를 둘러싸고 있는 생식수관 즉 수란관과 자궁조직세포의 분화와 관련하여 MT-MMP가 어떤 역할을 하는가를 조사하는 것으로써, 제1차 년도에는 생쥐의 각 발생단계별 배아를 대상으로 먼저 기존에 알려진 MT-MMP 유전자들의 발현 여부를 조사한다. 체외에서 생쥐 포배의 탈각을 인위적으로 억제시켰을 때 나타나는 MT-MMP의 발현양상을 조사하여 정상배와의 것과 비교분석함으로써 탈각과 MT-MMP간의 상호 관계를 알아본다.
본 연구의 목표는 사람을 포함한 포유류의 배발생 과정에서 MT-MMP가 최초로 발현되는 시기를 알아보고 나아가서 배아를 둘러싸고 있는 생식수관 즉 수란관과 자궁조직세포의 분화와 관련하여 MT-MMP가 어떤 역할을 하는가를 조사하는 것으로써, 제1차 년도에는 생쥐의 각 발생단계별 배아를 대상으로 먼저 기존에 알려진 MT-MMP 유전자들의 발현 여부를 조사한다. 체외에서 생쥐 포배의 탈각을 인위적으로 억제시켰을 때 나타나는 MT-MMP의 발현양상을 조사하여 정상배와의 것과 비교분석함으로써 탈각과 MT-MMP간의 상호 관계를 알아본다. 또한 미생물에서 metalloprotease를 분리하기 위해 gealtin이 포함된 배지에서 gelatin을 액화하는 능력이 있는 미생물을 탐색하고, protease를 생산하는 균주를 선별하여 그 미생물을 대량 배양하여 extracellular protease를 분리하여 metalloprotease를 정제한다. 제2차 년도에는 생식주기에 따른 사람과 생쥐의 자궁조직 및 생쥐의 수란관 조직을 대상으로 MT-MMP의 발현 여부를 조사한다. 특히 정상적인 착상시기를 전후로 한 생쥐의 자궁조직과 인위적으로 착상이 억제된 자궁조직 간의 MT-MMP 유전자 발현 양상을 살펴보고 또한 임신이 진행된 생쥐의자궁조직에서 착상이 일어난 부위 및 착상이 일어나지 않은 부위에서의 MT-MMP의 발현 양상을 비교 분석함으로써 체내에서의 착상과 관련한 MT-MMP의 역할을 규명한다. 순수분리된 metalloprotease를 azocasein을 기질로 하여 단백질 분해활성을 측정하고 물리화학 생화학적 특성을 분석하고 산업적인 응용가능성을 검토한다.
Abstract
▼
Throughout their reproductive cycle, mammalian ovaries and oviducts undergo extensive tissue remodeling. Since the remodeling accompanies changes of characteristic extracellular matrix(ECM) components, it has been well known that various types of matrix metalloproteinases are involved in the degrada
Throughout their reproductive cycle, mammalian ovaries and oviducts undergo extensive tissue remodeling. Since the remodeling accompanies changes of characteristic extracellular matrix(ECM) components, it has been well known that various types of matrix metalloproteinases are involved in the degradation of ovarian and oviductal ECM. In the present study, the possible roles of membrane-type matrix metalloproteinase 1(MT1-MMP) and 2(MT2-MMP) in the remodeling process of ovarian and oviductal tissues were investigated during periovulatory period using RT-PCR and western blot techniques. Their gene expression in mouse oocytes during meiotic maturation was also examined. When mouse ovarian tissues obtained at prepubertal, 48 hr post PMSG, 10 or 15 hr post hCG were examined, all of the specimen revealed the distinct expression of both types of MT-MMP mRNA. The amount of RT-PCR products of both MT1- and MT2-MMP appeared to be the same regardless whether the ovaries were either before or after ovulation by hormone. After western blot against anti-mouse MT1- or MT2-MMP antibody, all tissue samples showed the presence of MT1-MMP protein having a molecular weight of 55kDA and MT2-MMP protein of 47kDa. However, the western blot result showed that the band intensity of both MT-MMP proteins from the ovaries just prior to ovulation, i.e., at 10 hr post hCG was the strongest compared to those without hCG stimulation or after ovulation at 15 hr post hCG. Similarly RT-PCR products of MT1- and MT2-MMP in oviductal tissues did not reveal any significant difference among the samples throughout periovulatory periods. The oviductal tissue obtained from the pregnant mice at 48 hr post hCG also gave the similar results. However, when the protein expression of the latter oviducts obtained from the pregnant mice were examined after western blot against anti-mouse MT1- or MT2-MMP antibodies, they gave the most intense signal compared to the other oviductal tissues obtained at periovulatory period. In mouse oocytes with a germinal vesicle or