2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656766摘要：惡性黑色素細胞瘤是台灣最致命的皮膚癌症。在西方國家，百分之62.6 的病人是第零或第一期，百分之23.1 是第二期，相反地，台灣的病人當中，只有百分之40是第一或二期。淺型黑色素細胞瘤(深度小於1mm)有很好的預後，5 年存活率超過九成，相反的深型黑色素細胞瘤(深度大於4mm 併有潰瘍)則有很高的風險與相對較差的預後。但是現在已經清楚，仍然有有一小群淺型黑色素細胞瘤的病人，發生惡性腫瘤轉移，而有一群深型黑色素細胞瘤的病人反而不會有轉移，現在仍無法回答為何有人黑色素細胞瘤會在很早期就轉移為了更精確地預測預後，免疫組織染色與微陣列分析已經被應用，但仍有其限制，包含背景噪音與個體間差異。因此我們要應用以飛蛾跳躍子為基礎之突變形成，來篩選黑色素細胞瘤之侵犯與轉移基因，這是一個以表現型為基礎的高通量、全基因體正向遺傳式篩選。藉由插入單套的使活化或使失去功能跳躍子，我們可以隨機每次操縱一個基因，其中，使活去的跳躍子帶有一全能啟動子而可活化插入的基因，使失去功能跳躍子則帶有poly-A 序列而可以使基因失能。高通量的侵犯篩選模式為體外Transwell，惡性轉移模式則為小鼠尾靜脈注射之體內實驗。另外，藉著跳躍子可以跳出的特性，我們可以進一步去探究基因與侵犯轉移之關係。由於目前台灣沒有太多轉移黑色素細胞瘤的微陣列資料，我們也將收集成對之原始與轉移黑色素色細胞瘤作微陣列分析，與跳躍子突變形成平台的結果作結合，找出協助腫瘤侵犯與轉移的基因，其參與之主要訊息路徑也將獲得探究。研究目標:目標一: 建立B16黑色素細胞瘤之使活化及使失去功能的突變形成庫目標二: 以高通量的方法篩選促進侵犯與轉移的基因目標三: 對發現的基因作驗證與功能 描述目標四: 評估發現基因的臨床意義<br> Abstract: Malignant melanoma is the leading cause of death among skin cancers in Taiwan. Inwestern countries, the majority of patients who present with melanoma have early stagedisease, with 62.6% presenting with stage 0 or I disease, and an additional 23.1% with stageII disease. Conversely, only 40% are in stage I or II in Taiwan. Thin melanomas (<1 mm)have an excellent prognosis with greater than 90% survival at 5 years; in contrast, thickmelanomas (>4 mm with ulceration) are associated with high risk for metastasis and arelatively poor prognosis. However, it has become clear that a minority of patients with thinmelanomas will succumb to metastatic disease, whereas a subset of patients with thickulcerated primary lesions never recur. The question why some melanomas metastasize inearly stage remains unanswered.In an effort to better predict prognosis for melanomas, immunohistochemical staining,and microarray-based technology have been applied. However, there are several limitationsof these assessments, including the background noise and inter-individual variability. Thus,we will use the piggyBac transposon-mediated mutagenesis to screen genes facilitatinginvasion and metastasis. It is a phenotype-driven genome-wide forward genetic screen in ahigh throughput manner. Single copy insertion of gain-of-function or loss-of-functiontransposon allows us to randomly manipulate a gene at once. The gain-of-functiontransposon contains a universal promoter for overexpression of the trapped gene. Theloss-of-function transposon contains poly-A sequence for disabling the trapped gene. Thehigh-throughput screen includes in vitro Transwell as the model for invasion and in vivo tailvein injection as the model of metastasis. With the advantage of reversal capability intransposon-transposase system, the mechanism of how the target gene leads to the phenotypecan be next to be explored.Due to the lack of available microarray data of metastatic melanoma in Taiwan, localpatients with paired primary and metastatic melanoma will be collected for microarrayanalysis. In combination of results from insertional mutagenesis, the genes facilitatinginvasion and metastasis will be discovered. The involved canonical signal pathway will alsobe explored.SPECIFIC AIMS:AIM 1: Generation of gain-of-function and loss-of-function insertional mutagenesis libraryof B16 melanoma cellsAIM 2: Forward genetic screen for genes facilitating melanoma invasion and metastasis in ahigh-throughput manner.AIM 3: Validation and functional characterization of the discovered genesAIM4: Assessment of the clinical significance for discovered genes