摘要
为了探讨细胞外基质对卵巢颗粒细胞功能的影响。从未成熟ＳＤ大鼠分离出卵巢颗粒细胞，以３×１０５细胞／孔的密度，培养在铺有不同浓度的层粘连蛋白（ＬＮ）或纤粘连蛋白（ＦＮ）的培养板上，观察细胞贴壁情况。４８小时后加入卵泡刺激素和睾酮，再培养４８小时，收集培养液，测定雌二醇和孕酮。另外在双池培养系统中，在铺有和未铺ｍａｔｒｉｇｅｌ的上池中分别加入不同浓度的颗粒细胞，利用３５硫标记蛋氨酸掺入法观察ｍａｔｒｉｇｅｌ对颗粒细胞合成蛋白的影响，并利用免疫印迹法鉴定颗粒细胞膜蛋白中ＬＮ结合蛋白的分子量。结果显示，４μｇＬＮ即可使颗粒细胞在４小时内贴壁及展开，雌二醇和孕酮的产量增加。ＦＮ却无显著影响。从ｍａｔｒｉｇｅｌ双池培养系统中取上池培养液，测定放射活性，铺有ｍａｔｒｉｇｅｌ组显著高于对照组。放射自显影图象显示ｍａｔｒｉｇｅｌ显著刺激颗粒细胞合成功能。免疫印迹结果显示颗粒细胞膜蛋白存在着ＬＮ的多结合位点。本实验证实：ＬＮ对卵巢颗粒细胞的附着、伸展。
The aim of this study is to explore the effect of extracellular matrix on rat granulosa cells.The granulosa cells isolated from immature rats were cultured with different concentration of laminin or fibronectin.The cell of adhesion and extension of are observed after 4 hours incubation.FSH and testosterone were added after 48 hours.The levels of estradiol and progesterone in the media were meas-ured after additional 48 hours culture.In another experiment, the granulosa cells were cultured in bicameral chambers coated With or with-out matrigel at various cell densities.The de novo protein synthesis by granulosa cells has been observed with 35S-methionine incorporation method,Further more laminin binding proteins in the plasma mem-brane of the granulosa cells were identified by method of immunoblot analysi.The results showed that attachment and extension occurred on the plate coated with only 4g laminin within 4 hours culture,but not on the plastic or fibronectin coated plate. Laminin could significantly in-crease the production of estradiol and progesterone by the granulosa cells in a dose-dependent manner.The radioactivities of 10 1 condition medium from matrigel-coated group were significantly higher than those matrigel-coating.The autoradiography results also indicated that matrigel significantly stimulated the de novo protein synthesis by granulosa cells,The immunoblot analysis showed that laminin has mul-tiple proteins binding sites.The present study confirmed that laminin enhances the function of attachment,hormone secretion and protein synthesis of the rat granulosa cells.
出处
《生殖医学杂志》
CAS
1994年第3期140-144,共5页
Journal of Reproductive Medicine
关键词
细胞外基质
性激素类
蛋白质
卵巢颗粒细胞
Extracellular matrix Granulosa cells Sex hormones Proteins Animals,laboratory
