Guilhon E, Voisin P, de Zwart J A, Quesson B, Salomir R, Maurange C, Bouchaud V, Smirnov P, de Verneuil H, Vekris A, Canioni P, Moonen C T W
Résonance Magnétique des Systèmes Biologiques, UMR 5536 CNRS/Victor Segalen Université Victor Segalen Bordeaux 2, Bordeaux, France.
J Gene Med. 2003 Apr;5(4):333-42. doi: 10.1002/jgm.345.
Among the techniques used to induce and control gene expression, a non-invasive, physical approach based on local heat in combination with a heat-sensitive promoter represents a promising alternative but requires accurate temperature control in vivo. MRI-guided focused ultrasound (MRI-FUS) with real-time feedback control allows automatic execution of a predefined temperature-time trajectory. The purpose of this study was to demonstrate temporal and spatial control of transgene expression based on a well-defined local hyperthermia generated by MRI-FUS.
Expression of the green fluorescent protein (GFP) marker gene was used. Two cell lines were derived from C6 glioma cells. The GFP expression of the first one is under the control of the CMV promoter, whereas it is under the control of the HSP70 promoter in the second one and thus inducible by heat. Subcutaneous tumours were generated by injection in immuno-deficient mice and rats. Tumours were subjected to temperatures varying from 42 to 50 degrees C for 3 to 25 min controlled by MRI-FUS and analyzed 24 h after the heat-shock. Endogenous HSP70 expression and C6 cell distribution were also analyzed.
The results demonstrate strong expression at 50 degrees C applied during a short time period (3 min) without affecting cell viability. Induced expression was also clearly shown for temperature in the range 44-48 degrees C but not at 42 degrees C.
Heating with MRI-FUS allows a tight and non-invasive control of transgene expression in a tumour.
在用于诱导和控制基因表达的技术中，基于局部加热与热敏启动子相结合的非侵入性物理方法是一种很有前景的替代方法，但需要在体内进行精确的温度控制。具有实时反馈控制的磁共振成像引导聚焦超声（MRI-FUS）允许自动执行预定义的温度-时间轨迹。本研究的目的是证明基于MRI-FUS产生的明确局部热疗对转基因表达的时空控制。
使用绿色荧光蛋白（GFP）标记基因的表达。两种细胞系源自C6胶质瘤细胞。第一种细胞系中GFP的表达受巨细胞病毒（CMV）启动子控制，而在第二种细胞系中受热休克蛋白70（HSP70）启动子控制，因此可受热诱导。通过将细胞注射到免疫缺陷小鼠和大鼠体内产生皮下肿瘤。通过MRI-FUS将肿瘤加热到42至50摄氏度，持续3至25分钟，并在热休克后24小时进行分析。还分析了内源性HSP70的表达和C6细胞分布。
结果表明，在短时间（3分钟）内施加50摄氏度的温度可使GFP强烈表达，且不影响细胞活力。在44-48摄氏度范围内也明显显示出诱导表达，但在42摄氏度时未观察到。
MRI-FUS加热可对肿瘤中的转基因表达进行严格的非侵入性控制。
