臺灣博碩士論文加值系統

色素細胞 (chromatophores) 分布於硬骨魚類之真皮層中並提供如對抗紫外線之照射、示警、溝通、保護、環境適應、性別、求偶或捕食等重要之功能。其色素之生成受相關基因之影響，如酪胺酸酶基因 (tyrosinase) 、酪胺酸相關蛋白1及2 (tyrosinase-related protein 1/2, TRP1/2) 、microphthalmia-associate transcripition factor (Mitf) 、sox10及dopachrome tautomerase (Dct) 等基因之作用可於硬骨魚類體表產生不同之色澤與圖紋。雌雄吳郭魚 (tilapia， Oreochromis mossambicus) 為世界認可之重要水產養殖模式經濟魚種，其魚體色斑主要為黑色素、紅色素及黃色素所組成。本研究室先前於性成熟吳郭魚性腺及其他組織中發現神經胜肽 (neuropeptide) - 腦垂體腺苷酸環化酶激活肽 (pituitary adenylate cyclase-activating polypeptide，PACAP，tpacap38) 及其受體 (tpac1-r) 之表現量可受ovine PACAP38或環單磷酸腺苷酸 (cyclic adenosine monophosphate，cAMP) 之誘導而增加，並參與調控cAMP-蛋白激酶A (protein kinase A，PKA) 之訊號傳遞路徑。目前於硬骨魚類中並無PACAP與色素細胞兩者相關性以及調控機制之報告，故本研究以ovine PACAP38藥物對體外培養之吳郭魚尾鰭組織進行劑量與時間相關之試驗，以探討此藥物對色素細胞之影響。利用半定量反轉錄酶-聚合連鎖反應 (semi-quantitative RT-PCR) 分析mitf、sox10、tyrosinase、Dct、tpacap38及其受體之表現量發現，以不同濃度之ovine PACAP38 (2.5、5.0及10.0 nM) 誘導魚體外培養之吳郭魚尾鰭組之2小時後，或以5.0 nM之ovine PACAP38濃度誘導經不同培養時間 (0、2、4、6及8 h) 後，mitf、sox10、tyrosinase、Dct、tpacap38及tpac1-r之mRNA表現量於5 nM之ovine PACAP38培養2小時後之表現量較高，並隨著誘導時間或培養濃度增加其表現量則有明顯下降之趨勢 (P  0.05) 。於劑量及時間相關中之型態分析 (morphological analysis) 與黑色素沉澱 (melanin assay) 之試驗，色素細胞及黑色素之擴散及表現量亦有類似之結果。而於cAMP含量及酪胺酸酶 (tyrosinase) 活性分析之試驗結果發現，以濃度5.0 nM之PACAP誘導體外培養吳郭魚尾鰭組織2小時後，其cAMP含量及酪胺酸酶活性皆達到最高。因此，由以上之試驗結果可說明，PACAP似參與色素細胞之色素形成，且可能透過cAMP/PKA/CREB/Mitf/Tyrosinase之路徑調控其色素生成，推測神經胜肽可能於色素細胞扮演調控者之角色。此結果除可奠定魚類色素細胞相關之研究基礎，更進一步探討其運用於哺乳類色素相關疾病研究及治療之可能性，亦有助於未來觀賞魚類產業之發展。

Chromatophores distribution in the dermis determines the pattern and which is important functions in protection against UV irradiation, camouflage, kin recognition, shoaling and sexual selection in fish. The pigmentation is affected by the expression of coloration related genes, such as tyrosinase, tyrosinase-related protein 1/2 (TRP1/2), microphthalmia-associate transcripition factor (Mitf), sox10 and dopachrome tautomerase (Dct). These genes can induce different colors and patterns in the dermis of teleosts. Tilapia (Oreochromis mossambicus) is one of the most common commercial fish in Taiwan and they have three chromatophore types - melanophores, xanthophores and erythrophore. In our previous studies, a neuropeptide - pituitary adenylate cyclase - activating polypeptide (PACAP, tpacap38) and its receptor (tpac1-r) were found expressed in the gonad and other tissues of tilapia and involved in the cAMP-protein kinase A (PKA) signaling pathway. However, literature concerning the relationship among tilapia color patterns, chromatophores and neuropeptide is still scant. Thus, in this study, semi-quantitative RT-PCR was applied to analyze the expression of mitf, sox10, tyrosinase, Dct, tpacap38 and tpac1-r in the melanophores and erythrophores of fin after in vitro culture with different concentration of neuropeptide for different periods. The dose-dependent experiment was conducted by addition of different concentrations of ovine PACAP38 (2.5, 5.0 and 10.0 nM) to fin cultured for 2 h in vitro, and a time-course experiment was conducted with ovine PACAP38 (5.0 nM) for 0 to 8 h (2, 4, 6 and 8 h). The mRNA levels of mitf, sox10, tyrosinase, Dct, tpacap38 and tpac1-r were low at 0 h and increased significantly at 2 h, and then decreased with prolonged cultured periods in both dose-dependent and time-course experiments. Same results were found in morphological analysis and melanin assay experiments. The result of cAMP and tyrosinase analyses show that cultured with 5 nM ovine PACAP38 for 2 h, the concentration of cAMP and activity of tyrosinase increased significantly in the tail fin of tilapia. These results suggested that PACAP involves in coloration of chromatophores and in the cAMP/PKA/CREB/Mitf/Tyrosinase signaling pathway. Expression of tpacap38 in the chromatophores of fin in tilapia suggested that neuropeptide may involve in coloration in the bony fish, and that could be applied in the development of ornamental fish.

