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題 名 | 利用β-Catenin啟動子之報導基因平台評估具抗血癌活性之中草藥(2-1)=Evaluation of Anti-leukemia Activities of Chinese Herbs by β-catenin Reporter Gene (2-1) |
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作 者 | 郭育綺; | 書刊名 | 中醫藥年報 |
卷 期 | 28:7 2010.09[民99.09] |
頁 次 | 頁361-389 |
分類號 | 414.32 |
關鍵詞 | 血癌; 報導質體; LEF/TCF轉錄因子; 中草藥; β-catenin; |
語 文 | 中文(Chinese) |
中文摘要 | 研究顯示,血癌細胞中具異常活化的Wnt訊息傳導途徑(Wnt signaling p a t h w a y ) , 它 會 促 使 下 游 c o - a c t i v a t o r β - c a t e n i n 蛋 白 質 鍵 結 於 L E F / T C F transcriptional factor上並促使調控細胞生長之基因例如c-myc與cyclin D1過度 表現,進而造成細胞轉化成癌細胞(cell transformation),甚至趨使已分化的細 胞再度獲得如同幹細胞(stem cells)快速分裂與永生不滅的特性,因此若能將 Wnt訊息傳導途徑阻斷,將會使得血癌細胞生長減緩或根本由幹細胞階段移 除,以達到避免復發的優勢。近年來,國內科學研究不斷推動中草藥、小分 子藥物的發展,因此本年度計畫目的在建立β-catenin/LEF啟動子之報導基因 平台,期望未來能利用該模式從中草藥或天然物中找尋具有潛力能抑制該訊 息傳導之先導藥物。實驗以TOPFLASH質體為模板,利用PCR的方式將含有3個LEF/TCF結合序列之262bps DNA(稱之為TOP element)擴增出來,接著該 DNA片段在經過膠體純化系統純化後進行3’端A tailing的實驗並將之構築在 pGEM-T easy質體上做保存。為建立β-catenin/LEF啟動子報導基因系統,我 們將TOP element構築於pGL4報導質體上,並將之轉染至HEK293T細胞中, 藉由上述實驗設計我們將可建立一個可以用於藥物篩選之stable clone。至報 告撰寫時,我們得到下列成果:(1)我們成功以PCR的方式將TOP element擴增 出來,並構築於pGL4.3報導質體上而得到pGL4-TOP質體。(2)利用限制酵素 圖譜構築也確認該質體確實包含有LEF/TCF DNA片段。(3)目前我們正在進行 pGL4-TOP質體之轉染,利用Wnt 3a作為刺激物來觀察該質體在細胞中是否具 有功能性,以作為下年度藥物篩藥之重要工具。 |
英文摘要 | Aberrant activation of the Wnt signaling pathway in leukemia has been shown to promote downstream co-activator β-catenin that bind to LEF/TCF transcription factor. LEF/TCF was known to upregulate the cell cycle gene, c-myc and cyclin D1. Activation of this pathway has been considered as a crucial signaling for cancer cells transformation, even let the committed progenitors to gain self-renewal ability like stem cell. The blockade of Wnt signaling pathway may inhibit the growth of leukemic cell or eliminate the cancer cells at stem cell level (cancer stem cell). This strategy may prevent a relapse. Recently, the improvement of studies of Chinese herbs and small molecule drugs in Taiwan is set into action constantly. So, the aim of this study is to establish the β-catenin/LEF reporter model to screen the leader compounds from Chinese herbs that probably blocking the Wnt and β-catenin signaling pathways. The TOPFLASH plasmid was used to amplify the TOP element that contains 3 LEF/TCF binding sequence by PCR. After purification by gel extraction system, the adenine was added to the 3’ end of TOP element in order to preserve in pGEM-T plasmid by TA cloning. The TOP element was also constructed into pGL4.3 reporter plasmid and transfected into HEK293 cells so as to establish the β-catenin/LEF reporter plasmid. By the above experiment design, we will establish the stable clone that will used to screen the drugs. In this study, we got the following results: (1)We successfully amplify the TOP element by PCR and construct into the pGL4.3 reporter plasmid, called pGL4-TOP. (2)The accuracy of this plasmid is confirmed by restriction map. (3)Presently, the function of this plasmid in cells is check by the stimulation of Wnt3a. This model will be an important tool for anti-leukemia drug screening. |
本系統中英文摘要資訊取自各篇刊載內容。