【作者】 冯敏；

【导师】 赵洪洋；

【作者基本信息】 华中科技大学 ， 神经外科， 2009， 博士

【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-FeCl₃诱导的方法来制备兔颈总动脉血栓模型，取九只家兔根据FeCl₃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FeCl₃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1．08×10¹⁰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6．5×10⁹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。更多还原

【Abstract】 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 sensitivityObjective: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 activityDPPC,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 activityNBD-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 stabilityRabbit thrombus model made by FeCl₃ infiltration in common carotid artery of rabbitwas set up.Six rabbits were randomly invided into three groups according to FeCl₃infiltration 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 meansThe 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 byFeCl₃ 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 vitroFour 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 activityCommon 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 1.08×10¹⁰/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 activityThrombus-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 6.5×10⁹/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 stabilityGross 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 meansCDFI 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 vitroMassive 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.更多还原