超声靶向微泡破碎介导EGFP基因转染肝癌细胞的影响因素研究

摘要

To study the transfection efficiency and the viability of delivering plasmid pEGFP - N3 to HepG2 cells by ultrasound targeted microbubble destruction(UTMD) using different conditions of transfection. The cultured HepG2 cells were divided into 4 groups; plasmid group.micromubble and plasmid .ultrasound and plasmid .ultrasound plus microbubble and plasmid. According to diffirent culture conditions, every group was divided into 2 groups;cell suspension and 24hrs culture for complete adherence. The suspension cells of ultrasound plus microbubble and plasmid group were divided into plasmid concentration group and microbubble contrast agent concentration group. The transfection efficiency was assessed by fluorescent microscopy and flow cytometry. The cell viability was measured with MTT. Compared with cells adherence, transfection efficiency was significantly increased in cells suspension at 2w/cm2 expo-sured 60s with a duty cycle 20%. (P <0.01). The increased transfection efficiency was related to plasmid and microbubble contrast agent concentration. The highest transfeclion efficiency was obtained when DNA concentration was 10 礸/ml, microbubble:cell(40:l) with a transfection efficiency of (15.63% ±1.81%) and over 80% of the transfected cells were viable after the transfection. The increased transfection efficiency is related to improve Plasmid and microbubble contrast agent concentration. Compared with cells adherence , the transfection efficiency and the viability is significantly increased in cells suspension.%观察超声靶向微泡破碎(ultrasound targeted microbubble destruction,UTMD)在不同转染条件和细胞状态下对肝癌细胞HepG2转染率及细胞活性的影响.体外培养HepG2细胞,随机分为4组:质粒组、微泡+质粒组、超声+质粒组和超声+微泡+质粒组,各组再分为贴壁和悬浮状态2组.悬浮状态的超声+微泡+质粒组根据质粒和微泡浓度不同分为微泡浓度组和质粒浓度组,转染24h后在倒置荧光显微镜下观察绿色荧光蛋白在HepG2细胞中的表达,流式细胞仪测定细胞转染率,MTT法检测细胞活性.结果 在超声声强2w/cm2、占空比20％、照射时间60s的超声参数作用下,质粒5μg/ml、微泡:细胞20∶1时,悬浮状态细胞转染率为7.33％±0.98％,存活率为90.37％±1.80％贴壁状态细胞转染率为1.56％±0.81％,存活率为81.10±1.26％,两者比较有显著差异(P＜0.01).悬浮状态下,提高质粒和微泡浓度至质粒10 μg/ml、微泡:细胞40∶1时转染效率增至15.63％±1.81％,且细胞生存率＞80％.结论 相同转染条件下悬浮状态细胞转染率及存活率明显优于贴壁状态.优化质粒、微泡浓度可进一步提高超声微泡介导的基因转染效率和存活率.