摘要
背景:
实体肿瘤的发生、发展和转移依赖肿瘤新生血管的生长。抗肿瘤血管生成作为一种新兴的肿瘤治疗手段,通过药物结合新生血管特异的靶点抑制肿瘤血管新生从而抑制其生长,但仍属于一种药物治疗手段,无法避免耐药性及毒、副作用的产生。目前超声治疗肿瘤的方法以高强度聚焦超声(HIFU)为代表,利用声强高达2000～10000 W/cm2聚焦超声束的热效应造成靶区组织的凝固性坏死,从而消融肿瘤组织。但是,由于人体适合大范围聚焦的理想声窗较少,以及伴随的热效应损伤等副作用,HIFU的临床应用受到了一定的限制。超声空化效应是热效应之外另一种物理效应,主要释放冲击波、微射流等机械能量形式。利用超声空化效应阻断肿瘤微血管、抑制肿瘤生长的设想和研究鲜见报道。
微泡在超声激发下可以进一步增强空化效应,其释放的机械能可能具有损伤血管内皮、靶向定位消融组织的潜在能力;而肿瘤新生血管发育不完全、结构薄弱、通透性高,对各种理化因素的作用比较敏感,这使得采用微泡增强的超声空化效应破坏肿瘤新生血管,从而抑制肿瘤生长、消融肿瘤成为可能。采用低能量超声空化破坏阻断肿瘤血管的方法可以避免HIFU的热效应副损伤,同时具有操作简单、可重复性好等潜在优点。
目的:
(1)探讨微泡增强的超声空化阻断肿瘤微血管的可行性。
(2)通过观察微泡联合超声辐照后肿瘤组织的病理变化,尤其是肿瘤微小血管及其周围结构的变化,探讨其阻断机理,进一步探索一种新的、针对肿瘤新生血管的非创伤性物理治疗方法。
方法:
1.实验材料:
实验仪器:①脉冲式聚焦超声治疗仪两台第三军医大学新桥医院超声科自行研制,其治疗头频率分别为1.2 MHz和831KHz,超声发射及间歇时间、发射脉冲宽度、脉冲重复频率、峰值声压等多档可调。②超声诊断仪,美国通用电气公司Logiq 9彩色多普勒超声诊断仪,配有9L高频线阵探头,具备低机械指数谐波造影成像功能。实验微泡试剂:第三军医大学新桥医院超声科自行研制的脂膜微泡“脂氟显”,其核心气体为全氟丙烷,微泡平均粒径2μm,其中98﹪小于8μm;微泡浓度约为4~9×109/ ml。
实验动物:健康新西兰大白兔30只,雌雄不拘,体质量2000±225 g;清洁级雄性Wistar大鼠32只,体质量180±20 g。所有实验动物均由第三军医大学新桥医院实验动物中心提供并检疫。
2.实验方法:
(1)兔VX2肿瘤治疗实验: 26只腿部皮下VX2肿瘤荷瘤兔随机分为超声微泡组、单纯超声组及假照组。超声微泡组经耳缘静脉推注微泡(0.2 ml/kg)联合超声辐照肿瘤,单纯超声组以生理盐水代替微泡,假照组推注微泡而超声假照。各组治疗前、治疗后即刻及超声微泡组治疗后30和60 min时间点重复对肿瘤进行超声造影检查,分析各时间点造影灌注峰值灰阶变化。
(2)大鼠Walker-256肿瘤治疗实验:32只雄性Wistar大鼠,采用细胞悬液注射法建立Walker-256皮下肿瘤,并随机分为假照组、单纯超声组、低声压超声联合微泡组及高声压超声联合微泡组,治疗方法同前。治疗前、治疗后即刻以及处死动物取材前分别对肿瘤进行超声造影检查并比较肿瘤造影增强变化。分别于治疗后即刻、60 min及治疗后24小时处死部分大鼠,经主动脉灌注固定肿瘤,取材制作切片并染色,采用光镜、电镜观察并比较肿瘤组织病理变化,尤其是微小血管及其周围组织结构变化;并将病理变化评分与取材前肿瘤造影增强视觉评分进行相关性分析。
结果:
(1)新西兰白兔VX2肿瘤治疗实验结果:微泡联合超声组肿瘤造影平均灰阶值从治疗前的67.8±13.3降至29.7±20.1(P<0.01),呈无灌注负性显影;至60 min时(34.3±20.7,P<0.05)仍无明显再通;而单纯超声组、假照组治疗前后肿瘤造影灰阶值无明显变化(P>0.05)。
(2)大鼠Walker-256肿瘤治疗实验结果:微泡联合超声组治疗后肿瘤组织主要病理变化包括微血管扩张、崩解,弥漫性充血和出血,红细胞及血浆蛋白渗出,局部血肿血栓形成,并伴有肿瘤组织水肿。肿瘤细胞坏死主要发生在损伤的血管周围。透射电镜观察见肿瘤微血管内皮崩解,基底膜不完整,内皮细胞核结构不清楚,染色质分布聚集成团,胞质均匀化改变,细胞器结构不清楚,可见大量小泡。Spearman相关分析显示肿瘤病理变化评分与取材前肿瘤造影增强视觉评分呈显著负相关(rs= -0.890,P<0.01)。
结论:
(1)微泡增强的超声空化能够有效阻断兔VX2和大鼠Walker-256皮下移植瘤的微循环。
(2)微泡增强的超声空化阻断肿瘤微循环的主要机制为肿瘤微小血管壁严重损伤并崩解,造成局部肿瘤组织充血、血肿、水肿,以及血栓形成。
Background:
