超声微泡造影剂对心肌组织的生物学效应及介导VEGF基因转染大鼠心肌的实验研究

目的探讨超声微泡造影剂对心肌组织的生物学效应及其介导VEGF基因转染大鼠心肌的有效性. 方法 18只健康雄性Wistar大鼠,取3只采用超声波在鼠胸壁破坏微泡造影剂,观察对心肌组织显微结构的影响.将另15只急性心肌梗死3天后的雄性Wistar大鼠分为3组,每组5只.第一组采用超声破坏微泡造影剂的方式,将pcD2VEGF121基因转染大鼠心肌至造影剂不再显影(约6min);第二组尾静脉输入同等剂量携pcD2VEGF121基因的造影剂;第三组为对照.2周后,取缺血心肌组织行VEGF免疫组织化学染色,观察心肌组织血管内皮生长因子(VEGF)蛋白表达情况.结果超声波破坏微泡造影剂能使心肌组织充血,产生大量空泡,并有部分心肌细胞坏死.采用超声微泡造影剂介导的VEGF基因转染,能明显增强大鼠心肌组织VEGF蛋白的表达.结论超声微泡造影剂能明显增强对组织的空化效应,其介导的VEGF基因治疗是一种无创、新型、高效的基因转移方法.     

超声微泡造影剂介导VEGF基因治疗大鼠心肌缺血的实验性研究

目的：探讨超声微泡造影剂介导血管内皮生长因子（VEGF）基因转染大鼠缺血心肌的有效性。方法：将15只急性心肌梗塞3天后的雄性Wistar大鼠分为3组，每组5只，第一组采用超声破坏微泡造影剂的方式将pcD2VEGF121基因转染大鼠心肌约6分钟；第二组尾静脉输入同等剂量携pcD2VEGF121基因的造影剂；第三组为对照。2周后，分别行免疫组织化学检测缺血心肌中的VEGF蛋白及毛细血管内皮细胞，结果：采用超声微泡造影剂介导的VEGF基因转染，能明显增强VEGF基因在大鼠心肌组织内的表达，并可促进缺血心肌组织新生血管，结论：超声微泡造影剂介导的VEGF基因治疗是一种无创、新型、高效的基因转移方法。     

超声微泡造影剂对心肌组织毛细血管通透性的影响实验研究

目的　研究超声破坏微泡造影剂对靶区内心肌组织毛细血管通透性的影响 ;探讨超声微泡造影剂增强基因转染的机制。方法　 2 4只健康雄性 Wistar大鼠 ,取 15只分为 3组 ,第 1组经静脉输入含有伊文思蓝 (Evans blue,EB)的微泡造影剂 ,并采用超声波在鼠胸壁辐照约 6 min破坏心肌组织内造影剂 ;第 2组采用超声照射 ,同时经静脉单纯输入 EB溶液 ;第 3组单纯经静脉输入 EB作为对照。照射完毕 2 h后放血处死大鼠 ,使用标准曲线和分光光度法测量各组大鼠心肌组织中 EB含量 (作为反映血管通透性的指标 )。另 9只大鼠随机分为 3组 ,每组 3只。第 1组经静脉输入微泡造影剂 ,并采用超声波在鼠胸壁辐照约 6 min破坏心肌组织内造影剂 ;第 2组单纯采用超声照射 ;第 3组不加任何处理 ,作为对照。照射完毕即刻处死大鼠 ,取心肌组织进行电镜观察毛细血管的改变。结果　采用超声照射 ,并经静脉输入微泡造影剂的大鼠 ,心肌组织中 EB含量为 (75 .33± 16 .80 )μg/ g,比单纯超声照射[(32 .2 1± 9.5 3)μg/ g]及心肌正常组织 EB含量 [(37.16± 7.98)μg/ g]明显增加 (P<0 .0 5 )。电镜结果显示超声破坏微泡后 ,能使毛细血管破裂 ,红细胞溢出于毛细血管外。结论　超声波触发破坏微泡造影剂后能使心肌组织毛细血管通透性增加 ,可     

