定量超声基因导入与效应控制

目的研究低频超声裸基因导入新方法与基因导入效应的控制,为疾病的基因治疗提供安全可靠、无细胞毒性的导入新方法。方法用不同参数的35.1kHz超声进行裸基因导入细胞实验,用激光共聚焦显微镜、荧光显微镜、流式细胞仪和分光光度法分析细胞膜形态、细胞膜通透性与损伤阈值、细胞存活率和基因表达。结果实验所见90%细胞存活点的超声能量积分是细胞膜通透安全控制参数,80%细胞存活点的能量积分是细胞的损伤阈值。绿色荧光蛋白(GFP)在安全超声参数下成功导入了S180细胞,细胞转染率为35.83%±2.53%(n=6),荧光表达强度明显高于病毒转染法和控制组(P<0.001)。结论安全参数下低频超声可有效地将裸基因导入S180肿瘤细胞,其基因表达强度随超声能量积累而增强,并有细胞凋亡倾向。

Quantitative Ultrasonic Naked Gene Delivery and the Effect Control

Objective: To study ultrasonic naked gene delivery and its effect control, and to investigate the new methods, which are easy-to-use, reliable and non-cytotoxic for application in gene therapy so as to build an optimal parameter set. Method: Suspensions of different kinds of cells were exposed to calibrated ultrasound field (35.1 kHz) with different parameters for gene delivery. Morphology, membrane permeability, naked gene expression efficiency, threshold of cell damage and cell viability were examined by laser scanning confocal microscopy, fluorescent microscopy, flow cytometry and spectrophotometer. Result: The plasmid of green fluorescent protein (GFP) was delivered into S180 cells under optimal parameters without cell damage. The transfection rate was about 35.83% +/- 2.53% (n=6) in viable cells. The GFP expression intensity in ultrasound exposure group had a higher fluorescent peak than AVV-GFP group and control group (P<0.001). Conclusion: Ultrasound can effectively deliver naked gene into cells using optimal parameters without cell damage. The gene uptake depends on energy accumulation at 90% of cell viability, and the tendency of apoptosis is found in S180 tumor cells.