開發桿狀病毒做為食道癌基因光動力療法之研究

Development of Baculovirus Based-gene Therapy for Esophageal Cancer

詹燕茹(YEN-JU CHAN)

指導教授 ： 吳宗遠

中原大學/理學院/奈米科技碩士學位學程/碩士(2011年)

https://doi.org/10.6840/cycu201100773

摘要

中文摘要
利用桿狀病毒進行癌症基因療法是桿狀病毒表現系統的新熱門領域之一。本研究將嘗試以桿狀病毒雙效表現載體共同表現於光敏基因 KillerRed 與抑癌基因 p53，以結合癌症的基因療法與光動力療法（Photodynamic Therapy；PDT）。
首先，我們以人類巨噬細胞病毒 CMV 啟動子與 RhPV IRES、EV71 IRES、EV71 IRES(3′△100)‧RP110 等 IRES 序列。建構帶有 KillerRed 基因與 EGFP 基因之桿狀病毒雙效表現載體，並利用此載體生產重組病毒vAcCMV-KillerRed-Rhir-EGFP、vAcCMV-KillerRed-EV71 IRES-EGFP、vAcCMV-KillerRed-EV71 IRES (3′△100)‧RP110-EGFP ，轉導於食道癌細胞（CE48T）後經可見光照射，可導致約 50 % 食道癌細胞死亡。接著我們也將腫瘤抑癌基因 p53 與 KillerRed 共構築於表現載體中，發現當此重組病毒轉導於食道癌細胞株時，其細胞在未照光之前有死亡現象，照光後所導致死亡率亦為 50 % 。由於KillerRed 受 CMV 調控時，會造成在轉導後表現於 KillerRed 蛋白而導致照光後細胞及死亡，我們可將 KillerRed 基因構築於由 IRES 所調控的轉譯起始之後（Translation initiation），再由金剛烷胺（amantadine）調控 IRES的轉譯起始活性，可於轉染細胞時，調控 KillerRed 的表現，以期更能精準的掌握較佳之表現時機。因此，我們期望在未來能發展以桿狀病毒表現系統為基礎之光動力療法。

關鍵字

光動力治療法 ； 桿狀病毒 ； 活性氧自由基 ； 細胞凋亡 ； 金鋼烷胺 ； 抑癌基因p53 ； 光敏基因KillerRed ； 線粒體

並列摘要

Abstract
The baculovirus-mammalian gene expression vector system is a widely used and an important molecular biology tool. This is the first report on a novel esophageal cancer gene therapy based on photodynamic baculovirus-based model. Phosensitizers are small molecular photosensitive proteins used for photodynamic therapy（PDT）. In 2005, a light sensitive gene, KillerRed was discovered from Antomedusae chromoproteinam 2CP. The Killer Red protein has an excitation wavelength of around 540-580 nm that can produce reactive oxygen species（ROS）and induce cell death. In this study, the baculovirus-based bicistronic vectors（vCMV-KillerRed-EV71 IRES-EGFP, vCMV-EGFP -EV71 IRES-KillerRed ）were generated and were transduced to esophageal cancer cell lines （CE48T） to develop a vehicle for esophageal cancer gene therapy. Upon transduction, the photodynamic red fluorescent protein was irradiated by visible light to activate the KillerRed protein so as to induce cytotoxicity that is controlled by CMV promoter and EV71 IRES in vCMV-KillerRed-EV71 IRES-EGFP, vCMV-EGFP-EV71 IRES-KillerRed respectively. Furthermore, amantadine hydrochloride was used as an IRES regulatory compound to evaluate the activity of the CMV- EGFP-EV71 IRES-KillerRed on the esophageal cancer cell line. Hrer, if the amantadine is washed away, the system favors IRES dependent translation thus the cells appear worescent and the KillerRed gene will be translated resulting to toxicity on cells. Lastly, the study could be a promising tool for the development of a novel esophageal cancer gene therapy based on photodynamic model using a baculovirus expression system.

並列關鍵字

p53 gene ； amantadine ； Photodynamic Therapy ； Baculovirus ； KillerRed ； Apoptosis ； reactive oxygen species ； mitochondrial

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