Tumor angiogenesis is of vital importance to the growth and metastasis of solid tumors. Anti-angiogenesis, widely considered as an emerging technique in cancer therapy, usually attack or inhibit certain targets of tumor angiogenesis using specific chemical or biological substances, thus may have certain side effects as a method of pharmacologic therapy. High-intensity focused ultrasound (HIFU), at the intensity of 2000～10000 W/cm2, usually inducing permanent coagulative necrosis damage to tissues, has become a new method for tumor thermal ablation. The clinical applications of HIFU, however, were limited because of rare adequate acoustic window to access the tumors, or inevitable injuries to the adjacent structures. Physical therapy applications using ultrasound cavitation on the disruption of tumor neovasculature had seldom been reported.
Microbubbles (MBs) can generate significant physical effects from cavitation under ultrasound excitation. The mechanical energy released by cavitation (non-thermal effects) has a potential ability to eliminate targeted living tissues. This hypothesis suggests a possibility of disrupting the immature, leaky and fragile tumor microvasculature. In addition, being a simple physical therapeutic method, MBs enhanced ultrasound cavitation to obstruct tumor microcirculation would have a good repeatability, and would be able to prevent the thermal side effects of HIFU treatment.
Objective:
To investigate the possibility of MBs enhanced ultrasound cavitation on tumor microcirculation obstruction, as well as the mechanism underneath through Pathological histology study on the neoplasm, especially the morphological changes of the tumor micro-vasculature. This new method might be a potential noninvasive physical therapeutic method on malignant tumor neovasculature.
Materials and Methods:
(1) Materials:
①Therapeutic ultrasound instrument: specifically designed pulsed focused ultrasound (PFUS) devices, with the transducers frequency of 1.2 MHz & 831 KHz, adjustable emission/pause time, pulse length, pulse recurrence frequency, and peak acoustic pressure.
②Diagnostic ultrasound imaging system: GE Logiq 9 ultrasound system, 9L probe, eligible to perform contrast enhanced ultrasonography (CEUS) with low MI.
③Microbubbles: Zhifuxian, a formulation of lipid-encapsulated MBs with perfluoropropane gas, with the concentra1tion of 4~9×109 /ml. The mean diameter of MBs was measured to be 2μm, and 98% of the MBs less than 8μm in diameter.
