目的 构建一种阳离子微泡(cationic microbubble,CMB),通过与传统微泡(definity microbubble,DMB)比较,探讨其提高超声波靶向击碎微泡(ultrasound-targeted microbubble destruction,UTMD)技术介导的体内基因转染效率及治疗效果.方法 体外实验:应用薄膜水化法制备CMB,观察其形态、测量其粒径、zeta电位以及基因携带能力,以DMB作为对照.体内实验:首先取16只大鼠以生物荧光素酶质粒作为报告基因,分别应用CMB和DMB进行心脏的靶向转染,动态观察转染效率及靶向性.随后取64只大鼠制备心肌缺血再灌注(ischemia-reperfusion,I/R)模型,第5天彩色超声检查明确60只大鼠成功制备I/R模型;随机分为3组(n=20),其中对照组大鼠接受DMB携带空质粒转染,DMB组接受DMB携带AKT质粒转染,CMB组接受CMB携带AKT质粒转染.治疗后心脏彩色超声以及组织学观测各组大鼠心肌灌注、心脏功能、梗死区面积和梗死区组织厚度;免疫组织化学染色检测毛细血管及小动脉密度,TUNEL染色观察心肌细胞凋亡;Western blot检测心肌AKT、磷酸化AKT (phospho-AKT,P-AKT)、生存素(Survivin)和磷酸化BAD (phospho-BAD,P-BAD)表达.结果 实验制备的CMB形状均一,其zeta电位显著高于DMB(t=28.680,P=0.000);DNA携带百分比显著高于DMB (P＜0.05).无论在体检测还是离体检测均显示,应用CMB转染后的荧光素酶活性均显著高于DMB转染后,差异有统计学意义(P＜0.05).I/R模型术后第5天,各组前、后壁信号强度比值以及心脏射血分数、左室短轴缩短率比较,差异均无统计学意义(P＞0.05);第21天,CMB组及DMB组以上指标较对照组增加,且CMB组高于DMB组,差异均有统计学意义(P＜0.05).术后第21天,CMB组梗死区域长度最小、厚度最大,其次为DMB组、对照组;CMB组及DMB组毛细血管、小动脉血管密度均较对照组明显增大,CMB组较DMB组增大,组间差异均有统计学意义(P＜0.05).基因转染后第3天,对照组凋亡细胞最多,其次为DMB组、CMB组;CMB组及DMB组AKT、P-AKT、Survivin和P-BAD蛋白相对表达量均明显高于对照组,CMB组高于DMB组;组间比较差异均有统计学意义(P＜0.05).结论 CMB在保留了普通微泡理化性质的同时,显著提高了携带质粒DNA的能力,提高了超声微泡靶向转染效率;应用CMB进行超声靶向AKT基因转染治疗大鼠心肌I/R损伤时,显著提高了基因转染率,改善了大鼠心脏功能.“,”Objective To construct a cationic microbubble (CMB),and investigate the enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction (UTMD) in vivo wit h CMB compared to definity MB (DMB).Methods In vitro,the CMB was prepared by the method of thin film hydration.The morphology,size,zeta potential,and gene-carrying capacity of CMB were compared with the DMB.In vivo,the firefly luciferase gene which was used as a reporter gene was targeted transfected into myocardium of 16 rats with CMB and DMB,respectively.The gene transfection efficiency and targeting were observed dynamically.Then,ischemia-reperfusion (I/R) model was performed on 64 rats.The models of 60 rats were successfully confirmed by using ultrasonography at 5 days after I/R.The rats were divided into 3 groups (n=20) randomly.The control group received DMB carrying empty plasmid for transfection;DMB group received DMB carrying AKT plasmid for transfection;and CMB group received CMB carrying AKT plasmid for transfection.The cardiac perfusion,cardiac function,infarct size,and infarct thickness were measured by ultrasonography and histological observations after treatment.In addition,the capillary and arteriolar densities were measured with immunohistochemical staining.The myocyte apoptosis was measured with TUNEL staining.The protein expressions of AKT,phospho-AKT (P-AKT),Survivin,and phospho-BAD (P-BAD) were measured by Western blot.Results The size of CMB was uniformly.The zeta potential of CMB was significantly higher than that of DMB (t=28.680,P=0.000).The CMB bound more plasmid DNA than the DMB (P＜0.05).The luciferase activity of myocardium were higher in CMB group than in DMB group both in vitro and in vivo measurements (P＜0.05).There was no significant difference between groups in the ratio of signal intensity in anterior wall to posterior wall,ejection fraction (EF),and fractional shortening (FS) at 5 days after I/R (P＞0.05),but the above indexes were significant higher in CMB and DMB groups than in control group at 21 days after I/R (P＜0.05).Besides,the above indexes were significant higher in CMB group than in DMB group at 21 days after I/R (P＜0.05).The infarct size was the smallest and infarct thickness was the thickest in the CMB group,followed by DMB group,control group at 21 days after I/R.The capillary and arteriolar densities of CMB and DMB groups were significant higher than those of control group at 21 days after I/R (P＜0.05).Besides,the capillary and arteriolar densities of CMB group were significant higher than those of DMB group (P＜0.05).The apoptotic ceils were the most in the control group,followed by DMB group,CMB group at 3 days after gene transfection,showing significant differences between groups (P＜0.05).The protein expressions of AKT,P-AKT,Survivin,and P-BAD were significant higher in CMB and DMB groups than those in control group at 3 days after gene transfection (P＜0.05).Besides,these protein expressions were significant higher in CMB group than those in DMB group (P＜0.05).Conclusion The DNA-carrying capacity and gene transfection efficiency are elevated by CMB,although its physicochemical property is the same as DMB.When ultrasound-targeted AKT gene transfection is used to treat myocardial I/R injury in rats,delivery of AKT with the CMB can result in higher transfection efficiency and greater cardiac functional improvements compared to the DMB.