血栓靶向脂质微泡的制备及其在动脉血栓检测中的应用
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摘要
研究背景：
随着社会的发展和人口老龄化的进程，血栓栓塞性疾病的发病率逐年增加。据统计，全球每年有1500万人死于血栓栓塞性疾病，已经成为全球总死亡率的第一位。我国每年的发病人数有1000万，病死人数有100万，致残率也很高。现在人类正面临着血栓栓塞性疾病的巨大挑战。
因而临床上早期准确地检出并治疗血栓对于降低死亡率及并发症发生率非常重要。数字减影血管造影(DSA)一直是诊断血管病变的金标准，但DSA是有创性的检查，有2％—4％的并发症，而且价格昂贵。磁共振血管成像(MRA)、CT血管成像(CTA)虽能较好的显示血管及其出血，但费用高，而且在显示较小的血栓方面有较大的局限。超声是一种被广泛应用于临床的影像检测手段，对血栓性疾病的早期诊断与随访观察具有重要的意义。但由于新鲜血栓的回声与血液回声基本相似，超声对新鲜血栓的检出率受到限制。
1968年美国的Gramiak首先提出超声对比显影的概念，他们在心内注射吲哚青蓝染料(indocyanine green dye)后超声心动图上可见云雾状声影。他们将产生的对比效果归因为注射吲哚青蓝染料溶液产生的气泡。从那以后，吲哚青蓝染料成为标准的超声造影剂。到目前已经发展到了第三代。第一代微泡的充填气体为空气，并实现了外周静脉注射，是微泡超声造影剂发展的一个里程碑，但它在血中维持时间短，不能满足临床的要求；第二代微泡充填气体为氟碳气体，稳定性好，是临床应用的主要产品；第三代微泡是一种靶向微泡，可以实现组织的定位显影，目前国内外尚处于实验室研究阶段。故靶向超声造影剂已成为近年国内外研究的热点，有良好的研究前景和巨大的商业价值。因此我们的课题组就期待制备出一种亲血栓性靶向超声造影剂，以改善血栓的超声显影效果，从而提高超声检出新鲜血栓的准确性和敏感性。
研究目的：
研究制备血栓靶向性脂质微泡超声造影剂的方法，测定其生物学活性，通过目测及视频分析法评价该靶向超声造影剂增强兔颈总动脉新鲜血栓显像的效果。
研究方法：
一、普通脂质氟碳微泡的制备及生物学活性的测定
将二棕榈酰磷脂酰胆碱(DPPC)、二棕榈酰磷脂酰甘油(DPPG)、二棕榈酰磷脂酰乙醇胺(DPPE)、胆固醇(Cholesterol)按一定摩尔比例经薄膜-水化法制备成脂质微泡。用普通显微镜观测微泡的稳定性，用Zetasizer Nano ZS90测量微泡的粒径及表面电位，采用血细胞计数仪检测微泡的浓度。
二、血栓靶向脂质氟碳微泡的制备及生物学活性的测定
将荧光标记二棕榈酰磷脂酰胆碱(NBD PC)、DPPG、生物素标记二棕榈酰磷脂酰乙醇胺(Biotinyl PE)、Cholesterol按一定摩尔比例经薄膜-水化法制备成脂质微泡。用普通显微镜及荧光显微镜观测微泡的外观和稳定性，用Zetasizer Nano ZS90测微泡的粒径及表面电位，采用血细胞计数仪检测微泡的浓度。
三、动物血栓模型的建立及稳定性的评价
本实验采取化学损伤-FeCl_3诱导的方法来制备兔颈总动脉血栓模型，取九只家兔根据FeCl_3浸润时间随机分成三组：20min(第1组)、40min(第2组)、60min(第3组)每组各三只，实验完成后取血管段进行石蜡包埋、HE染色作病理检测来评价血栓模型。
四、不同的检测方法对血栓模型的影像学评价
血栓模型的建立：取家兔二十只，游离家兔右侧颈总动脉约2cm，模型建立前用彩色多普勒血流显像((Color Doppler Flow Imaging，CDFI)来获取正常血管的超声图像并储存，然后采用70%的FeCl_3溶液浸润40min，诱发右侧颈总动脉非梗阻性血栓形成。用CDFI来检测模型建立后的血栓超声图像并储存。注入造影剂的超声造影：经耳缘静脉团注0．5ml／kg普通脂质微泡，连续观察10min；爆破残留的微泡5min后再团注0．5ml／kg Sono Vue，连续观察10min；爆破残留的微泡5min后再依次注入生物素化的兔抗人纤维蛋白原0．1ml(1：200稀释)、30min后注入0．1ml亲和素(1：20稀释)、30min后团注0．5ml／kg靶向脂质微泡超声造影剂，连续观察10min；造影前后适时动态图像储存。血栓模型的MRI检测：超声检查完成后将模型进行三维时间飞越法磁共振血管成像Three-Dimensional Time-Of-Flight Magnetic Resonance Angiography，3D TOF MRA)并储存图像。造影结果评价：分别让五位不同的超声科医师对视频图像进行影像学的评价。
五、血栓靶向脂质微泡的体内外靶向结合实验
取家兔四只用同样的方法建立颈总动脉非梗阻性血栓模型，随机选取两只家兔取下病变血管段置于2ml生理盐水中依次加入生物素化的兔抗人纤维蛋白原0．1ml(1：200稀释)、30min后加入0．1ml亲和素(1：20稀释)、30min后加入0．5ml／kg靶向脂质微泡超声造影剂，将一部分标本快速冷冻切片后作荧光检测，将另一部分标本做透射电镜检测，观察靶向脂质微泡在血栓内的分布情况。另外两只家兔经耳缘静脉依次注入生物素化的兔抗人纤维蛋白原0．1ml(1：200稀释)、30min后注入0．1ml亲和素(1：20稀释)、30min后团注0．5ml／kg靶向脂质微泡超声造影剂，将一部分标本快速冷冻切片后作荧光检测，将另一部分标本做透射电镜检测，观察靶向脂质微泡在血栓内的分布情况。
研究结果：
一、普通脂质氟碳微泡的制备及生物学活性的测定
普通脂质氟碳微泡制备后外观呈乳白色，静置后可见微泡浮于液面之上，平均粒径为0．8-2．5μm，部分微泡的粒径达到了纳米级。微泡表面的Zeta电位(Zeta Potential)约为-11 mV，微泡浓度约为1．08×10~(10)个／mL。室温下放置一周左右微泡的粒径及表面的Zeta电位无明显的变化，但微泡的浓度有所降低，室温下放置半个月左右显微镜下只可见微泡数量明显减少，室温下放置一个月左右显微镜下只可见散在的微泡。
二、血栓靶向脂质氟碳微泡的制备及生物学活性的测定
血栓靶向脂质氟碳微泡的制备后外观呈橘黄色，静置后可见微泡浮于液面之上，平均粒径为1．1-2．5μm，微泡表面的平均Zeta电位(Zeta Potential)约为-11 mV，微泡浓度为6．5×10~9个／mL。室温下放置一周左右微泡的粒径及表面的Zeta电位无明显的变化，但微泡的浓度明显降低，室温下放置半个月左右显微镜下只可见散在的微泡存在，其稳定性比普通的脂质微泡相比略差。
三、动物血栓模型的建立及稳定性的评价
肉眼观：第1组模型血管管腔内未见明显的血栓形成。第2组模型血管官腔可见非梗阻性血栓形成，血栓约占管腔1／4-1／3。第3组模型可见梗阻闭塞性血栓形成，血栓占据整个管腔。镜下观：第2、3组为混合型血栓，血栓结构致密。
四、不同的检测方法对血栓模型的影像学评价
实验的二十只家兔建模型前CDFI显示右侧颈总动脉血流信号通畅，管腔完整。模型建立后CDFI图像只有四只家兔血管中可见较为明显的充盈缺损及血流信号的不连续，其余的图像显示欠佳。普通脂质微泡团注后3s在右侧颈总动脉腔内显影，10s左右显影达到峰值，可见血栓局部管腔内明显的充盈缺损，管壁欠光滑，管腔内血流信号不连续，显影持续60s左右，只有十只家兔均显影尚可，其余图像显示欠佳。SonoVue团注后3s在右侧颈总动脉腔内显影，20s左右显影达到峰值，可见血栓局部管腔内明显的充盈缺损，管壁欠光滑，管腔内血流信号不连续，显影持续150s左右，二十只家兔均显影良好。靶向脂质微泡团注后3s在右侧颈总动脉腔内显影，25s左右显影达到峰值，可见血栓局部管腔内明显的充盈缺损，管壁欠光滑，管腔内血流信号不连续，显影持续200s左右，二十只家兔均显影良好。3D-TOF-MRA的图像只有三只家兔右侧颈总动脉血栓局部血流信号变窄，管壁欠光滑，其余的图像显示欠佳。
五、血栓靶向脂质微泡的体内外寻靶实验
体内外两组实验快速冰冻切片后荧光显微镜观察均显示管腔内血栓局部有靶向脂质微泡聚集带，部分微泡聚集在血栓内部，不同的是体外实验组靶向脂质微泡聚集量较体内实验组较多，发出的荧光更明显。体内外两组实验电镜图片均显示血栓内有靶向脂质微泡聚集，体外实验组靶向脂质微泡聚集量仍较体内实验组为多。
研究结论：
一、本实验初步成功制备了血栓靶向脂质微泡并成功的将其应用于血栓的靶向性检测上，为国内开发自主的靶向超声造影剂奠定了良好的基础，有良好的研究前景和巨大的商业价值。
二、血栓靶向脂质微泡在血栓的超声检测中比普通脂质微泡、CDFI、MRA有明显的优势，其影像学表现清楚直观，造影时间长。
三、体内外实验表明，血栓靶向脂质微泡在体内外可以较快在新鲜血栓部位实现较高浓度聚集，并可深入到血凝块内部。不仅实现了血栓的靶向检测而且为进一步研究血栓的靶向溶栓治疗提供了理论基础。
Background:
The morbility of thromboembolic disease is increasing year by year due to the rapiddevelopment of the society and the intensified process of aging.It has been estimated thatabout 15 million people die of such disease each year globally,which has become the firstcause of the global mortality.Every year the morbility is about 10 million and the mortalityis 1 million in our country.In a word,we have faced with great challenges against thethromboembolic disease.Thus,detecting and treating thrombus exactly as early as possibleare of great significance for decreasing mortality and morbility of complications.
Although DSA is the primary imaging modality for detecting vascular lesions,it isinvasive,expensive and may cause complications at 2%-4% possibility.MRA and CTAmay show vessels and the thrombus better,but they are limited in manifesting smallthrombus.As a widely clinically applied imaging detected means,ultrasound is importantin the early diagnosis and follow-up observation of thrombus diseases.However,it islimited in the fresh thrombus detection due to the similarity of resonance between freshthrombus and blood.
In 1968,Gramiak first proposed the concept of ultrasound contrast imaging.Theyobserved blooming artifacts in echocardiogram after intracardial injection of indocyaninegreen dye and attributed the effect to the gas bubbles generated by injecting the green dye.Thus,indocyanine green dye has become the standard ultrasound contrast agent anddeveloped three generations currently.The first generation is a milestone in the contrastagent development,such microbubbles were filled with air and could be injected viaperipheral vein.But it could not be used clinically due to the short time maintenance in the blood.The second generation was filled with perfluorocarbon gas,has great stability andhas been widely applied clinically.The third generation is reported as targeted microbubbleand can be imaged in localized tissue.It is now in the stage of lab experiment domestically.Therefore,targeted contrast agent has become the hot spot in the few future and has broadprospect as well as potential market value.So we are expected to prepare suchthrombus-targeted contrast agent to improve the image effects of thrombus and increase theaccuracy and sensitivity
Objective:
The purpose of this study was designed to prepare a thrombus-targetedphospholipid-coated microbubbles,investigate its biologic activity and evaluate itsimaging effect on the enhancement on acute thrombus made in carotid artery of rabbitthrough visual examination and video analysis.
Methods:
1.To prepare common lipid-coated fluorocarbon microbubble and determinate itsbiologic activity
DPPC,DPPG,DPPE,cholesterol were prepared as phospholipid-coated microbubblefollowing membrane-hydration method.The stability of microbubbles was observed bylight microscope,the diameter and surface potential were measured by Zetasizer NanoZS90 and the concentration was detected by blood cell count machines.
2.To prepare thrombus-targeted lipid-coated fluorocarbon microbubbles anddeterminate its biologic activity
NBD-PC,DPPG,biotinyl PE,cholesterol were prepared as phospholipid-coatedmicrobubble following membrane-hydration method.The stability and appearance ofmicrobubbles were observed by light and fluorescent microscope,the diameter and surfacepotential were measured by Zetasizer Nano ZS90 and the concentration were detected byblood cell count machines.
3.To establish thrombus model and evaluate its stability
Rabbit thrombus model made by FeCl_3 infiltration in common carotid artery of rabbitwas set up.Six rabbits were randomly invided into three groups according to FeCl_3infiltration time:20min (the first group),40min (the second group),60min (the third group).After sacrificing the animals,vessels were obtained for HE staining to evaluate the thrombus model.
4.To evaluate the thrombus model by various imaging means
The establishment of thrombus model:Normal vessel ultrasound imaging wereobtained and saved by CDFI (Color Doppler Flow Imaging) before experiment.Rightcommon carotid artery of 20 healthy rabbits dissociated about 2cm were infiltrated byFeCl_3 about 30min to induce the formation of non-obstuctive thrombus.Thrombusultrasound imaging was obtained and saved by CDFI after the establishment of thrombusmodel.Ultrasound contrast imaging after contrast agent injection:common lipidmicrobubble through ear via intravenous bolus injection at a dose of 0.5ml/kg was observedconsecutively about 10 min,then Sono Vue at a dose of 0.5ml/kg was injected again andobserved 10min after clearing the residual microbubbles.Finally,we injected thebiotinylated rabbit anti-human fibrinogen (diluted as 1:200) 0.1ml,30min later,avidin(diluted as 1:20) 0.1ml and after 30 min,injected targeted lipid-coated contrast agent at adose of 0.5ml/kg,then we observed about 10min consecutively.MRI evaluation:thethrombus model was imaged and saved by 3D-TOF-MRA (3D-Time-Of-Flight MagneticResonance Angiography).The video images were assessed qualitatively by fiveindependent observers.