(2) Methods:
①26 New Zealand rabbits bearing subcutaneous VX2 tumor were randomly assigned into 3 groups for the factors including MBs infusion and ultrasound exposure. PFUS was delivered directly to the tumor surface for 10 minutes during intravenous infusion of MBs at 0.2 ml/kg in the experimental group(PFUS +MBs). The control groups were applied with only PFUS exposure or MBs injection. The tumor perfusion was imaged using CEUS before and 0 minute, 30 minutes, and 60 minutes after treatment. The gray scale values (GSV) of tumor contrast perfusion were compared statistically.
②32 male Wistar rats bearing subcutaneous Walker-256 tumor were randomly assigned into 4 different groups (Low acoustic pressure-PFUS +MBs, High acoustic pressure-PFUS +MBs, PFUS +NS, and Sham +MBs), and received respective treatments similar as described above. CEUS were performed before and after treatment, and prior to the scheduled point-of-time for animals sacrifice. After a 100 ml 4% paraformaldehyde intravascular perfusion fixation, the tumors were removed, fixed, sectioned, and stained for review with a light microscope (LM) and a transmission electron microscope (TEM). Qualitative and quantitative descriptions of the histological changes of the neoplasm, especially the morphological changes of the tumor micro-vasculature were noted and compared. A Spearman rank correlation analysis was performed to establish whether there was a relationship between the quantitative histological change and the visual score of the pre-sacrifice CEUS of the tumors.
Results:
①The contrast perfusion of VX2 tumors almost vanished immediately after treatment in the PFUS +MBs group, with the GSV reduced from 67.8±13.3 (before treatment) to 29.7±20.1 (0 minute post treatment, P﹤0.01), 37.3±19.9 (30 minutes post treatment), and 34.3±20.7 (60 minutes post treatment). No statistically significant were found before and after treatment (P >0.05) in both control groups (PFUS +NS & Sham +MBs).
②The predominant pathological findings of insonating the subcutaneous Walker-256 neoplasms appeared to be microvascular dilation, destruction of tumor capillaries associated with hemorrhage and increased intercellular edema, and in situ thrombosis. Necrosis of neoplastic cells occurred mainly in the areas around the damaged vasculatures. Injury of the endothelial cells of tumor capillaries was confirmed by TEM. A high correlation between the quantitative histological change and the visual score of the CEUS of the tumors were demonstrated with a Spearman rank correlation analysis (rs = -0.890, P﹤0.01).
Conclusions:
The tumor micro-circulation can be blocked by MBs enhanced ultrasound cavitation.
The main effects of MBs enhanced ultrasound cavitation over tumor micro-circulation was histological changes in the tumor vasculature, including damage of the endothelia, hemorrhage, increased intercellular fluid, and in situ thrombosis.
引文
1. Folkman J. Tumor angiogenesis: therapeutic implications. New Eng J Med, 1971, 285: 1182-1186.
2. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature, 2000, 407: 249-257.
3. Folkman J. Angiogenesis and apoptosis. Seminars in Cancer Biology, 2003, 13: 159-167.
4. Kerbel RS. Tumor Angiogenesis. New Eng J Med, 2008, 358: 2039-2049.
5. Wu F, Chen WZ, Bai J, et al. Pathological changes in human malignant carcinoma treated with high intensity focused ultrasound. Ultrasound in Medicine and Biology, 2001, 27: 1099-1106.
6. Wu F, Chen WZ, Bai J, et al. Tumor vessel disruption resulting from high- intensity focused ultrasound in patients with solid malignancies. Ultrasound in Medicine and Biology, 2002, 28: 535-542.
7. Diederich CJ, Stafford RJ, Nau WH, et al. Transurethral ultrasound applicators with directional heating patterns for prostate thermal therapy: In vivo evaluation using magnetic resonance thermometry. Med Phys, 2004, 31: 405-413.
8. Dogra VS, Zhang M, Bhatt S. High-Intensity Focused Ultrasound (HIFU) Therapy Applications. Ultrasound Clinics, 2009, 4: 307-321.
9. Fischer K, Gedroyc W, Jolesz FA. Focused ultrasound as a local therapy for liver cancer. Cancer Journal. 2010, 16: 118-24.
