诊断超声介导微泡造影剂助溶兔静脉血栓的体内实验研究

目的 研究诊断超声结合自制白蛋白微泡造影剂在体内的溶栓效应,并探讨其与尿激酶联用在溶栓方面的协同作用及溶栓机制.方法 32只健康家兔建立股静脉血栓模型,分为单纯尿激酶组、超声+微泡造影剂组、超声+微泡造影剂+尿激酶组和阳性对照组.根据前期的体外实验所确定的最佳参数组合,并联合尿激酶进行溶栓,利用彩色多普勒超声技术对各组不同时间点15 min、30 min、60 min的血管再通情况进行检测,评价血管的再通率.取溶栓治疗后的兔股静脉标本进行免疫组化检测,比较各组管腔内部结构及溶栓效果,并对其溶栓机制进行探讨.结果 治疗15 min与30 min后,超声+微泡造影剂+尿激酶组的血管再通率较单纯尿激酶组、超声+微泡造影剂组均显著增加,差异有统计学意义(P<0.05);治疗60 min后,超声+微泡造影剂+尿激酶组管再通评分要明显优于其他两组.免疫组化检测结果示超声+微泡造影剂+尿激酶组治疗60 min后股静脉内血栓结构崩解,纤维素断裂,血管腔大部分融通,优于治疗15 min、30 min时的溶栓效果.结论 诊断超声结合自制微泡造影剂在体内具有明显的溶栓效应,与尿激酶联用在溶栓方面有很好的协同作用.     

微泡超声爆破助溶血栓机制的离体研究

目的用荧光分子探针标记脂膜超声微泡,荧光显微镜下观察并追踪荧光微泡在超声作用下于血栓表面及内部分布情况,为超声联合微泡对体外血栓的助溶机制提供病理学依据。方法将染料标记于脂膜微泡,结合发射频率1MHz、输出声功率1.0W/cm2治疗超声用于离体溶栓实验。实验分为单纯微泡组和超声联合微泡两组,称量血栓失重量比较两组溶栓率,荧光显微镜观察两组血栓表面及内部的荧光分布强度及密度,最后血栓标本进行HE染色后光镜观察。结果单纯微泡组和超声联合微泡组的溶栓率分别为(21.73±2.78)%、(29.51±5.17)%,组间比较差异有统计学意义(P<0.01)。荧光显微镜下单纯微泡组血栓表面呈明亮荧光带,内部仅见几个细小荧光颗粒;超声联合微泡组血栓周边形成更为明亮宽大的荧光带,内部有较为密集的荧光颗粒分布。光镜下超声联合微泡组血栓表面见微孔和撕裂,内部可见不规则的裂隙,而单纯微泡组血栓无此改变。结论脂膜微泡在治疗超声连续爆破作用下有渗入血栓内部的倾向,这可能更有助于超声发挥助溶效应。     

诊断超声介导自制微泡造影剂的溶栓效应及参数优化体外实验研究

目的 研究诊断超声介导自制微泡造影剂对血栓的溶解效应以及对影响溶栓效果的超声频率、机械指数、照射时间、微泡浓度四个参数进行探讨,确定最佳的溶栓参数组合.方法 利用统计学L9(34)正交表确定不同的超声频率(1.8 MHz、5 MHz、11 MHz)、不同机械指数(0.5、1.2、1.6)、不同照射时间(10 min、30 min、60 min)、不同微泡浓度(3.5×109/ml、4.2×109/ml、5.0×109/ml)各参数不同水平间的组合,按照正交表所示组合参数条件进行体外溶栓,计算各组间的溶栓率P=[(W0- W1)/ W0]×100%,所得数据进行极差分析,切片HE染色及电镜下观察溶栓后的血凝块结构.结果 作用时间对其溶栓率的影响最大,其溶栓的最佳条件组合为超声频率A=1.8 MHz、机械指数B=1.6、作用时间C=60 min、微泡浓度D=5.0×109/ml.HE切片染色及扫描电镜1000倍观察溶栓后血凝块内部结构松散,并伴有大量纤维素断裂,出现较大的空洞与裂隙.结论 诊断超声介导下的超声微泡造影剂具有助溶作用,并在低频率、高机械指数、延长作用时间、高微泡浓度的参数条件下溶栓效果最佳.     

