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研究生:范姜懿群
研究生(外文):Yi‐Chun Fan‐Chiang
論文名稱:黑鯛腦發育與生殖腺性別分化之關係研究
論文名稱(外文):The association of brain development with gonadal sex differentiation in protandrous black porgy, Acanthopagrus schlegeli
指導教授:張清風張清風引用關係
指導教授(外文):Ching‐Fong Chang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:59
中文關鍵詞:環化酵素放射神經膠細胞神經新生作用視前區雌激素腦源神經滋養因子
外文關鍵詞:AromataseRadial-glial cellNeurogenesisPro-optic areaEstrogenBrain-derived neurotrophic factor
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黑鯛在生殖腺性別分化的時期,其腦部組織中cyp19a1b 會大量表現,然而在生殖腺發育過程中,腦部組織的cyp19a1b 隨著生殖腺成熟會有增加的趨勢,因此腦部的環化酵素(aromatase)會跟生殖腺的分化、發育息息相關,而且腦部的環化酵素會表現在放射神經膠細胞(radial glial cell),可以藉由腦源性滋養因子來促進神經新生作用(neurogenesis)。因此本研究之目的在探討腦部神經膠質細胞(glial cell)促進腦部發育以及和生殖腺之關聯性。在生殖腺分化時期,腦部組織中cyp19a1b 會在第105 天至120 天大量表現,從腦部組織化學免疫染色發現在第120 天時,標定神經膠質細胞的Blbp、S-100 在視前區域有神經纖維(radial glial fibers)產生,然而表現Aromatase 的細胞在第120天時並沒有明顯增多但是會隨著時間有增加的趨勢。藉由雌激素的刺激,雌激素會促進神經膠質細胞的生長,然而在腦部可以藉由環化酵素和雌激素受體(estrogen receptors,ER)的相互作用,迅速利用雌激素來調控神經元的分化,因此雌激素會促進腦部的發育也會對於生殖腺具有促進分化的作用。從腦部組織化學免疫染色發現,雌激素刺激後,會使表現Aromatase 的細胞明顯增多而且有神經纖維的產生,可以導引神經元的遷移,並且進行神經的分化。根據結果推論,雌激素是一種腦神經營養源,可以針對神經細胞及神經膠質細胞的發育。黑鯛腦部的環化酵素對於生殖腺內分泌系統應該具有重要的功能,因此環化酵素在生殖軸線中對於腦的發育和生殖腺性別分化扮演重要的角色。
關鍵字:環化酵素、放射神經膠細胞、神經新生作用、視前區、雌激素、腦源神經滋養因子。
The gonadal sex differentiation in black porgy(Acanthopagrus schlegeli), the gene cyp19a1b will be high expression in brain , but in the gonad development, the cyp19a1b in the brain will increase as the trend during gonads mature. Therefore, aromatase is closely related to the differentiation and development of gonads, aromatase is located in radial glial cell, which it can be promoted the neurogenesis by brain-derived neurotrophic factor.The objectives of this study were to investigate the association of glial cell to promote brain development with gonads. In the gonadal differentiation, the cyp19a1b will be high expression in the 105 to 120 days,from the brain immunohistochemical in the 120 days, the Blbp, S-100 which are glial cell marker,it will have radial glial fibers in pre-optic area, however will display aromatase the cell in the 120 days weather not to increase obviously, but will have the increase tendency along with the time. Because of the estrogen stimulation, the estrogen can promoted glial cell to growth, however in the brain may because of the aromatase and the estrogen receptors (ER) the interaction, regulates neuron differentiation rapidly using the estrogen, therefore the estrogen can promote brain growth also to have the promotion differentiation function regarding the gonad. From brain immunohistochemical, after estrogen stimulation, will cause to display aromatase the cell to increase obviously moreover brightly after the textile fiber production, may guide neuron migration, and will carry on the nerve the differentiation.
According to the result, the estrogen is one kind of brain nerve nutrition source, may aim at the nerve cell and glial cell growth. The black porgy aromatase in the brain should have the important function regarding the gonad endocrine system, therefore brain aromatase might be related to play an important role for brain development and gonadal sex
differentiation.
Key words:Aromatase、Radial-glial cell、Neurogenesis、Pro-optic area、Estrogen、Brain-derived neurotrophic factor。
中文摘要 i
英文摘要 iii
目錄 v
圖目錄 viii
第一章 文獻整理 1
1. 脊椎動物的生殖內分泌 1
2. 生殖腺的性別分化 1
3. 神經新生作用 1
4. 雌激素和神經活動性 2
5. 神經膠細胞(glial cell) 4
6. 腦源神經滋養因子(Brain-derived neurotrophic factor,BDNF) 5
7. 環化酵素(Aromatase) 6
8. 視前區pre-optic area (POA) 7
9. 研究緣起 8
(1)研究生物 8
(2)目前實驗室對於黑鯛性別分化之研究 8
(3)研究目的 9
第二章 黑鯛生殖腺分化的時期腦部神經膠質細胞的變化 10
一、前言 10
二、實驗材料與方法 11
1. 實驗魚種 11
2. 實驗設計 11
3. 實驗方法 12
(1) 全量核醣核酸 (RNA)之純化 12
(2) cDNA之合成 12
(3) 聚合酶連鎖反應 (PCR) 13
(4) DNA萃取 13
a. 洋菜膠體之萃取法 (gel extraction) 13
b. DNA重組接合 (ligation) 14
c. 勝任細胞 (competent cell) 14
d. 質體轉型 (Transformation) 14
e. 基因定序 15
f. 微量質體DNA抽取 (plasmid extraction) 15
(5)即時定量系統之建立(Quantification Real-time PCR) 16
(6)組織切片 17
a. Paraformadehyde固定 17
b.免疫染色(Immunohistochemiscal stain) 18
(7)統計分析 18
三、結果 19
(1)幼齡黑鯛在生殖腺分化時腦相關基因表現量 19
a. 幼齡黑鯛腦bdnf表現量 19
b. 幼齡黑鯛腦blbp表現量 19
c. 幼齡黑鯛腦cyp19a1b表現量 20
(2)組織免疫化學染色 20
四、討論 22
第三章 雌二醇的誘導對於黑鯛腦部神經膠質細胞的影響 31
一、前言 31
二、實驗材料與方法 32
1. 實驗魚種 32
2. 實驗設計 32
a. 幼齡黑鯛投餵雌二醇對於腦部神經膠質細胞相關基因表現之影響 32
b. 注射雌二醇探討黑鯛腦部神經膠質細胞數量、形態的變化 33
3. 實驗方法 33
三、結果 34
(1) 幼齡黑鯛在雌二醇的誘導下腦相關基因之表現量 34
a. 幼齡黑鯛在雌二醇的誘導腦bdnf表現量 34
b. 幼齡黑鯛在雌二醇的誘導腦blbp表現量 34
c. 幼齡黑鯛在雌二醇的誘導腦cyp19a1b表現量 35
(2)組織免疫化學染色 35
四、討論 38
第四章 結論 48
參考文獻 49

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