10. Xie F, Tsutsui JM, Lof J, et al. Effectiveness of Lipid Microbubbles and Ultrasound in Declotting Thrombosis. Ultrasound in Medicine and Biology, 2005, 31: 979-85.
11. Wang B, Wang L, Zhou XB, et al. Thrombolysis effect of a novel targeted microbubble with low-frequency ultrasound in vivo. Thromb Haemost, 2008, 100: 356-361.
12. Xie F, Lof J, Matsunaga T, et al. Diagnostic ultrasound combined with glycoproteinⅡb/Ⅲa targeted microbubbles improve microvascular recovery following acute coronary thrombotic occlusions. Circulation, 2009, 119: 1358-1360.
13. Taniyama Y, Tachibana K, Hiraoka K, et al. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation, 2002, 105:1233-1239.
14. Vassilis Sboros. Response of contrast agents to ultrasound. Advanced Drug Delivery Reviews, 2008, 60: 1117-1136.
15. Meijering BD, Juffermans LJ, Wamel A, et al. Ultrasound and Microbubble Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation. Circ Res, 2009, 104: 679- 687.
16. Roberts WW, Hall TL, Ives K, et al. Pulsed Cavitational Ultrasound: A Noninvasive Technology for Controlled Tissue Ablation (Histotripsy) in the Rabbit Kidney. The Journal of Urology, 2006, 175: 447-453.
17. Parsons JE, Cain CA, Abrams GD, et al. Pulsed cavitational ultrasound therapy for controlled tissue homogenization. Ultrasound in Medicine and Biology, 2006, 32: 115-129.
18. Hwang JH, Brayman AA, Reidy MA, et al. Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treatments in vivo. Ultrasound in Medicine and Biology, 2005, 31(4): 553-564.
19.李佩倞,左松,刘政,等.间歇式超声发射对超声空化损伤小血管的增强作用.临床超声医学杂志,2007,9: 577-580.
20.李秋颖,刘政,李佩倞,等.微泡介导下脉冲式聚焦超声空化导致血管损伤的实验研究.中国超声医学杂志, 2008, 24(4): 297-299.
1. Folkman J. Tumor angiogenesis: therapeutic implications. New England Journal Med, 1971, 285: 1182-1186.
2. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature,2000, 407: 249-257.
3. Folkman J. Angiogenesis and apoptosis. Seminars in Cancer Biology, 2003, 13:159-167.
4. Carmeliet P, Conway EM. Growing better blood vessels. Nat Biotechnol, 2001, 19: 1019-1020.
5. Jain RK. Tumor angiogenesis and accessibility: Role of vascular endothelial growth factor. Semin Oncol, 2002, 29(Suppl 16): 3-9.
6. Hwang JH, Brayman AA, Reidy MA, et al. Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treatments in vivo. Ultrasound in Medicine and Biology, 2005, 31(4): 553-564.
7.李佩倞,左松,刘政,等.间歇式超声发射对超声空化损伤小血管的增强作用.临床超声医学杂志,2007,9: 577-580.
8.李秋颖,刘政,李佩倞,等.微泡介导下脉冲式聚焦超声空化导致血管损伤的实验研究.中国超声医学杂志, 2008, 24(4): 297-299.
9.冯若.超声空化与超声医学.中华超声影像学杂志,2004, 13: 63-65.
10. Vassilis Sboros. Response of contrast agents to ultrasound. Advanced Drug Delivery Reviews, 2008, 60(10): 1117-1136.
11. Meijering BD, Henning RH, Van Gilst WH, et al. Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells. Journal of Drug Targetting, 2007, 15(10): 664-671.
12. Yu T, Xiong S, Mason TJ, et al. The use of a microbubble agent to enhance rabbit liver destruction using high intensity focused ultrasound. Ultrasonics Sonochemistry, 2006, 13:143-149
13.肖雁冰. SonoVue在超声诊断中的应用.临床超声医学杂志, 2004, 6(4): 239-241.
