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研究生:朱慈文
研究生(外文):Ci-wen Zhu
論文名稱:日本鰻在人工催熟過程中添加磷酸二脂酶三型抑制劑(Cilostazol) 對其卵巢與紅體發育之影響
論文名稱(外文):Effects of the Cilostazol (a specific phosphodiesterase type Ⅲ inhibitor) on ovarian and gas gland development in the induced female Japanese eels
指導教授:黃永森黃永森引用關係
指導教授(外文):Yung-sen Huang
學位類別:碩士
校院名稱:國立高雄大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:111
中文關鍵詞:人工繁殖磷酸二酯酶磷酸二脂酶三型抑制劑血管新生卵巢發育
外文關鍵詞:artificial propagationPDECilostazolangiogenesisovary development
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  在日本鰻 (Anguilla japonica) 的養殖產業中,人工繁殖一直是難以克服的問題,使用鮭魚腦下垂體研磨液行腹腔注射可促使鰻魚的卵巢成熟。訊息傳遞分子cAMP在內分泌激素的作用中扮演著相當重要的角色。促性腺激素作用是藉由提高細胞內 cAMP 的含量以刺激卵細胞的發育、顆粒細胞 (granulosa cells) 中雌性素 (estrogen) 的生成等。cAMP的合成與分解是經由腺苷酸環化酶 (AC) 以及磷酸二酯酶 (PDE) 的作用,在不同物種的卵巢濾泡中偵測到了磷酸二酯酶家族 (PDEs) 的表現,其中,PDE3 在卵細胞中表現。Cilostazol(PletalR)是磷酸二酯酶Ⅲ型的選擇性抑制劑,藉由此提高細胞內cAMP的含量。在哺乳類中,Cilostazol可促使血管新生的發生,但其對魚類的類似作用未知。在卵巢發育的過程中,其生殖腺體重指數 (GSI) 會顯著提升,在此過程中,卵巢所需的養分又須經由血液運輸,因此,新的血管網路的建立是必要的。所以本實驗欲研究Cilostazol在鰻魚中是否可藉由促使血管新生而影響卵巢的發育與藉由提高cAMP的量使得SPE刺激卵巢發育之效果提高。因此,本實驗分別將鮭魚腦下垂體研磨液 (SPE) 與Cilostazol,以及上述兩者混合後施打入鰻魚體內,之後將其犧牲並分析生理數據及卵巢與紅體成長相關基因之變化。本實驗使用日本鰻 (Anguilla japonica) 進行試驗,分成六組:腦下垂體組 (SPE)、腦下垂體與藥物處理組 (SPE +Cilostazol) 、藥物處理組 (Cilostazol)、葡萄糖食鹽水組 (control)、腦下垂體組轉打腦下垂體與藥物處理組 (SPE 轉 SPE +Cilostazol) 以及腦下垂體與藥物處理組轉打腦下垂體組 (SPE +Cilostazol 轉 SPE )。實驗結果,在血糖指數方面,SPE +Cilostazol組有高血糖的情況發生,其平均值為control組的1.52倍。而生殖腺體重指數方面,SPE 轉 SPE +Cilostazol組有顯著提升 (5.4倍),SPE組 (1.68倍) 與SPE +Cilostazol 轉 SPE 組 (1.11倍) 有提升。而Cilostazol組 (0.55倍) 與SPE +Cilostazol 組 (0.83倍) 則低於control組。紅體指數方面相較於control組,SPE +Cilostazol 組增加1.72倍 、SPE (1.6倍) 及 Cilostazol組 (1.17倍) 皆有提昇。眼重指數方面相較於control組,SPE +Cilostazol (1.37倍)、SPE (1.34倍) 及 Cilostazol組 (1.31倍) 皆有提昇。之後也對其卵巢與紅體內血管新生相關基因以qRT-PCR作表現量分析。由實驗結果發現,在卵巢發育過程中,VEGF表現量會隨GSI大小增大而上升 (r2=0.320)。而其餘的基因表現量有隨GSI大小增大而下降的趨勢。由各組的組織切片圖可觀察到,施打SPE可使卵發育更成熟 (相較於control組);而施打Cilostazol會抑制卵的發育 (相較於control組);同時施打SPE與Cilostazol則使得卵發育度差異大,卵徑大小不均。而先施打SPE 7針後轉打SPE +Cilostazol 4針可使卵巢發育至GSI =10.05 %;然而,若先施打SPE +Cilostazol 7針後轉打SPE 4針的效果則不如前者。總而言之,本實驗顯示日本鰻人工催熟早期添加Cilostazol或單獨施打會抑制其卵巢發育,而藉由調整Cilostazol加入藥劑中的時間點則可以有效改善施打Cilostazol的抑制效果,推測Cilostazol對卵巢發育的影響在卵巢發育不同的階段上有差異。
In the Japanese eel aquaculture, the most important problem is how to reproduce them artificially. It is critical for a solution of Japanese eel sustainable development. Eels received injections with Salmon Pituitary extracts (SPE) is known to induce its ovary maturation. Cyclic nucleotide phosphodiesterases (PDEs) are critical for ovarian function by their role in controlling cyclic nucleotide levels. PDEs modulate gonadotropin responses in the somatic compartment of the ovarian follicle. The synthesis and degradation of cAMP is due to the function of adenylyl cyclase (AC) and phosphodiesterase (PDE). The expression of PDEs have been detected in the follicle of different species. PDE3 was found in the oocytes. Cilostazol (PletalR) is a selective inhibitor of phosphodiesterase type III and mediates its effects by increasing cellular cAMP levels. Furthermore, cilostazol-induced vasculo-angiogenesis effects have also been shown, but there is no data on its effects in fish. During the process of ovarian maturation, its GSI will significantly increase. In this process, necessary nutrients to ovarian development came from the blood. So, the establishment of a network of new blood vessels is necessary. We address this question in the Japanese eel, since its ovarian development could be manipulated to study if cilostazol can ameliorate induced ovarian development or reinforce ovarian angiogenesis. Therefore, the present study was to inject with SPE and Cilostazol, and both of them into the eel. We analyze changes in physiological data, and the expression levels of angiogenesis genes were tested by qPCR. In the present study, Japanese eels (Anguilla japonica) were divided into six groups: pituitary group (SPE), the pituitary and the Cilostazol-treated group (SPE +Cilostazol), Cilostazol group (Cilostazol), glucose saline group (control), first treated with SPE then switched into SPE+cilostazol group (SPE->SPE +Cilostazol), and first treated with SPE +cilostazol then switched into SPE group (SPE +Cilostazol ->SPE). GSI, RMSI, and OI were all stimulated by SPE while the level of blood glucose (BG) significantly (x1.52) increased only in the SPE+Cilostazol group as compared with controls. The positive effect of SPE (x1.34) or Cilostazol (x1.31) alone on RMSI was shown, and an added positive effect on RMSI was shown in the SPE+Cilostazol (x1.37) group. The positive effect on GSI was shown in the SPE->SPE +Cilostazol group (x5.4), SPE group (x1.68) and SPE +Cilostazol ->SPE group (x1.11). Cilostazol alone inhibited GSI significantly (x0.55) as compared with the controls.The expression level of PTEN long, PTEN short, VEGF-Flk, and Tie2-Angpt1 were tested in ovary and gas gland by qPCR. During the ovaries development, VEGF expression will vary with the increase GSI (r2 = 0.320). While the expression of rest genes will decrease with the increase of GSI. The tissue slices show that SPE induced ovary maturation (compared to the control group); while Cilostazol inhibited ovary maturation (compared to the control group). Simultaneously injected with SPE +Cilostazol is making ovary maturity differences, egg size uneven. A modified experimental protocol got a better result. The result showed that after first treated with SPE 7 injections then switched into SPE +Cilostazol 4 injections enables ovarian development to GSI = 10.05%. However, the effect of first treated with SPE +Cilostazol 7 injections then switched into SPE 4 injections is not as effective as the former. In sμm, the present study had proved that a prolonged administration of Cilostazol had inhibitory effects on the induced ovarian development in the Japanese eel. But, a special regimen might improve this situation. We hypothesized that the effect of Cilostazol on ovarian development is distinct in different stages of ovarian development.
目錄
中文摘要 IX
英文摘要 XI
一、前言
1.1 鰻魚繁養殖現況與瓶頸
1.2 鰻魚生活史
1.3 鰻魚卵巢發育
1.4 動物組織生長與血管新生的關係
1.5 本論文欲研究之鰻魚卵巢發育相關基因
1.5.1血管內皮細胞生長因子VEGF (vascular endothelial cell growth factor)
1.5.2腫瘤抑制基因PTEN (phosphatase and tensin homology deleted from chromosome 10)
1.5.3血管生成素-1 (Angiopoietin-1)
1.6 環核苷酸訊息傳遞 (Cyclic nucleotide signaling) 與磷酸二酯酶 (phosphodiesterases)
1.7 Cilostazol
1.8 實驗目的
二、材料與方法
2.1 血管新生相關基因選殖
2.2 活體實驗
2.2.1預備實驗
2.2.2日本鰻實驗
2.3 GSI%、RMSI‰與OI‰的測量
2.4 組織全RNA的抽取
2.5 cDNA的製備
2.6 RT-PCR (reverse transcription PCR) 分析表現量
2.7 Real-time PCR分析表現量
2.8 日本鰻卵巢組織石蠟組織切片染色的製備
2.8.1日本鰻卵巢組織之固定液處理
2.8.2日本鰻卵巢組織之脫水至石蠟包埋的製備
2.8.3石蠟切片
2.9日本鰻卵巢石蠟組織切片染色之蘇木精-伊紅染色
2.10組織切片卵面積的測量
2.11卵徑測量
2.12統計方法
三、結果
3.1 預備實驗
3.1.1眼重指數與血糖濃度
3.1.2 RT-PCR分析眼睛中各處理下血管新生基因表現量
3.1.3眼重指數與Plk3、PTEN long的相關性
3.1.4 Quantitative real-time PCR (qRT-PCR)
3.1.5小結
3.2 日本鰻實驗
3.2.1體重變化率
3.2.2生殖腺體重指數與血糖濃度
3.2.3紅體體重指數與眼重指數
3.2.4日本鰻體重與其RMSI的關係
3.2.5組織切片
3.2.6測量組織切片截卵面積
3.2.7測量卵徑
3.2.8卵面積與菲列直徑 (Feret’s diameters) 之關係
3.2.9卵巢基因表現量 (qRT-PCR)
3.2.10紅體基因表現量 (qRT-PCR)
3.2.11卵巢血管新生基因表現量與GSI的關係
3.2.12紅體血管新生基因表現量與RMSI的關係
四、討論
4.1 預備實驗與日本鰻實驗死亡率
4.2 以不同處理對鰻魚人工催熟後血液中血糖濃度的影響
4.3 以不同處理對鰻魚人工催熟後卵巢、眼睛與紅體發育的影響
4.4 由卵巢組織切片結果討論不同處理對鰻魚人工催熟的影響
4.5 催熟過程中卵巢成長與血管新生基因表現之關係
五、結論
六、圖表
七、參考文獻
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