超声介导下重组腺相关病毒载体转染心肌的最佳滴度的实验研究

目的 探讨超声促进携带增强型绿色荧光蛋白报告基因(EGFP)的重组腺相关病毒载体2(rAAV2)心肌转染过程中rAAV2的最佳滴度.方法 健康成年SD大鼠21只随机分为实验组与对照组.实验组给予连接于SonoVue微泡表面的rAAV2-EGFP尾静脉注入,滴度为1.5×109～3.0×101111 vg/ml(virus genome/ml),超声爆破造影剂,促进rAAV2-EGFP心肌内转染.对照组只给予尾静脉注射造影剂并超声爆破(无rAAV2-EGFP).2周后处死大鼠并制作心肌组织冰冻切片.荧光显微镜下检测荧光蛋白的表达量,代表rAAV2转染率.结果 随着注入的rAAV2滴度的增加,心肌内荧光表达量亦增加,至滴度达到1.5×1011vg/ml时荧光的表达量显著增加(P<0.01).结论 促进心肌组织转染的rAAV2的最佳滴度为1.5×1011vg/ml.

Study on the best titer of rAAV2 delivered to myocardium by ultrasound mediated microbubbles

Objective To study the best titer of recombinant adeno-associated virus serotype 2 (rAAV2) delivered to myocardium by targeted ultrasound microbubbles. Methods Twenty one adult SD rats were divided into seven groups. SonoVue attached with different titer 1.5 × 109~ 3.0 × 1011 vg/ml (virus genome/ml)] of rAAV2-EGFP was infused into the tail vein of rats, following ultrasound mediated microbubbles destruction,as experiment groups. Normal saline was infused into the tail vein of rats as the control group (without rAAV2). Rats were killed after 14 days and hearts were harvested. GFP protein expression which showed rAAV2 transfer was observed under fluorescence microscope in frozen section and integrate optical density(IOD) was measured by Image Pro Plus software. Results When the titer of rAAV2 was 1.5 × 1011 vg/ml infused into the tail vein of rats there was much more GFP expression in myocardium than lower titers (P <0.01). Conclusions The best titer of rAAV2 delivered to myocardium by targeted ultrasound microbubbles is 1.5 × 1011 vg/ml in the tail vein infusion of rats.