5.Thrombus-targeted lipid-coated microbubbles' experiment in vivo and in vitro
Four rabbits were established non-obstructive thrombus model following the same way.Two rabbits' artery were placed in 2ml saline and we added the biotinylated rabbitanti-human fibrinogen (diluted as 1:200) 0.1ml,30min later avidin (diluted as 1:20) 0.1mland after 30min,added targeted lipid-coated contrast agent at a dose of 0.5ml/kg in theartery by order,then we frozen some of samples rapidly for fluorescence detection anddetected a part of samples by transmission electron microscope to observe the division ofmicrobubbles in the thrombus.Another two rabbits were injected the biotinylated rabbitanti-human fibrinogen (diluted as 1:200) 0.1 ml,30min late avidin (diluted as 1:20) 0.1mland after 30 min,injected targeted lipid-coated contrast agent at a dose of 0.5ml/kg via earvein by order,then we frozen some of samples rapidly for fluorescence detection anddetected a part of samples by transmission electron microscope to observe the division ofmicrobubble in the thrombus.
Results:
1.To prepare common lipid-coated fluorocarbon microbubble and determinate itsbiologic activity
Common lipid-coated fluorocarbon microbubble showed white appearance and theaverage diameter ranged from 0.8-2.5 um,some of which arrived at nm stage.The zetaelectric potential was about-11mv and the concentration was about 1.08×10~(10)/mL.Thediameter and potentials were changed little,while concentration has decreased after a weekunder room temperature.Amounts of microbubbles were decreased obviously after abouthalf a month,while after about one month,just a few scattered microbubbles were observedunder the microscope.
2.To prepare thrombus-targeted lipid-coated fluorocarbon microbubbles anddeterminate its biologic activity
Thrombus-targeted lipid-coated fluorocarbon microbubbles showed yellow appearanceand the average diameter ranged from 1.1-2.5 um.The zeta electric potential was about-11mv and the concentration was about 6.5×10~9/ml.The diameter and potentials werechanged little,while concentration has decreased after a week under room temperature.Justa few scattered microbubbles were observed under the microscope after about a month andits stability was a little weak than common lipid-coated microbubble.
3.To establish the thrombus model and evaluate its stability
Gross appearance:the first group:no significant thrombosis;the second group:non-obstructive thrombosis and thrombus holds about 1/3-1/4 of vessel;The third group:obstructive-occluding thrombosis and thrombus holds the total vessel.Microscope:Thesecond and third group could be imaged as mixed thrombus.
4.To evaluate the thrombus model by various imaging means
CDFI showed smooth signals and completed lumens in the right common carotidartery of 20 rabbits before the establishment of thrombus model,while only 4 rabbits'arteries were detected as obvious filling defect and inconsecutive blood signals by CDFI,the rest showed worse image in the established thrombus model.Common lipid-coatedmicrobubble developed an image in the right common carotid artery 3 second afterintravenous bolus injection and such image persisted about 60 second,arrived at peak inabout 10 second.Just 10 rabbits' ultrasound showed obvious filling defect,inconsecutive blood signals and insufficient smooth vessel wall in local lumens.SonoVue developed animage in the right common carotid artery 3 second after intravenous bolus injection andsuch image persisted about 150 second,arrived at peak in about 20 second.Ultrasoundshowed the same image as above.Targeted lipid-coated microbubbles developed an imagein the right common carotid artery 3 second after intravenous bolus injection and suchimage persisted about 200 second,arrived at peak in about 25 second.Ultrasound showedthe same image as above.As compared with above,3D-TOF-MRA showed inconsecutiveblood signals and insufficient smooth vessel wall in local lumens in only 3 rabbits,the restshowed worse.
5.Thrombus-targeted lipid-coated microbubbles' experiment in vivo and in vitro
Massive targeted lipid-coated microbubbles flocked around and some of themaggregated within the fresh blood clots under light and fluorescence microscope after rapidfrozen sections both in vivo and in vitro.While amounts of aggregated microbubbles invitro were larger than that in vivo,the fluorescence ejected in vitro was more obvious thanin vivo.The electron microscope showed the same image.
Conclusions:
1.The preparation of thrombus-targeted phospholipid-coated microbubbles and itsapplication in the arterial thrombus-targeted detection have been successfully performed,which establish a sound foundation for exploitation of targeted contrast agent domesticallyas well as provide the potential market value.
2.The imaging of thrombus-targeted phospholipid-coated microbubbles wasperformed more direct-viewing and longer time than common lipid microbubbles,CDFIand MRA.
3.Thrombus-targeted phospholipid-coated microbubbles could aggregate rapidly withhigher concentration in fresh blood clot and deep into the internal part both in vitro and invivo.Thus,it may realize the targeted thrombus detection as well as provide the theoreticfoundation for investigating targeted thrombolysis therapy.
随着社会的发展和人口老龄化的进程，血栓栓塞性疾病的发病率逐年增加。据统计，全球每年有1500万人死于血栓栓塞性疾病，已经成为全球总死亡率的第一位。我国每年的发病人数有1000万，病死人数有100万，致残率也很高。现在人类正面临着血栓栓塞性疾病的巨大挑战。
因而临床上早期准确地检出并治疗血栓对于降低死亡率及并发症发生率非常重要。数字减影血管造影(DSA)一直是诊断血管病变的金标准，但DSA是有创性的检查，有2％—4％的并发症，而且价格昂贵。磁共振血管成像(MRA)、CT血管成像(CTA)虽能较好的显示血管及其出血，但费用高，而且在显示较小的血栓方面有较大的局限。超声是一种被广泛应用于临床的影像检测手段，对血栓性疾病的早期诊断与随访观察具有重要的意义。但由于新鲜血栓的回声与血液回声基本相似，超声对新鲜血栓的检出率受到限制。
1968年美国的Gramiak首先提出超声对比显影的概念，他们在心内注射吲哚青蓝染料(indocyanine green dye)后超声心动图上可见云雾状声影。他们将产生的对比效果归因为注射吲哚青蓝染料溶液产生的气泡。从那以后，吲哚青蓝染料成为标准的超声造影剂。到目前已经发展到了第三代。第一代微泡的充填气体为空气，并实现了外周静脉注射，是微泡超声造影剂发展的一个里程碑，但它在血中维持时间短，不能满足临床的要求；第二代微泡充填气体为氟碳气体，稳定性好，是临床应用的主要产品；第三代微泡是一种靶向微泡，可以实现组织的定位显影，目前国内外尚处于实验室研究阶段。故靶向超声造影剂已成为近年国内外研究的热点，有良好的研究前景和巨大的商业价值。因此我们的课题组就期待制备出一种亲血栓性靶向超声造影剂，以改善血栓的超声显影效果，从而提高超声检出新鲜血栓的准确性和敏感性。
研究目的：
研究制备血栓靶向性脂质微泡超声造影剂的方法，测定其生物学活性，通过目测及视频分析法评价该靶向超声造影剂增强兔颈总动脉新鲜血栓显像的效果。
研究方法：
一、普通脂质氟碳微泡的制备及生物学活性的测定
将二棕榈酰磷脂酰胆碱(DPPC)、二棕榈酰磷脂酰甘油(DPPG)、二棕榈酰磷脂酰乙醇胺(DPPE)、胆固醇(Cholesterol)按一定摩尔比例经薄膜-水化法制备成脂质微泡。用普通显微镜观测微泡的稳定性，用Zetasizer Nano ZS90测量微泡的粒径及表面电位，采用血细胞计数仪检测微泡的浓度。
二、血栓靶向脂质氟碳微泡的制备及生物学活性的测定
将荧光标记二棕榈酰磷脂酰胆碱(NBD PC)、DPPG、生物素标记二棕榈酰磷脂酰乙醇胺(Biotinyl PE)、Cholesterol按一定摩尔比例经薄膜-水化法制备成脂质微泡。用普通显微镜及荧光显微镜观测微泡的外观和稳定性，用Zetasizer Nano ZS90测微泡的粒径及表面电位，采用血细胞计数仪检测微泡的浓度。
三、动物血栓模型的建立及稳定性的评价
本实验采取化学损伤-FeCl_3诱导的方法来制备兔颈总动脉血栓模型，取九只家兔根据FeCl_3浸润时间随机分成三组：20min(第1组)、40min(第2组)、60min(第3组)每组各三只，实验完成后取血管段进行石蜡包埋、HE染色作病理检测来评价血栓模型。
四、不同的检测方法对血栓模型的影像学评价
血栓模型的建立：取家兔二十只，游离家兔右侧颈总动脉约2cm，模型建立前用彩色多普勒血流显像((Color Doppler Flow Imaging，CDFI)来获取正常血管的超声图像并储存，然后采用70%的FeCl_3溶液浸润40min，诱发右侧颈总动脉非梗阻性血栓形成。用CDFI来检测模型建立后的血栓超声图像并储存。注入造影剂的超声造影：经耳缘静脉团注0．5ml／kg普通脂质微泡，连续观察10min；爆破残留的微泡5min后再团注0．5ml／kg Sono Vue，连续观察10min；爆破残留的微泡5min后再依次注入生物素化的兔抗人纤维蛋白原0．1ml(1：200稀释)、30min后注入0．1ml亲和素(1：20稀释)、30min后团注0．5ml／kg靶向脂质微泡超声造影剂，连续观察10min；造影前后适时动态图像储存。血栓模型的MRI检测：超声检查完成后将模型进行三维时间飞越法磁共振血管成像Three-Dimensional Time-Of-Flight Magnetic Resonance Angiography，3D TOF MRA)并储存图像。造影结果评价：分别让五位不同的超声科医师对视频图像进行影像学的评价。
五、血栓靶向脂质微泡的体内外靶向结合实验
取家兔四只用同样的方法建立颈总动脉非梗阻性血栓模型，随机选取两只家兔取下病变血管段置于2ml生理盐水中依次加入生物素化的兔抗人纤维蛋白原0．1ml(1：200稀释)、30min后加入0．1ml亲和素(1：20稀释)、30min后加入0．5ml／kg靶向脂质微泡超声造影剂，将一部分标本快速冷冻切片后作荧光检测，将另一部分标本做透射电镜检测，观察靶向脂质微泡在血栓内的分布情况。另外两只家兔经耳缘静脉依次注入生物素化的兔抗人纤维蛋白原0．1ml(1：200稀释)、30min后注入0．1ml亲和素(1：20稀释)、30min后团注0．5ml／kg靶向脂质微泡超声造影剂，将一部分标本快速冷冻切片后作荧光检测，将另一部分标本做透射电镜检测，观察靶向脂质微泡在血栓内的分布情况。
研究结果：
一、普通脂质氟碳微泡的制备及生物学活性的测定
普通脂质氟碳微泡制备后外观呈乳白色，静置后可见微泡浮于液面之上，平均粒径为0．8-2．5μm，部分微泡的粒径达到了纳米级。微泡表面的Zeta电位(Zeta Potential)约为-11 mV，微泡浓度约为1．08×10~(10)个／mL。室温下放置一周左右微泡的粒径及表面的Zeta电位无明显的变化，但微泡的浓度有所降低，室温下放置半个月左右显微镜下只可见微泡数量明显减少，室温下放置一个月左右显微镜下只可见散在的微泡。
二、血栓靶向脂质氟碳微泡的制备及生物学活性的测定
血栓靶向脂质氟碳微泡的制备后外观呈橘黄色，静置后可见微泡浮于液面之上，平均粒径为1．1-2．5μm，微泡表面的平均Zeta电位(Zeta Potential)约为-11 mV，微泡浓度为6．5×10~9个／mL。室温下放置一周左右微泡的粒径及表面的Zeta电位无明显的变化，但微泡的浓度明显降低，室温下放置半个月左右显微镜下只可见散在的微泡存在，其稳定性比普通的脂质微泡相比略差。
三、动物血栓模型的建立及稳定性的评价
肉眼观：第1组模型血管管腔内未见明显的血栓形成。第2组模型血管官腔可见非梗阻性血栓形成，血栓约占管腔1／4-1／3。第3组模型可见梗阻闭塞性血栓形成，血栓占据整个管腔。镜下观：第2、3组为混合型血栓，血栓结构致密。
四、不同的检测方法对血栓模型的影像学评价
实验的二十只家兔建模型前CDFI显示右侧颈总动脉血流信号通畅，管腔完整。模型建立后CDFI图像只有四只家兔血管中可见较为明显的充盈缺损及血流信号的不连续，其余的图像显示欠佳。普通脂质微泡团注后3s在右侧颈总动脉腔内显影，10s左右显影达到峰值，可见血栓局部管腔内明显的充盈缺损，管壁欠光滑，管腔内血流信号不连续，显影持续60s左右，只有十只家兔均显影尚可，其余图像显示欠佳。SonoVue团注后3s在右侧颈总动脉腔内显影，20s左右显影达到峰值，可见血栓局部管腔内明显的充盈缺损，管壁欠光滑，管腔内血流信号不连续，显影持续150s左右，二十只家兔均显影良好。靶向脂质微泡团注后3s在右侧颈总动脉腔内显影，25s左右显影达到峰值，可见血栓局部管腔内明显的充盈缺损，管壁欠光滑，管腔内血流信号不连续，显影持续200s左右，二十只家兔均显影良好。3D-TOF-MRA的图像只有三只家兔右侧颈总动脉血栓局部血流信号变窄，管壁欠光滑，其余的图像显示欠佳。
五、血栓靶向脂质微泡的体内外寻靶实验
体内外两组实验快速冰冻切片后荧光显微镜观察均显示管腔内血栓局部有靶向脂质微泡聚集带，部分微泡聚集在血栓内部，不同的是体外实验组靶向脂质微泡聚集量较体内实验组较多，发出的荧光更明显。体内外两组实验电镜图片均显示血栓内有靶向脂质微泡聚集，体外实验组靶向脂质微泡聚集量仍较体内实验组为多。
研究结论：
一、本实验初步成功制备了血栓靶向脂质微泡并成功的将其应用于血栓的靶向性检测上，为国内开发自主的靶向超声造影剂奠定了良好的基础，有良好的研究前景和巨大的商业价值。
二、血栓靶向脂质微泡在血栓的超声检测中比普通脂质微泡、CDFI、MRA有明显的优势，其影像学表现清楚直观，造影时间长。
三、体内外实验表明，血栓靶向脂质微泡在体内外可以较快在新鲜血栓部位实现较高浓度聚集，并可深入到血凝块内部。不仅实现了血栓的靶向检测而且为进一步研究血栓的靶向溶栓治疗提供了理论基础。
Background:
The morbility of thromboembolic disease is increasing year by year due to the rapiddevelopment of the society and the intensified process of aging.It has been estimated thatabout 15 million people die of such disease each year globally,which has become the firstcause of the global mortality.Every year the morbility is about 10 million and the mortalityis 1 million in our country.In a word,we have faced with great challenges against thethromboembolic disease.Thus,detecting and treating thrombus exactly as early as possibleare of great significance for decreasing mortality and morbility of complications.
