超声联合聚乙烯亚胺增强MCF-7癌细胞基因表达的转染参数优化

The optimization of parameters on DNA transfection in MCF-7 cancer cells combining ultrasound with polyethyleneimine

目的探讨超声辐照并超声造影剂联合聚乙烯亚胺（PEI）增强MCF-7乳腺癌细胞质粒DNA转染的最优条件及协同作用。方法制备PEI／荧光素酶质粒（pCMV—luciferase—GL3）复合物，用于MCF-7癌细胞基因转染，超声辐照前添加超声造影剂SonoVue。通过荧光素酶活性和细胞存活率的测定，对超声辐照参数进行优化，对质粒浓度、孵育时间、血清、溶媒类型、培养基体积等因素进行分析。结果细胞活力和荧光素酶活性均受超声强度、辐照时间和占空比的影响，适当条件的超声辐照可促进PEI／DNA复合物渗透入胞内，从而提高质粒DNA的转染率。最优超声辐照条件为1W／cm^2，10％占空比，辐照3min。超声辐照并超声造影剂联合PEI的转染效率显著高于单纯超声辐照和PEI转染（P〈0.01）。在超声辐照前将细胞与PEI／DNA复合物共孵育2h时，荧光素酶活性显著增强（P〈0．01）。此外，血清、培养基体积和溶媒类型也对转染效率有影响。结论优化的超声和转染参数能显著提高MCF-7癌细胞的基因表达效率。超声辐照并超声造影剂联合PEI对DNA转染效率有协同作用，是一种增强质粒DNA基因表达简单而有应用前景的方法。

Objective To study the optimized condition of transfection efficiency for MCF-7 cells enhanced by ultrasound（US） irradiation and contrast agent combined with polyethyleneimine（PEI） and observe whether the combination can have a synergistic effect to increase DNA transfection. Methods MCF-7 cells were transfected with the compounds prepared by the vector of plasmid DNA encoding luciferase （pCMV luciferase-GL3） and PEI. SonoVue microhubble was added to the cell suspension to serve as nucleation sites for acoustic cavitation before US irradiation. The DNA expression of luciferase plasmid and viability of ceils were evaluated. The strategy of US irradiation was optimized. Furthermore, the influencing factor, such as the concentration of plasmid, incubation time,serum,the type of solvent and the volume of culture media,were examined. Results The viability of cells and US-induced enhancement of luciferase activity were influenced by the US intensity,exposure time and duty cycle. US irradiation under an appropriate condition enables cells to accelerate the permeation of the PEI/DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. Optimal US condition for the enhancement was determined to be 1 W/cm^2, 10% DC for 3 min. In contrast to the PEI/DNA complex alone without US irradiation or US irradiation alone,the combination of US irradiation with contrast agent and PEI had a significantly enhanced luciferase activity （ P〈0.01）. The 2 h pre-irradiation incubation with PEI/DNA complex for MCF-7 cells exhibited a significantly enhanced luciferase activity （ P 〈0.01）. Besides, serum, type of solvent and the volume of culture media did affect the transfection efficiency. Conclusions The optimized parameters of US and transfection provide efficient gene delivery in MCF-7 cancer cells. The combination of US irradiation with contrast agent and PEI has a synergistic effect to increase DNA transfection. This is a simple and promising method to enhance the gene expression of plasmid DNA.