第四章 討論
4.9 紅體細胞以類固醇(Steroids)處理 72 小時，Angiopoietin -1 與相
4.9.3 以黃體酮(progesterone)處理
將黃體酮注射至切除卵巢之老鼠體內，透過結合核內受器 PR a 與 PR b (Tibbetts et al. 1998)後可刺激 VEGF 表現量增加，促使子宮內膜血管 內皮細胞大量增生且血管新生的累積相當顯著(Ma et al., 2001)，而發現另 外注射雌二醇的組別其內皮細胞生長受到抑制(Walter et al., 2005)，以往 研究指出 PR 基因可能受到雌二醇經由位於 PR 基因促進子(promoter)上 estrogen response elements 所調控(Kraus et al., 1993)，故一般認為雌二醇 為影響黃體酮刺激內皮細胞增生之必要因子，但在哺乳類活體實驗中發 現單獨注射黃體酮亦有刺激子宮內膜內皮細胞增生的作用，而雌二醇為 非必要因子(Walter et al., 2005) ，此外在小鼠研究中體內注射黃體酮的組 別，其血管週邊細胞與平滑肌細胞數量明顯增加，推測與血管成熟有相 關性(Girling et al., 2007)。本實驗中可看出黃體酮刺激 Angiopoietin-1 與
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FLK 表現，VEGF 在黃體酮濃度 10-7M 時表現有上升趨勢，推測黃體酮 在日本鰻紅體細胞中調控情形與哺乳類研究相似，其作用機制是否與哺 乳類相同仍需探討。
4.10 紅體細胞以生長因子(Growth factors)處理 24 小時，
Angiopoietin-1 與相關血管生長因子表現 4.10.1 以胰島素(Insulin)處理
在人類視網膜上皮細胞研究中，發現胰島素可誘發HIF α次單元使 HIF蛋白表現，增進VEGF基因轉錄以刺激血管新生，VEGF表現在短時 間內(6小時)隨胰島素濃度增加成正比(Treins et al., 2002)，胰島素增加HIF α次單元與刺激VEGF轉錄為透過PI3K-Akt與Ras / MAPK傳遞路徑所誘導 (Saltiel et al., 2002) (Baron et al., 2001) 。在鰻魚紅體細胞中，VEGF、
Angiopoietin-1表現趨勢受胰島素刺激而升高在蛋白質免疫螢光染色亦有 類似結果(圖74 -1)，以VEGF增加較為顯著，但FLK無明顯變化。在胰島 素調控下，VEGF表現增加並促進Angiopoietin-1表現以誘導血管新生，但 是否與哺乳類研究有類似機制，透過PI3-Akt路徑或誘導HIF因子，須再 比較探討。
4.10.2 以鹼性纖維母細胞生長因子 ( Basic fibroblast growth factor )處理
利用bFGF處理人類上皮細胞株PNT1培養三天，發現bFGF會刺激 PNT1的VEGF表現增加(Sordello et al., 1998) ，在許多哺乳類血管內皮細
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胞研究中，bFGF與受器結合後活化Akt/PKB途徑(Forough et al., 2005)，
促進纖維母細胞及內皮細胞之有絲分裂，為一良好的分裂素(mitogen) (Klein et al., 1996)。本實驗中Angiopoietin-1在bFGF濃度大於10-8M時有刺 激效果，VEGF表現隨bFGF濃度增加而提升且FLK表現在bFGF濃度10-8M 有升高趨勢，podocalyxin濃度10-8M表現受抑制，而Eph表現似乎無太大 差異，推測鰻魚紅體細胞以bFGF調控下可能活化Akt/PKB途徑，促使內 皮細胞分裂並刺激Angiopoietin-1、VEGF表現，與哺乳類研究有類似角 色。
4.10.3 以吳郭魚類胰島素生長因子-1 (Tilapia Insulin-Like growth factor-1)處理
在哺乳類研究中 IGF-1 與膜上受器 IGF1-R 結合後，透過磷酸化 Akt、
Src 等訊息傳遞分子，以活化血管新生及淋巴新生(Jackson et al., 2005); 將 小鼠視網膜血管內皮細胞 IGF-1 受器基因剔除後發現血管新生顯著減少，
故 IGF-1 被認為是引起糖尿病視網膜病變的重要因子(Smith et al., 1999)，
而大鼠神經元中 IGF-1 可引起 HIF α 表現(Robert et al., 2003)以增加 VEGF 表現。在對胰島素敏感的組織中，IGF-1 可調控多種生理代謝途徑，如 PI3K-Akt、MAPK 傳遞路徑等，活化抗細胞凋亡、細胞生長及遷移、細 胞分化等功能。本實驗中 Angiopoietin-1 與 Podocalyxin 表現在 IGF-1 濃 度 10-8M 時表現達高峰且蛋白質免疫螢光染色亦有類似結果(圖 74-2) ; VEGF 則隨 IGF-1 濃度增加而有下降趨勢，Eph 在 IGF-1 濃度 10-8M 及 10-7M 時表現量達高峰。鰻魚紅體細胞內以 IGF-1 調控可能活化