Although DSA is the primary imaging modality for detecting vascular lesions,it isinvasive,expensive and may cause complications at 2%-4% possibility.MRA and CTAmay show vessels and the thrombus better,but they are limited in manifesting smallthrombus.As a widely clinically applied imaging detected means,ultrasound is importantin the early diagnosis and follow-up observation of thrombus diseases.However,it islimited in the fresh thrombus detection due to the similarity of resonance between freshthrombus and blood.
In 1968,Gramiak first proposed the concept of ultrasound contrast imaging.Theyobserved blooming artifacts in echocardiogram after intracardial injection of indocyaninegreen dye and attributed the effect to the gas bubbles generated by injecting the green dye.Thus,indocyanine green dye has become the standard ultrasound contrast agent anddeveloped three generations currently.The first generation is a milestone in the contrastagent development,such microbubbles were filled with air and could be injected viaperipheral vein.But it could not be used clinically due to the short time maintenance in the blood.The second generation was filled with perfluorocarbon gas,has great stability andhas been widely applied clinically.The third generation is reported as targeted microbubbleand can be imaged in localized tissue.It is now in the stage of lab experiment domestically.Therefore,targeted contrast agent has become the hot spot in the few future and has broadprospect as well as potential market value.So we are expected to prepare suchthrombus-targeted contrast agent to improve the image effects of thrombus and increase theaccuracy and sensitivity
Objective:
The purpose of this study was designed to prepare a thrombus-targetedphospholipid-coated microbubbles,investigate its biologic activity and evaluate itsimaging effect on the enhancement on acute thrombus made in carotid artery of rabbitthrough visual examination and video analysis.
Methods:
1.To prepare common lipid-coated fluorocarbon microbubble and determinate itsbiologic activity
DPPC,DPPG,DPPE,cholesterol were prepared as phospholipid-coated microbubblefollowing membrane-hydration method.The stability of microbubbles was observed bylight microscope,the diameter and surface potential were measured by Zetasizer NanoZS90 and the concentration was detected by blood cell count machines.
2.To prepare thrombus-targeted lipid-coated fluorocarbon microbubbles anddeterminate its biologic activity
NBD-PC,DPPG,biotinyl PE,cholesterol were prepared as phospholipid-coatedmicrobubble following membrane-hydration method.The stability and appearance ofmicrobubbles were observed by light and fluorescent microscope,the diameter and surfacepotential were measured by Zetasizer Nano ZS90 and the concentration were detected byblood cell count machines.
3.To establish thrombus model and evaluate its stability
Rabbit thrombus model made by FeCl_3 infiltration in common carotid artery of rabbitwas set up.Six rabbits were randomly invided into three groups according to FeCl_3infiltration time:20min (the first group),40min (the second group),60min (the third group).After sacrificing the animals,vessels were obtained for HE staining to evaluate the thrombus model.
4.To evaluate the thrombus model by various imaging means
The establishment of thrombus model:Normal vessel ultrasound imaging wereobtained and saved by CDFI (Color Doppler Flow Imaging) before experiment.Rightcommon carotid artery of 20 healthy rabbits dissociated about 2cm were infiltrated byFeCl_3 about 30min to induce the formation of non-obstuctive thrombus.Thrombusultrasound imaging was obtained and saved by CDFI after the establishment of thrombusmodel.Ultrasound contrast imaging after contrast agent injection:common lipidmicrobubble through ear via intravenous bolus injection at a dose of 0.5ml/kg was observedconsecutively about 10 min,then Sono Vue at a dose of 0.5ml/kg was injected again andobserved 10min after clearing the residual microbubbles.Finally,we injected thebiotinylated rabbit anti-human fibrinogen (diluted as 1:200) 0.1ml,30min later,avidin(diluted as 1:20) 0.1ml and after 30 min,injected targeted lipid-coated contrast agent at adose of 0.5ml/kg,then we observed about 10min consecutively.MRI evaluation:thethrombus model was imaged and saved by 3D-TOF-MRA (3D-Time-Of-Flight MagneticResonance Angiography).The video images were assessed qualitatively by fiveindependent observers.
5.Thrombus-targeted lipid-coated microbubbles' experiment in vivo and in vitro
Four rabbits were established non-obstructive thrombus model following the same way.Two rabbits' artery were placed in 2ml saline and we added the biotinylated rabbitanti-human fibrinogen (diluted as 1:200) 0.1ml,30min later avidin (diluted as 1:20) 0.1mland after 30min,added targeted lipid-coated contrast agent at a dose of 0.5ml/kg in theartery by order,then we frozen some of samples rapidly for fluorescence detection anddetected a part of samples by transmission electron microscope to observe the division ofmicrobubbles in the thrombus.Another two rabbits were injected the biotinylated rabbitanti-human fibrinogen (diluted as 1:200) 0.1 ml,30min late avidin (diluted as 1:20) 0.1mland after 30 min,injected targeted lipid-coated contrast agent at a dose of 0.5ml/kg via earvein by order,then we frozen some of samples rapidly for fluorescence detection anddetected a part of samples by transmission electron microscope to observe the division ofmicrobubble in the thrombus.
Results:
1.To prepare common lipid-coated fluorocarbon microbubble and determinate itsbiologic activity
Common lipid-coated fluorocarbon microbubble showed white appearance and theaverage diameter ranged from 0.8-2.5 um,some of which arrived at nm stage.The zetaelectric potential was about-11mv and the concentration was about 1.08×10~(10)/mL.Thediameter and potentials were changed little,while concentration has decreased after a weekunder room temperature.Amounts of microbubbles were decreased obviously after abouthalf a month,while after about one month,just a few scattered microbubbles were observedunder the microscope.
2.To prepare thrombus-targeted lipid-coated fluorocarbon microbubbles anddeterminate its biologic activity
Thrombus-targeted lipid-coated fluorocarbon microbubbles showed yellow appearanceand the average diameter ranged from 1.1-2.5 um.The zeta electric potential was about-11mv and the concentration was about 6.5×10~9/ml.The diameter and potentials werechanged little,while concentration has decreased after a week under room temperature.Justa few scattered microbubbles were observed under the microscope after about a month andits stability was a little weak than common lipid-coated microbubble.
3.To establish the thrombus model and evaluate its stability
Gross appearance:the first group:no significant thrombosis;the second group:non-obstructive thrombosis and thrombus holds about 1/3-1/4 of vessel;The third group:obstructive-occluding thrombosis and thrombus holds the total vessel.Microscope:Thesecond and third group could be imaged as mixed thrombus.
4.To evaluate the thrombus model by various imaging means
CDFI showed smooth signals and completed lumens in the right common carotidartery of 20 rabbits before the establishment of thrombus model,while only 4 rabbits'arteries were detected as obvious filling defect and inconsecutive blood signals by CDFI,the rest showed worse image in the established thrombus model.Common lipid-coatedmicrobubble developed an image in the right common carotid artery 3 second afterintravenous bolus injection and such image persisted about 60 second,arrived at peak inabout 10 second.Just 10 rabbits' ultrasound showed obvious filling defect,inconsecutive blood signals and insufficient smooth vessel wall in local lumens.SonoVue developed animage in the right common carotid artery 3 second after intravenous bolus injection andsuch image persisted about 150 second,arrived at peak in about 20 second.Ultrasoundshowed the same image as above.Targeted lipid-coated microbubbles developed an imagein the right common carotid artery 3 second after intravenous bolus injection and suchimage persisted about 200 second,arrived at peak in about 25 second.Ultrasound showedthe same image as above.As compared with above,3D-TOF-MRA showed inconsecutiveblood signals and insufficient smooth vessel wall in local lumens in only 3 rabbits,the restshowed worse.
5.Thrombus-targeted lipid-coated microbubbles' experiment in vivo and in vitro
Massive targeted lipid-coated microbubbles flocked around and some of themaggregated within the fresh blood clots under light and fluorescence microscope after rapidfrozen sections both in vivo and in vitro.While amounts of aggregated microbubbles invitro were larger than that in vivo,the fluorescence ejected in vitro was more obvious thanin vivo.The electron microscope showed the same image.
Conclusions:
1.The preparation of thrombus-targeted phospholipid-coated microbubbles and itsapplication in the arterial thrombus-targeted detection have been successfully performed,which establish a sound foundation for exploitation of targeted contrast agent domesticallyas well as provide the potential market value.
2.The imaging of thrombus-targeted phospholipid-coated microbubbles wasperformed more direct-viewing and longer time than common lipid microbubbles,CDFIand MRA.
3.Thrombus-targeted phospholipid-coated microbubbles could aggregate rapidly withhigher concentration in fresh blood clot and deep into the internal part both in vitro and invivo.Thus,it may realize the targeted thrombus detection as well as provide the theoreticfoundation for investigating targeted thrombolysis therapy.
引文
[1]周永昌，郭万学。超声医学[M]。北京：科学技术文献出版社，1994： 5-6
[2]Gramiak．Ultrasoundcardiography： contraststudiesinanatomyandfunction．Radiology， 1969，92：929
[3]Feinstein SB，Ten Cate FJ，Zwehl W，et al．Two-dimensional contrast echocardiography：I．In vitro development and quantitative of echo contrast agents．JACC，1984， 3：14
[4]Kelley MW，Feinstein SB，Watson DD．Successful left ventricular opacification following peripheral venous injection of sonicated contrast agent：An experimental evaluation．Am Heart J，1987，114：570
[5]Feinstein SB，Cheirif J，Ten Cate FJ．Safety and efficacy of a new tranpulmonary ultrasound contrast agent： Initial mu l ticenter clinical resul ts．JACC， 1990， 16：316
[6]Barnhart J，Levene H，Villapando E，et al．Characteristics of Albunex：Air-filled albumin microspheres for echocardiography contrast enhancement．Invest．Radiol，1990，25(1)：S162
[7]Christiansen JP，Leong-Poi H，Xu F，Klibanov AL，et al．Non-invasive imaging of myocardial reperfusion injury using leukocyte-targeted contrast echocardiography．Circulation，2002，105：1764-1767
[8]UngerEC，MeCereyrT，SweitzerR，etal．MRX501：a novel ultrasound contrast agent with terapeutic properties．Acad Rdaiol．1998，5：s247-s249．
[9]PeterAF，ShuyunaC，Ralph VS，etal．DNA-loaded albumin microbubbles enhance ultrasound-mediated transfection in vitro．Ulsound Med Biol．2002，28：817-822
[10]Korpanty G，Grayburn PA，Shohet RV，et al Targeting vascular endothelium with avidin microbubbles．BrekkenUltrasound in Medicine&Biology，2005，31，(9)： 1279-1283
