超声介导微泡空化靶向传输系统促血管新生作用的初探
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摘要
一．前言
1．研究背景
冠心病是世界范围内公认的威胁人类身心健康及引起死亡的第一杀手，冠心病的实质是缺血和缺氧。近二十年来，人们不断努力，取得了许多成就，循证医学指导下的新的更为合理的心血管药物以及以冠状动脉旁路搭桥术(CABG)和血管成形术(PTCA)为代表的血运重建心肌再灌注疗法，使冠心病尤其是急性心肌梗死的治疗和预后取得了长足的进步。然而，仍有诸多重要问题未予解决。例如，对于一些由于自身冠状动脉病变复杂、严重、弥散、终末小分枝病变、以及合并其它不适合介入治疗的全身性疾病如糖尿病、肿瘤等，则不能进行PTCA和CABG冠脉重建；有些病人虽可进行冠脉重建，但重建后血供仍不充分，缺血心肌濒临坏死、凋亡、纤维化；另外PTCA和CABG术后再狭窄问题，使心肌再灌注疗法面临挑战。因此，寻求其它血运重建策略迫在眉睫，人们寄厚望于称为“分子搭桥”的治疗性血管新生(Therapeutic angiogensis)以及心肌干细胞移植。
缺氧心肌微循环的代偿包括微血管扩张、征募储备血管、微血管新生及动、静脉侧支形成，严重冠心病患者即使在合并使用硝酸酯和钙拮抗剂等药物治疗基础上，内源性的促血管生长调节下的微循环代偿已达极限。因此，在药物治疗、血运重建基础上，通过输入外源性的促血管生长因子，促进心肌血管新生(Therapeutic Angiogenesis)、改善心肌缺血即心肌血管分子搭桥，是人们正在努力探索的研究热点。研究证实，Angiogenesis是由多种有丝分裂原相互作用、调控的瀑布式生物反应，一系列血管生长的促进和拮抗因子，如VEGF(血管内皮生因子)、FGF(纤维生长因子)、Angiopoietin(血管生长素)、Ephrins、Thrombospondin-1(凝血栓蛋白)等，通过血管内皮高亲合力受体及信号传导途径参与血管生成。分子生物学在心血管领域的应用及心肌分子搭桥基因治疗的飞速发展，使分子搭桥已进入临床进行初步实践；如开胸心肌注射VEGF_(121)重组腺病毒、经心内膜和心包注射重组VEGF_(165)和VEGF_(121)真核表达质
粒、冠脉内注射bFGF、VEGF16。进行血运重建的临床试验等。这些研
究虽然取得了一些令人振奋的效果，但有创、高风险的基因传输方式明
显限制了分子搭桥疗法的深入开展。
超声在基因和活性药物靶向传输中的应用是正在兴起的研究热点，。
其核心是应用超声波及声学造影剂微气泡发展基因和药物定向释放技
术，即超声介导微泡空化靶向传输系统。该系统的原理是将声学造影剂
微气泡耦连或包载靶向药物或基因，使载药微泡在显影心肌的同时，利
用声学造影剂微气泡在超声场内发生的空化效应，将基因和药物运载和
释放到特定的组织和器官，使局部组织达到有效的治疗浓度。己有充分
研究证实该系统的可行性和应用前景，其全身给药少、局部浓度高、无
创、简便、并可在床边进行等优点，赋予当前靶向性差、正处于低迷状
态的基因疗法以新的希望。
2．目的和意义
本研究拟以上述理论、研究背景为依据，利用分子生物学、细胞生物
学及声学造影技术，以蛋白质靶向微泡在组织水平探索超声介导微泡空
化靶向传输放射性核素标记的大分子物质BSA的可行性，以蛋白质微泡。
脂质体微泡在细胞水平初步探索超声介导微泡空化效应促内皮细胞报告
基因转染、表达的可能性；以自制脂质体靶向微泡向大鼠梗塞心肌靶向
传输自行构建、克隆的血管内皮生长因子基因VEGF165，并在心肌梗塞动
物模型评价VEGF16；分子搭桥血管新生的效果：为超声介导微泡空化靶
向传输系统的深入研究以及冠心病分子搭桥基因治疗服务于临床，奠定
理论基础和提供实践经验。
第一军医大学南方医院率先研制的第一。二代造影剂填补了国内
心肌声学造影的一项空白，如果研制成功能进行心肌靶向传输的第三代
造影剂，则能紧跟国际前沿，进一步丰富超声介导微泡空化靶向传输系
统这一涉及多个学科的复杂理论体系。未来超声介导微泡空化靶向传输
系统的成功实践，不但会给心肌血管新生的分子搭桥基因治疗提供有效
的基因靶向投送手段，而且还有助于攻克目前基因治疗靶向性差的世界
难题。同时，靶向微泡的成功研制还将用于诸如肿瘤、外用血栓病的治
疗，因此本研究具有重要的经济和社会意义。
3．研究内容
一6－
本课题主要进行以下几个方面的的研究：
＊）在国内应用超声介导微泡空化靶向传输的动物及细胞模型，首次进行
靶向传输研究；
。c）向大鼠心肌传输放射性核素标记的牛血清白蛋白，取得预期心肌靶向
一传输效果；
m 向培养的人血管内皮细胞传输LacZ报告基因，证明脂质体靶向微泡
有良好的靶向传输及促基因转染、表达效果；
O） 向大鼠梗塞心肌传输自行构建的血管内皮生长因子基因VEGF165；取
得一定的促血管生成效应：
门在血管内皮生长因于基因表达载体的构建过程中，发现一新的血管内
皮生长因子基因的剪接形式
Introduction 1. Background
Coronary Heart Disease (CHD),which essence is ischmia and hxpoxia,is publicly recognized as main threat for human health and cause the leading death worldwide. With the guidance of evidence medicine,much achievement have been made on the treatment of CAD,especially acute myocardial infarction over the past decades due to lots of newly-found drugs and the reperfusion therapies represented by coronary artery bypass graft (CABG) and percutaneous transluminal coronary angioplasty (PTCA);however,a lot of issues remain resolved. A growing number of patients neither are suitable for candidates for conventional intervention therapies secondary to anatomical constraints imposed by the severity or extent of their coronary artery disease and some systematical condition such as diabetes and tumor,nor can they get enough blood supply under intervention. If such condition developed,the ischemic myocardium would be doomed to apoptosis,necrosis and fibrosis. It's urgent for people to seek other biological revascularization