超声造影剂介导血管内皮生长因子基因治疗心肌缺血的实验研究

目的探讨超声破坏造影剂微泡介导血管内皮生长因子(VEGF)基因转染兔缺血心肌的有效性。方法新西兰白兔48只，结扎兔冠状动脉左旋支，建立急性心肌梗死模型。术后3d，经胸超声检查左室壁运动情况，明确心肌缺血区域，然后将VEGF真核表达质粒与新型超声造影剂JD-95混合后，经耳缘静脉输入兔体内，同时进行超声实时造影成像，照射心脏缺血区。其他对照组包括超声和造影剂组、单独基因组、基因和超声组、基因和造影剂组以及空白对照组。术后17d处死动物，取动物缺血心肌、肝、肾及下肢骨骼肌组织，用免疫组化方法检测VEGF蛋白表达。结果在超声破坏造影剂微泡介导基因转染的实验组中5只兔的缺血心肌中VEGF蛋白的表达，2只兔肾有VEGF表达。而其他对照组中无VEGF蛋白表达。结论超声破坏造影剂微气泡的方法是将体外生物活性物质传递至心肌中的一种有效途径。     

靶向超声介导VEGF165cDNA基因治疗大鼠心肌缺血的实验研究

目的探讨靶向超声介导VEGF165 cDNA基因治疗大鼠心肌缺血的可行性.方法将60只心肌缺血的雄性SD大鼠分为3组超声介导基因组(采用超声破坏微泡造影剂的方法将VEGF165 cDNA基因转染大鼠)、单纯基因组(尾静脉输入同等量携带VEGF165cDNA基因的造影剂)、对照组.每组又分为24 h后处死组和14d后处死组,每小组为10只.处死后分别行坏死区中性粒细胞记数,梗死区质量比测定,免疫组织化学检测缺血心肌中的VEGF蛋白表达,缺血心肌区域毛细血管记数.结果24 h处死组超声介导基因组坏死区中性粒细胞平均为(59.30±7.70)个/高倍视野,单纯基因组为(62.53±7.50)个/高倍视野.14 d后处死组超声介导基因组梗死区质量比明显小于单纯基因组,单纯基因组又明显小于对照组.超声介导基因组有大量VEGF蛋白阳性反应的棕褐色颗粒,而单纯基因组VEGF蛋白阳性反应的棕褐色颗粒少于超声介导基因组.结论超声介导微泡造影剂携带VEGF165cDNA基因能提高基因转染率,促进缺血区毛细血管再生,减少心肌梗死面积,对缺血区炎性细胞的浸润也有一些影响,且基因的转染＞24 h.     

靶向超声介导VEGF_(165)cDNA基因治疗大鼠心肌缺血的实验研究

目的探讨靶向超声介导VEGF165cDNA基因治疗大鼠心肌缺血的可行性。方法将60只心肌缺血的雄性SD大鼠分为3组:超声介导基因组(采用超声破坏微泡造影剂的方法将VEGF165cDNA基因转染大鼠)、单纯基因组(尾静脉输入同等量携带VEGF165cDNA基因的造影剂)、对照组。每组又分为24h后处死组和14d后处死组,每小组为10只。处死后分别行坏死区中性粒细胞记数,梗死区质量比测定,免疫组织化学检测缺血心肌中的VEGF蛋白表达,缺血心肌区域毛细血管记数。结果24h处死组:超声介导基因组坏死区中性粒细胞平均为(59.30±7.70)个/高倍视野,单纯基因组为(62.53±7.50)个/高倍视野。14d后处死组:超声介导基因组梗死区质量比明显小于单纯基因组,单纯基因组又明显小于对照组。超声介导基因组有大量VEGF蛋白阳性反应的棕褐色颗粒,而单纯基因组VEGF蛋白阳性反应的棕褐色颗粒少于超声介导基因组。结论超声介导微泡造影剂携带VEGF165cDNA基因能提高基因转染率,促进缺血区毛细血管再生,减少心肌梗死面积,对缺血区炎性细胞的浸润也有一些影响,且基因的转染>24h。     

超声破坏微泡造影剂促进大鼠心肌血管新生的实验研究

目的探讨超声破坏微泡刺激大鼠心肌内源性VEGF分泌、促进血管生成的新途径。方法正常成年Wistar大鼠30只随机分为3组。第1组超声破坏微泡组，第2组单纯超声辐照组，第3组对照组。每组均进行3次处理。第一次处理3d后每组各取1只大鼠断颈处死后取其心肌，常规HE染色光镜观察心肌结构变化。剩余大鼠于2周后取材，免疫组化检测心肌组织中VEGF表达，CD34免疫组化检测评定超声破坏微泡促进心肌血管新生疗效。结果超声破坏微泡组心肌组织中见大量VEGF和CD34表达，新生血管较多。单纯超声组心肌组织中有较少VEGF和CD34表达，新生血管较少。对照组仅有极少量VEGF和CD34表达，未见明显新生血管。结论超声破坏微泡可刺激心肌内源性VEGF分泌，促进血管新生。     