[11]Bekeredjian R，Shuyuan Chen，Grayburn PA，et al．Augmentation of cardiac protein deliveryusingultrasoundtargetedmicrobubbledestruction．Ultrasoundin Medicine&Biologv， 2005，31(5)：687-691
[12]ShohetRV，Chen S，Zhou YT，et al．Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium．Circulation，2000：2554-2556
[13]Klibanov AL．Targeted delivery of gas-filled micropheres contrast agents for ultrasound imaging．Adv Drug Deliv Rev， 1999，37：139-157
[14]Klibanov A．L，Rychak J．J，Kaul S，et al．Fold-Carrying Particles： Improvement of Targeted Microbubble Attachment．Academic Radiology，Supplement 1，2005，12(5)，43-44
[15]Bekeredjian R，Shuyuan Chen，Wentong Pan，et al．Effects of ultrasound-targeted microbubble destruction on cardiac gene expression Ultrasound in Medicine&Biology，2004，30(4)：539-543
[16]Fisher NG，Christiansen JP，Leong PH，et al．Myocardial and microcirculatory kinetics of BR 14，a novel third generation intravenous ultrasound contrast agent．J Am Coll Cardiol，2002，39：530-537
[17]Klibanov AL，Hughes MS，Villanueva FS，et al．Targeting and ultrasound imaging of microbubble-based contrasagent．Magma， 1999，8(3)：177-184
[18]卞爱娜，高云华，夏红梅，等。免疫脂质体超声造影剂增强荷肝癌裸鼠肿瘤显像 的实验研究。中国超声医学杂志，2005， 21(6)： 413-416
[19]王志刚，凌智瑜，冉海涛，等。超声微泡造影剂对心肌组织的生物学效应介导VEGF基因转染大鼠心肌的实验研究。中国医学影像技术，2003， 19(6)： 656-658
[20]Klibanov AL．Targeted delivery of gas-filled microspheres，contrast agents for ultrasound imaging．Advanced Drug Delivery Reviews， 1999，5：139-157
[21]陈志良，陈树元，金伟军，等。心脏靶向超声造影剂空气白蛋白微球的研究(1)。中国生化药物杂志，1999， 20(1)： 1-3
[22]逯敏飞，程永清，李丽君，等。微泡超声造影剂：一种新型的药物靶向载体。中国医学影像技术，2005， 21(4) ： 513-515
[23]LindnerJR， Dayton PA，Coggins MP，et al．Non invasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles．Circulation 2000；102(5)：531-538
[24]Tan PH，Manunta M，Ardjomand N，et al．Antibody targeted gene transfer to endothelium．J Gene Med，2003，5：311-323
[25]卞爱娜，高云华，谭开彬。人肝癌靶向脂质体微泡造影剂的鉴定及免疫学性质研究。中华超声影像学杂志，2004， 13： 696-699
[26]Klibanov AL，Hughes MS，Villanueva FS，et al．Targetingand ultrasound imaging of microbubbles based contrast agents．MAGMA，1999，8(3)：177-184
[27]夏红梅，高云华，卞爱娜，等。亲血栓性靶向超声造影剂的鉴定及体外寻靶实验研究。中国超声医学杂志，2004， 20：321-323
[28]王向涛，李沙，张小滨，等。血栓靶向尿激酶脂质体的制备及其体内溶栓效果。药学学报，2003，38：231-235
[29]Orth Reid N，Clark TG，Craighead HG．Avidin-Biotin Micropatterning Methods for Biosensor Applications．Biomedical Microdevices．2003，5：29-34
[30]Wilchek M，Bayer EA，Yamada S，et al．Introduction to avidin-biotin technology．Methods Enzymol，1990，184(1)：5-13
[31]Wilchek M，Bayer EA，Yamada S，et al．Application of avidin-biotin technology： literature survey．Methods Enzymol，1990，184(1)：14-25
[32]Chen L，Schechter BA，Arnon RL，etal．Tissue selective affinity targeting using the avidin-biotin system．Drug Dev Res，2000，50(1)：258-271
[33]李成文．生物素亲合素的免疫化学．生物化学与生物物理进展．1990 ，17(1) ：27
[1][1]Runge MSR，Haber E．Importance of experimental models in study of thrombosis and thrombolysis．Circulation 199 1；83 (suppl Ⅳ)：Ⅳ 1-2
[2]Kurz KD，ain BW，Sandusky GE．Rat model of arterial thrombosis induced by ferric chloride．Thromb Res，1990：60：269-280
[3]阮长耿。血栓形成机理。江苏医药1983；(5)：24
[4]Luci M，Mattei FHA，Nader H B et al．Effect of different glycosaminoglycans in a guinea-pig carotid artery thrombosis model．Thromb Res 1994；75：591-599
[5]李叔庚，高建华，王铁霞。实验性主动脉血栓形成的超微结构．湖南医科大学学报1991；16：29-32
[6]Badylak SF，Voytik S，Klabunde RE，et al．Bolusdose response characteristics of single chain urokinase collasminogen activator and tissue plasminogen activator in a dog model of arterial thrombosis．Thromb Res 1988；52：295-312
[7]陈铁军，陈瑞芳，杨果杰。一种家兔股动脉血管内制作血栓模型的简便方法。实验动物科学与管理，1997，14(2)：33-34
[8]万卫星。血栓动物模型的~(99m)TC2重组水蛭素显像研究。中华核医学杂， 1999，19(4)：201-203
[9]张丽萍，牟善初，余霞君。蚓激酶对实验性血栓的预防作用。中国循环杂志，1995，10(1)：679-680
[10]Reyer L．Failure of asprin at different doses to modify experimental thrombosis in rat．Thromb Res，1980，18：669
[11]陈奇。中药药理研究方法学。北京人民卫生出版社，1993．511
[12]夏红梅，高云华，卞爱娜，等。动脉血栓靶向超声显像的体内实验研究[J]。中国医学影像技术，2007，23(1)：13-16
[13]Yu FJ，Wei YL，Ding QH，et al ．A study of arterial thrombosis model induced by galvanic stimulation [J]．Chinese Journal of Physics Medical and Rest，2001 ，23(1)：41-42于富军，卫亚利，丁庆华，等．直流电刺激法制备动脉血栓模型的实验研究[J]．中华物理医学与康复杂志，2001 ，23 (1) ：41-42
[14]Li CJ，Huang J，Yang GP，et al ．Femoral arterial thrombosis model in rabbit [J]．Acta Academiae Medicinae Nanking，2002，21 (4)：277-280李春坚，黄峻，杨国平，等．球囊损伤致兔股动脉血．栓形成模型[J]．南京医科大学学报，2002 ，21(4)：277-280
[15]郭丹．中国黑眼镜蛇毒蛋白酶natrahagin抑制血小板凝集和动脉血栓形成的作用．中国药理学与毒理学杂志，2001 ，15 (1)：27-30
[16]Imura Y， Terashita Z，Nishikawa K．The role of thromboxane(TXA_2) in rabbit arterial thrombosis induced by endothelial damage．Thromb Res，1990 ，59(1)：195-205
[17]谢协驹．降低红细胞压积对家兔颈总动脉血栓形成的影响．中国病理生理学杂志，1995 ，5(2)：198-200
[18]胡三觉．一种新的体内血栓形成动物模型．中华血液杂志，1993，14(10) ：541-542
[19]Rosa M D．Biological properties of carrageenan．J Pharm Pharmac，1972 ，24 ：89
[20]杨文清．绿光光照诱导大鼠脑血栓模型后脑组织病理与行为学改变的研究．现代康复，2001 ，5(4) ：38-39
[21]Aoki T， CoxD， Senzaki K， et al．Comparison of the antithrombotic effects of FK 633， GP Ⅱb/Ⅲa antagonist, and aspirin in a guinea pig thrombosis model [J].Thromb Res,1998,89(3):129-136
[22] Chen L Y, Jokela R, LiD Y, et al. Effect of stable fish oil on arterial thrombogenesis,platelet aggregation, and superoxide dismutase activity[J]. J Cardiovasc Pharmacol,2000, 35(3): 502-505
[23] Farrehi PM, Ozaki C K, Carmeliet P, et al. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice[J]. Circulation,1998,97(10):1002-1008
[24] Friedman MF , Vander Bovenkamp EJ . The pat hogenesis of coronary thrombus[J].Am J Pathol, 1966 ,48(1): 19-44
[25] Wang XK, Xu L. An optimized murine model of ferric chloride induced arterialthrombosis for thrombosis research [J].Thromb Res ,2005,115(122) :95-100
[1]Blebea J， Strothman G， Fowl R．Bilateral lower-extremity US for deep venous thrombosis[J]．Radiology， 1995， 197(3)： 315-316
[2]Kurz KD， Main BW， Sandusky GE， et al．Rat model of arterial thrombosis induced by ferric chloride[J]．Thromb Res， 1990，2(2)：269-280
[3]MattreyRF，AguirreDA．Advancesincontrastmedia research[J]．AcadRadiol，2003，10(12)：1450-1460
[4]Zheng YY， Wang ZG， Ran HT，et al ．Preliminary experimental study of a new self-madehighmolecularultrasoundcontrastagent[J]．ChineseJ UltrasoundMed ，2004 ，20(12) ：888-890郑元义，王志刚，冉海涛，等．自制高分子材料超声造影剂及初步实验研究[J]．中国超声医学杂志，2004，20 (12) ：888-890
[5]MorawskiAM，Lanza GA，Wickline SA．Targetedcontrastagentsfor Magnetic resonance imaging and ultrasound[J]．Curr Opm in Biotechnol，2005，16(1)：1-4
[6]Lanza GA，Wickline SA．Targeted ultrasonic contrast agents for molecular imaging and therapy[J]．Curr Probl Cardiol，2003，28(12)：625-653
[7]Oeffinger BE，Wheatley MA．Development and characterization of a nano-scale contrast agent [J]．Ultrasonics，2004，42(5)：343-347
[8]Unger E，McCreery TP，Sweitzer RH，et al．In vitro studies of a new thrombus-specific ultrasound contrast agent ．Am J Cardiol，1998 ，81 ：58G-61G
[9]Unger E，Metzger P，Krupinski E，et al．The use of a thrombus specific ultrasound contrast agent to detect thrombus in arteriovenous fistulae．Invest Radiol ，2000 ，35：86-89
[10]Takeuchi M，Ogunyankin K， Pandian NG， et al．Enhanced visualization of intravascular and left atrial appendage thrombus with the use of a thrombus2targeting ultrasonographiccontrastagent(MRX-408A1)：invivoexperimental echocardiographic studies．J Am Soc Echocardiogr，1999，12：1015-1021
[11]夏红梅，高云华，卞爱娜，等。亲血栓性靶向超声造影剂的鉴定及体外寻靶实验研究。中国超声医学杂志，2004， 20：321-323
[12]Szebeni J，Baranyi L，Savay S，et al ．Role of complement activation in hyper-sensitivity reactions to doxil and hynic PEG liposomes：experimental and clinical studies[J]．J Liposome Res ，2002，12(122)：165-172
[13]Ding CF，Xing D，Guo H．Detection of imaging difference in different tissues with sonolummescence of focusing ultrasound[J]．Chin J Med Imaging Technol ，2005 ，21 (4) ：504-506丁春峰，邢达，郭洪．利用聚焦超声的声致发光技术检测不同组织的成像差异[J]．中国医学影像技术，2005，21(4)：504-506
[14]Guzman HR，McNamara AJ，Nguyen DX，et al ．Bioeffects caused by changes in acoustic cavitation bubble density and cell concent ration：a united explanation based on cell-to-bubble ratio and blast radius[J]．Ultrasound Med Biol，2003 ，29(8)：1211-1222
[15]Miller DL，Pislaru SV，Greenleaf J E．Sonoporation：mechanical DNA delivery by ultrasounic cavitation[J]．Somat Cell Mol Genet ，2002 ，27(126)：115-134
[16]Wible J H J r，Galen KP，Wojdyla J K， et al．Microbubbles induce renal hemorrhage when exposed to diagnostic ultrasound in anesthetized rats [J]．Ultrasound Med Biol， 2002．28(11212)：1535-1546
[1]Kurz KD，ain BW，Sandusky GE．Rat model of arterial thrombosis induced by ferric chloride．Thromb Res， 1990：60：269-280
[2]Gramiak．Ultrasoundcardiography： contraststudiesinanatomyandfunction．Radiology， 1969，92：929
[3]Mattrey RF，Aguirre DA．Advances in contrast media research[J]．Acad Radiol，2003， 10(12)：1450-1460
[4]Unger E，McCreery TP，Sweitzer RH，et al．In vitro studies of a new thrombus-specific ultrasound contrast agent ．Am J Cardiol，1998 ，81 ：58G-61 G
[5]Unger E，Metzger P，Krupinski E，et al．The use of a thrombus specific ultrasound contrast agent to detect thrombus in arteriovenous fistulae．Invest Radiol ，2000 ，35：86-89
[6]Takeuchi M，Ogunyankin K， Pandian NG， et al．Enhanced visualization of intravascular and left atrial appendage thrombus with the use of a thrombus2targeting ultrasonographic contrast agent (MRX-408A 1)：in vivo experimental echocardiographic studies．J Am Soc Echocardiogr，1999，12：1015-1021
[7]夏红梅，高云华，卞爱娜，等。亲血栓性靶向超声造影剂的鉴定及体外寻靶实验研究。中国超声医学杂志，2004， 20：321-323
[8]Klibanov AL，Hughes MS，Villanueva FS，et al．Targetingand ultrasound imaging of microbubbles based contrast agents．MAGMA， 1999，8(3)：177-184
[9]Orth Reid N，Clark TG，Craighead HG．Avidin-Biotin Micropatterning Methods for Biosensor Applications．Biomedical Microdevices．2003，5：29-34
[10]Wilchek M，Bayer EA，Yamada S，et al．Introduction to avidin-biotin technology．Methods Enzymol，1990，184(1)：5-13
[11]Wilchek M，Bayer EA，Yamada S，et al．Application of avidin-biotin technology： literature survey．Methods Enzymol，1990，184(1)：14-25
[12]Chen L，Schechter BA，Arnon RL，etal．Tissue selective affinity targeting using the avidin-biotin system．Drug Dev Res，2000，50(1)：258-271
[13]李成文．生物素亲合素的免疫化学．生物化学与生物物理进展．1990，17(1) ：27
[14]陆伟跃，刘敏，潘俊，等．叶酸-脂质体制备及对HeLa细胞靶向作用．上海医科大学学报，2000， 27： 4-8
[15]卞爱娜，高云华，谭开彬．人肝癌靶向脂质体微泡造影剂的鉴定及免疫学性质研究．中华超声影像学杂志，2004， 13： 696-699
[16]李占山，周瑞华，周冀平，等．RGD多肽结合型长循环脂质体的肿瘤靶向性研究．中国药学，2003， 14： 393-394
[1]R．Gramiak， P．M．Shah， D．H．Kramer， Radiology 92(1969) 939-948
[2]Correas JM， Bridal L， Lesavre A et al (2001)Ultrasound contrast agents：properties， principles of action， tolerance，and artifacts．Eur Radiol 1 1：1316-1328
[3]Hauff P， Fritsch T， Reinhardt M et al(1997) Delineation of experimental liver tumors in rabbits by a new ultrasound contrast agent and stimulated acoustic emission．Invest Radiol 32：94-99
[4]Blomley MJK， Albrecht T， Cosgrove DO et al(1998) Stimulated acoustic emission in liver parenchyma with Levovist．Lancet 351：568-569
[5]Forsberg F， Goldberg BB， Liu JB et al (1999) Tissue specific US contrast agent for evaluation of hepatic andsplenic parenchyma．Radiology 210：125-132
[6]Quaia E， Blomley MJK， Patel S et al (2002) Initial observations on the effect of irradiation on the liver-specific uptake of Levovist．Eur J Radiol 41：192-199
[7]Kindberg GM， Tolleshaung H， Roos N， Skotland T (2003) Hepatic clearance of Sonazoid perfluorobutane microbubble by Kupffer cells does not reduce the ability of liver to phagocytose or degrade albumin microspheres．Cell Tissue Res 312(1)：49-54