strategy,and a large body of work involved in both therapeutic angiogensis and stem cell graft are hopefully under way.
Normally microcirculation compensation of ischemic heart includes microvessel enlargement,angiogensis and collateral vessel development between artery and vein. However,even nitrates and calcium antagonist were fully administrated,intrinsic microcirculation adjustment still could not reach beyond the basic demand of oxygen and blood of ischemic myocardium,therefore therapeutic angiogenesis induced by exogenesis growth factor which can initiate the formation of a plexus of collateral vessels in zones of ischemic myocardium serve to relieve angina and to improve advanced myocardial ischemia at the base of drug therapy and revascularization intervention. Angiogenesis,which is called "molecular graft of vessels",is a complex and cascade process that involves in stimulation of endothelial cell proliferation and migration by a huge amount of antagonist and agonist
growth factors,including the fibroblast growth factor (FGF) family,vascular endothelial growth factor (VEGF) family,angiopoietin,ephrins and thrombospondin-1 through stimulating high affinity receptors and signal transduction passway of endothelial cells,and those mitogen factors and cascades seems to act in a coordinated way to achieve this process. The vascular endothelial growth factor(VEGF) was identified as endothelial cell specific mitogen. It has been shown to be involved in endothelial cell proliferation and blood vessel formation. VEGF mediates its function through its specific receptor on the vascular endothelial cell surface. VEGF plays important roles in variety of physiological and pathological processes including embryogenesis,tumor and some cardiovascular diseases. The rapid development of molecular biology in myocardiovascular scope and growth factor therapy offers to clinical practices. Although several clinical trials involved various endovascular delivery devices have been designed for angiogenesis,such as bolus injection of naked plasmid DNA encoding phVEGF165,phVEGF121 and bFGF of recombinated virus,either in pericardium,direct myocardium at the time of thoracotomy or intracoronary injection and endoluminal delivering,the limitations of such delivery including the possibility of increased vascular trauma,high risk,low efficiency of localization,inconsistency of delivery,and rapid washout of the agents from the vascular wall after delivery prevent the progres of further development of angiogenesis.