长脉冲超声联合脂质体微泡溶解微血栓的体外实验研究

目的评价长脉冲超声联合脂质体微泡造影剂溶解微血栓的疗效。方法构建评价溶栓效能的体外循环系统,通过造成200~300μm的微血栓阻塞于滤网来模仿体内的微循环栓塞。然后治疗组给予脂质体微泡加超声治疗15min,超声频率1MHz、声压1.5MPa、脉冲间隔3s,包括100、1 000、5 000 3种周期,对照组给予超声照射但无声学微泡。结果治疗组溶栓的效果随着脉冲的延长而增加,且对应的惯性空化效应强度也随之增加。1 000和5 000周期组的溶栓率显著高于单纯超声组(P均<0.01)。结论长脉冲超声联合脂质体微泡能实现良好的溶栓效果,且在一定范围内溶栓的效果随着脉冲的延长而增加。惯性空化可能是长脉冲超声介导微泡溶栓的重要机制之一。     

脂质体微泡促进超声波溶栓作用的体外实验

目的在体外循环条件下，评价脂质体造影剂微泡对超声溶栓的影响及其与超声波频率、强度之间的关系。方法以健康成人静脉全血37℃恒温水浴孵育2h制备体外血栓80份。40份样本被分为单纯超声组、单纯微泡组、微泡＋超声组和对照组。辐照超声为2MHz-1．8W／cm^2、占空比95％的脉冲式超声波，脂质体微泡用量为50μl，各组处理时间为10min。比较各组溶栓率。其余血栓样本分为4组，加入50μl微泡后分别给予2MHz-0．7W／cm^2、2、MHz-1．4W／cm^2、1、MHz-0．7W／cm^2和0．5MHz-0．7W／cm^2的脉冲式超声波辐照10min。计算溶栓率与频率、声强之间的相关性。结果微泡＋超声组溶栓率明显高于单纯超声组（P〈0．05）、单纯微泡组（P〈0．01）和对照组（P〈0．01）。溶栓率与超声频率呈负相关（r1=1．000，P〈0．01），与声强呈正相关（r=-1．000，P〈0．01）。结论在体外循环条件下，脂质体微泡能显著增强超声波溶栓作用，且其效果与超声频率成反比，与声强成正比。     

超声微泡造影剂经导管直接溶栓治疗犬股静脉血栓的实验研究

目的研究超声联合微泡造影剂在体内的溶栓效应,并探讨其与尿激酶的协同作用。方法 13只健康犬建立血栓模型,分为对照组,尿激酶组,尿激酶加超声和微泡组,给予相应的治疗,应用超声多普勒,X线血管造影对血管的再通率进行评价,并进行病理学检查,比较各组溶栓效果。结果治疗后血管再通率尿激酶组与尿激酶加超声及微泡组均为100%,尿激酶加超声及微泡组再通等级优于尿激酶组。对照组血管再通率为0%,差异有统计学意义。光镜下观察,对照组股静脉管腔完全梗阻,血管壁见附壁血栓。尿激酶组、尿激酶加超声及微泡组股静脉管腔内部部分融通,局部区域内皮细胞脱落。结论超声联合微泡造影剂有一定的溶栓效应,与尿激酶共同应用,可以起协同作用。     

超声和超声微泡造影剂的溶栓作用

急性脑卒中是临床常见的严重急症，主要分为缺血性卒中和出血性卒中。目前对缺血性卒中治疗的主要方法是药物溶栓，早期快速的溶栓治疗能挽救生命，恢复生理功能。但由于治疗时间窗的限制和一些并发症的出现，如出血、血管再闭塞等，其疗效令人不甚满意。自近年发现超声作用可以促进血栓溶解以来，对超声及微泡造影剂溶栓作用的研究日益深入，     