14. Ralph M Bunte, Sara Ansaloni, Chandra M Sehgal, et al. Histopathological observations of the antivascular effects of physiotherapy ultrasound on a murine neoplasm. Ultrasound in Medicine and Biology, 2006, 3(3): 453-461.
15. Andrew KW Wood, Ralph M Bunte, Jennie D Cohen, et al. The antivascular action of physiotherapy ultrasound on a murine tumor: role of a microbubble contrast agent. Ultrasound in Medicine and Biology, 2007, 33(12): 1901-1910.
1. Folkman J. Tumor angiogenesis: therapeutic implications. New England Journal Med, 1971, 285: 1182-1186.
2. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature, 2000, 407: 249-257.
3. Kerbel RS. Tumor Angiogenesis. New England Journal Med, 2008, 358: 2039-2049.
4. Carmeliet P, Conway EM. Growing better blood vessels. Nature Biotechnology, 2001, 19: 1019-1020.
5. Van Leenders GJLH, Beerlage HP, Ruijter ET, et al. Histopathological changes associated with high intensity focused ultrasound (HIFU) treatment for localized adenocarcinoma of the prostate. Journal of clinical pathology, 2000, 53: 391-394.
6. Wu F, Chen WZ, Bai J, et al. Tumor vessel destruction resulting from high-intensity focused ultrasound in patients with solid malignancies. Ultrasound in Medicine and Biology, 2002, 28: 535-542
7. Diederich CJ, Stafford RJ, Nau WH, et al. Transurethral ultrasound applicators with directional heating patterns for prostate thermal therapy: In vivo evaluation using magnetic resonance thermometry. Medical Physics, 2004, 31: 405-413.
8. Fischer K, Gedroyc W, Jolesz FA. Focused ultrasound as a local therapy for liver cancer. Cancer Journal. 2010, 16: 118-24.
9. Rosenthal I, Sostaric JZ, Riesz P. Sonodynamic therapy- a review of the synergistic effects of drugs and ultrasound. Ultrasonic Sonochemistry, 2004, 11: 349-363.
10. Yu TH, Wang ZB, Mason TJ. A review of research into the uses of low level ultrasound in cancer therapy. Ultrasonic Sonochemistry, 2004, 11: 95-103.
11. Yu TH, Xiong SH, Mason TJ, et al. The use of a microbubble agent to enhance rabbit liver destruction using high intensity focused ultrasound. Ultrasonic Sonochemistry, 2006, 13: 143-149.
12. Miller DL, Thomas RM. Contrast-agent gas bodies enhance hemolysis induced by lithotripter shock waves and high-intensity focused ultrasound in whole blood. Ultrasound in Medicine and Biology, 1996, 22: 1089-1095.
13. Miller DL, Li P, Dou CY, et al. Influence of contrast agent dose and ultrasoundexposure on cardiomyocyte injury induced by myocardial contrast echocardiography in rats. Radiology, 2005, 237: 137-143.
14. Bertuglia S. Increase in capillary perfusion following low-intensity ultrasound and microbubbles during post-ischemic reperfusion. Critical Care Medicine, 2005, 33: 2061-2067.
15. Bunte RM, Ansaloni S, Sehgal CM, et al. Histopathological observations of the antivascular effects of physiotherapy ultrasound on a murine neoplasm. Ultrasound in Medicine and Biology, 2006, 32(3): 453-461.
16. Wood AK, Bunte RM, Price HE, et al. The Disruption of Murine Tumor Neovasculature by Low intensity Ultrasound: Comparison Between 1 and 3 MHz Sonication Frequencies. Academic Radiology, 2008, 15: 1133-1141.
17.李秋颖,刘政,李佩倞,等.微泡介导下脉冲式聚焦超声空化导致血管损伤的实验研究.中国超声医学杂志, 2008, 24: 297-299.
18. Chappell JC, Klibanov AL, Price RJ. Ultrasound–microbubble -induced neovascularization in mouse skeletal muscle. Ultrasound in Medicine and Biology, 2005, 31: 1411-1422.