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Angiopoietin-1 與 Podocalyxin 較顯著，而 VEGF 在鰻魚紅體細胞內可能 受 IGF-1 負調控，造成此種差異之因素需進一步探討是否為哺乳動物與 魚類間演化過程或生活環境上差異有關。
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總結
1. 選殖出日本鰻 Angiopoietin-1 部分基因。
2. 在不同體重日本鰻的紅體中，Angiopoietin-1 在體重較大之組別中表現 量達高峰，推測體型較大的鰻魚其血管新生較體重較小之組別更趨完 整、成熟，故 Angiopoietin-1 表現量較高。
3. 以腦下垂體催取液催熟之日本鰻紅體，Angiopoietin-1 在雄鰻中，在注 射第 0 針至第 6 針過程中，Angiopoietin-1 表現量逐漸升高至第 9 針下 降，推測在在雄鰻催熟發育過程後期 Angiopoietin-1 促進紅體血管成 熟。
4. 以腦下垂體催取液催熟之雄鰻紅體，個體之 Angiopoietin-1、FLK、Eph、
DLL-4、Adam 與 RMI 值呈正相關; 而雌鰻紅體個體之 Angiopoietin-1、
VEGF 與 RMI 值呈正相關，但 FLK 與 RMI 值呈負相關。
5. 以腦下垂體萃取液催熟之雌鰻卵巢，在注射第 0 針至第 9 針過程中，
GSI 值逐漸上升，但個體之 Angiopoietin-1、VEGF、FLK、DLL-4、Adam 表現量隨 GSI 值增加呈負相關趨勢; 但合併注射睪固酮 9 針之組別，
其 GSI 值較只注射腦下垂體萃取液有顯著提升， VEGF、FLK、Eph、
Podocalyxin、DLL-4、Adam 亦有提升現象。在卵巢組織切片以 VEGF 一級抗體標定染色，發現其蛋白質與 mRNA 表現情形相似。推測睪固
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酮對於卵巢內血管新生因子確實有提升現象，使血管新生更旺盛以促 進卵巢發育完全。
6. 日本鰻紅體細胞無加藥處理培養 2 至 8 週，Angiopoietin-1 在第 2 週至 第 6 週表現提升達穩定，至第 7 週後表現有升高趨勢，與 VEGF、FLK、
Eph、Podocalyxin 在第 6 週後表現有下降趨勢，與 Angiopoietin-1 表現 趨勢有互補現象，推測 Angiopoietin-1 與 VEGF 等因子表現高峰有時間 上的差異。
7. PPAR β agonist 刺激日本鰻紅體細胞 Angiopoietin-1、VEGF 表現量升高;
PPARγagonist 刺激 VEGF 表現，抑制 Angiopoietin-1 的表現量。
8. 日本鰻紅體細胞以內皮細胞生長補充劑(ECGs)處理 12 小時後，
Angiopoietin-1、VEGF、FLK 表現量在 ECGs 濃度 150 μg 與 300 μg 隨 著升高; 處理 24 小時後，Angiopoietin-1、VEGF 在 ECGs 濃度 75 μg 時表現最高，在濃度 150 μg 以上其刺激效果反而下降，推測可能是 ECGs 濃度已達飽和。
9. 日本鰻紅體細胞以鈷、銅、鋅離子處理，鈷離子刺激 VEGF 表現，對 於 Angiopoietin-1 刺激不明顯; 銅離子刺激對本實驗之血管新生因子無 顯著影響; 鋅離子可誘導 Angiopoietin-1、VEGF、FLK 及 Podocalyxin 表現，鋅離子對於鰻魚紅體內皮細胞之血管新生因子的刺激效果，與
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鈷離子和銅離子相較之下，刺激作用較佳。
10. 日本鰻紅體細胞以睪固酮、雌二醇、黃體酮處理，睪固酮可刺激 Angiopoietin-1 並抑制 VEGF 與 FLK 表現; 雌二醇刺激 Angiopoietin-1、
VEGF、FLK 與 Podocalyxin 表現;黃體酮刺激 Angiopoietin-1、VEGF 與 FLK 表現，推測此三種類固醇在日本鰻紅體細胞中調控情形可能與 哺乳類研究類似，但是否透過相同機制則需進一步探討。
11. 日本鰻紅體細胞以胰島素、bFGF、IGF-1 處理，胰島素可使 VEGF 表現增加並促進 Angiopoietin-1 等血管生長因子表現; bFGF 可促使血 管內皮細胞分裂並刺激 Angiopoietin-1、VEGF、FLK 表現，與哺乳類 研究有類似角色。而 IGF-1 刺激 Angiopoietin-1、FLK、Eph 及 Podocalyxin 表現，但抑制 VEGF 表現，此現象與哺乳類研究有所不同，造成此種 差異之因素，推測是否為哺乳動物與魚類間演化過程或生活環境上差 異有關。
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未來展望
1. 擴增出日本鰻 Angiopoietin-1 完整片段與選殖出相關 Angiopoietins，
並了解其在日本鰻紅體組織受調控情形。
2. 純化出 Angiopoietin-1 重組蛋白，運用於鰻魚人工繁殖，以解決因血 管生長不完全所造成之鰻魚生長缺陷問題。
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