超声介导白蛋白微泡破裂对外源性基因在ECV304细胞和BALB/c鼠心肌中的表达

Expression studies of foreign gene in ECV304 cell and BALB/c mouse myocyte cell via ultrasound-mediated microbubble destruction

目的探讨经超声介导白蛋白微泡破裂对外源性基因在体外ECV304细胞的转染效率及表达情况和在BALB/c鼠心肌中的表达及显像效果。方法18只BALB/c小鼠分为裸质粒组(P组)、质粒+超声介导转化组(P+U组)、质粒+白蛋白微泡+超声介导转化组(P+M+U组),每组6只。选择携带增强绿色荧光蛋白基因(EGFP)的质粒DNA并与自制的白蛋白微泡相混,以超声介导白蛋白微泡破裂方法分别在体外对ECV304细胞及体内对BALB/c鼠心肌细胞进行基因转染,并测定基因转染和表达效率。结果体内和体外研究表明,超声介导的白蛋白微泡破裂组(P+M+U组)与P和P+U组相比可明显提高外源性基因转染及表达效率(P<0.01)。体外采用频率0.8MHz、声强1.0W/cm2、10%占空比,并30s两次的超声介导白蛋白微泡破裂可有效稳定地转染EGFP基因在ECV304细胞中的表达,并对细胞无毒副作用;体内超声介导白蛋白微泡破裂后具有良好的心肌灌注显像效果。结论超声介导的白蛋白微泡破裂是一种安全且有效的基因转染方法,在一定的超声条件下可明显提高外源性基因在体内和体外的转染和表达。

Objective To investigate transfection efficacy and expression of foreign gene in ECV304 cell in vitro and in expression and image of BALB/c mouse myocyte cell in vivo by ultrasound-mediated microbubble destruction. Methods Ultrasound-mediated microbubble destruction was used respectively in ECV304 cell in vitro and in BALB/c mouse myocyte cell in vivo after mixture of plasmid DNA with EGFP gene and albumin microbubble made by ourselves. The transfection efficacy and expression were measured. Results The results showed that the transfection efficacy and expression in plasmid ＋ albuminmicrobubble ＋ ultrasound （ P ＋ M ＋ U） group were significantly improved, compared with P group and P ＋ U group （P 〈 0.05）. EGFP gene could be effectively transfected with 0.8 MHz, 1.0 W/cm^2, 10% duty cycle, 30 seconds twice in ECV304 cell in vitro by ultrasound-mediated microbubble destruction, and no side effects were found to ECV304 cell. The good myocardial perfusion- image was obtained in BALB/c myocyte cell in vivo by ultrasound-mediated microbubble destruction. Conclusions Ultrasound-mediated microbubble destruction is a safe, effective gene transfection method. The transfection efficacy and expression in vitro and in vivo can be improved in certain situation. It may provide foundation of high efficiency, safety and easy practice for gene therapy.