polar body, any discernible RT-PCR product of mRNA of MT1-MMP was not found whether they were grown or not. In contrast, RT-PCR product of mRNA of MT2-MMP was observed in both GV and PB oocytes matured either in vivo or in vitro. Mammalian uterus is one of the most important organs involved in the reproduction and is known to undergo extensive tissue remodeling during reproductive cycle and pregnancy. Since tissue remodeling requires various hydrolases including matrix metalloproteinases which hydrolyse extracellular components surrounding a cell, the present study aimed to see if MT-MMPs might be involved in tissue remodeling of mouse uterus during estrous cycle and preimplantation period and during the early development of mouse enbryos. mRNA expression patterns of both MT1-MMP and MT2-MMP were investigated usign RT-PCR and protein expressons were examined using western blotting techniques. In mouse uterus, expression of both mRNAs of MT1- and MT2-MMP was observed throughout the extrous cycles, namely proestrus, estrus, metestrus and diestrus. However, nosignificant difference was observed among the band intensities of each cycle sample. The expression of MT1- and MT2-MMP proteins by uterine tissue was observed to occur throughout estrous cycle. Western blot results using antibodies against mouse MT1- and MT2-MMP, showed uterine MT1-MMP as a 55kDa and MT2-MMP as a 47kDa. The band intensities of each cycle tissue was not different from each other. Preimplantation stage-mouseuteri examined at day 1, 2, 3, and 4 of pregnancy also revealed distinct mRNA expressions of both MT1- and MT2-MMP. And there was no difference among the band intensities. However, as pregnancy progressed, the amounts of both MT1- and MT2-MMP protein expression appeared to increase in such a way that uterine tissue of day 4 pregnancy gave the most intense band while that of day 1 gave the least one. In early embryos, mRNA of MT2-MMP was found to be expressed by 4-cell stage embryo, morula, early blastocyst and hatched blascyst. However, no discernible RT-PCR product of MT2-MMP was observed in 2-cell stage embryo. In contrast, RT-PCR product of MT1-MMP was never found in the same embryos at all. Based upon these observations, it is suggested that both MT1- and MT2-MMP proteins might play a important role in the remodeling process of mouse ovarian tissues, particularly, in the follicular rupture prior to ovulation and possibly be involved in the remodeling of oviductal tissues at the time of early pregnancy. The physiological meaning of the mRNA expression of MT2-MMP but not MT1-MMP in the oocytes in relation to the meiotic maturation needs to be studied further. Also, it is suggested that both MT1- and MT2-MMP proteins might play an important role in the remodeling process of mouse uterine tissue during preimplantation period while they might not be involved in the tissue remodeling events during estrous cycle. In addition, MT2-MMP but not MT1-MMP is also suggested to be involved in the early differentiation of mouse embryos.
목차 Contents
- 제1장 서론...15
- 제2장 국내외 기술개발 현황...17
- 제3장 연구개발수행 내용 및 결과...18
- 제1절 실험방법...18
- 1. 생쥐 난자와 배아 및 생식기관에서의 MT-MMP의 발현양상 분석...18
- 2. 미생물 metalloproteinase...19
- 제2절 연구결과...20
- 1. 생쥐배아 및 생식기관에서의 MT-MMP 발현양상 분석...20
- 2. 미생물 metalloprotease의 순수분리 및 특성...23
- 제4장 연구개발목표 달성도 및 대외기여도...27
- 제5장 연구개발결과의 활용계획...28
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