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

目的探讨人肝细胞癌靶向脂质体超声造影剂对荷肝癌裸鼠肿瘤的增强显像效果。方法采用人肝癌细胞株HHCC接种于裸鼠皮下,建立荷人肝癌裸鼠模型;将靶向脂质体超声造影剂或普通脂质体超声造影剂经尾静脉注入荷瘤裸鼠体内,使用二次谐波显像模式观察并记录造影过程;采用目测观察和视频灰阶分析技术,以时间-强度曲线分析来定量评价肿瘤显像的增强效果。结果造影后目测观察,靶向造影剂对肿瘤有延迟增强显像效果,靶向造影剂组肿瘤的增强显影约在8min达到峰值,峰值灰阶强度值为(39.545±10.099)dB;普通造影剂组肿瘤增强显影达峰值时间约为10s,峰值灰阶强度值为(22.438±5.108)dB,与靶向造影剂造影后灰阶强度峰值比较相差显著(P<0.01)。结论人肝细胞癌靶向脂质体超声造影剂可以增强荷人肝癌裸鼠的肿瘤超声显像效果。     

小鼠视网膜母细胞瘤的靶向超声造影成像研究

目的评估携带单纯疱疹病毒Ⅰ型胸苷激酶（HSV1-tk）质粒和血管内皮细胞生长因子受体2（VEGFR2）抗体的靶向超声造影剂Targesphere SA对视网膜母细胞瘤的造影效果。方法建立小鼠眼视网膜母细胞瘤模型,选取10例明显突出眼表的肿瘤,直径〉5 mm时行超声造影检查。制备携带HSV1-tk质粒和VEGFR2抗体的靶向微泡Targesphere SA。超声造影采用Sequoia 512型彩色多普勒超声诊断仪。先行声诺维超声造影,眼球后注射约50μl,同步录像并计时,观察至造影剂完全消退。15 min后,采用与前者同样的方法进行靶向造影剂成像。回放录像,由3名有经验的超声医师比较2种造影剂对肿瘤的增强时间、达峰时间、达峰强度及消退特征。结果 2种造影剂注入后,肿瘤内部均很快（1~2 s）可见有造影剂进入,声诺维组6~9 s增强达高峰,靶向造影剂组5~9 s增强达高峰,2种造影剂对肿瘤的增强程度一致,均呈高增强。60 s后声诺维组造影剂明显消退,而靶向造影剂组仍呈高增强。2 min后声诺维组肿瘤内的造影剂明显消退,而靶向造影剂组仍呈高增强。5 min后声诺维组肿瘤内造影剂完全消退,而靶向造影剂组仍呈高增强。10 min后靶向造影剂组肿瘤内呈高增强,15 min后造影剂明显消退。结论靶向造影剂能对视网膜母细胞瘤进行有效的超声造影成像,与声诺维比较,在2 min内无明显差异,2 min后增强效果更好,持续时间更长。     

微泡造影剂在肝脏疾病诊治中的研究进展

新型造影剂的发展,使肝脏的超声造影正发生巨大的变化.当静脉注射不同类型的超声造影剂时,微泡通过肺循环,在特定的影像程序下,不仅可以增强多普勒信号、改善灰阶超声图像的质量,而且可以实时地观察微血管及组织的血流灌注.通过在微泡上偶联肿瘤特异性抗体等方法使微泡具有靶向功能,大大提高了对肿瘤诊断的准确性,同时还可以携带药物与基因用于肿瘤的治疗.随着靶向超声微泡技术的发展,靶向超声微泡必将为超声显像与临床治疗提供一种有效的方法.     