[8]Harvey CJ， Blomley MJK， Eckersley RJ， Cosgrove DO (2001)Developments in ultrasound contrast media．Eur Radiol 11：675-689
[9]Morel DR， Schwieger I， Hohn L et al (2000) Human pharmacokinetics and safety evaluation of SonoVue， a new contrast agent for ultrasound imaging．Invest Radiol 35：80-85
[10]N．de Jong， A．Bouakaz， P．Frinking， Echocardiography 19 (3) (2002) 229-240
[11]Torzilli G (2005) Adverse effects associated with SonoVue use．Expert Opin Drug Saf 4(3)：399-401
[12]D．H．Simpson， P．N．Burns， M．A．Averkiou， IEEE Trans．Ultrason．Ferroelectr．Freq．Control 48 (6) (2001)1483-1494
[13]P．Philips， Proc．IEEE Ultrason．Symp．(2001)1739-1745
[14]陈立新．造影剂在声场中的表现形式与声学造影新技术，国外医学生物医学工程分册，2004， 27 (3)：188-192
[15]Simlason HD，Chin CT，Burns PV，el al．Pulse Inversion Dolalaler：A new method for detecting nonlinear echoes from miorobubble contrast agents.IEEE Transaction UFFC,1999,46: 372-382
[16] Burns PN,Wilson SR, Muradali D el al.Microbobble destruction is the origin of harmonic signals from FS069(abstr).Radiology,1996, 201: 158-161
[17] M. Reinhardt, P. Hauff, A. Briel, V. Uhlendorf, R.A. Linker, M. M(?)urer, M.Schirner,Invest. Radiol. 40 (1) (2005) 2-7
[18] R. Baggs, D.P. Penney, C. Cox, S.Z. Child, C.H. Raeman, D. Dalecki, E.L.Carstensen,Ultrasound Med. Biol. 22 (1) (1996) 119-128
[19] P.A. Van der Wouw, A.C.Brauns, S.E. Bailey, et al.,J. Am. Soc.Echocardiogr. 13 (2000)288-294
[20] S. Schafer, S. Kliner, L. Klinghammer, H. Kaarmann, I. Lucic, U. Nixdorff,U.Rosenschein, W.G. Daniel, F.A. Flachskampf, Ultrasound Med. Biol. 31 (6) (2005) 841-847
[21] C.A. Molina, M. Ribo, M. Rubiera, J. Montaner, E. Santamarina, R. Delgadoederos, J.F. Arenillas, R. Huertas, F. Purroy, P. Delgado, J. Alvarez-Sabin,Stroke 37 (2) (2006) 425-429. Epub 2005, December 22
[22] J.R. Lindner, M.P. Coggins, S. Kaul, A.L. Klibanov, G.H. Brandenburger, K.Ley,Circulation 101 (6) (2000) 668-675
[23] J.R. Lindner, J. Song, F. Xu, A.L. Klibanov, K. Singbartl, K. Ley, S. Kaul,Circulation 102 (22) (2000) 2745-2750
[24] B.A. Kaufmann, J.R. Lindner, Curr. Opin. Biotechnol. 18 (1) (2007) 11-16. Epub 2007, January 22. Review
[25] P. Hauff, M. Reinhardt, A. Briel, N. Debus, M. Schirner, Radiology 231 (3) (2004) 667-673. Epub 2004, April 29
[26] G.M. Lanza, D.R. Abendschein, C.S. Hall, M.J. Scott, D.E. Scherrer, A.Houseman,J.G. Miller, S.A. Wickline, J. Am. Soc. Echocardiogr. 13 (6) (2000) 608-614
[27] D.B. Ellegala, H. Leong-Poi, J.E. Carpenter, A.L. Klibanov, S. Kaul, M.E. Shaffrey,J.Sklenar, J.R. Lindner, Circulation 108 (3) (2003) 336-341. Epub 2003, June 30
[28] H. Leong-Poi, J. Christiansen, P. Heppner, C.W. Lewis, A.L. Klibanov, S. Kaul,J.R.Lindner, Circulation 111(24) (2005) 3248-3254. Epub 2005, June 13
[29] S.J. Lee, E.N. Benveniste, J. Neuroimmunol. 98 (2) (1999) 77-88
[30] M. Laschinger, B. Engelhardt, J. Neuroimmunol. 102 (1) (2000) 32-43
[31] J.R. Lindner, J. Song, J. Christiansen, A.L. Klibanov, F. Xu, K. Ley, Circulation 104 (17)(2001)2107-2112
[32] A.J. Hamilton, S.L. Huang, D. Warnick, M. Rabbat, B. Kane, A. Nagaraj,M.Klegerman, D.D. McPherson, J. Am. Coll. Cardiol. 43 (3) (2004) 453-460
[33] F.S. Villanueva, R.J. Jankowski, S. Klibanov, M.L. Pina, S.M. Alber, S.C.Watkins,G.H. Brandenburger, W.R. Wagner, Circulation 98 (1) (1998) 1-5
[34] B.A. Kaufmann, J.M. Sanders, C. Davis, A. Xie, P. Aldred, I.J. Sarembock,J.R.Lindner, Circulation 116 (3) (2007) 276-284. Epub 2007, June 25
[35] E.C.Unger, T.P. McCreery, R.H. Sweitzer, D. Shen, G. Wu, Am. J. Cardiol. 81 (12A) (1998)58G-61G
[36] P.A. Schumann, J.P. Christiansen, R.M. Quigley, T.P. McCreery, R.H. Sweitzer,E.C.Unger, J.R. Lindner, T.O. Matsunaga, Invest. Radiol. 37 (11) (2002) 587-593
[37] A. Hamilton, S.L. Huang, D. Warnick, A. Stein, M. Rabbat, T. Madhav, B. Kane,A.Nagaraj, M. Klegerman, R. MacDonald, D. McPherson, Circulation 105 (23) (2002) 2772-2778
[38] Berry JD, Sidhu PS (2004) Microbubble contrast-enhanced ultrasound in liver transplantation. Eur Radiol 14 (Suppl 8):P96-P103
[39] Albrecht T, Blomley M, Bolondi L et alEFSUMB Study group (2004) Guidelines for the use of contrast agents in ultrasound. Ultraschall Med 25:249-256
[40] Dill-Macky M, Burns P, Khalili K,Wilson S (2002) Focal hepatic masses:enhancement patterns with SH U 508 And pulse inversion US. Radiology 222:95-102
[41] Quaia E, Bertolotto M, Calderan L,Mosconi E, Pozzi Mucelli R (2003) US characterization of focal hepatic lesions with intermittent high acoustic power mode and contrast material. AcadRadiol 10:739-750
[42] Catalano O, Sandomenico F, Raso MM,Siani A (2004) Low mechanical index contrast-enhanced sonographic findings of pyogenic hepatic abscesses. AJR Am J Roentgenol 182(2):447-450
[43] Nicolau C, Catala V, Vilana R, Gilabert R, Bianchi L, Sole M, Pages M, Bru C(2004) Evaluation of hepatocellular carcinoma using SonoVue, a secondgeneration ultrasound contrast agent:correlation with cellular differentiation.Eur Radiol 14:1092-1099
[44] Quaia E, Bartolotta TV, Midiri M,Bertolotto M, Belgrano M, Cova M (2006) Analysis of different contrast enhancement patterns after microbubble-based contrast agent injection in liver hemangiomas with atypical appearance at baseline scan.Abdom Imag 31(l):59-64
[45] Quaia E, Calliada F, Bertolotto M et al (2004) Characterization of focal liver lesions by contrast-specific US modes and a sulfur hexafluoride-filled microbubble contrast agent: diagnostic performance and confidence.Radiology 232:420-430
[46] Kim SH, Lee JM, Lee JY, Lee JY, Yang HK, Lee HJ, Shin KS, Choi BI (2005)Value of contrast-enhanced sonography for the characterization of focal hepatic lesions in patients with diffuse liver disease: Receiver operating characteristic analysis. AJR Am J Roentgenol 184:1077-1084
[47] Nicolau C, Vilana R, Catala V, Bianchi L, Gilabert R, Garcia A, Bru C (2006) Importance of evaluating all vascular phases on contrast-enhanced sonography in the differentiation of benign from malignant focal liver lesions. AJR Am J Roentgenol 186:158-167
[48] Leen E, Ceccotti P, Kalogeropoulou C,Angerson WJ, Moug SJ, Horgan PJ (2006) Prospective multicenter trial evaluating a novel method of characterizingfocal liver lesions using contrast-enhanced sonography. AJR Am J Roentgenol 186:1551-1559
[49] Quaia E, Palumbo A, Rossi S, Degobbis F, Cernic S, Tona G, Cova M (2006)Characterization of liver tumors insonated at low transmit power after microbubble contrast agent injectionxomparison of visual and quantitative analysis in diagnostic performance. AJR Am J Roentgenol 186:1560-1570
[50] Wu W, Chen MH, Yin SS et al (2006) The role of contrast-enhanced sonography of focal liver lesions before percutaneous biospy. AJR Am J Roentgenol 187:752-761
[51] Wilson SR, Burns P (2006) An algorithm for the diagnosis of focal liver masses using microbubble contrast-enhanced pulse-inversion sonography. AJR Am J Roentgenol 186(5):1401-1412
[52] Bipat S, van Leeuwen MS, Comans EF et al (2005) Colorectal liver metastases:CT, MR imaging, and PET for diagnosis-meta-analysis.Radiology 237:123-1312
[53] Albrecht T, Blomley MJK, Burns PN et al (2003) Improved detection of hepatic metastases with pulse-inversion US during the liver-specific phase of SHU 508A:multicenter study. Radiology 227:361-370
[54] Oldenburg A, Hohmann J, Foert E et al (2005) Detection of hepatic metastases with low MI real time contrast enhanced sonography and SonoVue.Ultraschall Med 26(4):277-284
[55] Dietrich CF, Kratzer W, Strobe D et al (2006) Assessment of metastatic liver disease in patients with primary extrahepatic tumors by contrast-enhanced sonography versus CT and MRl.World J Gastroenterol 12(11):1699-1705
[56] Konopke R, Kersting S, Bergert H,Bloomenthal A, Gastmeier J, Saeger HD, Bunk A (2006) Contrast-enhanced ultrasonography to detect liver metastases:A prospective trial to compare transcutaneous unenhanced and contrast-enhanced ultrasonography in patients undergoing laparotomy. Int J Colorectal Dis May 30
[57] Quaia E, D'Onofrio M, Palumbo A,Rossi S, Bruni S, Cova M (2006) Comparison of contrast-enhanced ultrasonography vs baseline ultrasound and contrast-enhanced computed tomography in metastatic disease of the liver: diagnostic performance and confidence.Eur Radiol 16(7): 1599-1609
[58] Siosteen AK, Elvin A (2004) Intraoperative uses of contrast-enhanced ultrasound. Eur Radiol 14(Suppl 8):P87-P95
[59] Torzilli G, Del Fabbro D, Palmisano A et al (2005) Contrast-enhanced intraoperative ultrasonography during hepatectomies for colorectal cancer liver metastases. J Gastrointest Surg 9(8):1148-1153
[60] Leen E, Cecotti P, Moug SJ et al (2006) Potential value of contrast-enhanced intraoperative ultrasonography during partial hepatectomy for metastases: an essential investigation before resection?Ann Surg 243(2):236-240
[61] Vilana R, Bianchi L, Varela M et al (2006) Is microbubble-enhanced ultrasonography sufficient for assessment of response to percutaneous treatment in patients with early hepatocellular carcinoma?Eur Radiol 16(1l):2454-2462
[62] Meloni MF, Livraghi T, Filice C,Lazzaroni S, Calliada F, Perretti L (2006) Radiofrequency ablation of liver tumors: the role of microbubble contrast agents. Ultrasound Q 22(l):41-47
[63] WHO handbook for reporting results of cancer treatment (1979) World Health Organization Offset Publication No. 48.Geneva [64] Therasse P, Arbuck SG, Eisenhauer EA et al (2000) New guidelines to evaluate the response to tratment in solid tumors. J Natl Cancer Inst 92:205-216
[65] Lim AK, Patel N, Eckersley RJ, Taylor-Robinson SD, Cosgrove DO, Blomley MJK (2004) Evidence for spleen-specific uptake of a microbubble contrast agent: a quantitative study in healthy volunteers. Radiology 231:785-788
[66] Harvey CJ, Lim AK, Lynch M, Blomley MJK, Cosgrove DO (2005) Application of ultrasound microbubbles in spleen.In: Quaia E (ed) Contrast media in ultrasonography:Basic principles and clinical applications. Springer, Berlin Heidelberg New York, pp 205-219
[67] Catalano O, Sandomenico F, Matarazzo I, Siani A (2005) Contrast-enhanced sonography of the spleen. AJR Am J Roentgenol 184:1150-1156
[68] Ferraioli G, Di Sarno A, Coppola C,Giorgio A (2006) Contrast-enhanced low mechanical index ultrasonography in hepatic splenosis. J Ultrasound Med 25(1):133-136
[69] Claudon M, Plouin PF, Baxter G et al (2000) Renal arteries in patients at risk of renal arterial stenosis: multicenter evaluation of the echo-enhancer SH U 508 A at color and spectral Doppler US.Levovist renal artery stenosis study group. Radiology 214:739-746
[70] Correas JM, Claudon M, Tranquart F,Helenon AO (2006) The kidney: imaging with microbubble contrast agents. Ultrasound Q 22(1 ):53-66
[71] Quaia E, Siracusano S, Bertolotto M et al (2003) Characterization of renal tumours with pulse inversion harmonic imaging by intermittent high mechanical index technique. Preliminary results. Eur Radiol 13:1402-1412
[72] Tamai H, Takiguchi Y, Oka M et al (2005) Contrast-enhanced ultrasonography in the diagnosis of solid renal tumors. J Ultrasound Med 24 (12): 1635-1640