Ultrasound,which has been playing a very important role in targeted delivering of gene and active agents,merits further exploration. The aim is to use ultrasound and ultrasound echo contrast agents to offer a targeting delivering procedure,called the delivery system of ultrasound-mediated destruction and cavitation of targeted microbubbles. The core of the approach,which depend on the cavitation effect of microbubble under ultrasound field,is locally delivered pharmacological drugs or t
1．研究背景
冠心病是世界范围内公认的威胁人类身心健康及引起死亡的第一杀手，冠心病的实质是缺血和缺氧。近二十年来，人们不断努力，取得了许多成就，循证医学指导下的新的更为合理的心血管药物以及以冠状动脉旁路搭桥术(CABG)和血管成形术(PTCA)为代表的血运重建心肌再灌注疗法，使冠心病尤其是急性心肌梗死的治疗和预后取得了长足的进步。然而，仍有诸多重要问题未予解决。例如，对于一些由于自身冠状动脉病变复杂、严重、弥散、终末小分枝病变、以及合并其它不适合介入治疗的全身性疾病如糖尿病、肿瘤等，则不能进行PTCA和CABG冠脉重建；有些病人虽可进行冠脉重建，但重建后血供仍不充分，缺血心肌濒临坏死、凋亡、纤维化；另外PTCA和CABG术后再狭窄问题，使心肌再灌注疗法面临挑战。因此，寻求其它血运重建策略迫在眉睫，人们寄厚望于称为“分子搭桥”的治疗性血管新生(Therapeutic angiogensis)以及心肌干细胞移植。
缺氧心肌微循环的代偿包括微血管扩张、征募储备血管、微血管新生及动、静脉侧支形成，严重冠心病患者即使在合并使用硝酸酯和钙拮抗剂等药物治疗基础上，内源性的促血管生长调节下的微循环代偿已达极限。因此，在药物治疗、血运重建基础上，通过输入外源性的促血管生长因子，促进心肌血管新生(Therapeutic Angiogenesis)、改善心肌缺血即心肌血管分子搭桥，是人们正在努力探索的研究热点。研究证实，Angiogenesis是由多种有丝分裂原相互作用、调控的瀑布式生物反应，一系列血管生长的促进和拮抗因子，如VEGF(血管内皮生因子)、FGF(纤维生长因子)、Angiopoietin(血管生长素)、Ephrins、Thrombospondin-1(凝血栓蛋白)等，通过血管内皮高亲合力受体及信号传导途径参与血管生成。分子生物学在心血管领域的应用及心肌分子搭桥基因治疗的飞速发展，使分子搭桥已进入临床进行初步实践；如开胸心肌注射VEGF_(121)重组腺病毒、经心内膜和心包注射重组VEGF_(165)和VEGF_(121)真核表达质
粒、冠脉内注射bFGF、VEGF16。进行血运重建的临床试验等。这些研
究虽然取得了一些令人振奋的效果，但有创、高风险的基因传输方式明
显限制了分子搭桥疗法的深入开展。
超声在基因和活性药物靶向传输中的应用是正在兴起的研究热点，。
其核心是应用超声波及声学造影剂微气泡发展基因和药物定向释放技
术，即超声介导微泡空化靶向传输系统。该系统的原理是将声学造影剂
微气泡耦连或包载靶向药物或基因，使载药微泡在显影心肌的同时，利
用声学造影剂微气泡在超声场内发生的空化效应，将基因和药物运载和
释放到特定的组织和器官，使局部组织达到有效的治疗浓度。己有充分
研究证实该系统的可行性和应用前景，其全身给药少、局部浓度高、无
创、简便、并可在床边进行等优点，赋予当前靶向性差、正处于低迷状
态的基因疗法以新的希望。
2．目的和意义
本研究拟以上述理论、研究背景为依据，利用分子生物学、细胞生物
学及声学造影技术，以蛋白质靶向微泡在组织水平探索超声介导微泡空
化靶向传输放射性核素标记的大分子物质BSA的可行性，以蛋白质微泡。
