超声微泡破裂法联合阳离子脂质体介导基因转染的实验研究

Ultrasound-mediated microbubble destruction accompanied with cationic liposome enhanced gene transfection in vitro

目的探讨超声微泡破裂法联合阳离子脂质体（cationic liposome，CL）介导绿色荧光蛋白质粒在肝癌细胞（HepG2）基因转染的可行性，并探索最佳转染条件。方法依次采用培养液中是否含有血清和不同的CL浓度、不同超声辐照时间点、不同纳米级脂质微泡造影剂（nano—liposomalbubble，NB）浓度等处理因素进行细胞基因转染。荧光显微镜和流式细胞仪检测基因转染效率，CCK-8法检测细胞活性，以获得优化的转染参数。结果血清能降低CL的细胞毒性，但对基因转染效率无明显影响，CL与质粒DNA质量比4：1时可以达到相对高效低毒的转染效果，转染率（17．71±0．79）%，存活率（91．28±0．76）％。CL联合1h时间点辐照超声可以提高转染率至（24．85±0．78）%（P〈0．01），加入10％的NB可进一步提高转染率至（32．47±4．01）％（P〈0．05）。结论超声微泡破裂法可以有效增强CL介导的基因转染，联合应用为基因治疗提供了新思路。

Objective To investigate whether ultrasound-mediated microbubble destruction（UMD） could enhance cationic liposome （CL） induced plasmid DNA delivery or not, and optimize the transfection conditions. Methods Multiple parameters were explored to obtain the optimal transgene efficiency by means of with or without serum in culture medium, various CL or nano-liposomal bubble （NB） concentrations, different time point of ultrasonic irradiation. The transfeetion efficiency was assessed by fluorescence microscopy and flow cytometer, and cell viability was evaluated by Cell Counting Kit-8 （CCK-8） assay. Results The serum could protect the cells but show little impact on transfection efficiency induced by CL. CL and plasmid DNA at a weight ratio of 4 ： 1 exhibited high transfection efficiency of （17.71 ± 0.79）% and high cell viability of （91.28 ± 0.76）%. CL combining with ultrasonic irradiation at the time point of 1 hour could increase the transfection efficiency to （24.85± 0.78）% （ P d0.01）. Higher transfection rate （32. 47 ± 4. 01）% was obtained by adding NB at the concentration of 10% （P〈0.05）.Conclusions UMD accompanied with CL could enhance gene delivery effectively, which would provide a new method for gene therapy.