[73] Quaia E (2005) Characterization and detection of renal tumors. In: Quaia E (ed) Contrast media in Ultrasonography:Basic principles and clinical applications.Springer, Berlin Heidelberg New York, pp 223-244
[74] Slabaugh TK, Machaidze Z, Hennigar R, Ogan K (2005) Monitoring radiofrequency renal lesions in real time using contrast-enhanced ultrasonography:a porcine model. J Endourol 19:579-583
[75] Albrecht T, Blomley MJ, Cosgrove DO et al (1999) Non-invasive diagnosis of hepatic cirrhosis by transit-time analysis of an ultrasound contrast agent.Lancet 353:1579-1583
[76] Blomley MJ, Lim AK, Harvey CJ et al (2003) Liver microbubble transit time compared with histology and Child-Pugh score in diffuse liver disease: a cross sectional study. Gut 52:1188-1193
[77] McCarville MB, Streck CJ, Dickson PV, Li CS, Nathwani AC, Davidoff AM (2006) Angiogenesis inhibitors in a murine neuroblastoma model: quantitative assessment of intratumoral blood flow with contrast-enhanced gray-scale US. Radiology 240(l):73-81
[78] Stieger SM, Bloch SH, Foreman O,Wisner ER, Ferrara KW, Dayton PA (2006) Ultrasound assessment of angiogenesis in a matrigel model in rats.Ultrasound Med Biol 32(5):673-681
[79] Holsher T, Wilkening W, Draganski B et al (2005) Transcranial ultrasound brain perfusion assessment with a contrast agent-specific imaging mode: results of a two-center trial. Stroke 36 (10):2283-2285
[80] Federlein J, Postert T, Meves S, Weber S, Przuntek H, Buttner T (2000) Ultrasonic evaluation of pathological brain perfusion in acute stroke using second harmonic imaging. J Neurol Neurosurg Psychiatry 69:616-622
[81] Wei K, Ragosta M, Thorpe J, Coggins M, Moos S, Kaul S (2001) Non-invasive quantification of coronary blood flow reserve in humans using myocardial contrast echocardiography.Circulation 103:2560-2565
[82] [82] Wei K, Tong KL, Belcik T, Rafter P,Ragosta M, Wang XQ, Kaul S (2005) Detection of coronary stenoses at rest with myocardial contrast echocardiography.Circulation 112(8):1154-1160
[83] Gasparini C, Bertolotto M, Croce SL,Perrone R,Quaia E, Tiribelli C (2003) Evaluation of liver parenchymal blood flow with contrast-enhanced US: preliminary results in healthy and cirrhotic patients. Acad Radiol 10:869-876
[84] Paltiel HJ, Kalish LA, Susaeta RA,Frausher F, O'Kane PL, Freitas-Filho LG (2006) Pulse inversion US imaging of testicular ischemia: Quantitative and qualitative analyses in a rabbit model.Radiology 239:718-729
[85] Wei K, Le E, Bin JP, Coggins M,Thorpe J, Kaul S (2001) Quantification of renal blood flow with contrastenhanced ultrasound. J Am Coll Cardiol 37(4):1135-1140
[86] Kim JH, Eun HW, Lee HJ, Goo DE,Choi DL (2005) Clinical use of renal perfusion imaging by means of harmonic sonography with a microbubble contrast agent in patients after renal transplantation: preliminary study.J Ultrasound Med 24(6):755-762
[87] Catalano O, Cusati B, Nunziata A,Siani A (2006) Active abdominal bleeding:contrast-enhanced sonography.Abdom Imaging 31(1):9-16
[88] Poletti PA, Platon A, Becker CD et al (2004) Blunt abdominal trauma: Does the use of a second generation sonographic contrast agent help to detect solid organs injuries? AJR Am J Roentgenol 183:1293-1301
[89] Takeshima K, Kumada K, Toyoda H et al (2005) Comparison of IV Contrastenhanced sonography and histopathology of pancreatic cancer. AJR Am J Roentgenol 185:1193-1200
[90] D'Onofrio M, Zamboni G, Tognolini A, Malago R, Faccioli N, Frulloni L,Pozzi Mucelli R (2006) Mass-forming pancreatitis: Value of contrast-enhanced ultrasonography. World J Gastroenterol 14(12):4181-4184
[91] D'Onofrio M, Mansueto G, Vasori S,Falconi M, Procacci C (2003) Contrastenhanced ultrasonographic detection of small pancreatic insulinoma.J Ultrasound Med 22(4):423-417
[92] Maconi G, Bianchi Porro G (2005) Intestinal pathology. In: Quaia E (ed) Contrast media in ultrasonography:Basic principles and clinical applications.Springer, Berlin Heidelberg New York, pp 349-358
[93] Marret H, Sauget S, Giraudeau B,Brewer M, Ranger-Moore J, Body G, Tranquart F (2004) Contrast-enhanced sonography helps in discrimination of benign from malignant adnexal masses. J Ultrasound Med 23:1629-1639
[94] Abramowicz JS (2005) Ultrasonographic contrast media: has the time come in obstetrics and gynecology? J Ultrasound Med 24:517-531
[95]Klauser A， Demharter J， De Marchi A et al (2005) Contrast enhanced grayscale sonography in assessment of joint vascularity in rheumatoid arthritis： results from the IACUS study group．Eur Radiol 15(12)：2404-2410
[96]Nishioka T，Luo H，Fishbein MC，et al．Dissolution of thrombotic arterial occlusion by high intensity，low frequency ultrasound and dodecafluoropentane emulsion：amn vitro and in vivo study．J Am Coll Cardiol，1997，30(2)：561-568
[97]Evan CU，Terry OM，Thomas MC，et al．Therapeutic applications of microbubbles．J Eur Radiol，2002，42：160-168
[98]吴巍，宁新宝，姜藻，等．低功率超声幅射Levovist试剂致家兔肝脏微血管栓塞研究[J]．东南大学学报，2003，33(3)：300-30
[2]Gramiak．Ultrasoundcardiography： contraststudiesinanatomyandfunction．Radiology， 1969，92：929
[3]Feinstein SB，Ten Cate FJ，Zwehl W，et al．Two-dimensional contrast echocardiography：I．In vitro development and quantitative of echo contrast agents．JACC，1984， 3：14
[4]Kelley MW，Feinstein SB，Watson DD．Successful left ventricular opacification following peripheral venous injection of sonicated contrast agent：An experimental evaluation．Am Heart J，1987，114：570
[5]Feinstein SB，Cheirif J，Ten Cate FJ．Safety and efficacy of a new tranpulmonary ultrasound contrast agent： Initial mu l ticenter clinical resul ts．JACC， 1990， 16：316
[6]Barnhart J，Levene H，Villapando E，et al．Characteristics of Albunex：Air-filled albumin microspheres for echocardiography contrast enhancement．Invest．Radiol，1990，25(1)：S162
[7]Christiansen JP，Leong-Poi H，Xu F，Klibanov AL，et al．Non-invasive imaging of myocardial reperfusion injury using leukocyte-targeted contrast echocardiography．Circulation，2002，105：1764-1767
[8]UngerEC，MeCereyrT，SweitzerR，etal．MRX501：a novel ultrasound contrast agent with terapeutic properties．Acad Rdaiol．1998，5：s247-s249．
[9]PeterAF，ShuyunaC，Ralph VS，etal．DNA-loaded albumin microbubbles enhance ultrasound-mediated transfection in vitro．Ulsound Med Biol．2002，28：817-822
[10]Korpanty G，Grayburn PA，Shohet RV，et al Targeting vascular endothelium with avidin microbubbles．BrekkenUltrasound in Medicine&Biology，2005，31，(9)： 1279-1283
[11]Bekeredjian R，Shuyuan Chen，Grayburn PA，et al．Augmentation of cardiac protein deliveryusingultrasoundtargetedmicrobubbledestruction．Ultrasoundin Medicine&Biologv， 2005，31(5)：687-691
[12]ShohetRV，Chen S，Zhou YT，et al．Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium．Circulation，2000：2554-2556
[13]Klibanov AL．Targeted delivery of gas-filled micropheres contrast agents for ultrasound imaging．Adv Drug Deliv Rev， 1999，37：139-157
[14]Klibanov A．L，Rychak J．J，Kaul S，et al．Fold-Carrying Particles： Improvement of Targeted Microbubble Attachment．Academic Radiology，Supplement 1，2005，12(5)，43-44
[15]Bekeredjian R，Shuyuan Chen，Wentong Pan，et al．Effects of ultrasound-targeted microbubble destruction on cardiac gene expression Ultrasound in Medicine&Biology，2004，30(4)：539-543
[16]Fisher NG，Christiansen JP，Leong PH，et al．Myocardial and microcirculatory kinetics of BR 14，a novel third generation intravenous ultrasound contrast agent．J Am Coll Cardiol，2002，39：530-537
[17]Klibanov AL，Hughes MS，Villanueva FS，et al．Targeting and ultrasound imaging of microbubble-based contrasagent．Magma， 1999，8(3)：177-184
[18]卞爱娜，高云华，夏红梅，等。免疫脂质体超声造影剂增强荷肝癌裸鼠肿瘤显像 的实验研究。中国超声医学杂志，2005， 21(6)： 413-416
[19]王志刚，凌智瑜，冉海涛，等。超声微泡造影剂对心肌组织的生物学效应介导VEGF基因转染大鼠心肌的实验研究。中国医学影像技术，2003， 19(6)： 656-658
[20]Klibanov AL．Targeted delivery of gas-filled microspheres，contrast agents for ultrasound imaging．Advanced Drug Delivery Reviews， 1999，5：139-157
[21]陈志良，陈树元，金伟军，等。心脏靶向超声造影剂空气白蛋白微球的研究(1)。中国生化药物杂志，1999， 20(1)： 1-3
[22]逯敏飞，程永清，李丽君，等。微泡超声造影剂：一种新型的药物靶向载体。中国医学影像技术，2005， 21(4) ： 513-515
[23]LindnerJR， Dayton PA，Coggins MP，et al．Non invasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles．Circulation 2000；102(5)：531-538
[24]Tan PH，Manunta M，Ardjomand N，et al．Antibody targeted gene transfer to endothelium．J Gene Med，2003，5：311-323
[25]卞爱娜，高云华，谭开彬。人肝癌靶向脂质体微泡造影剂的鉴定及免疫学性质研究。中华超声影像学杂志，2004， 13： 696-699
[26]Klibanov AL，Hughes MS，Villanueva FS，et al．Targetingand ultrasound imaging of microbubbles based contrast agents．MAGMA，1999，8(3)：177-184
[27]夏红梅，高云华，卞爱娜，等。亲血栓性靶向超声造影剂的鉴定及体外寻靶实验研究。中国超声医学杂志，2004， 20：321-323
[28]王向涛，李沙，张小滨，等。血栓靶向尿激酶脂质体的制备及其体内溶栓效果。药学学报，2003，38：231-235
[29]Orth Reid N，Clark TG，Craighead HG．Avidin-Biotin Micropatterning Methods for Biosensor Applications．Biomedical Microdevices．2003，5：29-34
[30]Wilchek M，Bayer EA，Yamada S，et al．Introduction to avidin-biotin technology．Methods Enzymol，1990，184(1)：5-13
[31]Wilchek M，Bayer EA，Yamada S，et al．Application of avidin-biotin technology： literature survey．Methods Enzymol，1990，184(1)：14-25
[32]Chen L，Schechter BA，Arnon RL，etal．Tissue selective affinity targeting using the avidin-biotin system．Drug Dev Res，2000，50(1)：258-271
[33]李成文．生物素亲合素的免疫化学．生物化学与生物物理进展．1990 ，17(1) ：27
[1][1]Runge MSR，Haber E．Importance of experimental models in study of thrombosis and thrombolysis．Circulation 199 1；83 (suppl Ⅳ)：Ⅳ 1-2
[2]Kurz KD，ain BW，Sandusky GE．Rat model of arterial thrombosis induced by ferric chloride．Thromb Res，1990：60：269-280
[3]阮长耿。血栓形成机理。江苏医药1983；(5)：24
[4]Luci M，Mattei FHA，Nader H B et al．Effect of different glycosaminoglycans in a guinea-pig carotid artery thrombosis model．Thromb Res 1994；75：591-599
[5]李叔庚，高建华，王铁霞。实验性主动脉血栓形成的超微结构．湖南医科大学学报1991；16：29-32
[6]Badylak SF，Voytik S，Klabunde RE，et al．Bolusdose response characteristics of single chain urokinase collasminogen activator and tissue plasminogen activator in a dog model of arterial thrombosis．Thromb Res 1988；52：295-312
[7]陈铁军，陈瑞芳，杨果杰。一种家兔股动脉血管内制作血栓模型的简便方法。实验动物科学与管理，1997，14(2)：33-34
[8]万卫星。血栓动物模型的~(99m)TC2重组水蛭素显像研究。中华核医学杂， 1999，19(4)：201-203
[9]张丽萍，牟善初，余霞君。蚓激酶对实验性血栓的预防作用。中国循环杂志，1995，10(1)：679-680
[10]Reyer L．Failure of asprin at different doses to modify experimental thrombosis in rat．Thromb Res，1980，18：669
[11]陈奇。中药药理研究方法学。北京人民卫生出版社，1993．511
[12]夏红梅，高云华，卞爱娜，等。动脉血栓靶向超声显像的体内实验研究[J]。中国医学影像技术，2007，23(1)：13-16
[13]Yu FJ，Wei YL，Ding QH，et al ．A study of arterial thrombosis model induced by galvanic stimulation [J]．Chinese Journal of Physics Medical and Rest，2001 ，23(1)：41-42于富军，卫亚利，丁庆华，等．直流电刺激法制备动脉血栓模型的实验研究[J]．中华物理医学与康复杂志，2001 ，23 (1) ：41-42
[14]Li CJ，Huang J，Yang GP，et al ．Femoral arterial thrombosis model in rabbit [J]．Acta Academiae Medicinae Nanking，2002，21 (4)：277-280李春坚，黄峻，杨国平，等．球囊损伤致兔股动脉血．栓形成模型[J]．南京医科大学学报，2002 ，21(4)：277-280
[15]郭丹．中国黑眼镜蛇毒蛋白酶natrahagin抑制血小板凝集和动脉血栓形成的作用．中国药理学与毒理学杂志，2001 ，15 (1)：27-30
[16]Imura Y， Terashita Z，Nishikawa K．The role of thromboxane(TXA_2) in rabbit arterial thrombosis induced by endothelial damage．Thromb Res，1990 ，59(1)：195-205
[17]谢协驹．降低红细胞压积对家兔颈总动脉血栓形成的影响．中国病理生理学杂志，1995 ，5(2)：198-200
[18]胡三觉．一种新的体内血栓形成动物模型．中华血液杂志，1993，14(10) ：541-542
[19]Rosa M D．Biological properties of carrageenan．J Pharm Pharmac，1972 ，24 ：89
[20]杨文清．绿光光照诱导大鼠脑血栓模型后脑组织病理与行为学改变的研究．现代康复，2001 ，5(4) ：38-39
[21]Aoki T， CoxD， Senzaki K， et al．Comparison of the antithrombotic effects of FK 633， GP Ⅱb/Ⅲa antagonist, and aspirin in a guinea pig thrombosis model [J].Thromb Res,1998,89(3):129-136
