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血球系幹細胞灌流培養系を用いた前胞状卵胞の体外成熟に関する研究

血细胞干细胞灌流培养系统体外成熟窦前卵泡的研究

基本信息

批准号：
15659390
负责人：
藤井俊策
金额：
$ 2.11万
依托单位：
Hirosaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Exploratory Research
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15659390/
关键词：
卵胞幹細胞

项目摘要

【目的】血球系幹細胞を持続灌流しながら卵巣組織を器官培養する系において,卵巣内の前胞状卵胞を体外発育させることができる至適条件を設定すること。【方法】排卵周期を有しホルモン動態がヒトに近いラット卵巣を用いて基礎実験を行なった。3週齢の近交系SHR雌ラットから片側卵巣を摘出し、ビブラトームを用いて薄切した。さらに骨髄を採取し,密度勾配遠心法で単核球を分取した。骨髄単核球はStemPro-34 SFM mediumに浮遊させ,幹細胞の分化を抑制しながら冷蔵保存した。灌流培養の基本培養液としてはHAS, ITS, L-glutamine添加M-199を用い,これにrhFSH(Serono International, Geneva)を10 IU/Lの濃度で添加した。骨髄単核球灌流群と非灌流群の2群を設定し,それぞれパーフュージョンディッシュチェンバー(Ieda Trading, Japan)に薄切した卵巣組織を置き、上記基本培養液で持続灌流しながら培養した。持続的灌流には2台のペリスタルティックポンプを改造して用いた。冷蔵保存しておいた骨髄単核球は用時に基本培養液に再浮遊させ,灌流群ではパーフュージョンディッシュチェンバーの別のチャンネルから骨髄単核球浮遊液を灌流した。非灌流群では基本培養液のみを灌流した。【結果】7日間,骨髄単核球浮遊液を灌流培養することにより,卵胞径の増大と卵胞腔の形成が認められた。しかし,結果に再現性が得られておらず,至適条件の設定にはまだ時間を要するものと思われる。また,ペリスタルティックポンプの回路が閉塞するなど灌流方法にも問題が生じており,培養方法の再検討も必要である。

[Objective] To establish optimal conditions for the development of precellular oocytes in vitro from hematopoietic stem cells by perfusion and organ culture. [Method] Ovulation cycle is dynamic, and it is essential to maintain the ovulation cycle. 3-week-old inbred strain SHR female, female Today, the density of the core is determined by the distance between the core and the core. Stem cell differentiation is inhibited by StemPro-34 SFM medium and preserved in cold storage. Perfusion culture medium was supplemented with HAS, ITS, L-glutamine M-199 and rhFSH(Serono International, Geneva) at a concentration of 10 IU/L. The two groups of bone marrow perfusion group and non-perfusion group were set up, and the two groups were divided into two groups: the first group was divided into three groups: the first group was divided into three groups: the second group was divided into three groups: the first group was divided into three groups: the first group was divided into three groups: the second group was divided into three groups: the third group was divided into three groups: the third group was divided into four groups: the third group was divided into three groups: the fourth group was divided into four groups: the fourth group was divided into three groups: the fourth group was divided into four groups: the fourth group was divided into four groups: the fourth group was divided into three groups: the fourth group was divided into four groups: the fourth group was divided into three groups: the fourth group was divided into four groups: the fourth group was divided into three groups: the fourth group was divided into four groups: the fourth group was divided into four groups: the fourth group was divided into four 2 sets of perfusion equipment When cryopreserved, the bone marrow spheres are re-cultured in the basic culture medium, and the perfusion group is re-cultured in the basic culture medium. Non-perfused group is composed of basic culture medium and perfusion medium. [Results] During the seventh day, the diameter of egg cell increased and the formation of egg cavity was recognized. The reproducibility of the results is determined by the time required to set the appropriate conditions. The problem of perfusion method is caused by the circuit of culture method, and it is necessary to reconsider the culture method.