脂质体微泡在细胞水平初步探索超声介导微泡空化效应促内皮细胞报告
基因转染、表达的可能性；以自制脂质体靶向微泡向大鼠梗塞心肌靶向
传输自行构建、克隆的血管内皮生长因子基因VEGF165，并在心肌梗塞动
物模型评价VEGF16；分子搭桥血管新生的效果：为超声介导微泡空化靶
向传输系统的深入研究以及冠心病分子搭桥基因治疗服务于临床，奠定
理论基础和提供实践经验。
第一军医大学南方医院率先研制的第一。二代造影剂填补了国内
心肌声学造影的一项空白，如果研制成功能进行心肌靶向传输的第三代
造影剂，则能紧跟国际前沿，进一步丰富超声介导微泡空化靶向传输系
统这一涉及多个学科的复杂理论体系。未来超声介导微泡空化靶向传输
系统的成功实践，不但会给心肌血管新生的分子搭桥基因治疗提供有效
的基因靶向投送手段，而且还有助于攻克目前基因治疗靶向性差的世界
难题。同时，靶向微泡的成功研制还将用于诸如肿瘤、外用血栓病的治
疗，因此本研究具有重要的经济和社会意义。
3．研究内容
一6－
本课题主要进行以下几个方面的的研究：
＊）在国内应用超声介导微泡空化靶向传输的动物及细胞模型，首次进行
靶向传输研究；
。c）向大鼠心肌传输放射性核素标记的牛血清白蛋白，取得预期心肌靶向
一传输效果；
m 向培养的人血管内皮细胞传输LacZ报告基因，证明脂质体靶向微泡
有良好的靶向传输及促基因转染、表达效果；
O） 向大鼠梗塞心肌传输自行构建的血管内皮生长因子基因VEGF165；取
得一定的促血管生成效应：
门在血管内皮生长因于基因表达载体的构建过程中，发现一新的血管内
皮生长因子基因的剪接形式
Introduction 1. Background
Coronary Heart Disease (CHD),which essence is ischmia and hxpoxia,is publicly recognized as main threat for human health and cause the leading death worldwide. With the guidance of evidence medicine,much achievement have been made on the treatment of CAD,especially acute myocardial infarction over the past decades due to lots of newly-found drugs and the reperfusion therapies represented by coronary artery bypass graft (CABG) and percutaneous transluminal coronary angioplasty (PTCA);however,a lot of issues remain resolved. A growing number of patients neither are suitable for candidates for conventional intervention therapies secondary to anatomical constraints imposed by the severity or extent of their coronary artery disease and some systematical condition such as diabetes and tumor,nor can they get enough blood supply under intervention. If such condition developed,the ischemic myocardium would be doomed to apoptosis,necrosis and fibrosis. It's urgent for people to seek other biological revascularization
strategy,and a large body of work involved in both therapeutic angiogensis and stem cell graft are hopefully under way.