19. Miller DL, Dou CY. The potential for enhancement of mouse melanoma metastasis by diagnostic and high -amplitude ultrasound. Ultrasound in Medicine and Biology, 2006, 32: 1097-1101.
20. Xing YF, Lu XC, Pua EC, et al. The effect of high intensity focused ultrasound treatment on metastases in a murine melanoma model. Biochemical and Biophysical Research Communications, 2008, 375: 645-650.
21. Deng J, Zhang Y, Feng J, et al. Dendritic cells loaded with ultrasound -ablated tumour induce in vivo specific antitumour immune responses. Ultrasound in Medicine and Biology, 2010, 36: 441-448.
1. Datta S, Coussios CC, McAdory LE, et al. Correlation of cavitation with ultrasound enhancement of thrombolysis. Ultrasound in Medicine and Biology, 2006, 32: 1257-1267.
2. Miller DL, Li P, Dou C, et al. Influence of contrast agent dose and ultrasound exposure on cardiomyocyte injury induced by myocardial contrast echocardiography in rats. Radiology, 2005, 237: 137-143.
3. Li P, Armstrong WF, Miller DL, et al. Impact of myocardial contrast echocardiography on vascular permeability: comparison of three different contrast agents. Ultrasound in Medicine and Biology, 2004, 30: 83-91.
4. Dalecki D, Rota C, Raeman CH, et al. Premature cardiac contractions produced by ultrasound and microbubble contrast agents in mice. Acoustical Research Letters Online, 2005, 6: 221-226.
5.王雅婕,刘德英,刘政,等.超声造影在心脏负荷实验中对心肌微血管的损伤效应.中国超声医学杂志,2008,24(2):106-109.
6. Hwang JH, Brayman AA, Reidy MA, et al. Vascular effects induced by combined
1-MHz ultrasound and microbubble contrast agent treatments in vivo. Ultrasound in Medicine and Biology, 2005, 31: 553-564.
7. Trubestein G, Engel C , Stumpff U , et a1. Embolism by ultrasound thrombolysis.Langenbecks Arch Chir, 1976, 340: 199-255.
8. Rosenschein U, Gaul G, Erbel R, et al. Percutaneous transluminal therapy of occluded saphenous vein grafts: can the challenge be met with ultrasound thrombolysis? Circulation. 1999, 99:26-29.
9. Pfaffenberger S, Devcic-Kuhar B, Kastl SP, et al. Ultrasound thrombolysis. Thrombosis and Haemostasis, 2005, 94: 26-36.
10. Rosenschein U, Furman V,Kerner E,et a1.Ultrasound imaging guided noninvasive ultrasound thrombolysis.Preclinical results. Circulation. 2000, 102:238-245.
11. Unger EC,Porter TR, Culp WC,et al. Therapeutic applications of lipid-Coatedmicrobubbles. Advanced Drug Delivery Reviews,2OO4,56:1291-1314.
12. Holland CK, Apfel RE. Thresholds for transient cavitation produced by pulsed ultrasound in a controlled nuclei environment. The Journal of Acoustical Society of America. 1990, 88: 2059-2069.
13. Tachibana K, Tachibana S. Albumin Microbubble Echo-contrast Material as an Enhancer for Ultrasound Accelerated Thrombolysis. Circulation, 1995, 92: 1148-1150.
14. Porter TR, LeVeen RF, Fox R, et al. Thrombolytic enhancement with perfluorocarbon exposed sonicated dextrose albumin microbubbles. The American Heart Journal, 1996, 132: 964-968.
15. Culp WC,Erdem E,Robertson PK,et al. Microbubbles potentiated ultrasound as a method of stroke therapy in a pig model:preliminary findings. Journal of Vascular and Interventional Radiology,2OO3,14:1433-1436.
16. Culp WC, Porter TR, McCowan TC, et al. Microbubble-augmented ultrasound declotting of thrombosed arteriovenous dialysis grafts in dogs. Journal of Vascular and Interventional Radiology,2003, 14: 343-347.