[22] Chen L Y, Jokela R, LiD Y, et al. Effect of stable fish oil on arterial thrombogenesis,platelet aggregation, and superoxide dismutase activity[J]. J Cardiovasc Pharmacol,2000, 35(3): 502-505
[23] Farrehi PM, Ozaki C K, Carmeliet P, et al. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice[J]. Circulation,1998,97(10):1002-1008
[24] Friedman MF , Vander Bovenkamp EJ . The pat hogenesis of coronary thrombus[J].Am J Pathol, 1966 ,48(1): 19-44
[25] Wang XK, Xu L. An optimized murine model of ferric chloride induced arterialthrombosis for thrombosis research [J].Thromb Res ,2005,115(122) :95-100
[1]Blebea J， Strothman G， Fowl R．Bilateral lower-extremity US for deep venous thrombosis[J]．Radiology， 1995， 197(3)： 315-316
[2]Kurz KD， Main BW， Sandusky GE， et al．Rat model of arterial thrombosis induced by ferric chloride[J]．Thromb Res， 1990，2(2)：269-280
[3]MattreyRF，AguirreDA．Advancesincontrastmedia research[J]．AcadRadiol，2003，10(12)：1450-1460
[4]Zheng YY， Wang ZG， Ran HT，et al ．Preliminary experimental study of a new self-madehighmolecularultrasoundcontrastagent[J]．ChineseJ UltrasoundMed ，2004 ，20(12) ：888-890郑元义，王志刚，冉海涛，等．自制高分子材料超声造影剂及初步实验研究[J]．中国超声医学杂志，2004，20 (12) ：888-890
[5]MorawskiAM，Lanza GA，Wickline SA．Targetedcontrastagentsfor Magnetic resonance imaging and ultrasound[J]．Curr Opm in Biotechnol，2005，16(1)：1-4
[6]Lanza GA，Wickline SA．Targeted ultrasonic contrast agents for molecular imaging and therapy[J]．Curr Probl Cardiol，2003，28(12)：625-653
[7]Oeffinger BE，Wheatley MA．Development and characterization of a nano-scale contrast agent [J]．Ultrasonics，2004，42(5)：343-347
[8]Unger E，McCreery TP，Sweitzer RH，et al．In vitro studies of a new thrombus-specific ultrasound contrast agent ．Am J Cardiol，1998 ，81 ：58G-61G
[9]Unger E，Metzger P，Krupinski E，et al．The use of a thrombus specific ultrasound contrast agent to detect thrombus in arteriovenous fistulae．Invest Radiol ，2000 ，35：86-89
[10]Takeuchi M，Ogunyankin K， Pandian NG， et al．Enhanced visualization of intravascular and left atrial appendage thrombus with the use of a thrombus2targeting ultrasonographiccontrastagent(MRX-408A1)：invivoexperimental echocardiographic studies．J Am Soc Echocardiogr，1999，12：1015-1021
[11]夏红梅，高云华，卞爱娜，等。亲血栓性靶向超声造影剂的鉴定及体外寻靶实验研究。中国超声医学杂志，2004， 20：321-323
[12]Szebeni J，Baranyi L，Savay S，et al ．Role of complement activation in hyper-sensitivity reactions to doxil and hynic PEG liposomes：experimental and clinical studies[J]．J Liposome Res ，2002，12(122)：165-172
[13]Ding CF，Xing D，Guo H．Detection of imaging difference in different tissues with sonolummescence of focusing ultrasound[J]．Chin J Med Imaging Technol ，2005 ，21 (4) ：504-506丁春峰，邢达，郭洪．利用聚焦超声的声致发光技术检测不同组织的成像差异[J]．中国医学影像技术，2005，21(4)：504-506
[14]Guzman HR，McNamara AJ，Nguyen DX，et al ．Bioeffects caused by changes in acoustic cavitation bubble density and cell concent ration：a united explanation based on cell-to-bubble ratio and blast radius[J]．Ultrasound Med Biol，2003 ，29(8)：1211-1222
[15]Miller DL，Pislaru SV，Greenleaf J E．Sonoporation：mechanical DNA delivery by ultrasounic cavitation[J]．Somat Cell Mol Genet ，2002 ，27(126)：115-134
[16]Wible J H J r，Galen KP，Wojdyla J K， et al．Microbubbles induce renal hemorrhage when exposed to diagnostic ultrasound in anesthetized rats [J]．Ultrasound Med Biol， 2002．28(11212)：1535-1546
[1]Kurz KD，ain BW，Sandusky GE．Rat model of arterial thrombosis induced by ferric chloride．Thromb Res， 1990：60：269-280
[2]Gramiak．Ultrasoundcardiography： contraststudiesinanatomyandfunction．Radiology， 1969，92：929
[3]Mattrey RF，Aguirre DA．Advances in contrast media research[J]．Acad Radiol，2003， 10(12)：1450-1460
[4]Unger E，McCreery TP，Sweitzer RH，et al．In vitro studies of a new thrombus-specific ultrasound contrast agent ．Am J Cardiol，1998 ，81 ：58G-61 G
[5]Unger E，Metzger P，Krupinski E，et al．The use of a thrombus specific ultrasound contrast agent to detect thrombus in arteriovenous fistulae．Invest Radiol ，2000 ，35：86-89
[6]Takeuchi M，Ogunyankin K， Pandian NG， et al．Enhanced visualization of intravascular and left atrial appendage thrombus with the use of a thrombus2targeting ultrasonographic contrast agent (MRX-408A 1)：in vivo experimental echocardiographic studies．J Am Soc Echocardiogr，1999，12：1015-1021
[7]夏红梅，高云华，卞爱娜，等。亲血栓性靶向超声造影剂的鉴定及体外寻靶实验研究。中国超声医学杂志，2004， 20：321-323
[8]Klibanov AL，Hughes MS，Villanueva FS，et al．Targetingand ultrasound imaging of microbubbles based contrast agents．MAGMA， 1999，8(3)：177-184
[9]Orth Reid N，Clark TG，Craighead HG．Avidin-Biotin Micropatterning Methods for Biosensor Applications．Biomedical Microdevices．2003，5：29-34
[10]Wilchek M，Bayer EA，Yamada S，et al．Introduction to avidin-biotin technology．Methods Enzymol，1990，184(1)：5-13
[11]Wilchek M，Bayer EA，Yamada S，et al．Application of avidin-biotin technology： literature survey．Methods Enzymol，1990，184(1)：14-25
[12]Chen L，Schechter BA，Arnon RL，etal．Tissue selective affinity targeting using the avidin-biotin system．Drug Dev Res，2000，50(1)：258-271
[13]李成文．生物素亲合素的免疫化学．生物化学与生物物理进展．1990，17(1) ：27
[14]陆伟跃，刘敏，潘俊，等．叶酸-脂质体制备及对HeLa细胞靶向作用．上海医科大学学报，2000， 27： 4-8
[15]卞爱娜，高云华，谭开彬．人肝癌靶向脂质体微泡造影剂的鉴定及免疫学性质研究．中华超声影像学杂志，2004， 13： 696-699
[16]李占山，周瑞华，周冀平，等．RGD多肽结合型长循环脂质体的肿瘤靶向性研究．中国药学，2003， 14： 393-394
[1]R．Gramiak， P．M．Shah， D．H．Kramer， Radiology 92(1969) 939-948
[2]Correas JM， Bridal L， Lesavre A et al (2001)Ultrasound contrast agents：properties， principles of action， tolerance，and artifacts．Eur Radiol 1 1：1316-1328
[3]Hauff P， Fritsch T， Reinhardt M et al(1997) Delineation of experimental liver tumors in rabbits by a new ultrasound contrast agent and stimulated acoustic emission．Invest Radiol 32：94-99
[4]Blomley MJK， Albrecht T， Cosgrove DO et al(1998) Stimulated acoustic emission in liver parenchyma with Levovist．Lancet 351：568-569
[5]Forsberg F， Goldberg BB， Liu JB et al (1999) Tissue specific US contrast agent for evaluation of hepatic andsplenic parenchyma．Radiology 210：125-132
[6]Quaia E， Blomley MJK， Patel S et al (2002) Initial observations on the effect of irradiation on the liver-specific uptake of Levovist．Eur J Radiol 41：192-199
[7]Kindberg GM， Tolleshaung H， Roos N， Skotland T (2003) Hepatic clearance of Sonazoid perfluorobutane microbubble by Kupffer cells does not reduce the ability of liver to phagocytose or degrade albumin microspheres．Cell Tissue Res 312(1)：49-54
[8]Harvey CJ， Blomley MJK， Eckersley RJ， Cosgrove DO (2001)Developments in ultrasound contrast media．Eur Radiol 11：675-689
[9]Morel DR， Schwieger I， Hohn L et al (2000) Human pharmacokinetics and safety evaluation of SonoVue， a new contrast agent for ultrasound imaging．Invest Radiol 35：80-85
[10]N．de Jong， A．Bouakaz， P．Frinking， Echocardiography 19 (3) (2002) 229-240
[11]Torzilli G (2005) Adverse effects associated with SonoVue use．Expert Opin Drug Saf 4(3)：399-401
[12]D．H．Simpson， P．N．Burns， M．A．Averkiou， IEEE Trans．Ultrason．Ferroelectr．Freq．Control 48 (6) (2001)1483-1494
[13]P．Philips， Proc．IEEE Ultrason．Symp．(2001)1739-1745
[14]陈立新．造影剂在声场中的表现形式与声学造影新技术，国外医学生物医学工程分册，2004， 27 (3)：188-192
[15]Simlason HD，Chin CT，Burns PV，el al．Pulse Inversion Dolalaler：A new method for detecting nonlinear echoes from miorobubble contrast agents.IEEE Transaction UFFC,1999,46: 372-382
[16] Burns PN,Wilson SR, Muradali D el al.Microbobble destruction is the origin of harmonic signals from FS069(abstr).Radiology,1996, 201: 158-161
[17] M. Reinhardt, P. Hauff, A. Briel, V. Uhlendorf, R.A. Linker, M. M(?)urer, M.Schirner,Invest. Radiol. 40 (1) (2005) 2-7
[18] R. Baggs, D.P. Penney, C. Cox, S.Z. Child, C.H. Raeman, D. Dalecki, E.L.Carstensen,Ultrasound Med. Biol. 22 (1) (1996) 119-128
[19] P.A. Van der Wouw, A.C.Brauns, S.E. Bailey, et al.,J. Am. Soc.Echocardiogr. 13 (2000)288-294
[20] S. Schafer, S. Kliner, L. Klinghammer, H. Kaarmann, I. Lucic, U. Nixdorff,U.Rosenschein, W.G. Daniel, F.A. Flachskampf, Ultrasound Med. Biol. 31 (6) (2005) 841-847
[21] C.A. Molina, M. Ribo, M. Rubiera, J. Montaner, E. Santamarina, R. Delgadoederos, J.F. Arenillas, R. Huertas, F. Purroy, P. Delgado, J. Alvarez-Sabin,Stroke 37 (2) (2006) 425-429. Epub 2005, December 22
[22] J.R. Lindner, M.P. Coggins, S. Kaul, A.L. Klibanov, G.H. Brandenburger, K.Ley,Circulation 101 (6) (2000) 668-675
[23] J.R. Lindner, J. Song, F. Xu, A.L. Klibanov, K. Singbartl, K. Ley, S. Kaul,Circulation 102 (22) (2000) 2745-2750
[24] B.A. Kaufmann, J.R. Lindner, Curr. Opin. Biotechnol. 18 (1) (2007) 11-16. Epub 2007, January 22. Review
[25] P. Hauff, M. Reinhardt, A. Briel, N. Debus, M. Schirner, Radiology 231 (3) (2004) 667-673. Epub 2004, April 29
[26] G.M. Lanza, D.R. Abendschein, C.S. Hall, M.J. Scott, D.E. Scherrer, A.Houseman,J.G. Miller, S.A. Wickline, J. Am. Soc. Echocardiogr. 13 (6) (2000) 608-614
[27] D.B. Ellegala, H. Leong-Poi, J.E. Carpenter, A.L. Klibanov, S. Kaul, M.E. Shaffrey,J.Sklenar, J.R. Lindner, Circulation 108 (3) (2003) 336-341. Epub 2003, June 30
[28] H. Leong-Poi, J. Christiansen, P. Heppner, C.W. Lewis, A.L. Klibanov, S. Kaul,J.R.Lindner, Circulation 111(24) (2005) 3248-3254. Epub 2005, June 13
[29] S.J. Lee, E.N. Benveniste, J. Neuroimmunol. 98 (2) (1999) 77-88
[30] M. Laschinger, B. Engelhardt, J. Neuroimmunol. 102 (1) (2000) 32-43
[31] J.R. Lindner, J. Song, J. Christiansen, A.L. Klibanov, F. Xu, K. Ley, Circulation 104 (17)(2001)2107-2112
[32] A.J. Hamilton, S.L. Huang, D. Warnick, M. Rabbat, B. Kane, A. Nagaraj,M.Klegerman, D.D. McPherson, J. Am. Coll. Cardiol. 43 (3) (2004) 453-460
[33] F.S. Villanueva, R.J. Jankowski, S. Klibanov, M.L. Pina, S.M. Alber, S.C.Watkins,G.H. Brandenburger, W.R. Wagner, Circulation 98 (1) (1998) 1-5
[34] B.A. Kaufmann, J.M. Sanders, C. Davis, A. Xie, P. Aldred, I.J. Sarembock,J.R.Lindner, Circulation 116 (3) (2007) 276-284. Epub 2007, June 25
[35] E.C.Unger, T.P. McCreery, R.H. Sweitzer, D. Shen, G. Wu, Am. J. Cardiol. 81 (12A) (1998)58G-61G
[36] P.A. Schumann, J.P. Christiansen, R.M. Quigley, T.P. McCreery, R.H. Sweitzer,E.C.Unger, J.R. Lindner, T.O. Matsunaga, Invest. Radiol. 37 (11) (2002) 587-593
[37] A. Hamilton, S.L. Huang, D. Warnick, A. Stein, M. Rabbat, T. Madhav, B. Kane,A.Nagaraj, M. Klegerman, R. MacDonald, D. McPherson, Circulation 105 (23) (2002) 2772-2778
[38] Berry JD, Sidhu PS (2004) Microbubble contrast-enhanced ultrasound in liver transplantation. Eur Radiol 14 (Suppl 8):P96-P103
[39] Albrecht T, Blomley M, Bolondi L et alEFSUMB Study group (2004) Guidelines for the use of contrast agents in ultrasound. Ultraschall Med 25:249-256
[40] Dill-Macky M, Burns P, Khalili K,Wilson S (2002) Focal hepatic masses:enhancement patterns with SH U 508 And pulse inversion US. Radiology 222:95-102
[41] Quaia E, Bertolotto M, Calderan L,Mosconi E, Pozzi Mucelli R (2003) US characterization of focal hepatic lesions with intermittent high acoustic power mode and contrast material. AcadRadiol 10:739-750
[42] Catalano O, Sandomenico F, Raso MM,Siani A (2004) Low mechanical index contrast-enhanced sonographic findings of pyogenic hepatic abscesses. AJR Am J Roentgenol 182(2):447-450
[43] Nicolau C, Catala V, Vilana R, Gilabert R, Bianchi L, Sole M, Pages M, Bru C(2004) Evaluation of hepatocellular carcinoma using SonoVue, a secondgeneration ultrasound contrast agent:correlation with cellular differentiation.Eur Radiol 14:1092-1099