超声波介导微泡破裂促EGFP基因转染大鼠脑组织的实验研究

目的 探讨超声波介导微泡造影剂破裂促外源基因在中枢神经系统转染的可行性.方法 15只大鼠分3组,1组经股静脉输入含绿色荧光蛋白质粒(pEGFP)的造影剂0.8 ml,立即用超声波照射大鼠颅骨3 min;第2组输入相同的造影剂0.8 ml,不采用超声波照射;第3组输入不含造影剂的pEGFP 0.2 ml,立即超声波照射3 min.48 h后处死大鼠,荧光显微镜下观察绿色荧光蛋白表达.结果 只在股静脉输入含pEGFP的造影剂,并经超声波照射的大鼠微血管壁上观察到绿色荧光蛋白表达.结论 以微泡造影剂为基因载体,通过超声波靶向破坏微泡,有可能在脑血管内皮细胞中获得基因转染.     

靶向超声介导VEGF_(165) cDNA基因及抗ICAM-1单克隆抗体治疗大鼠心肌缺血的对比性研究

目的 探讨靶向超声介导VEGF_(165) cDNA、抗ICAM-1单克隆抗体及两者协同作用于受损心肌的有效性及可行性.方法 将80只心肌缺血的雄性SD大鼠随机分为四组:第一组采用超声击破微泡造影剂的方法将VEGF_(165) cDNA基因及抗ICAM-1单克隆抗体混合物定向于受损心肌;第二组采用超声击破微泡造影剂的方法将VEGF_(165) cDNA基因定向于受损心肌;第三组采用超声击破微泡造影剂的方法将抗ICAM-1单克隆抗体定向于受损心肌;第四组为对照组只注射生理盐水1 ml,不采用超声波照射.每组分别于24 h和14 d后处死10只.处死后分别行坏死区中性粒细胞记数,梗死区测定,免疫组织化学检测缺血心肌中的VEGF蛋白表达,缺血坏死心肌区域毛细血管记数.结果 第一组大鼠心肌缺血治疗效果较第二、第三组效果显著,第一组中性粒细胞浸润以及心肌梗死区域均明显低于其他各组,VEGF蛋白表达以及毛细血管记数明显高于其他三组.结论 采用超声触发破坏含靶基因及抗体的微泡造影剂的方式,可明显对抗心肌再损伤,促进缺血心肌的新生血管再生,改善心肌缺血,且两者协同作用比单独作用效果更佳.     

超声靶向破坏阳离子微泡介导SDF-1α基因转染提高外源性血管新生效应的研究

目的通过证实超声介导阳离子微泡能显著提高微泡的携基因量及体内转染后的血管新生效应来探讨阳离子微泡作为基因载体的价值。方法紫外分光光度法检测阳离子微泡与声诺维微泡在体外的基因携带效率。构建兔子急性心肌梗死模型并分组:阳离子微泡组(超声靶向破坏携SDF-1α基因的阳离子微泡并进行转染)、声诺维微泡组(超声靶向破坏携SDF-1α基因的声诺维微泡并进行转染)和对照组(不进行转染)。转染后3 d后3组各取3只兔子处死,ELISA检测目的基因蛋白表达情况;转染后4周行超声心动图和心肌超声造影,检测兔子心功能及心肌灌注情况,之后全部处死,免疫组化检测心肌血管新生效应。结果阳离子微泡组基因携带率是声诺维微泡组的11倍。阳离子微泡组SDF-1α蛋白表达是声诺维微泡组的4倍,对照组的9倍。阳离子微泡组在心肌毛细血管新生效应、心肌血流灌注和心功能方面均优于声诺维微泡组和对照组(P<0.05)。结论阳离子微泡能明显提高在体外的基因携带效率,从而使体内转染效率增强,更好地促进心肌血管新生,是一种高效的基因载体。     