17. Xie F, Tsutsui JM, Lof J, et al. Effectiveness of Lipid Microbubbles and Ultrasound in Declotting Thrombosis. Ultrasound in Medicine and Biology, 2005, 31: 979-985.
18. Tsutsui JM, Xie F, Johanning J, et al. Treatment of deeply located acute intravascular thrombi with low frequency guided ultrasound and intravenous microbubbles. Journal of Ultrasound in Medicine, 2006, 25: 1161-1168
19. Tiukinhoy-Laing SD, Huang S, Klegerman M, et al. Ultrasound-facilitated thrombolysis using tissue-plasminogen activator-loaded echogenic liposomes. Thrombosis Research, 2007, 119: 777-84.
20. Kawata H, Naya N, Takemoto Y, et al. Ultrasound accelerates thrombolysis of acutely induced plateletrich thrombi similar to those in acute myocardial infarction. Circulation Journal, 2007, 71: 1643-1648
21. Culp WC, Porter TR, Lowery J, et al. Intracranial clot lysis with intravenous microbubbles and transcranial ultrasound in swine. Stroke, 2004, 35: 2407-2411.
22. Wang B, Wang L, Zhou XB, et al. Thrombolysis effect of a novel targeted microbubblewith low-frequency ultrasound in vivo. Thrombosis and Haemostasis, 2008, 100:356-361.
23. Xie F, Lof J, Matsunaga T, et al. Diagnostic ultrasound combined with glycoproteinⅡb/Ⅲa targeted microbubbles improve microvascular recovery following acute coronary thrombotic occlusions. Circulation, 2009, 119: 1358-1360.
24. Everbach EC, Francis CW. Cavitational mechanisms in ultrasound-accelerated thrombolysis at 1 MHz. Ultrasound in Medicine and Biology, 2000, 26: 1153-1160.
25. Prokop AF, Soltani A, Roy RA. Cavitational mechanisms in ultrasound accelerated fibrinolysis. Ultrasound in Medicine and Biology, 2007, 33: 924-933.
26. Datta S, Couissios CC, Ammi AY, et al. Ultrasound-enhanced thrombolysis using definity as a cavitation nucleation agent. Ultrasound in Medicine and Biology, 2008, 34: 1421-1433.
27. Tsivgoulis G, Eggers J, Ribo M, et al. Safety and Efficacy of Ultrasound -Enhanced Thrombolysis: A Comprehensive Review and Meta-Analysis of Randomized and Nonrandomized Studies. Stroke, 2010, 41: 280-287.
28. Kassan DG, Lynch AM, Stiller MJ. Physical enhancement of dermatologic drug delivery: iontophoresis and phonophoresis. Journal of the American Academy of Dermatology, 1996, 34: 657-666.
29. Iwanaga K, Tominaga K, Yamamoto K, et al. Local delivery system of cytotoxic agents to tumors by focused sonoporation. Cancer Gene Therapy, 2007, 14: 354-363.
30. Meairs S, Alonso A. Ultrasound, microbubbles and the blood-brain barrier. Progress in Biophysics and Molecular Biology, 2007, 93: 354-362.
31. Stride EP,Coussios CC. Cavitation and contrast: the use of bubbles in ultrasound imaging and therapy. Journal of Engineering in Medicine, 2010, 224: 171-191.
32. Lindner JR, Coggins MP, Kaul S, et al. Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement- mediated adherence to activated leukocytes. Circulation, 2000, 101: 668-675
33. Klibanov AL. Ultrasound molecular imaging with targeted microbubble contrast agents. Journal of Nuclear Cardiology, 2007, 14: 876-884.
34. Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: The art of turning infectious agents into vehicles of therapeutics. Nature Medicine, 2001, 7: 33-40.
35. Taniyama Y, Tachibana K, Hiraoka K, et al. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation, 2002, 105: 1233-1239.
36. Frenkel PA, Chen S, Thai T, et al. DNA-loaded albumin microbubbles enhance Ultrasound mediated transfection in vitro. Ultrasound in Medicine and Biology, 2002, 28: 817-822.