微泡增强超声助溶兔股动脉血栓的实验研究

目的 探索自制脂膜微泡对超声助溶兔股动脉血栓的增强效果。 方法 对16只股动脉血栓模型兔进行分组溶栓，包括正常对照组、阳性对照组、单纯超声组及超声联合微泡组，采用CDFI、X-线血管造影进行再通率检测和血流再通情况评分，并进行病理光镜检测，比较各组溶栓效果。 结果 血管再通率显示阳性对照组无1例再通，单纯超声组溶栓治疗后血管再通率为25％，血流评分仅达Ⅰ级；超声联合微泡组血管再通率为87．5％，血流评分超过Ⅱ级。单纯超声组与超声联合微泡组再通率比较，差异有统计学意义（P〈0．05）。光镜下股动脉血栓呈完全梗阻型并以红细胞成分为主，伴部分纤维蛋白；单纯超声治疗后血栓内部结构部分变疏松；超声联合微泡治疗后股动脉管腔大部分溶通，仅残留少量附壁血栓。 结论 超声微泡能显著增强治疗超声对兔股动脉血栓的助溶作用。     

超声微泡造影剂的肿瘤靶向治疗研究进展

近几年,国内外着眼于靶向微泡造影剂的研究,利用超声波与微泡造影剂的相互作用及所产生的生物学效应,可实现微泡所携带的基因/药物等向目标组织的转移释放,介导肿瘤细胞的凋亡及肿瘤微血管的栓塞阻断等,从而起到靶向治疗的作用。随着各种兼具诊断治疗双重作用的超声探头和靶向微泡造影剂包括纳米级微泡造影剂的研制,以及对超声生物学效应的深入研究,超声微泡介导肿瘤靶向治疗将为临床肿瘤的治疗带来新的希望。     

超声联合微泡溶解体外血栓声像图改变与病理对照研究

目的观察超声联合微泡对体外血栓溶栓治疗后的声像图变化,对照病理结果,探讨溶栓治疗后血栓声像图与病理改变之间的关系。方法将DiO荧光分子探针标记脂膜微泡,联合1 MHz治疗超声进行血栓溶栓治疗10 min。分组:单纯超声辐照组;单纯微泡组;超声联合微泡组。对各组溶栓治疗后血栓进行超声显像并计算溶栓率。血栓标本HE染色后光镜下观察,微泡治疗组血栓制成冷冻切片在荧光显微镜下观察。结果超声联合微泡组溶栓率(29.51±5.17)%与单纯超声组(24.13±3.93)%、单纯微泡组(21.73±2.78)%比较差异显著(P<0.01,P<0.01)。超声显示治疗前血栓呈均质弱回声团块,溶栓治疗后单纯超声组血栓呈不均质弱回声伴内部少数强光点;单纯微泡组表面可见较多强光点,内部仍呈低回声;超声联合微泡组血栓呈不均质强回声团块。光镜下单纯超声组血栓内部可见几个小裂隙,而单纯微泡组血栓内结构较均一,未见裂隙,超声联合微泡组可见血栓表面空泡和内部不规则裂隙。荧光显微镜下显示单纯微泡组血栓表面呈明亮荧光带,内部几无荧光颗粒分布,超声联合微泡组血栓表面呈更为宽大明亮荧光带,内部的荧光颗粒分布较密集。结论溶栓治疗后血栓的超声声像图改变与病理学结果关系密切。     

The source of ultrasound contrast effect

Evidence that microbubbles are the main sources of ultrasound contrast in injected solutions has been largely indirect. To investigate this directly, we examined freshly agitated indocyanine green, freshly agitated water, commercially prepared precision microbubbles (diameter 75 +/- 25 mu) in gelatin, carbonated water, "degassed" indocyanine green solution, and "degassed" water in one or more of four different assay systems. Only fluids with microbubbles produced ultrasound contrast. Injected contrast material rose in a water bath at a rate that identified it as being caused by microbubbles. Indocyanine green and gelatin surface tensions were measured and found to be low (43 dynes/cm2), thus explaining their tendency to stabilize the microbubbles that cause ultrasound contrast effect when injected and to hold foam after agitation. The force of hand injections (force similar to that used clinically through catheters and 19-gauge or 23-gauge needles) was below the force needed to cause cavitation or ultrasound contrast effect. Microbubble content could be quantified by the decrease in amplitude of the echo from a structure distant to the microbubbles. We conclude that that the ultrasound contrast effect seen in peripherally injected fluids is caused by microbubbles present in the injectant. The contrast is not due to cavitation at needle tips, and it can be quantified over a limited range. Improved design for a peripheral contrast agent is suggest.     