经多功能心腔内超声导管超声辐照破坏微泡对犬心肌组织的生物学效应

目的观察经多功能心腔内超声(ICE)导管超声辐照破坏微泡对动物心肌产生的生物学效应,探索基因治疗缺血性心脏病的新方法。方法 15只犬随机分为US+MB组、US组、对照组3组,每组5只。以介入法将多功能ICE导管送入犬心室。对US+MB在ICE监控下向左心室游离壁注射0.5ml微泡,并以1 W/cm2的声能对注射部位辐照1min;对US组以相同条件行左心室壁辐照,但不注射微泡;对照组在插入导管后不进行任何处理。术后3天处死动物,观察心肌组织大体改变,并行HE染色观察细微结构变化。结果ICE能对注射针的进针深度、微泡注射及辐照过程进行实时监控。观察期内所有动物均正常存活。US+MB组心肌辐照部位出现充血、心肌细胞间隙增宽、少量炎性细胞浸润等改变,US组心肌组织仅出现轻微充血;对照组动物心肌无异常变化。结论经ICE导管超声辐照破坏微泡能在靶区域产生相应生物学效应,内置ICE可对心肌内微泡注射、超声辐照过程进行实时监控。此款新型多功能导管可能为基因治疗缺血性心脏病提供新的、更加安全有效的途径。     

Spatial Distribution of Ultrasound Targeted Microbubble Destruction Increases Cardiac Transgene Expression But Not Capillary Permeability

Ultrasound targeted microbubble destruction (UTMD) has evolved as a promising tool for organ specific gene and drug delivery. Using DNA-loaded microbubbles, cardiac transfection has been shown to be feasible. However, two-dimensional properties of the ultrasound beam limit cardiac transgene expression to the focal zone, thus, reducing its potential therapeutic effect. The aim of this study was to test if spatial distribution of ultrasound targeted microbubble destruction in the heart could lead to augmented transgene expression or increased capillary permeability. Lipid microbubbles containing plasmids with a luciferase transgene were used to target rat hearts. The diagnostic ultrasound probe was fixed in a mid-short axis view with a gel stand-off between the chest and probe. Ultrasound (1.3 MHz) with a mechanical index of 1.6 was intermittently applied to rats during microbubble infusion. Rats were randomized to either stay in that position or move horizontally in a cranio-caudal direction (3 mm sweep) relative to the ultrasound probe during UTMD. After 4 days, organs were harvested and analyzed for reporter gene expression. Another group of rats received Evans Blue, followed by UTMD with unloaded microbubbles. Again, rats were randomized into a static or moving group. Hearts were harvested to evaluate extravasation of Evans Blue. Moving rats in a cranio-caudal direction significantly increased transgene expression by 19-fold in the anterior heart, by sixfold in the posterior heart and by 32-fold in the apex. Interestingly, Evans Blue extravasation was not augmented in the moving group. Spatial distribution of UTMD may increase transgene expression due to sonication of larger areas in the heart. In contrast, capillary permeability does not increase, indicating less capillary damage. (E-mail: raffi.bekeredjian@med.uni-heidelberg.de)     

超声破坏微泡促进骨骼肌血管新生的时间-效应关系研究

目的 探讨超声破坏微泡促进大鼠骨骼肌血管新生的时间-效应关系.方法 将48只SD大鼠随机分为4组(超声破坏微泡组、单纯超声组、单纯微泡组、对照组),超声破坏微泡组经尾静脉输入自制的脂质全氟丙烷微泡造影剂0.5 ml,同时用1.0 MHz、2.0 W/cm2的超声在其大腿骨骼肌局部作用3min;单纯超声组仅在骨骼肌局部用同等的超声能量作用;单纯微泡组仅由尾静脉输入脂质全氟丙烷微泡造影剂0.5 ml;对照组不作任何处理.实验结束后的第3、7、10、14、21、28 d,各组分别取两只大鼠处死,取局部骨骼肌行石蜡切片HE染色观察显微结构的变化,CD34免疫组化计数微血管密度(MVD),酶联免疫吸附试验(ELISA)定量评价血管内皮生长因子(VEGF)的表达.结果 超声破微泡组有较多的血管新生;单纯超声组新生血管较少;单纯微泡组和对照组几乎无血管新生.随着时间的变化,超声破坏微泡组的MVD值和VEGF表达在第10 d达到高峰;单纯超声组的高峰出现在第14 d.结论 超声破坏微泡可刺激骨骼肌中内源性VEGF较快、较多地分泌,从而更好地促进新生血管生成.     

