中華鱉腦垂體甘丙氨激素之研究：cDNA選殖、表現及調控

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龎慶弘(Chin-Hon Pan) 查詢紙本館藏

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生命科學系

論文名稱

中華鱉腦垂體甘丙氨激素之研究：cDNA選殖、表現及調控
(Studies on pituitary galanin of Chinese mud turtle: cDNA clonig, expression and regulation)

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摘要(中)

甘丙胺激素是一種C端有乙醯化的神經內泌素，已知其具有調控脊椎動物行為與食慾的功能。為了對爬行類腦垂體甘丙胺激素的物種歧異度與基因之演化有更進一步之了解，本研究以中華鱉為實驗動物，利用快速核酸兩端放大之技術，定序腦垂體甘丙胺激素互補核苷酸序列，所得的序列共有672個核苷酸，5’-未轉錄區99 個；前原甘丙胺激素有324個核苷酸；3’-未轉錄區有249 個。前原甘丙胺激素有108個胺基酸；本體為29個胺基酸，與其他脊椎動物之間的同源性為72% ~ 96%。半定量聚合酶連鎖反應實驗結果顯示，前原甘丙胺激素於腦、下丘腦、胃、肝臟、胰臟、睪丸、卵、子宮、大腸與小腸皆有表現，而在心臟、脾臟、肌肉與脂肪組織則無法偵測到其表現。雌性前原甘丙胺激素之表現量似乎高於雄性。離體腦垂體組織培養研究顯示：雌二醇(10-6M~10-8M)、性釋素(10-7M~10-9M) 隨著濃度增加會明顯促進前原甘丙胺激素之表現；而瘦素(10-6M~10-8M)與神經月太 Y(10-6M~10-8M)隨著濃度增加，抑制甘丙胺激素基因表現之情形更趨明顯。綜觀上述結果顯示脊椎動物之前原甘丙胺激素具高度之保守性，且與雌二醇、性釋素、瘦素及神經月太 Y在中華鱉腦垂體的生理功能上具交互作用。

摘要(英)

Galanin is a C-terminally-amidated neurohormone known to regulate various aspects of behavior and appetite of vertebrates. To better understanding the phylogenetic diversity and evolution of galanin genes in reptilie, we here conducted an analysis of galanin cDNA from Chinese mud turtle pituitary. Using 3’- and 5’- RACE techniques, we have isolated and sequenced a novel cDNA derived from pituitary total RNA. Sequence analysis revealed a 672-bp cDNA containing a 99-bp 5’-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3’-untranslated region. The deduced protein is a 108-amino acid preprogalanin molecule with [Ser23]-galanin-29 at the C-terminus of the coding region, making its homology to other vertebrates in the range of 72%～96%. Semiquantitative PCR revealed that preprogalanin was expressed in the brain, hypothalamus, pituitary, stomach, liver, pancreas, testis, ovary, uterus, small intestine and large intestine, but undetected in the heart, spleen, muscle and adipose tissue. Females seemed to express more preprogalanin than males. In vitro pituitary tissue culture study indicated that E2 (10-6M~10-8M) or GnRH (10-7M~10-9M) treatment significantly increased the levels of preprogalaninin a dose- dependent manner. However, Lep (10-6M~10-8M) or NPY (10-7M~10-9M) treatment decreased the levels of preprogalanin also in a dose-dependent manner. These results suggest that preprogalanin is conserved in vertebrates and that a physiological role of galanin interacting with E2, GnRH, Lep and/or NPY may exist in the pituitary of mud turtle.

關鍵字(中)

★ 中華鱉
★ 甘丙胺激素

關鍵字(英)

