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In vitro maturation of granulose cell by adenovirus mediated gene transduction employing follicle stimulating hormone receptor gene.

利用卵泡刺激素受体基因通过腺病毒介导的基因转导使颗粒细胞体外成熟。

基本信息

批准号：
13470346
负责人：
TANAKA Kenichi
金额：
$ 6.27万
依托单位：
NIIGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470346/
关键词：
adenoviral vector estradiol granulosa cells in vitro maturation 卵成熟顆粒膜細膜

项目摘要

Since the granulosa cells rapidly lose the expression of follicle stimulating hormone receptor (FSHR) and the function of steroidogenesis in primary culture conditions, co-culture of immature oocytes with granulosa cell has limitation to support oocyte maturation satisfactory for clinical application. Then, we tried to maintain the in-vitro granulosa cell function by adenoviral-mediated FSHR gene transduction. The number of FSHR on the surface of the granulosa cells prepared from the rat primed by diethylstilbestrol was 4,874 per cell immediately after sacrifice, then it decreased to 2,176 in by the third day, and further reduced to 828 and 693 on the fifth and seventh days, respectively. During the third day of culture, the amount of production of estradiol by FSH stimulation also decreased to about one-quarter of the first day's culture. Then, we created the FSHR expressing recombinant adenovirus. When it was introduced into the granulosa cells on the first day of culture, the expression of FSHR was enhanced up to 7,206 per cell and the amount of estradiol produced was remarkably increased up to 5,940 pg/ml on the third day. The co-culture of the immature oocytes with FSHR introduced granulosa cells is supposed to improve their developmental competence.

由于颗粒细胞在原代培养条件下迅速丧失促卵泡激素受体（FSHR）的表达和类固醇生成功能，因此未成熟卵母细胞与颗粒细胞的共培养在支持卵母细胞成熟以满足临床应用方面存在局限性。然后，我们尝试通过腺病毒介导的FSHR基因转导来维持体外颗粒细胞功能。处死后立即用己烯雌酚致敏的大鼠制备的颗粒细胞表面的FSHR数量为4,874个/细胞，然后到第三天减少到2,176个，并在第五天和第七天分别进一步减少到828个和693个。在培养的第三天，FSH刺激产生的雌二醇的量也减少到第一天培养的约四分之一。然后，我们创建了表达 FSHR 的重组腺病毒。当在培养第一天将其引入颗粒细胞时，每个细胞的 FSHR 表达增强至 7,206，并且产生的雌二醇量在第三天显着增加至 5,940 pg/ml。未成熟卵母细胞与引入 FSHR 的颗粒细胞的共培养有望提高其发育能力。