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    Title: 核醣核酸還原??與甲型前胸素原在皮膚癌化過程中所扮演的角色
    Other Titles: Biological Roles of Ribonucleotide Reductase M2 and Prothymosin Alpha during Skin Carcinogenesis
    Authors: 陳曜鴻
    Contributors: 淡江大學化學學系
    Date: 2011
    Issue Date: 2012-05-02 09:50:51 (UTC+8)
    Abstract: 本計畫是以分子生物與發育生物學的角度,探討皮膚癌化的機制與核苷酸還原酶 (ribonucleotide reductase M2, rrm2)、基因胸腺素原(prothymosin alpha, ptma)的基因、 Gli 轉錄因子、Shh 訊息傳遞途徑之關係。Shh 訊息傳遞途徑在人類與老鼠的基底細胞癌 中均有過度表現的現象。本實驗室先前發表之研究結果顯示,當過度表現Shh 訊息傳遞 途徑時,rrm2 及ptma 的基因表現均有顯著增加的趨勢。rrm2 與ptma 在正常生理功能 與病理癌化過程中皆扮演相當重要的角色,然而其參與皮膚癌化的分子機制至今仍然所 知有限。本研究欲利用斑馬魚胚胎及人類皮膚細胞探討皮膚癌化之分子機制,其研究結 果在學術上及應用上,都有助於了解皮膚癌化以及抗癌藥物治療皮膚癌的分子機制。這 些研究項目十分繁重,擬分三年來完成下列目標。 第一年計畫的目標在於建立 keratin18:rrm2:RFP 與keratin18:ptma:RFP 基因轉殖魚品 系,再利用分子生物方法驗證這些魚是否罹患皮膚癌。內容涵蓋:(1) 構築rrm2 或ptma 致癌蛋白分別與紅螢光蛋白(RFP)報導基因融合的表現質體;(2) 顯微注射法將上述 質體植入斑馬魚胚胎內;(3) 穩定遺傳品系的獲得;(4) 切片技術與組織染色;(5) 轉殖 品系的量產。 第二年計畫的目標在於選殖出 rrm2 與ptma 這兩個基因的上游調控區,並找出哪些 功能區可能是轉錄因子Gli 的結合位置。內容涵蓋:(1) keratin18:rrm2:RFP 與 keratin18:ptma:RFP 基因轉殖魚的飼育;(2) 切片技術與組織染色;(3) 抗體免疫螢光染 色實驗的建立;(4) 染色質免疫共同沉澱的操作;(5) 基因上游調控區的選殖。 第三年計畫的目標在於利用細胞轉染方式來直接證明 Shh、Gli 如何調控rrm2 與 ptma 而引起皮膚癌,甚至引發其他的癌症。內容涵蓋:(1) 細胞培養;(2) 切片技術與 組織染色;(3) 抗體免疫螢光染色實驗的建立;(4) 染色質免疫共同沉澱的操作;(5) 細 胞轉染;(6) 冷光分析儀之操作;(7) 定點突變技術的操作。
    This project aims to elucidate the mechanism of skin carcinogenesis from the perspectives of molecular and developmental biology, in particular to investigate the roles of ribonucleotide reductase m2 gene (rrm2), prothymosin alpha gene (ptma), Gli transcription factor, and Sonic hedgehog (Shh) signaling pathway involved in skin carcinogenesis. Overexpression of Shh signaling pathway has been shown in basal cell carcinoma of mice and human. A significant up-regulation of rrm2 and ptma genes upon Shh overexpression was revealed in our previous studies. Both rrm2 and ptma play important roles in physiological function and pathological carcinogenesis. However, their roles in the molecular mechanism of skin carcinogenesis are still unclear. This project thus intends to use zebrafish embryos and human skin cells to elucidate the molecular mechanism of skin carcinogenesis and provide insights to development of anti-cancer drugs. The project objectives to be accomplished in three years are as follows: The first-year objective is to establish two transgenic lines of zebrafish, Tg(keratin18:rrm2:RFP) and Tg(keratin18:ptma:RFP), and to examine if these transgenic zebrafish are associated with skin cancer by using molecular biological approaches. The specific aims include: (1) Constructing expression plasmids of fusion protein encoding red fluorescent protein (RFP) reporter gene along with either rrm2 or ptma; (2) Microinjection of above-mentioned expression plasmids into zebrafish embryos; (3) Generating stable transgenic lines; (4) Cryosectioning and immunohistological staining; (5) Large-scale production of transgenic lines. The second-year objective is to isolate the upstream regulatory region of rrm2 and ptma genes, in particular to characterize putative binding sites for Gli transcription factor. The specific aims include: (1) Maintenance and breeding of the transgenic zebrafish, Tg(keratin18:rrm2:RFP) and Tg(keratin18:ptma:RFP); (2) Cryosectioning and immunohistological staining; (3) Immunofluorescence staining; (4) Chromatin immunoprecipitation; (5) Cloning and characterization of upstream regulatory region of rrm2 and ptma genes. The third-year objective is to directly assess if Shh and Gli regulate rrm2 and ptma consequently leading to skin or other cancer by using cell transfection experiments. The specific aims include: (1) Cell culture; (2) Cryosectioning and immunohistological staining; (3) Immunofluorescence staining; (4) Chromatin immunoprecipitation; (5) Cell transfection; (6) Luciferase reporter assay; (7) Site-directed mutagenesis.
    Appears in Collections:[化學學系暨研究所] 研究報告

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