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目的建立一套包括经皮介入心肌内基因投递、心腔内超声辐照增强基因表达的技术体系,并探讨其安全性。方法研制心腔内超声辐照导管(intracardiac ultrasound,US),经常规介入法置于活体犬左心室,并经导管中特制的微型针刺入心肌注射报告基因增强绿色荧光蛋白(enhanced green fluorescence protein,EGFP)质粒;12月龄健康杂种犬12只,体质量15～20 kg,雌雄不拘,分为EGFP+US组、单纯EGFP组。在EGFP+US组,注射质粒500μg后,心腔内超声辐照1 min(1 MHz、1 W/cm2、60s)。单纯EGFP组仅注射质粒,继续饲养犬,测定照射前、照射过程中、照射后心肌酶谱变化,7 d后,测定心肌、肝、肾、肺脏、肌肉中EGFP mRNA及蛋白质表达,观察心肌及上述脏器的病理形态变化。结果经超声导管成功实现了经皮心肌内注入EGFP质粒,心肌形态正常,心腔内超声辐照后明显增加EGFP基因在心肌表达,与单纯EGFP组相比,EGFP+US组EGFP mRNA表达增加6.5倍,蛋白表达增加3.4倍(P<0.01)。心肌无损伤、出血,辐照前后心肌酶谱没有变化(P>0.05)。光镜下肺脏、肝脏、肾脏形态无改变,也未见EGFP表达,心肌外未见质粒沉积。结论心腔内超声辐照能增强裸质粒在活体心肌组织中转染表达,未见超声辐照对心脏的毒副作用,心腔内超声辐照增强基因表达安全有效。
Objective To establish a set of technical systems including percutaneous gene delivery in myocardium and intracardiac ultrasound irradiation to enhance gene expression and to explore its safety. Methods An intracardiac ultrasound catheter (US) was developed. After conventional interventional therapy, the left ventricle of living dogs was punctured and the gene expression of enhanced green fluorescence protein , EGFP) plasmid; 12-month-old healthy mongrel dogs 12, weight 15 ~ 20 kg, male or female, divided into EGFP + US group, simple EGFP group. In the EGFP + US group, 500 μg of plasmids were injected intracardiacly for 1 min (1 MHz, 1 W / cm2, 60 s). The EGFP mRNA and protein expression in myocardium, liver, kidney, lung and muscle were measured 7 days after exposure to, EGFR-treated and irradiated dogs. EGFP mRNA and protein expression in myocardium, liver, kidney, Pathological changes in the above organs. Results The EGFP plasmid was successfully injected into the percutaneous myocardium by transcatheter ultrasound. The morphology of myocardium was normal. The expression of EGFP gene in the myocardium was increased after intracardiac ultrasound irradiation. Compared with the EGFP group, EGFP mRNA expression increased by 6.5 Fold, and protein expression increased by 3.4 times (P <0.01). Myocardial injury, bleeding, myocardial enzyme did not change before and after irradiation (P> 0.05). Under the light microscope, there was no change in the morphology of lung, liver and kidney, and no EGFP expression was observed. No plasmids were found outside the myocardium. Conclusions Intracardiac ultrasound irradiation can enhance the expression of naked plasmid in live myocardium. No toxic or side effects of ultrasound irradiation on heart can be observed. Intracardiac ultrasound irradiation can enhance gene expression safely and effectively.