目錄

封面內頁
簽名頁
中文摘要 iii
英文摘要 v
誌謝 vii
目錄 ix
圖目錄 xiv
表目錄 xvii
附錄 xviii

1. 前言 1
2. 文獻回顧 3
2.1 色素細胞 (chromatophroes) 3
2.1.1 色素細胞之簡介 3
2.1.2 黑色素細胞 (melanophore) 之發育過程 3
2.1.3 色素細胞之分子調控 4
2.1.4 幹細胞因子 (SCF) 之調控路徑 5
2.1.5 黑色素刺激激素 (MSH) 之調控路徑 6
2.1.6 色素合成之機制 7
2.1.7 環腺苷酸 (cAMP) 於色素細胞之調控關鍵 8
2.2 腦垂體腺苷酸環化酶激活肽 (PACAP) 9
2.2.1 腦垂體腺苷酸環化酶激活肽之簡介 9
2.2.2 腦垂體腺苷酸環化酶激活肽之分布 12
2.2.3 腦垂體腺苷酸環化酶激活肽於生物體之影響作用及相關研究 12
2.2.4 腦垂體腺苷酸環化酶激活肽 (PACAP) 於生理功能之調控 14
2.3 吳郭魚 (tilapia，Oreochromis mossambicus) 之簡介 15
2.4 研究目的 16
試驗架構 18
3. 材料與方法 19
3.1 試驗材料 19
3.1.1 試驗動物 19
3.2 試驗方法 19
3.2.1 雄性吳郭魚尾鰭組織之體外預培養 (pre-culture) 19
3.2.2 神經胜肽 (PACAP) 對體外培養雄性吳郭魚魚鰭色素細胞呈色之誘導分析 20
3.2.2.1 劑量相關 (dose-dependent) 之誘導試驗 20
3.2.2.2 時間相關 (time-course) 之誘導試驗 21
3.2.3 體外培養雄性吳郭魚尾鰭之Mitf、sox10、Dct、tyrosinase、tpacap38及tpac1-r mRNA表現量分析 21
3.2.3.1 吳郭魚尾鰭組織之核醣核酸 (RNA) 萃取 21
3.2.3.2 互補DNA (complementary DNA，cDNA) 之合成 22
3.2.3.3 聚合酶連鎖反應 (polymerase chain reaction，PCR) 23
3.2.3.4 洋菜膠體製備與電泳分析 (electrophoresis) 23
3.2.4 半定量 (semi-quantitation) 統計分析 24
3.2.5 雄性吳郭魚尾鰭之黑色素與紅色素細胞 (melanophore and erythrophore) 呈色量統計分析 24
3.2.6 雄性吳郭魚尾鰭之黑色素含量分析 25
3.2.6.1 雄性吳郭魚尾鰭黑色素之萃取 25
3.2.6.2 黑色素含量數據之統計分析 26
3.2.7 雄性吳郭魚尾鰭二次訊號 (cAMP) 之功能性分析 26
3.2.7.1 雄性吳郭魚尾鰭添加藥劑誘導之組織前處理 27
3.2.7.2 分析尾鰭之環磷酸腺苷 (cyclic adenine monophosphate，cAMP) 表現 27
3.2.7.3 環磷酸腺苷 (cyclic adenine monophosphate，cAMP) 之濃度統計分析 28
3.2.8 雄性吳郭魚尾鰭酪胺酸酶 (tyrosinase) 之活性分析 28
3.2.8.1 酪胺酸酶之萃取及純化 29
3.2.8.2 酪胺酸酶之定量 29
3.2.8.3 酪胺酸酶之活性測定及統計分析 30
4. 結果 31
4.1 色素細胞型態變化及呈色表現量分析 31
4.1.1 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其色素細胞型態變化及呈色表現量分析 31
4.1.2 吳郭魚尾鰭組織經定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其色素細胞型態變化及呈色表現量分析 32
4.2 色素細胞之黑色素含量分析 32
4.2.1 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其黑色素含量分析 32
4.2.2 吳郭魚尾鰭組織經定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其黑色素含量分析 33
4.3 色素細胞相關基因 (Mitf、sox10、tyrosinase、Dct、tpacap38及pac1-r) 之mRNA表現量分析 33
4.3.1 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其Mitf、sox10、tyrosinase、Dct、tpacap38及pac1-r之mRNA表現量分析 34
4.3.1.1 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其Mitf之mRNA表現量分析 34
4.3.1.2 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其sox10之mRNA表現量分析 34
4.3.1.3 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其tyrosinase之mRNA表現量分析 35
4.3.1.4 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其Dct之mRNA表現量分析 35
4.3.1.5 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其tpacap38之mRNA表現量分析 36
4.3.1.6 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其pac1-r之mRNA表現量分析 36
4.3.2 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其Mitf、sox10、tyrosinase、Dct、tpacap38及pac1-r之mRNA表現量分析 37
4.3.2.1 吳郭魚尾鰭組織經以定量之ovine PACAP38誘導不同時間後其Mitf之mRNA表現量分析 37
4.3.2.2 吳郭魚尾鰭組織經以定量之ovine PACAP38誘導不同時間後其sox10之mRNA表現量分析 37
4.3.2.3 吳郭魚尾鰭組織經以定量之ovine PACAP38誘導不同時間後其tyrosinase之mRNA表現量分析 38
4.3.2.4 吳郭魚尾鰭組織經以定量之ovine PACAP38誘導不同時間後其Dct之mRNA表現量分析 48
4.3.2.5 吳郭魚尾鰭組織經以定量之ovine PACAP38誘導不同時間後其tpacap38之mRNA表現量分析 39
4.3.2.6 吳郭魚尾鰭組織經以定量之ovine PACAP38誘導不同時間後其pac1-r之mRNA表現量分析 39
4.4 吳郭魚尾鰭組織之cAMP濃度分析 40
4.4.1 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其cAMP之濃度分析 40
4.4.2 吳郭魚尾鰭組織經定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其cAMP之濃度分析 41
4.5 吳郭魚尾鰭組織之酪胺酸酶 (tyrosinase) 活性分析 41
4.5.1 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其酪胺酸酶之活性分析 41
4.5.2 吳郭魚尾鰭組織經定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其酪胺酸酶之活性分析 42
5. 討論 43
6. 結論 47
參考文獻 72