[44] Quaia E, Bartolotta TV, Midiri M,Bertolotto M, Belgrano M, Cova M (2006) Analysis of different contrast enhancement patterns after microbubble-based contrast agent injection in liver hemangiomas with atypical appearance at baseline scan.Abdom Imag 31(l):59-64
[45] Quaia E, Calliada F, Bertolotto M et al (2004) Characterization of focal liver lesions by contrast-specific US modes and a sulfur hexafluoride-filled microbubble contrast agent: diagnostic performance and confidence.Radiology 232:420-430
[46] Kim SH, Lee JM, Lee JY, Lee JY, Yang HK, Lee HJ, Shin KS, Choi BI (2005)Value of contrast-enhanced sonography for the characterization of focal hepatic lesions in patients with diffuse liver disease: Receiver operating characteristic analysis. AJR Am J Roentgenol 184:1077-1084
[47] Nicolau C, Vilana R, Catala V, Bianchi L, Gilabert R, Garcia A, Bru C (2006) Importance of evaluating all vascular phases on contrast-enhanced sonography in the differentiation of benign from malignant focal liver lesions. AJR Am J Roentgenol 186:158-167
[48] Leen E, Ceccotti P, Kalogeropoulou C,Angerson WJ, Moug SJ, Horgan PJ (2006) Prospective multicenter trial evaluating a novel method of characterizingfocal liver lesions using contrast-enhanced sonography. AJR Am J Roentgenol 186:1551-1559
[49] Quaia E, Palumbo A, Rossi S, Degobbis F, Cernic S, Tona G, Cova M (2006)Characterization of liver tumors insonated at low transmit power after microbubble contrast agent injectionxomparison of visual and quantitative analysis in diagnostic performance. AJR Am J Roentgenol 186:1560-1570
[50] Wu W, Chen MH, Yin SS et al (2006) The role of contrast-enhanced sonography of focal liver lesions before percutaneous biospy. AJR Am J Roentgenol 187:752-761
[51] Wilson SR, Burns P (2006) An algorithm for the diagnosis of focal liver masses using microbubble contrast-enhanced pulse-inversion sonography. AJR Am J Roentgenol 186(5):1401-1412
[52] Bipat S, van Leeuwen MS, Comans EF et al (2005) Colorectal liver metastases:CT, MR imaging, and PET for diagnosis-meta-analysis.Radiology 237:123-1312
[53] Albrecht T, Blomley MJK, Burns PN et al (2003) Improved detection of hepatic metastases with pulse-inversion US during the liver-specific phase of SHU 508A:multicenter study. Radiology 227:361-370
[54] Oldenburg A, Hohmann J, Foert E et al (2005) Detection of hepatic metastases with low MI real time contrast enhanced sonography and SonoVue.Ultraschall Med 26(4):277-284
[55] Dietrich CF, Kratzer W, Strobe D et al (2006) Assessment of metastatic liver disease in patients with primary extrahepatic tumors by contrast-enhanced sonography versus CT and MRl.World J Gastroenterol 12(11):1699-1705
[56] Konopke R, Kersting S, Bergert H,Bloomenthal A, Gastmeier J, Saeger HD, Bunk A (2006) Contrast-enhanced ultrasonography to detect liver metastases:A prospective trial to compare transcutaneous unenhanced and contrast-enhanced ultrasonography in patients undergoing laparotomy. Int J Colorectal Dis May 30
[57] Quaia E, D'Onofrio M, Palumbo A,Rossi S, Bruni S, Cova M (2006) Comparison of contrast-enhanced ultrasonography vs baseline ultrasound and contrast-enhanced computed tomography in metastatic disease of the liver: diagnostic performance and confidence.Eur Radiol 16(7): 1599-1609
[58] Siosteen AK, Elvin A (2004) Intraoperative uses of contrast-enhanced ultrasound. Eur Radiol 14(Suppl 8):P87-P95
[59] Torzilli G, Del Fabbro D, Palmisano A et al (2005) Contrast-enhanced intraoperative ultrasonography during hepatectomies for colorectal cancer liver metastases. J Gastrointest Surg 9(8):1148-1153
[60] Leen E, Cecotti P, Moug SJ et al (2006) Potential value of contrast-enhanced intraoperative ultrasonography during partial hepatectomy for metastases: an essential investigation before resection?Ann Surg 243(2):236-240
[61] Vilana R, Bianchi L, Varela M et al (2006) Is microbubble-enhanced ultrasonography sufficient for assessment of response to percutaneous treatment in patients with early hepatocellular carcinoma?Eur Radiol 16(1l):2454-2462
[62] Meloni MF, Livraghi T, Filice C,Lazzaroni S, Calliada F, Perretti L (2006) Radiofrequency ablation of liver tumors: the role of microbubble contrast agents. Ultrasound Q 22(l):41-47
[63] WHO handbook for reporting results of cancer treatment (1979) World Health Organization Offset Publication No. 48.Geneva [64] Therasse P, Arbuck SG, Eisenhauer EA et al (2000) New guidelines to evaluate the response to tratment in solid tumors. J Natl Cancer Inst 92:205-216
[65] Lim AK, Patel N, Eckersley RJ, Taylor-Robinson SD, Cosgrove DO, Blomley MJK (2004) Evidence for spleen-specific uptake of a microbubble contrast agent: a quantitative study in healthy volunteers. Radiology 231:785-788
[66] Harvey CJ, Lim AK, Lynch M, Blomley MJK, Cosgrove DO (2005) Application of ultrasound microbubbles in spleen.In: Quaia E (ed) Contrast media in ultrasonography:Basic principles and clinical applications. Springer, Berlin Heidelberg New York, pp 205-219
[67] Catalano O, Sandomenico F, Matarazzo I, Siani A (2005) Contrast-enhanced sonography of the spleen. AJR Am J Roentgenol 184:1150-1156
[68] Ferraioli G, Di Sarno A, Coppola C,Giorgio A (2006) Contrast-enhanced low mechanical index ultrasonography in hepatic splenosis. J Ultrasound Med 25(1):133-136
[69] Claudon M, Plouin PF, Baxter G et al (2000) Renal arteries in patients at risk of renal arterial stenosis: multicenter evaluation of the echo-enhancer SH U 508 A at color and spectral Doppler US.Levovist renal artery stenosis study group. Radiology 214:739-746
[70] Correas JM, Claudon M, Tranquart F,Helenon AO (2006) The kidney: imaging with microbubble contrast agents. Ultrasound Q 22(1 ):53-66
[71] Quaia E, Siracusano S, Bertolotto M et al (2003) Characterization of renal tumours with pulse inversion harmonic imaging by intermittent high mechanical index technique. Preliminary results. Eur Radiol 13:1402-1412
[72] Tamai H, Takiguchi Y, Oka M et al (2005) Contrast-enhanced ultrasonography in the diagnosis of solid renal tumors. J Ultrasound Med 24 (12): 1635-1640
[73] Quaia E (2005) Characterization and detection of renal tumors. In: Quaia E (ed) Contrast media in Ultrasonography:Basic principles and clinical applications.Springer, Berlin Heidelberg New York, pp 223-244
[74] Slabaugh TK, Machaidze Z, Hennigar R, Ogan K (2005) Monitoring radiofrequency renal lesions in real time using contrast-enhanced ultrasonography:a porcine model. J Endourol 19:579-583
[75] Albrecht T, Blomley MJ, Cosgrove DO et al (1999) Non-invasive diagnosis of hepatic cirrhosis by transit-time analysis of an ultrasound contrast agent.Lancet 353:1579-1583
[76] Blomley MJ, Lim AK, Harvey CJ et al (2003) Liver microbubble transit time compared with histology and Child-Pugh score in diffuse liver disease: a cross sectional study. Gut 52:1188-1193
[77] McCarville MB, Streck CJ, Dickson PV, Li CS, Nathwani AC, Davidoff AM (2006) Angiogenesis inhibitors in a murine neuroblastoma model: quantitative assessment of intratumoral blood flow with contrast-enhanced gray-scale US. Radiology 240(l):73-81
[78] Stieger SM, Bloch SH, Foreman O,Wisner ER, Ferrara KW, Dayton PA (2006) Ultrasound assessment of angiogenesis in a matrigel model in rats.Ultrasound Med Biol 32(5):673-681
[79] Holsher T, Wilkening W, Draganski B et al (2005) Transcranial ultrasound brain perfusion assessment with a contrast agent-specific imaging mode: results of a two-center trial. Stroke 36 (10):2283-2285
[80] Federlein J, Postert T, Meves S, Weber S, Przuntek H, Buttner T (2000) Ultrasonic evaluation of pathological brain perfusion in acute stroke using second harmonic imaging. J Neurol Neurosurg Psychiatry 69:616-622
[81] Wei K, Ragosta M, Thorpe J, Coggins M, Moos S, Kaul S (2001) Non-invasive quantification of coronary blood flow reserve in humans using myocardial contrast echocardiography.Circulation 103:2560-2565
[82] [82] Wei K, Tong KL, Belcik T, Rafter P,Ragosta M, Wang XQ, Kaul S (2005) Detection of coronary stenoses at rest with myocardial contrast echocardiography.Circulation 112(8):1154-1160
[83] Gasparini C, Bertolotto M, Croce SL,Perrone R,Quaia E, Tiribelli C (2003) Evaluation of liver parenchymal blood flow with contrast-enhanced US: preliminary results in healthy and cirrhotic patients. Acad Radiol 10:869-876
[84] Paltiel HJ, Kalish LA, Susaeta RA,Frausher F, O'Kane PL, Freitas-Filho LG (2006) Pulse inversion US imaging of testicular ischemia: Quantitative and qualitative analyses in a rabbit model.Radiology 239:718-729
[85] Wei K, Le E, Bin JP, Coggins M,Thorpe J, Kaul S (2001) Quantification of renal blood flow with contrastenhanced ultrasound. J Am Coll Cardiol 37(4):1135-1140
[86] Kim JH, Eun HW, Lee HJ, Goo DE,Choi DL (2005) Clinical use of renal perfusion imaging by means of harmonic sonography with a microbubble contrast agent in patients after renal transplantation: preliminary study.J Ultrasound Med 24(6):755-762
[87] Catalano O, Cusati B, Nunziata A,Siani A (2006) Active abdominal bleeding:contrast-enhanced sonography.Abdom Imaging 31(1):9-16
[88] Poletti PA, Platon A, Becker CD et al (2004) Blunt abdominal trauma: Does the use of a second generation sonographic contrast agent help to detect solid organs injuries? AJR Am J Roentgenol 183:1293-1301
[89] Takeshima K, Kumada K, Toyoda H et al (2005) Comparison of IV Contrastenhanced sonography and histopathology of pancreatic cancer. AJR Am J Roentgenol 185:1193-1200
[90] D'Onofrio M, Zamboni G, Tognolini A, Malago R, Faccioli N, Frulloni L,Pozzi Mucelli R (2006) Mass-forming pancreatitis: Value of contrast-enhanced ultrasonography. World J Gastroenterol 14(12):4181-4184
[91] D'Onofrio M, Mansueto G, Vasori S,Falconi M, Procacci C (2003) Contrastenhanced ultrasonographic detection of small pancreatic insulinoma.J Ultrasound Med 22(4):423-417
[92] Maconi G, Bianchi Porro G (2005) Intestinal pathology. In: Quaia E (ed) Contrast media in ultrasonography:Basic principles and clinical applications.Springer, Berlin Heidelberg New York, pp 349-358
[93] Marret H, Sauget S, Giraudeau B,Brewer M, Ranger-Moore J, Body G, Tranquart F (2004) Contrast-enhanced sonography helps in discrimination of benign from malignant adnexal masses. J Ultrasound Med 23:1629-1639
[94] Abramowicz JS (2005) Ultrasonographic contrast media: has the time come in obstetrics and gynecology? J Ultrasound Med 24:517-531
[95]Klauser A， Demharter J， De Marchi A et al (2005) Contrast enhanced grayscale sonography in assessment of joint vascularity in rheumatoid arthritis： results from the IACUS study group．Eur Radiol 15(12)：2404-2410
[96]Nishioka T，Luo H，Fishbein MC，et al．Dissolution of thrombotic arterial occlusion by high intensity，low frequency ultrasound and dodecafluoropentane emulsion：amn vitro and in vivo study．J Am Coll Cardiol，1997，30(2)：561-568
[97]Evan CU，Terry OM，Thomas MC，et al．Therapeutic applications of microbubbles．J Eur Radiol，2002，42：160-168
[98]吴巍，宁新宝，姜藻，等．低功率超声幅射Levovist试剂致家兔肝脏微血管栓塞研究[J]．东南大学学报，2003，33(3)：300-30
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