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目的 利用人热休克蛋白基因启动子结合加热选择性诱导目的基因在肿瘤局部表达 ,建立肿瘤基因治疗新方法。方法 分离不同大小的人热休克蛋白 70基因 5’端调控顺序作为启动子 ,以绿色荧光蛋白 (GFP)作报告基因 ,构建热诱导型GFP表达质粒。转染体外培养的肿瘤细胞 ,或将稳定转导GFP表达质粒的肿瘤细胞移植到大鼠皮窗内 ,或将热诱导型GFP腺病毒注射到肿瘤内 ,加热后直接利用荧光显微镜观察GFP表达水平。结果 40 0bp人热休克蛋白 70基因 5’端调控顺序背景表达水平低 ,加热后可被显著激活 ,能作为启动子有效驱动下游目的基因的表达。GFP作报告基因使体内外肿瘤细胞内目的基因的表达水平仅通过荧光显微镜便能直接观察到。结论 利用热诱导型启动子结合加热能选择性诱导目的基因在肿瘤局部表达 ,为肿瘤基因治疗提供了一种新方法。
OBJECTIVE: To use the human heat shock protein gene promoter combined with heat to selectively induce the expression of the target gene in the tumor and establish a new method for tumor gene therapy. METHODS: Different sizes of human heat shock protein 70 gene 5’-end regulatory sequences were used as promoters, green fluorescent protein (GFP) was used as reporter gene to construct heat-inducible GFP expression plasmids. Transfection of cultured tumor cells in vitro, or transplantation of tumor cells stably transduced with the GFP expression plasmid into the rat skin window, or injection of heat-inducible GFP adenovirus into the tumor, and direct observation of GFP expression levels by fluorescence microscopy after heating . Results The background expression level of the 5’-end human heat shock protein 70 gene was low and could be activated after heating. It could be used as a promoter to effectively drive the expression of downstream target genes. GFP as a reporter gene enables the expression level of the target gene in tumor cells in vivo and in vitro to be directly observed only by a fluorescence microscope. Conclusion The heat-inducible promoter combined with heat can selectively induce the expression of the target gene in the tumor, providing a new method for tumor gene therapy.