冠心病血管新生治疗的进展

冠心病是威胁人类健康的疾病。新近研究发现，血管生长因子经安全有效的载体导入或直接注入缺血心肌，可以促进血管新生。干细胞研究的进展和基因治疗技术以及在心血管的应用，为缺血性心脏病的治疗提供了新策略。超声介导微泡造影剂携药物或基因进行缺血性疾病的治疗是一种新型的靶向治疗技术。本文就各种因子和细胞及各种方法在血管新生中的作用作一综述。     

Augmentation of cardiac protein delivery using ultrasound targeted microbubble destruction

Gas-filled microbubbles have become an important tool as ultrasonic contrast agents. We have previously shown that ultrasound-targeted microbubble destruction (UTMD) can direct plasmids to the heart. The aim of this study was to evaluate UTMD for protein delivery. Six different groups of rats received 1 microg of luciferase protein with varying protocols: (1) luciferase-loaded microbubbles and ultrasound; (2) luciferase only; (3) luciferase and ultrasound; (4) luciferase-loaded microbubbles; (5) unloaded microbubbles incubated with luciferase and ultrasound; (6) unloaded microbubbles with ultrasound followed by luciferase. Relative luminescence units per mg protein per s were determined in hearts and control organs. The rats that received ultrasound and luciferase-loaded bubbles showed a six-fold higher cardiac luciferase uptake compared with control groups that did not include bubbles. None of the other groups significantly augmented cardiac luciferase activity. We conclude that ultrasound-targeted microbubble destruction can substantially and noninvasively augment organ-specific delivery of proteins.     

超声微泡造影剂在冠状动脉粥样硬化性心脏病治疗中的应用进展

冠状动脉粥样硬化性心脏病(CHD)是威胁人类健康和生命的疾病之一,药物、介入及外科治疗均不能根治.外源性给予某些促血管生成因子或治疗基因能促进缺血心肌血管再生和侧支循环建立,改善心肌缺血.超声微泡造影剂作为促血管生成因子或治疗基因的载体,联合超声辐照能更安全、高效、靶向地导入缺血心肌组织,为CHD的治疗开辟了一个新的应用前景.本文就近年来超声微泡造影剂在CHD治疗中的应用进展做一综述.     

Safety of Image-Guided Treatment of the Liver with Ultrasound and Microbubbles in an in Vivo Porcine Model

Ultrasound and microbubbles are useful for both diagnostic imaging and targeted drug delivery, making them ideal conduits for theranostic interventions. Recent reports have indicated the preclinical success of microbubble cavitation for enhancement of chemotherapy in abdominal tumors; however, there have been limited studies and variable efficacy in clinical implementation of this technique. This is likely because in contrast to the high pressures and long cycle lengths seen in successful preclinical work, current clinical implementation of microbubble cavitation for drug delivery generally involves low acoustic pressures and short cycle lengths to fit within clinical guidelines. To translate the preclinical parameter space to clinical adoption, a relevant safety study in a healthy large animal is required. Therefore, the purpose of this work was to evaluate the safety of ultrasound cavitation treatment (USCTx) in a healthy porcine model using a modified Philips EPIQ with S5-1 as the focused source. We performed USCTx on eight healthy pigs and monitored health over the course of 1 wk. We then performed an acute study of USCTx to evaluate immediate tissue damage. Contrast-enhanced ultrasound exams were performed before and after each treatment to investigate perfusion changes within the treated areas, and blood and urine were evaluated for liver damage biomarkers. We illustrate, through quantitative analysis of contrast-enhanced ultrasound data, blood and urine analyses and histology, that this technique and the parameter space considered are safe within the time frame evaluated. With its safety confirmed using a clinical-grade ultrasound scanner and contrast agent, USCTx could be easily translated into clinical trials for improvement of chemotherapy delivery. This represents the first safety study assessing the bio-effects of microbubble cavitation from relevant ultrasound parameters in a large animal model.     

Activation of microbubbles by short-pulsed ultrasound enhances the cytotoxic effect of cis-diamminedichloroplatinum (II) in a canine thyroid adenocarcinoma cell line in vitro

Ultrasound targeted microbubble destruction has succeeded in delivering drugs and genes. This study was designed to explore characteristics of ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound. Canine thyroid adenocarcinoma cells were exposed to short-pulsed diagnostic ultrasound in the presence of cis-diamminedichloroplatinum (II) (cisplatin) and ultrasound contrast agent Sonazoid^® microbubbles. The cytotoxic effect of cisplatin was enhanced by short-pulsed diagnostic ultrasound and microbubbles. Incubation time with microbubbles influenced the cytotoxic effect of cisplatin. However, exposure duration did not affect the cytotoxic effect of cisplatin. Therefore, short-pulsed diagnostic ultrasound may activate microbubbles near cells and deliver cisplatin into cells. In addition, activation of microbubbles may be concluded in a short time. Our results suggest that short exposure duration could be potentially sufficient to induce efficient drug delivery by ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound.