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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48845

Title: E3 泛素接合酶 RNF149 對生殖母細胞分化之標的與功能
Authors: Lee, Yun-Jia
李云嘉
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 不孕症;男性生殖;精原幹細胞;生殖母細胞;RNF149
RNF149;spermatogonial stem cell;gonocyte;RNF149;infertility
Date: 2014
Issue Date: 2018-08-22T06:38:35Z
Abstract: 泛素蛋白酶體系統 (Ubiquitin Proteasome System),由一連串酵素性反應,導致蛋白質的泛素化,造成蛋白質的功能降解或改變。男性精子的生成需依賴一群精原幹細胞作為基礎,精原幹細胞為一群具多功能 (pluripotent) 分化特性早期幹細胞,其可以自體新生 (self-renew),若分化為精細胞,則攜帶遺傳基因,產生子代,形成世代交替。當 gonocyte 分化異常時則會引發睪丸癌發生。已知 retinoic acid (RA) 為 gonocyte 分化所必需且若改變 UPS 的基因與睪丸癌及男性不育相關。本研究目的為探討 UPS 系統中 E3 ubiquitin ligase RNF149 基因overexpression 對於 RA 誘導 gonocyte 分化之影響。本實驗分為兩個模式分別以細胞株 LC540 Leydig cell 及初代細胞 gonocyte 探討過度表現 RNF149 下對於 RA 刺激 Leydig cell 睪固酮相關生合成酵素及初代細胞 gonocyte 分化指標影響為何。實驗以 20 隻雄性 SD 大鼠為出生三天,取其睪丸分離出 gonocyte 及細胞株 Leydig cell 各分為兩組 (1) 控制組 (2) retinoic acid (1 μM ) 而後藉由 pExpress-1 將 RNF149 cDNA 送入生殖母細胞中過度表現。最後,測量 Leydig cell 睪固酮相關生合成酵素 StAR、CYP11A1、CYP17A1、17#westeur032#-HSD 及初代細胞 gonocyte分化指標 Oct-4、PLZF、VASA。結果顯示,在RA的刺激下Leydig cell 睪固酮相關生合成酵素 StAR、CYP11A1、17#westeur032#-HSD 會因RNF149過度表現下降。而在初代細胞 gonocyte 部分其分化指標 Oct-4、VASA 也顯示相同情形,且抑制分化指標之 PLZF 表現上升。本研究證實 E3 ubiquitin ligase RNF149 會抑制 retinoic acid 刺激細胞分化,兩者具有拮抗作用。
The ubiquitin proteasome system (UPS) consists of a cascade of enzymatic reactions leading to the ubiquitination of proteins with consequent degradation or altered functions of the proteins. Alterations in UPS genes have been associated with male infertility suggesting the role of UPS in spermatogenesis. The protein is a RING domain-containing E3 ubiquitin ligase involving control of gene transcription, translation, cytoskeletal organization, cell adhesion, and epithelial development. Spermatogenesis relies on the existence of a pool of germline stem cells, the spermatogonial stem cells (SSCs), that will support the production of mature spermatozoa throughout the lifetime of a male. E3 ubiquitin ligase RNF149 (RING finger protein 149) is a RING finger domain and a specialized type of zinc finger of 40–60 residues that binds two atoms of zinc. Full-length rat RNF149 cDNA, the transcriptional activation domain was inserted into a pExpress-1 vector. A rat leydig cell cell line LC540 and primary cell gonocyte was used to establish stable transformants. Our results suggested that testosterone synthesis pathway may induce by treating retinoic acid (RA). While E3 ubiquitin ligase RNF149 was overexpression, the RA induction would be eliminated. Then the protein expression such as StAR, CYP11A1 and 17#westeur032#-HSD would be decreased. Under 1 µM RA stimulation E3 ubiquitin ligase RNF149 overexpression would inhibit gonocyte relative differentiation markers like OCT-4 and VASA, but promote PLZF expression. This study demonstrated that E3 ubiquitin ligase RNF149 may block RA induced differentiation showing the antagonism with each other.  
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010132027.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48845
Appears in Collections:[食品科學系] 博碩士論文

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