Normally microcirculation compensation of ischemic heart includes microvessel enlargement,angiogensis and collateral vessel development between artery and vein. However,even nitrates and calcium antagonist were fully administrated,intrinsic microcirculation adjustment still could not reach beyond the basic demand of oxygen and blood of ischemic myocardium,therefore therapeutic angiogenesis induced by exogenesis growth factor which can initiate the formation of a plexus of collateral vessels in zones of ischemic myocardium serve to relieve angina and to improve advanced myocardial ischemia at the base of drug therapy and revascularization intervention. Angiogenesis,which is called "molecular graft of vessels",is a complex and cascade process that involves in stimulation of endothelial cell proliferation and migration by a huge amount of antagonist and agonist
growth factors,including the fibroblast growth factor (FGF) family,vascular endothelial growth factor (VEGF) family,angiopoietin,ephrins and thrombospondin-1 through stimulating high affinity receptors and signal transduction passway of endothelial cells,and those mitogen factors and cascades seems to act in a coordinated way to achieve this process. The vascular endothelial growth factor(VEGF) was identified as endothelial cell specific mitogen. It has been shown to be involved in endothelial cell proliferation and blood vessel formation. VEGF mediates its function through its specific receptor on the vascular endothelial cell surface. VEGF plays important roles in variety of physiological and pathological processes including embryogenesis,tumor and some cardiovascular diseases. The rapid development of molecular biology in myocardiovascular scope and growth factor therapy offers to clinical practices. Although several clinical trials involved various endovascular delivery devices have been designed for angiogenesis,such as bolus injection of naked plasmid DNA encoding phVEGF165,phVEGF121 and bFGF of recombinated virus,either in pericardium,direct myocardium at the time of thoracotomy or intracoronary injection and endoluminal delivering,the limitations of such delivery including the possibility of increased vascular trauma,high risk,low efficiency of localization,inconsistency of delivery,and rapid washout of the agents from the vascular wall after delivery prevent the progres of further development of angiogenesis.
Ultrasound,which has been playing a very important role in targeted delivering of gene and active agents,merits further exploration. The aim is to use ultrasound and ultrasound echo contrast agents to offer a targeting delivering procedure,called the delivery system of ultrasound-mediated destruction and cavitation of targeted microbubbles. The core of the approach,which depend on the cavitation effect of microbubble under ultrasound field,is locally delivered pharmacological drugs or t
引文
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24.Yuhong Xu, Francis C,Szoka Jr. Mechanism of DNA Release from Cationic Liposome/DNA Complexes Used in Cell Transfection. Biochemistry. 1996;35:5616-5623
25.Guzma R J, Lemarchand P, Crystal RG, et al. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Cir Res. 1993 ;73:1202-1207
26.Alexander LK, Michael SH, Flordeliza SV, et al. Targeting and ultrasound imaging of microbubble-based contrast agents. Magnetic Resonance Materials in Physics, Biology and Medicine. 1999,8:177-184
27.Mark WK, Steven SS, Sanjiv K, et al. The behavior of sonicated albumin microbubbles within the microcirculation: a basis for their use during myocardial contrast echocardiography. Circulation Research. 1989,65(2):458-467
28.Alexander L.Klibanov. Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging. Advanced Drug Delivery Reviesws. 1999,37:139-157
29.Jonathan R, Lindner MD, Sanjiv K, et al. Delivery of drugs with ultrasound.Echocardiogaphy. 2001,18(4):329-337
30．范义湘．硕士论文：亲合素促排用于~(99m)Tc-C13-Bt大肠癌放免显像的实验研究．第一军医大学．2000，6
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39.Skyba DM, Price RJ, Linka AZ, Skalak TC, Kaul S. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue. Circulation. 1998;98:290-293.
40.Wong J, Mukharjee D, Porter T, Young D, Sen S, Thomas J. Ultrasound enhances PESDA linked oligonucleotide deposition into myocardial tissue. J Am Soc Echocardiog. 1998;11 (5):498.
41.Porter TR, Iverson PL, Li SP, Xie F. Interaction of diagnostic ultrasound with synthetic oligonucleotide-labeled perfluorocarbon-exposed sonicated dextrose albumin microbubbles, J Ultrasound Med 1996; 15:577-584.
42.Unger EC, Hersh E, Vannan M, Matsunaga TO, McCreery T. Local drug and gene delivery through microbubbles. Prog Cardiovasc Dis. 2001;44(1):45-54.
43.Mitragotri S, Blankschtein D, Langer R. Ultrasound-mediated transdermal protein delivery. Science. 1995;269:850-852.