★ galanin
★ Chinese mud turtle

論文目次

目錄
中文摘要 1
英文摘要 2
縮寫與全名對照 3
第一章：簡介 4
壹、中華鱉 4
貳、甘丙胺激素 4
1. 歷史 4
2. 種類 4
3. 結構 4
4. 基因序列 4
5. 組織分佈與血中含量 5
6. 接受器 5
7. 生物功能 6
8. 內分泌調控甘丙胺激素之表現 6
參、研究目的 7
第二章：材料與方法 8
壹、實驗材料 8
貳、實驗動物 8
參、實驗方法 8
一、快速核酸兩端放大 (RACE) 8
1. RNA萃取 (RNA Extraction) 9
2. 3´RACE 9
A. 第一股cDNA合成 9
B. 聚合酶連鎖反應(PCR) 10
3. 5´RACE 11
A. 第一股cDNA合成 11
B. 聚合酶連鎖反應(PCR) 11
二、組織特異性實驗 11
1. RNA萃取 (RNA Extraction) 11
A. 第一股cDNA合成 12
B. 聚合酶連鎖反應(PCR) 12
2. 第一股cDNA合成 12
3. 聚合酶連鎖反應(PCR) 12
三、激素對甘丙胺激素表達情形影響之實驗 13
1. 鱉腦垂體於M199離體培養實驗 13
2. 激素處理 13
第三章：結果 14
壹、中華鱉腦垂體甘丙胺激素cDNA序列 14
貳、組織特異性 14
參、激素對甘丙胺激素基因表達之影響 14
1. 甘丙胺激素基因於M199離體培養之表現情形 14
2. 雌二醇促進甘丙胺激素基因的表現 15
3. 瘦素抑制甘丙胺激素基因的表現 15
4. 雌二醇與瘦素之交互影響甘丙胺激素基因的表現 15
5. 性釋素促進甘丙胺激素基因的表現 16
7. 神經月太 Y抑制甘丙胺激素基因的表現 16
8. 性釋素與神經月太 Y之交互影響甘丙胺激素基因的表現 17
第四章：討論 18
壹、中華鱉腦垂體甘丙胺激素cDNA序列 18
貳、組織特異性 19
參、激素影響中華鱉腦垂體甘丙胺激素基因的表現 19
第五章：結論與展望 20
第六章：參考文獻 21
第七章：附錄 27
圖目錄 Ⅳ
表目錄 Ⅵ
圖目錄
圖一、哺乳動物甘丙胺激素的基因表現受到其他激素的調控 28
圖二、本研究所採用之引子於甘丙胺激素的相對位置 29
圖三、鱉腦垂體甘丙胺激素cDNA序列 30
圖四、中華鱉甘丙胺激素的組織特異性 31
圖五、離體培養中華鱉腦垂體甘丙胺激素基因表現之時間型態 32
圖六、雌二醇 (10-6M) 對中華鱉腦垂體甘丙胺激素mRNA表現的影響 33
圖七、雌二醇 (10-8 M) 對中華鱉腦垂體甘丙胺激素mRNA表現的影響 34
圖八、瘦素對中華鱉腦垂體甘丙胺激素mRNA表現的影響 35
圖九、雌二醇 (10-8M) 與瘦素 (10-8M) 交互處理6小時對中華鱉腦垂體甘丙胺激素mRNA表現的影響 36
圖十、雌二醇 (10-8M) 與瘦素 (10-8M) 交互處理12小時對中華鱉腦垂體甘丙胺激素基因表現的影響 37
圖十一、性釋素 (GnRH) 對中華鱉腦垂體甘丙胺激素mRNA表現的影響 38
圖十二、性釋素 (GnRH, 10-9 M) 對中華鱉腦垂體中甘丙胺激素mRNA表現的影響 39圖十三、神經月太 Y (NPY) 對中華鱉腦垂體甘丙胺激素mRNA表現的影響 40
圖十四、性釋素 (GnRH,10-9 M) 與神經月太 Y (NPY, 10-9 M) 交互處理12小時對中華鱉腦垂體甘丙胺激素mRNA表現的影響 41
圖十五、脊椎動物甘丙胺激素之演化樹 (phylogenic tree) 42
圖十六、激素對中華鱉腦垂體甘丙胺激素的調控 43
表目錄
表一：甘丙胺激素對脊椎動物荷爾蒙的影響 44
表二：本研究所採用之引子 (primer) 序列 45表三：中華鱉之前原甘丙胺激素(preprogalanin) cDNA序列與其他脊椎動物之同源性 46
表四：中華鱉甘丙胺激素胺基酸序列與脊椎動物之同源性 47
表五：脊椎動物甘丙胺激素的胺基酸序列 48

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指導教授

高永旭(Yun-Hsi Kao)

審核日期

2002-7-18

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