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.

 

摘要 I
Abstract II
目錄 III
圖目錄 VI
附錄目錄 VII
名詞縮寫 VIII
壹、緒論 1
貳、文獻回顧 3
一、Ubiquitin Proteasome System (UPS) 3
二、RNF149 3
三、幹細胞 4
3-1. 胚胎幹細胞 (embryonic stem cell; ESC) 4
3-2. 成體幹細胞 (adult stem cell) 5
3-3. 精原幹細胞 5
3-3-1. 原生殖細胞 (primordial germ cell ; PGC) 6
3-3-2. 前精原幹細胞 (gonocyte) 6
3-3-3. 精原幹細胞 (spermgonia stem cell; SSC) 6
3-4. 精子的生成與發育 7
3-4-1. 精原幹細胞期/精原細胞期 (spermatogonia) 7
3-4-2. 精母細胞期 (spermatocyte) 7
3-4-3. 精細胞期 (spermatid) 7
3-5. 精原幹細胞的標記物 8
3-5-1. Plzf (promyelocyte leukaemia zinc-finger factor) 8
3-5-2. Oct-4 (octamer-binding transcription factor 4) 8
3-5-3. c-kit 8
3-5-4. Dazl 8
3-5-5. Piwi 9
3-5-6. GCNF 9
四、Retinoic acid (RA) 9
五、男性生殖與不孕症 10
5-1. 不孕症 10
5-1-1. 內分泌系統 10
5-1-2. 睪丸狀態 10
5-1-3. 先天性染色體的異常 10
5-1-4. 環境 10
5-1-5. 生活因素 10
5-1-6. 疾病 11
5-2. 男性生殖系統 11
5-3. SOX家族與不孕症 12
六、睪丸癌 12
6-1. 睪丸癌簡介 12
6-1-1. 精原細胞癌 (seminoma) 13
6-1-2. 非精原細胞癌 （non-seminoma) 13
6-2. 睪丸癌與精原幹細胞 14
參、實驗設計 15
一、Gonocyte isolation 15
二、Gene transfection 16
三、轉染 48 小時後分析項目 17
肆、實驗材料與方法 18
一、實驗材料 18
1-1. 細胞株 18
1-2. 實驗動物 18
1-3. 藥品 18
1-3-1. LC540 18
1-3-2. Gonocyte Isolation 18
1-3-3. Gene transfection 18
1-3-4. Retinoic acid 18
1-3-5. 細胞存活率測定 (MTT assay) 19
1-3-6. 蛋白質膠體電泳 (SDS-PAGE) 19
1-3-7. Western blot 19
1-4. 儀器設備 19
二、實驗方法 20
2-1. LC540 細胞培養 20
2-2. Gonocyte Isolation 20
2-3. Retinoic acid 21
2-4. 抽取 Plasmid DNA 21
2-5. 質體DNA 的大量製備 21
2-6. Gene transfection (RNF149) 21
2-7. Q-PCR 分析 RNF149 基因表現 22
2-7-1. Total RNA 之抽取 22
2-7-2. 反轉錄反應 22
2-7-3. Q-PCR 22
2-7-4. DNA Electrophoresis 23
2-7-5. 細胞存活率測定 (MTT assay) 23
三、統計分析 24
伍、結果與討論 25
一、重組質體中 RNF149 基因鑑定 25
二、以體外實驗探討 RNF149 過度表現對於細胞株 LC540 睪固酮生合成酵素之影響 25
2-1. RA 對 LC-540 細胞存活率探討 25
2-2. 以Q-PCR 確認 RNF149 過度表現情形 26
2-3. 過度表現 RNF149 對 LC-540 細胞存活率探討 26
2-4. 過度表現 RNF149 對於其下游蛋白表現之探討 26
2-5. 過度表現 RNF149 對於睪固酮生合成酵素表現之探討 26
三、以體外實驗探討 RNF149 過度表現對於初代細胞 gonocyte 分化之影響 27
3-1. RA 對 gonocyte 細胞存活率探討 27
3-2. 以Q-PCR 確認 RNF149 過度表現情形 27
3-3. 以流式細胞儀觀察 RNF149 過度表現後對於 OCT-4 表現之 探討 28
3-4. 過度表現 RNF149 對生殖母細胞分化指標 PLZF 及 VASA 蛋白之影響 28
3-5. 過度表現 RNF149 後對 gonocyte 及 LC540 相關蛋白影響 之比較 29
陸、結論 31
柒、參考文獻 32
捌、圖表 42
玖、附錄 57
圖目錄
圖一、含 RNF149 基因之 E. coli DH10#westeur032# 轉殖株之純化質體經 NotI 和 EcoRV.限制酶剪切後之 1 % 洋菜膠電泳圖。 42
圖二、E. coli DH10#westeur032# 轉殖株與 RNF149 基因序列比對。 43
圖三、不同濃度之 RA 對 LC-540 大鼠萊氏細胞存活率之影響。 44
圖四、RNF149 基因轉染 48 小時後對 LC540 RNF149 mRNA 之影響。 45
圖五、RNF149 基因轉染 48 小時後對 LC-540 大鼠萊氏細胞存活率之影響。 46
圖六、RNF149 基因轉染 48 小時後對 LC-540 大鼠萊氏細胞 B-Raf 蛋白之影 響。 47
圖七、RNF149 基因轉染 48 小時後對 LC-540 大鼠萊氏細胞睪固酮生合成酵素 StAR 蛋白之影響。 48
圖八、RNF149 基因轉染 48 小時後對 LC-540 大鼠萊氏細胞睪固酮生合成酵素 CYP11A1 蛋白之影響。 49
圖九、RNF149 基因轉染 48 小時後對 LC-540 大鼠萊氏細胞睪固酮生合成酵素 CYP17A1 蛋白之影響。 50
圖十、RNF149基因轉染 48 小時後對 LC-540 大鼠萊氏細胞睪固酮生合成酵17#westeur032#-HSD 蛋白之影響。 51
圖十一、不同濃度之 RA 對生殖母細胞存活率之影響。 52
圖十二、RNF149基因轉染 48 小時後對生殖母細胞 RNF149 mRNA 之影響 53
圖十三、RNF149基因轉染 48 小時後對生殖母細胞分化指標 OCT4 之影響。 54
圖十五、RNF149基因轉染 48 小時後對生殖母細胞分化指標 VASA 之影響。 56
 
附錄目錄
附錄一、RNF149 在人類器官中之表現。 58
附錄二、RNF149 在癌症中之表現。 59


 

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