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45.Lindner JR, Song J, Christiansen J, Klibanov AL, Xu F, Ley K. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation. 2001; 104(17):2107-12.
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21.Koch S, Pohl P, Cobet U, et al.. Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects. Ultrasound Med Biol. 2000 ;26(5):897-90
22.Shohet RV, Chen S,Zhou YT, et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation. 2000; 101 (22):2554-6
23.Lawrie A, Brisken AF, Francis SE, et al. Microbubble-enhanced ultrasound for vascular gene delivery. Gene therapy. 2000;7:2023-2027
24.Yuhong Xu, Francis C,Szoka Jr. Mechanism of DNA Release from Cationic Liposome/DNA Complexes Used in Cell Transfection. Biochemistry. 1996;35:5616-5623
25.Guzma R J, Lemarchand P, Crystal RG, et al. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Cir Res. 1993 ;73:1202-1207
26.Alexander LK, Michael SH, Flordeliza SV, et al. Targeting and ultrasound imaging of microbubble-based contrast agents. Magnetic Resonance Materials in Physics, Biology and Medicine. 1999,8:177-184
27.Mark WK, Steven SS, Sanjiv K, et al. The behavior of sonicated albumin microbubbles within the microcirculation: a basis for their use during myocardial contrast echocardiography. Circulation Research. 1989,65(2):458-467
28.Alexander L.Klibanov. Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging. Advanced Drug Delivery Reviesws. 1999,37:139-157
29.Jonathan R, Lindner MD, Sanjiv K, et al. Delivery of drugs with ultrasound.Echocardiogaphy. 2001,18(4):329-337
30．范义湘．硕士论文：亲合素促排用于~(99m)Tc-C13-Bt大肠癌放免显像的实验研究．第一军医大学．2000，6
31.Schwarz SW, Connett JM, Anderson C J, et al. Evaluation of a direct method for technetium labeling intact and F(ab)1A3, an anticolorectal monoclonal antibody. Nucl Med Biol. 1994,21 (4):619-626
32.Castiglia SG, Duran A, Fiszman G, et al.~(99m)Tc direct labeling of anti-CEA monoclonal atidodies:quality control and preclinical studies. Nucl Med Biol. 1995,22(3):367-372
33.Verma IM, Somia N. Grene therapy: Promises, problems, and prospects. Nature 1997;389:239-242.
34.Kay MA, Liu D, Hoogerbrugge PM. Gene therapy. Proc Natl Acad Sci USA. 1997;94:12744-12746.
35.Anderson WF. Human gene therapy. Nature. 1998;392:25-30.
36.Langer R. Drug delivery and targeting. Nature. 1998;392:5-10.
37.Price R J, Skyba DM, Kaul S, Skalak TC. Delivery of colloidal particles and red blood cells to tissue through microvessel ruptures created by targeted microbubble destruction with ultrasound. Circulation. 1998;98:1264-1267.
38.Shohet RV, Chen S, Zhou YT, Wang Z, Meidell RS, Unger RH, Grayburn PA. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation. 2000; 101:2554-2556.
39.Skyba DM, Price RJ, Linka AZ, Skalak TC, Kaul S. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue. Circulation. 1998;98:290-293.
40.Wong J, Mukharjee D, Porter T, Young D, Sen S, Thomas J. Ultrasound enhances PESDA linked oligonucleotide deposition into myocardial tissue. J Am Soc Echocardiog. 1998;11 (5):498.
41.Porter TR, Iverson PL, Li SP, Xie F. Interaction of diagnostic ultrasound with synthetic oligonucleotide-labeled perfluorocarbon-exposed sonicated dextrose albumin microbubbles, J Ultrasound Med 1996; 15:577-584.
42.Unger EC, Hersh E, Vannan M, Matsunaga TO, McCreery T. Local drug and gene delivery through microbubbles. Prog Cardiovasc Dis. 2001;44(1):45-54.
43.Mitragotri S, Blankschtein D, Langer R. Ultrasound-mediated transdermal protein delivery. Science. 1995;269:850-852.
