在分子基因影像上應用於轉譯醫學之最理想第一型單純泡疹病毒胸腺嘧啶激脢/綠色螢光蛋白質融合報導基因之研發

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题名:	在分子基因影像上應用於轉譯醫學之最理想第一型單純泡疹病毒胸腺嘧啶激脢/綠色螢光蛋白質融合報導基因之研發
其它题名:	Development of an Optimal TKGFP Fusion Reporter Gene for Use in Translation Medicine in Molecular-Genetic Imaging
作者:	謝佳宏(Chia-Hung Hsieh)
贡献者:	醫學院基礎醫學研究所;中國附醫醫學研究部共同實驗室
日期:	2009-07-31
上传时间:	2009-09-01 15:55:04 (UTC+8)
摘要:	在轉譯醫學方面，多重影像整合方法並結合報導基因的使用對於用來偵測分子治療中體內內生性的訊息傳遞活性或是轉殖基因的表現扮演不可或缺的角色。透過一融合基因結合兩個不同的影像技術(例如:正子斷層影像和光學影像)其優點能快速且容易在細胞或小型動物上確認基因的表現並依序轉譯到人類上。此外，此方法也可以克服每一個照影方法的缺點及限制。為了符合如此的目的，第一型單純皰疹病毒胸腺嘧啶激酶/綠色螢光蛋白質(TKGFP)雙向報導基因就此產生。此雙向報導基因結合光學影像和核子影像一直為常用的方法用來監測內生性的生物過程或轉殖基因的表現。儘管TKGFP 是被廣泛使用在分子基因影像的報導基因，但尚有一些未知的問題和潛在性應用於轉譯醫學的問題: (1).TKGFP 融合蛋白似乎在哺乳動物細胞內是很穩定的，因此可能有較低的時間解析度對於監測動態的內生性訊息傳導活性或轉殖基因的表現， (2). 細胞毒性和低總細胞內的TK 酵素活性限制了它在轉譯醫學方面的應用。因此，需要進一步的研究去更深入探討這些未知的問題和克服這些潛在性應用在轉譯醫學的問題。本研究計畫中，我們計劃去評估TKGFP 雙向報導基因系統應用於轉錄基因調節上動態的研究以及產生新的TKGFP 報導基因其有較高的時間解析度及較低的細胞毒性可以立即監測時間上的動態變化及空間異質性對於內生性訊息傳導活性或轉殖基因的表現。此計畫中，我們擬定三個重要研究方向來達到此研究目標。在研究方向一，我們提議評估在體外和體內實驗下TKGFP 為一報導基因的細胞毒性，蛋白質降解以及時間解析度。我們希望藉此能回答一些關於TKGFP 為一報導基因在分子基因影像方面未定論的問題。雖然關於TKGFP 當作報導基因的細胞毒性和時間解析度這方面的詳細資訊還不是完全清楚，早期的研究以及我們的初步研究結果顯示出TKGFP 當作報導基因可能在監測基因表現方面有較高的細胞毒性和低的時間解析度。在研究方向二，我們企圖創造新型的突變TKGFP 融合基因其具備低細胞毒性以及高時間解析度，如此的報導基因可用來即時監測內生性的生物過程或轉殖基因的表現。藉由此研究，我們企圖研發出最理想的TKGFP 融合基因來當作報導基因以適合應用在轉譯醫學研究上。在研究方向三，我們計畫應用此研發的TKGFP 報導基因用來監測體內動態的內生性訊息傳導活性變化。我們所設計實驗會專注在體外和體內的研究用來測試是否我們研發的TKGFP 融合基因當作報導基因有足夠的時間解析度可以用來即時監測在腫瘤缺氧和復氧狀態下所導致動態的HIF-1 訊息傳導活性變化及它表現的空間異質性。我們期望在3 年研究時間內從我們的研究衍生出的資訊可以更深入瞭解關於在分子基因影像上TKGFP 當作報導基因的一些問題，並且研發出應用於轉譯醫學上之最理想TKGFP 融合報導基因。 In the translation medicine, a multimodality imaging approach together with using reporter genes plays an important role for detecting endogenous signal transduction activity or transgene expression for molecular therapy. Combining two different technologies (e.g., PET with optical) through a fusion gene would have the advantage of speed and ease of validating approaches in cells or small animals that in turn can be translated to humans. Furthermore, this approach can also overcome the shortcomings or limitations of each imaging modality. For such purpose, the HSV1-tk/GFP (TKGFP) dual reporter gene was created. This dual reporter gene combined with optical imaging and nuclear imaging modalities has been utilized for monitoring endogenous biological processes or transgene expression. Despite the TKGFP is widely used reporter in the molecular-genetic imaging, there are several unaddressed issues and potential problems associated with its applications in the translation medicine: (1) TKGFP fusion protein seems to be highly stable in mammalian cells and therefore, may has low temporal resolution for monitoring temporal dynamics of endogenous signal transduction activity or transgene expression , (2) The cytototoxicity and low total cellular TK enzymatic activity trigger by nuclear tropism of HSV1-TK limited in its applications in translation medicine. Therefore, it needs further studies to give an insight into these issues and overcome these potential problems of TKGFP as a reporter for use in the translation medicine. In this project, we are aim at evaluating the application of TKGFP dual reporter system in dynamic studies of transcriptional gene regulation and creating novel mutant forms of TKGFP reporter genes with high temporal resolution and low cytototoxicity for real-time monitoring the temporal dynamics and spatial heterogeneity of endogenous signal transduction activity or transgene expression. Three specific aims are involved in this project to achieve these study goals. In the specific aim 1, we propose to evaluate the cytototoxicity, protein degradation and temporal resolution of TKGFP as a reporter in vitro and in vivo. We hope to address some issues regarding TKGFP as a reporter in the molecular-genetic imaging. Although the detail information about the cytotoxicity and temporal resolution of TKGFP as reporter is not fully clear, the earlier studies and our preliminary results indicate TKGFP as a reporter may be high cytotoxicity and low temporal resolution for monitoring dynamic gene expression. In the specific aim 2, we attempt to create new mutant forms of TKGFP fusion gene with low cytototoxicity and high temporal resolution for real-time monitoring temporal dynamics of gene expression evens or signal transduction activities. By such studies, we attempt to develop an optimal TKGFP fusion gene as a reporter for the applications in the translation medicine. In the specific aim 3, we propose to apply our developed TKGFP reporters in monitoring temporal dynamics of endogenous signal transduction activity. Our design experiments will focus on in vitro and in vivo investigating whether our developed TKGFP fusion genes as reporters have enough temporal resolution for real-time monitoring the temporal dynamics and spatial heterogeneity of HIF-1 signal transduction activity in tumor hypoxia and reoxygenation. We expect that the information derived from our research could address some issues regarding TKGFP as a reporter in the molecular-genetic imaging and develop an optimal TKGFP fusion reporter for use in the translation medicine within the period of three years research.
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