連續性造影技術監測第一型泡疹病毒胸腺嘧啶激酶基因轉導的老鼠肉瘤細胞在活體內之標的轉移

Serial in vivo imaging of the targeted migration of HSV1-tk gene transduced murine sarcoma

李建志(Jian-Chih Lee)

指導教授 ： 鄧文炳

臺北醫學大學/口腔學院/生物醫學材料研究所/碩士(2004年)

摘要

以非侵入性造影追蹤活體內的基因表現，可說是一種有效監測基因治療的方法。第一型泡疹病毒胸腺嘧啶激酶基因(HSV1-tk)被廣泛使用在基因表現之造影，原因是它不但可當作報導基因(reporter gene)；同時也可當作具有治療性的”自殺基因”(suicide gene)，應用在癌症的基因治療上。許多放射性標幟的核甘類似物(nucleoside analogue)可當作HSV1-thymidine kinase的受質探針，藉由被磷酸化而滯留在細胞內。運用核子分子造影技術，除了可依據HSV1-tk基因轉導之細胞累積受質探針的程度來反應HSV1-tk基因表現之外，也能以受質探針為指標來追蹤細胞在活體內的位置及移動。 本研究的主要目的是使用非侵入性造影方式，監測具轉移性(metast-atic)的老鼠肉瘤細胞在活體內的位置、移動及存活，來建立一個穩定的肺部腫瘤模式，以應用在癌症基因治療的研究上。我們先確定所使用的HSV1-tk基因轉導肉瘤細胞株YD-SML-TK，在體外可以選擇性地累積放射性標幟的類核甘藥物[131I]FIAU，並且在攝取[131I]FIAU後仍然可維持其存活。藉由讓YD-SML-TK細胞在體外及體內攝取[131I]FIAU的方式，我們使用SPECT進行平面伽瑪攝影(planar gamma camera imaging)來追蹤細胞在FVB/N小鼠體內的轉移。結果顯示，攝取[131I]FIAU後的YD-SML-TK仍保有其轉移至肺部的能力。在RT-PCR的分析上，也顯示出YD-SML-TK細胞在肺部形成腫瘤後還繼續進行HSV1-tk基因的表現。此外，每天從腹腔給予前驅藥物GCV治療的老鼠，從平面伽瑪攝影的影像可看出，肺部腫瘤在第7天有明顯的消退。

關鍵字

非侵入性造影 ； 基因治療 ； 第一型泡疹病毒胸腺嘧啶激酶報導基因 ； 自殺基因 ； 轉移性 ； [131I]FIAU ； GCV

並列摘要

Noninvasive imaging of gene expression is a useful method for monitoring gene therapy in vivo. Herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) is widely used as a reporter gene for imaging gene expression as well as a therapeutic “suicide gene” for cancer gene therapy. Many radiolabed nucleoside analogues are specific substrate of HSV1-TK and, upon entering the cell, can be phosphorylated by HSV-1-thymidine kinase, resulting in the intracellular retention of the radiolabeled products. Thus, accumulation of the phosporylated nucleotides would reflect HSV1-tk gene expression and can be determined by nuclear molecular imaging technologies for localizing and tracking these cells in the living subjects. In this research I have taken the experimental approach of noninvasive imaging to monitor the location, migration, and survival of metastatic murine sarcoma in living mice for the establishment of a stable lung tumor model of cancer gene therapy. First I demonstrated HSV1-tk transduced YD-SML-TK sarcoma cells incubated with [131I]FIAU in vitro could selectively accumlate this radiolabeled substrate with minimal effect on their viability. Also, YD-SML-TK cells labeled with [131I]FIAU in vitro or in vivo could be tracked in FVB/N mice by serial planar gamma camera imaging. Future more, radiolabed YD-SML-TK cells still retained their capability of metastasis to the lungs. Expression of the HSV-tk gene in YD-SML-TK cells-induced lung tumor could be demonstrated by RT-PCR analysis. Planar gamma camera images showed regression of YD-SML-TK lung tumor at day 7 of consecutive daily treatment with the prodrug ganciclovir.

並列關鍵字

Noninvasive imaging ； gene therapy ； HSV1-TK ； reporter gene ； suicide gene ； metastatic ； [131I]FIAU ； ganciclovir

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