44.Lindner JR, Coggins MP, Kaul S, Klibanov AL, Brandenburger GH, Ley K.Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin-β and complement-mediated adherence to activated leukocytes. Circulation. 2000 15; 101 (6):668-75.
45.Lindner JR, Song J, Christiansen J, Klibanov AL, Xu F, Ley K. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation. 2001; 104(17):2107-12.
46.Liu J, Lewis TN, Prausnitz MR. Non-invasive assessment and control of ultrasound-mediated membrane permeabilization. Pharmaceutical Res. 1998;15:918-924.
47.Lanza GM, Wickline SA. Targeted ultrasonic contrast agents for molecular imaging and therapy. Prog Cardiovasc Dis. 2001 ;44( 1): 13-31.
48.Miller DL, Gies RA. The interaction of ultrasonic heating and cavitation in vascular bioeffects on mouse intestine. Ultrasound in Med & Biol. 1998;24:123-128.
49.Dayton P, Morgan K, Allietta M, Klibanov A, Brandenburger G, Ferrara K. Simultaneous optical and acoustical observations of contrast agents. IEEE Ultrasonics Symposium. 1997; 1583-1591.
50.Fisher NG, Leong-Poi H, Sakuma T, Rim S J, Bin JP, Kaul S.Detection of coronary stenosis and myocardial viability using a single intravenous bolus injection of BR14. J Am Coll Cardiol. 2002(in press).
51.Fisher NG, Christiansen JP, Klibanov A, Taylor RP, Kaul S, Lindner JR. Surface charge of microbubbles influences their capillary retention and consequently their myocardial contrast effect. 20002 J Am Coll Cardiol.
52.Wei K, Skyba DM, Firschke C, et al. Interactions between microbubbles and ultrasound: in vitro and vivo observations. J Am Coll Cardiol, 1997, 29:1081-1088
53.Ward M, Wu J, Chiu JF. Experimental study of the effects of Optison concentration on sonoportion in vitro. Ultrasound Med Biol, 2000, 26:1169-1175
54.Qian Z, Sangers RD, Pill WG. Investigations of the mechanism of the bioacoustic effect. J Biomed Maler Res, 1999, 44:198-205
55.Miller DL, Quddus J. Sonoporation of monolayer cells by diagnostic ultrasound activation of contrast-agent gas bodies. Ultrasound Med Biol. 2000,26:661-667
56.Jon AW, Robert W, Malone P, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990,247(23):1465-1468
57．李建军，夏豪，李庚山．基因转导现状．现代诊断与治疗．199910(5)：294-296
58．何维，吴鹤龄．细胞谱系的一种标记基因—LacZ基因．1995—1996
59.Alexander MY, Webster KA, McDonald PH, et al. Gene transfer and models of gene therapy for the myocardium. Clin Exp Pharmacol Physiol, 1999, 26:661-668
60．赵海霞，郭兴奎，孔德亮，等．脂质体制备技术．中药制药新技术．山东中医杂志．19(7)：435—437
61．刘启光，吴旻，沈子威，等．包裹质粒DNA及线状DNA脂质体的制备．中国医学科学院学报．1992，14(3)：220-223
62．卢圣栋，主编．现代分子生物学实验技术．第二版．中国协和医科大学出版社．1999，12
63．颜子颖，王海林，译．精编分子生物学实验指南．科学出版社．1998，6
64.Kim H J, Greenleaf JF, Kinnck RR, et al. Ultrasound-mediated transfection of mammalian cells. Hum Gene Ther, 1996,7:1339-1346
65.Mukherjee D, Wong J, Griffin B, et al. Tenfold augmentation of endothelial uptake of vascular endothelial growth factor with ultrasound after systemic administration. J Am Cardiol, 2000, 35:1678-1686
66.Manome Y, Nakamura M, Ohno T, et al. Ultrasound facilitates transductio of naked plasmid DNA into colon carcinoma cells in vitro and in vivo. Hum Gene, 2000, 11:1521-1528
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