利用 beta-Catenin 啟動子之報導基因平台評估具抗血癌活性

郭育綺
輔仁大學
根據衛生署近十年來的統計資料顯示，惡性腫瘤為台灣十大死因之首位，細分這些癌症可發現肺癌、肝癌、結腸直腸癌、女性乳癌、胃癌、口腔癌等皆為實體腫瘤 (solid tumor)，因此可藉由手術來切除與合併化學治療以清除癌細胞。然而，血癌 (leukemia) 屬於遊走全身之血液疾病，只能藉由副作用高的化學或放射線療法為治療策略，於療效不佳的情況下往往造成復發的現象，所以研發更有效且專一性高的抗血癌藥物極為重要。根據研究報導顯示，血癌細胞中具異常活化的 Wnt訊息傳導途徑 (Wnt signaling pathway)，它會促使下游之傳訊因子 (transcription factor) b-catenin 鍵結於 LEF/TCF 啟動子 (LEF/TCF promoter ) 上，促使調控細胞生長之基因表現例如 c-myc 與 cyclins 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。因此本計畫最終目標為建立一個 b-catenin 啟動子之報導基因平台，利用該模式篩選出具有抗血癌的先導藥物，並評估該藥物體外與體內之藥理活性。整體研究計畫將分三年進行，第一年計畫將以建立「b-catenin 啟動子之報導基因平台」為重點，實驗內容包括：(1)將 Wnt signaling 所主導之傳訊因子β-catenin，其 binding promoter 稱之為 LEF/TCF promoter，構築於 pGL4 質體上 (稱之為 pGL4-TOP)；(2)將 pGL4-TOP 質體送入HEK293T 細胞，且利用Hygromycin B 篩選出穩定細胞株 (稱之為 HEK293-TOPrep)；(3)利用 HEK293-TOPrep進行中草藥抽出物或化合物初篩；(4)以 Jurkat 血癌細胞株與血癌病患周邊血細胞進行體外活性確認。第二年計畫將以篩選出的有效成分進行體內藥理活性分析，實驗內容包括：(1)利用血癌 Jurkat 細胞注射入 NOD/SCID 小鼠建立類似血癌之動物模式；(2)進行有效成分一週之連續餵食；(3)小鼠犧牲後採取血清進行肝功能與腎功能檢測；(4)分離小鼠周邊血淋巴細胞進行血球外型特性觀察、細胞週期分析與 β-catenin 活性偵測。第三年計畫將進行有效成分藥理活性機轉評估，實驗內容包括：(1)將 Jurkat 細胞株處理以有效成分，利用 Colony forming assay觀察細胞處理藥物後增生與轉化的情況；(2)以流式細胞技術偵測 Jurkat 細胞株細胞週期與細胞凋亡現象，是否受到有效成分影響；(3)以 RT-PCR 分析 Wnt signaling 與 b-catenin 下游相關基因是否受到抑制或活化；(4) 以 Western blotting、EMSA、Immunoprecipation 等方法探討 Wnt 訊息傳導過程中相關蛋白質磷酸化、表現量、蛋白質之間交互作，以說明其作用機轉。藉由本計畫體外到體內的實驗執行，期盼能從中草藥中找尋出具發展潛力的抗血癌藥物或輔佐療法，進而提升血癌的治療效果與病患的生活品質。
關鍵字：血癌；Wnt 訊息傳導途徑；β-catenin 啟動子；報導基因；中草藥

Evaluation of anti-leukemia activities of Chinese herbs by -catenin reporter gene

Yuh-Chi Kuo
Fu Jen Catholic University
Over the past decade, data from the Department Of Health shows that the top one in ten highest mortality of diseases in Taiwan is the malignant carcinoma. After further classification, carcinomas such as lung cancers, hepatoma, colorectal carcinoma, gastric cancers, breast cancers, cervical cancers, oral cancers, prostate cancers, and pancreatic cancers are so-called "solid tumors". It is possible to eliminate these carcinomas by surgical operation and combination with chemotherapy. However, leukemia belongs to the blood diseases, both chemotherapy and radiotherapy are the first and only choice to clean the cancer cells. Relapsing is also frequently observed in leukemia patients. So, it is important to develop new anti-leukemia drugs with higher activities and specificity. Aberrant activation of the Wnt signaling pathway and downstream transcription factor b-catenin binding to its promoter LEF/TCF which upregulate the cell cycle gene, c-myc and cyclins, has been reported in leukemia cells and considered as a crucial signaling for cancer cells transformation, even let the committed progenitors to gain self-renewal ability like stem cell. The aim of this study is to establish the LEF/TCF reporter model to screen the leader compounds from Chinese herbs that probably blocking the Wnt and b-catenin signaling pathway, then evaluate the drug’s activity in vivo and action mechanism of drugs in vitro. We designed 3 years proposal.
關鍵字：Leukemia；Wnt signaling pathway；β-catenin promoter；reporter gene；Chinese herbs