37. Vassilis Sboros. Response of contrast agents to ultrasound. Advanced Drug Delivery Reviews, 2008, 60: 1117-1136.
38. Meijering BD, Henning RH, Van Gilst WH, et al. Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells. Journal of Drug Targetting, 2007, 15: 664-671.
39. Meijering BD, Juffermans LJ, Van Wamel A, et al. Ultrasound and Microbubble Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation. Circulation Research, 2009, 104: 679-687.
40. Eisenbrey JR, Burstein OM, Kambhampati R, et al. Development and optimization of a doxorubicin loaded poly (lactic acid) contrast agent for ultrasound directed drug delivery. Journal of Controlled Release, 2010, 143: 38-44.
41. Yu TH, Xiong SH, Mason TJ, et al. The use of a microbubble agent to enhance rabbit liver destruction using high intensity focused ultrasound. Ultrasonic Sonochemistry, 2006, 13: 143-149.
42. Rosenthal I, Sostaric JZ, Riesz P. Sonodynamic therapy: a review of the synergistic effects of drugs and ultrasound. Ultrasonic Sonochemistry, 2004, 11: 349-363.
43. Yu TH, Wang ZB, Mason TJ. A review of research into the uses of low level ultrasound in cancer therapy. Ultrasonic Sonochemistry, 2004, 11: 95-103.
44.郑元义,王志刚,冉海涛,等.诊断超声加微泡造影剂对新生血管的作用.中国医学影像技术, 2004, 20: 349-150.
45.张群霞,王志刚,冉海涛,等.超声微泡促进肿瘤细胞报告基因转染.临床超声医学杂志,2005, 7:78-80.
46. Roberts WW, Hall TL, Ives K, et al. Pulsed Cavitational Ultrasound: A Noninvasive Technology for Controlled Tissue Ablation (Histotripsy) in the Rabbit Kidney. The Journal of Urology, 2006, 175: 447-453.
47. Parsons JE, Cain CA, Abrams GD, et al. Pulsed cavitational ultrasound therapy for controlled tissue homogenization. Ultrasound in Medicine and Biology, 2006, 32: 115-129.
48. Hwang JH, Brayman AA, Reidy MA, et al. Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treatments in vivo. Ultrasound in Medicine and Biology, 2005, 31: 553-564.
49.李佩倞,左松,刘政,等.间歇式超声发射对超声空化损伤小血管的增强作用.临床超声医学杂志,2007,9: 577-580.
50.李秋颖,刘政,李佩倞,等.微泡介导下脉冲式聚焦超声空化导致血管损伤的实验研究.中国超声医学杂志, 2008, 24: 297-299.
51. Folkman J. Tumor angiogenesis: therapeutic implications. New England Journal Med, 1971, 285: 1182-1186.
52. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature, 2000, 407: 249-257.
53. Kerbel RS. Tumor Angiogenesis. New England Journal Med, 2008, 358: 2039-2049.
54. Bunte R, Ansaloni S, Sehgal C, et al. Histopathological observations of the antivascular effects of physiotherapy ultrasound on a murine neoplasm. Ultrasound in Medicine and Biology, 2006, 32: 453-61.
55. Wood A, Bunte R, Cohen J, et al. The antivascular action of physiotherapy ultrasound on a murine tumor: role of a microbubble contrast agent. Ultrasound in Medicine and Biology, 2007, 33: 1901-1910.
56. Wood A, Bunte R, Price H, et al. The Disruption of Murine Tumor Neovasculature by Low-intensity Ultrasound—Comparison Between 1 MHz and 3 MHz Sonication Frequencies. Academic Radiology, 2008, 15: 1133-1141.
57.高顺记,李佩倞,赵洋,等.微泡增强的超声空化阻断肿瘤微循环的初步研究.中国超声医学杂志, 2010,26:110-112.
58.赵洋,高顺记,刘政,等.超声破坏微泡效应抑制兔VX2皮下肿瘤生长的实验研究. 中华超声影像学学杂志,2010,4: 352-355.