建構膜蛋白形式的報告基因用以進行非侵犯性照影在活體內偵測轉殖基因的表現

Construction of membrane reporter gene for noninvasive imaging of gene expression in vivo

林志鴻(Jhih-Hong Lin)

指導教授 ： 鄭添祿

高雄醫學大學/生命科學院/醫藥暨應用化學研究所/碩士(2005年)

摘要

進行基因治療時最重要的目標是將治療基因表現在特定的組織或器官。於是研究者們發展出報告基因來觀察治療基因的表現位置。本篇論文的內容就是發展一個新的報告基因進行非侵犯性的活體內照影來達到這個目的。我們建構膜蛋白形式的老鼠葡萄糖醛酸酵素(Mem-βGlu)作為報告基因。造影劑(fluorescein di-β-D-glucuronide, FDGlcU)會被葡萄糖醛酸酵素活性催化，再利用合適的照影儀器照出報告基因的表現位置。再藉由反轉錄病毒讓Mem-βGlu基因被穩定表現在CT26細胞中。以流式細胞儀確認Mem-βGlu酵素能被表現於細胞膜上。並證實Mem-βGlu酵素仍保有活性且能催化造影劑。接下來從植入穩定表現Mem-βGlu的腫瘤團塊老鼠尾巴靜脈注射入造影劑以進行活體照影。短時間內就能在表現Mem-βGlu的腫瘤團塊處偵測到相當明顯的螢光反應，而不表現Mem-βGlu的腫瘤團塊處則沒有螢光反應。以攜帶Mem-βGlu基因的腺病毒感染HCC36細胞的活體外及活體內實驗也都有類似的結果。由這些實驗結果我們可以瞭解以膜蛋白形式的葡萄糖醛酸酵素作為報告基因的非侵犯性造影系統是可行的。這套報告基因系統將能用來測試各種基因治療載體傳輸基因或治療基因表現時的組織特異性。使未來基因治療的相關研究更加方便。

關鍵字

膜蛋白 ； 報告基因 ； 非侵犯性照影 ； 轉殖基因 ； 葡萄糖醛酸酵素

並列摘要

The most important target of gene therapy is to express the therapeutic genes in specific tissues or organs. Hence the researchers developed reporter genes to monitor the expression locations of therapeutic genes. This paper relates developing a novel reporter gene to proceed in vivo noninvasive imaging for the same purpose. We had constructed membrane form mouse β-glucuronidase(Mem-βGlu) as a reporter gene. The imaging probe (fluorescein di-β-D-glucuronide, FDGlcU) will be catalyzed by β-glucuronidase enzyme activity, and then we can employ suitable imaging apparatus to monitor the expression site of this reporter gene. The Mem-βGlu gene is persistently expressed in CT26 cells by retrovirus infection. We confirmed that the Mem-βGlu enzyme is expressed on the cell membrane by flow cytometry. And we proved that the Mem-βGlu enzyme retains activity to catalyze imaging probe. After that in vivo imaging was exercised in Mem-βGlu expressed tumor implanted mouse with i.v. injection of imaging probes. Significant fluorescence is detected at the location of Mem-βGlu expressed tumor in short time but not the site of control tumor. Similar results were also shown in in vitro and in vivo experiments of HCC36 cells infected with adenovirus carrying Mem-βGlu gene. According to these results, we knew the noninvasive imaging system based on Mem-βGlu enzyme is workable. This reporter gene system could be used to test the tissue specificity of therapeutic genes' expression or transmitting genes by kinds of gene therapy vectors. It will enable the related researches of gene therapy to be more convenient in future.

並列關鍵字

reporter gene ； membrane protein ； noninvasive imaging ； transgenic gene ； glucuronidase

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