超声微泡与辐射防护的相关研究进展

放射治疗是一种重要的医疗手段,其产生的电离辐射在发挥治疗效果的同时,也会对局部机体或全身造成不同程度的损伤。现有的辐射防护剂因价格高昂、具有副作用等因素应用受到限制。近年来,超声微泡作为药物等治疗性物质的理想载体,其载药特性及通过超声辐照爆破靶向药物传递的特性在生物医学领域有极大的应用潜力。本文就有望载入超声微泡进行抗辐射作用的药物和材料进行综述,为研发理想的新型抗辐射药物及方式提供参考。     

包膜微泡超声造影剂的研究进展

越来越多的研究表明,包膜微泡造影剂以其优越的显影和携载等功能而逐渐成为造影剂发展的主流.按膜材料的不同,包膜微泡造影剂可分为白蛋白类、非离子表面活性剂类、脂质体类和多聚体类.本文主要对各类造影剂的研究状况及优缺点进行了介绍,并经过比较得出结论:多聚体是目前乃至将来造影剂研制中最有前途的包膜材料.     

Self-assembled liposome-loaded microbubbles: The missing link for safe and efficient ultrasound triggered drug-delivery

Liposome-loaded microbubbles have been recently introduced as a promising drug delivery platform for ultrasound guided drug delivery. In this paper we design liposome-loaded (lipid-shelled) microbubbles through the simple self-assembly of the involved compounds in a single step process. We thoroughly characterized the liposome-loading of the microbubbles and evaluated the cell killing efficiency of this material using doxorubicin (DOX) as a model drug. Importantly, we observed that the DOX liposome-loaded microbubbles allowed killing of melanoma cells even at very low doses of DOX. These findings clearly prove the potential of liposome-loaded microbubbles for ultrasound targeted drug delivery to cancer tissues.     

诊断超声联合微泡对兔VX2肿瘤的血流增强效应

目的探讨不同机械指数(MI)的诊断超声联合微泡对兔VX_2肿瘤的血流增强效应。方法选取健康雄性新西兰白兔40只,采用单侧大腿内侧瘤组织块接种法接种VX_2肿瘤,造模成功后将其随机分为实验1组(MI=0.3)、实验2组(MI=0.7)、实验3组(MI=1.4)及对照组,每组各10只。抽取0.2 ml"脂氟显"用5.0 ml生理盐水稀释后经耳缘静脉通道匀速推入,同时分别经不同机械指数辐照肿瘤,对照组予以超声假照,时间均为5 min。储存治疗前后动态造影图像,并用造影分析软件分析,记录峰值强度(PI)和曲线下面积(AUC)。结果实验1组、实验3组治疗前后PI比较差异均有统计学意义(P=0.028、0.018),实验2组、对照组治疗前后PI比较差异无统计学意义(P=0.994、0.978);实验3组治疗前后AUC值比较差异有统计学意义(P=0.009),实验1、2组及对照组治疗前后AUC值比较差异均无统计学意义(P=0.099、0.497、0.898)。结论低能量诊断超声(MI=0.3)联合微泡可丰富兔VX_2肿瘤血供,高能量诊断超声(MI=1.4)可减少血流灌注。     

Microbubbles induce renal hemorrhage when exposed to diagnostic ultrasound in anesthetized rats

The generation of ultrasound (US) bioeffects using a clinical imaging system is controversial. We tested the hypothesis that the presence of microbubbles in the US field of a medical imager induces biologic effects. Both kidneys of anesthetized rats were insonified for 5 min using a medical imaging system after the administration of microbubbles. One kidney was insonified using a continuous mode (30 Hz) and the opposite kidney was insonified using an intermittent (1 Hz) technique. The microbubbles were exposed to three different transducer frequencies and four transducer output powers. After insonification, the animals were euthanized, the kidneys were removed and their gross appearance scored under "blinded" conditions using a defined scale. After the administration of microbubbles, US imaging of the kidney caused hemorrhage in the renal tissue. The severity and area of hemorrhage increased with an increase in the transducer power and a decrease in the transducer frequency. Intermittent insonification in the presence of microbubbles produced a greater degree of renal hemorrhage than continuous imaging techniques.     