圖目錄

圖1. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其色素細胞之型態變化 49
圖2. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其色素細胞之型態變化 50
圖3. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後，以Image J分析其色素細胞之呈色表現量 51
圖4. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後，以Image J分析其色素細胞之呈色表現量 52
圖5. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其黑色素細胞之含量 53
圖6. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其黑色素細胞之含量 54
圖7. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後，以半定量RT-PCR分析其Mitf mRNA之表現量 55
圖8. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後，以半定量RT-PCR分析其sox10 mRNA之表現量 56
圖9. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後，以半定量RT-PCR分析其tyrosinase mRNA之表現量 57
圖10. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後，以半定量RT-PCR分析其Dct mRNA之表現量 58
圖11. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後，以半定量RT-PCR分析其tpacap38 mRNA之表現量 59
圖12. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後，以半定量RT-PCR分析其tpac1-r mRNA之表現量 60
圖13. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後，以半定量RT-PCR分析其Mitf mRNA之表現量 61
圖14. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後，以半定量RT-PCR分析其sox10 mRNA之表現量 62
圖15. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後，以半定量RT-PCR分析其tyrosinase mRNA之表現量 63
圖16. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後，以半定量RT-PCR分析其Dct mRNA之表現量 64
圖17. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後，以半定量RT-PCR分析其tpacap38 mRNA之表現量 65
圖18. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後，以半定量RT-PCR分析其tpac1-r mRNA之表現量 66
圖19. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其cAMP之濃度 67
圖20. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其cAMP之濃度 68
圖21. 吳郭魚尾鰭組織經不同濃度之ovine PACAP38誘導2小時後其酪胺酸酶之活性 69
圖22. 吳郭魚尾鰭組織經以定量之ovine PACAP38 (5.0 nM) 誘導不同時間後其酪胺酸酶之活性 70

表目錄

表1. Mitf、sox10、tyrosinase、tpacap38及pac1-r應用於RT-PCR之專一性引子 71

附錄

1. 幹細胞因子 (SCF) 及黑色素刺激激素 (MSH) 於色素細胞之調控路徑 84
2. 黑色素於色素細胞之合成機制 85
3. 腦垂體腺苷酸環化酶激活肽 (PACAP) 於顆粒細胞內之調控機制 86

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