臺灣博碩士論文加值系統

Sonic hedgehog (shh) 是Hedgehog 蛋白質家族的一員，在動物的胚胎發育與皮膚組織的形成具有關鍵性的角色。為了了解shh在皮膚癌發生時所扮演的角色，我們以斑馬魚當作我們的模式物種，以皮膚組織特定的驅動子(Keratin18,K18)及(Keratin5,K5)來使shh過度表現，並接以紅螢光的報導基因(Red Fluorescent Protein,RFP)，以此建立K18-shh-RFP及K5-shh-RFP的紅螢光品系，用來觀察皮膚細胞的癌化過程。受精後3天大的K18-shh-RFP品系的斑馬魚與野生種斑馬魚在外觀上並無顯著差異，然而到了5天大時K18-shh-RFP品系的斑馬魚在頭部、眼睛、胸鰭的基部以及軀幹的皮膚發生隆起的現象。由冷凍切片與蘇木精與伊紅的染色法(Hematoxylin and Eosin stain) 的結果，發現這些隆起的皮膚組織的細胞核具有不規則的形狀、細胞生長的排列方式也不規則、細胞的數量也有變多的趨勢，證明了這些組織屬於惡化的皮膚癌細胞。另外，我們以K18-shh-RFP質體注射入Fli-1品系，結果發現，惡化的皮膚癌細胞處，有血管增生的現象。為了進一步研究shh造成皮膚癌化的分子機轉我們使用可作為癌化標記的抗體(cyclin D1、PCNA、MYC及Ki-67)證明，在K18-shh-RFP品系斑馬魚頭部的變異部位，有較強的cyclin D1、PCNA、MYC及Ki-67表現。這些結果顯示，過量表現SHH蛋白質會誘導cyclin D1、PCNA、MYC及Ki-67的表現最後導致癌化。而在K5-shh-RFP品系中則可以發現到頭部及胸鰭的變異情形，並影響其存活比例，在經過了cyclopamine及triparanol控制後，則可抑制變異情形及提高存活率。由以上實驗證明K18-shh-RFP及K5-shh-RFP這兩種品系，可作為研究皮膚癌及篩選治療藥物之新模式物種。

Constitutive expression of sonic hedgehog (shh) signaling pathway are pivotal to development of basal cell carcinomas (BCC). The study of BCC gene expression not only may elucidate mechanisms by which aberrant tumor behavior were produced but also can provide data on in vivo shh target gene control. To examine in vivo the effect of excess shh signaling, we generated the transgenic line Tg(k18:shh:RFP) and (k5:shh:RFP) that over-express SHH restrictively in the epidermis. We fused keratin 18 (k18) and keratin5(k5) promoter with SHH coding region and in frame with a red fluorescent protein (RFP) reporter. By 5 days-post-fertilization (dpf), many epidermal lesions were easily observed, including a swollen yolk sac, epidermolysis bullosa around the eyes and at the basement of the pectoral fins. Skin histology revealed that embryos derived from Tg(k18:shh:RFP) displayed highly Nuclear/Cytoplasmic (N/C) ratio and pleomorphic nuclears compared to their wild type littermates, indicating that the epidermal lesions on Tg(k18:shh:RFP) were dysplasia. Moreover, we stained the embryos with various monoclonal antibodies to further analyze the signaling pathways underlying epidermolysis bullosa. Our data revealed that over-express SHH restrictively in the epidermis of Tg(k18:shh:RFP) led to up regulation the amount of PCNA-, Cyclin D1-, c-myc ,and Ki-67-positive cells. On the basis of these findings, we suggest that shh signaling promotes cell cycle progression and consequently causes epidermis dysplasia of the zebrafish embryos. We believe that Tg(k18:shh:RFP) and (k5:shh:RFP) fish should be an excellent experimental animal for analyzing the molecular mechanisms of BCC, and should be a good material to develop anti-cancer new drugs.

中文摘要--------------------------------------------------I
英文摘要-------------------------------------------------II
目 錄------------------------------------------------III
圖表目錄--------------------------------------------------V
第一章 前言-----------------------------------------------1
第二章 材料與方法----------------------------------------10
第一節 建立在皮膚過度表現shh的斑馬魚品系---------------10
2.1.1 斑馬魚飼養及胚胎的收集-----------------------------10
2.1.2 小量質體DNA抽取------------------------------------10
2.1.3 顯微注射實驗---------------------------------------11
2.1.4 建立K18-shh-RFP品系之基因轉殖斑馬魚----------------11
2.1.5 阿爾襄藍軟骨染色(Alcain Blue staining)-------------12
第二節 shh的過度表現造成頭部及皮膚的變異-----------------13
2.2.1 抗體染色方法---------------------------------------13
2.2.2 冷凍切片前處理-------------------------------------16
2.2.3 冷凍切片方法---------------------------------------16
2.2.4 Oil red O 染色方法---------------------------------17
2.2.5 西方墨漬法-----------------------------------------17
第三節 頭部及皮膚的變異是由於細胞不正常增生--------------20
2.3.1 蘇木紫與伊紅染色(H&E staining)---------------------20
第三章 結果----------------------------------------------21
第一節 建立在皮膚過度表現shh的斑馬魚品系-----------------21
第二節 shh的過度表現造成頭部及皮膚的變異---------------- 23
第三節 頭部及皮膚的變異是由於細胞不正常增生--------------26
第四節 變異部位細胞的不正常增生之分子機轉探討------------27
第五節 變異部位細胞的不正常增生伴隨著血管增生------------30
第六節 預防及治療之藥物篩選------------------------------31
第四章 討論----------------------------------------------34
第一節 過度表現shh訊息造成胚胎死亡-----------------------34
第二節 斑馬魚與鼠科動物發生基底細胞癌之比較--------------35
第三節 與人類之基底細胞癌之比較--------------------------36
第四節 膽固醇與shh訊息路徑-------------------------------37
參考文獻-------------------------------------------------39
圖 表-------------------------------------------------44
附 錄-------------------------------------------------62

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