Delivery of oligodeoxynucleotides into human saphenous veins and the adjunct effect of ultrasound and microbubbles

Therapy with naked oligodeoxynucleotides (ODNs, molecular weight: 3000 to 7500) provides an elegant means of modulating gene expression without the problems associated with conventional gene therapy, but the relatively low transfer efficiency on intravascular administration is a limitation to clinical application. Ultrasound, which can be potentiated by microbubbles, shows promise as a method of delivering macromolecules such as plasmid DNA and other transgenes into cells. Since uptake of molecules into cells depends on their molecular weight, it might be expected that the delivery of ODNs, which are relatively small, will be facilitated by ultrasound and microbubbles. In the present study, we delivered ODNs into veins using ultrasound and microbubbles. First, we quantified the uptake of fluorescent-labeled ODNs into intact ex vivo human saphenous veins and isolated smooth muscle cells from the veins, evaluating the effect of ultrasound and microbubbles on uptake. Ultrasound potentiated the delivery of ODN in cells, except at high concentrations. When intact veins were studied, we achieved nuclear localization of fluorescent-labeled ODNs in cells. This increased with increasing concentration and incubation time and was not potentiated by ultrasound, even when microbubbles were used. We then applied a therapeutic ODN (antisense to intercellular adhesion molecule 1, ICAM-1) to vein samples and documented a functional inhibition of gene expression in a sequence-specific manner at the protein level with immunohistochemistry and western blot analysis. Again, no significant difference was seen with adjunct ultrasound. These observations suggest high diffusion of ODNs into human saphenous veins in this ex vivo model, indicating potential applications to inhibition of vascular bypass graft occlusion and other vasculopathies. Although microbubble-ultrasound was of value with cells in culture, it was not beneficial with intact veins.     

Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles

The aim of this study was to assess the relative efficacy and mechanism of gene transfection by ultrasound (US) destruction of plasmid-bearing microbubbles. Luciferase reporter plasmid was charge-coupled to cationic lipid microbubbles. Rat hindlimb skeletal muscle was exposed to intermittent high-power US during dose-adjusted intra-arterial (IA) or IV administration of plasmid-bearing microbubbles via the carotid artery or jugular vein, respectively. At 4 days, luciferase activity in US-exposed skeletal muscle was 200-fold greater with IA than with IV administration of plasmid-bearing microbubbles, and was similar to transfection achieved by IM injection of plasmid (positive control). No transfection occurred with US and IA injection of plasmid alone. Intravital microscopy of the cremaster muscle in mice following administration of microbubbles and US exposure demonstrated perivascular deposition of fluorescent plasmid, the extent of which was twofold greater for IA compared to IV injection. Electron microscopy demonstrated a greater extent of myocellular microporations in US-exposed muscle after IA injection of microbubbles. We conclude that muscle transfection by US destruction of plasmid-bearing cationic microbubbles is amplified by IA, rather than IV, injection of microbubbles due to greater extravascular deposition of plasmid and to greater extent of myocellular microporation. (E-mail: jlindner@virginia.edu)     

微泡超声造影剂:一种新型的药物靶向载体

药物携载是目前重要的研究领域,如何使得药物安全、有效、靶向性地导入体内特定器官、组织并使其释放在靶细胞内是研究的重点.微泡超声造影剂作为一种新型的体内药物载体,受到国内外学者的广泛关注.本文对有关微泡超声造影剂作为药物载体的作用原理、制备要求、制备方法、特点和应用等方面的研究作了介绍.