抗VEGF抗体介导靶向超声造影剂的体外实验研究

目的以抗血管内皮因子（VEGF）抗体为配体研制能与血管内皮细胞特异性结合的靶向脂质体超声造影剂并检测其体外寻靶能力。 方法以静电吸附法将抗VEGF抗体连接到脂质体造影剂微泡的表面；体外培养ECV304人血管内皮细胞，用免疫荧光法检测靶向造影剂与其体外结合能力，以普通造影剂为对照组。 结果所制备的靶向超声造影剂与普通微泡无显著差异；免疫荧光实验结果显示靶向造影剂能在体外与血管内皮细胞特异性结合。 结论携抗VEGF抗体的脂质体靶向造影剂能通过静电吸附法成功制备，且在体外能与血管内皮细胞特异性结合。     

携VEGFR2靶向超声微泡在肾癌裸鼠模型中的显像研究

目的评价携血管内皮生长因子受体2(VEGFR2)抗体的靶向超声微泡在原位移植裸鼠肾癌模型中的显像效果。方法建立人肾透明细胞癌原位移植裸鼠模型,制备携VEGFR2抗体的靶向微泡(MBV)及携同型抗体IgG的对照微泡(MBC),每只裸鼠分别注射MBV及MBC(顺序随机,注射时间间隔30min)后行超声造影检查,比较注射两种微泡后10min内肿瘤的增强强度,并对肿瘤组织行VEGFR2免疫组化检测。结果超声造影示注射微泡后10s时两种微泡增强强度上升百分比比较差异无统计学意义,注射微泡后1,2,4,6,8,10min时MBV增强强度上升百分比明显高于MBC(P<0.05)。免疫组化显示肾肿瘤血管内皮VEGFR2高表达。结论应用携VEGFR2抗体的靶向微泡与肾癌新生血管内皮靶点特异性结合,能持续增强显像,可作为评价肿瘤新生血管及监测抗血管治疗疗效的重要分子成像方法。     

能量多普勒显像定量评价兔肾炎症组织靶向造影实验研究

目的用能量多普勒显像（PDI）定量评价免缺血再灌注损伤肾靶向造影效果。 方法将自制活性白细胞靶向造影剂和普通造影剂对兔缺血再灌注损伤肾行超声造影，采集造影前及造影后15min PDI图，用国产DFY型超声图像定量分析诊断仪对肾彩色与总面积比（CP／TP值）及彩色强度（CI值）进行定量分析，并与肾组织髓过氧化物酶活性作相关性分析。 结果造影后15min，靶向组CP／TP值及CI值显著大于普通组（P均＜0．001），且与反映肾组织炎症程度的髓过氧化物酶活性有良好相关性（P＜0．05，r=0．842和r=0．823）。 结论PDI可定量评价免缺血再灌注损伤肾靶向造影的增强效果。     

新型靶向超声造影剂的研究应用进展

自从1968年Gramiak等[1]首次提出超声造影显像的概念以来,超声造影剂的研发取得突飞猛进的发展.靶向超声造影剂是指超声造影剂表面结合或连接有特异性配体或抗体,此种超声造影剂可通过血液循环积聚到特定的靶组织,从而使靶组织在超声显像中得到特异性增强.与普通超声造影剂相比,靶向超声造影剂能从分子水平识别并结合于病灶,对病灶进行精确定位,在靶点产生特异性显影,显著提高了超声诊断的灵敏感度和特异度.同时,靶向超声造影剂还可用于引导治疗性物质局部聚集和释放.研制安全有效、可满足各种疾病检查需要的超声造影剂是超声分子成像最重要的方面.近年来,新型超声造影剂取得了巨大的进步,因其较普通超声造影剂具有更多的功能,更广阔的应用空间,因而成为当前超声医学研究的热点和前沿.本文对新型靶向超声造影剂的研究现状及发展趋势进行综述.     

靶向造影剂对卵巢癌血管生成拟态的超声评价

目的探讨携带抗金属基质蛋白酶2(MMP2)单克隆抗体的靶向超声造影剂(MBt)对裸鼠卵巢癌移植瘤血管生成拟态(VM)的应用价值。方法以生物素-链霉素桥连法构建MBt,免疫荧光法体外检测造影剂与抗体的链接情况;建立小鼠卵巢癌SKOV3细胞株模型,分别注射MBt和普通超声造影剂(MBc)进行超声造影检查。分析各组的时间-强度曲线(TIC)相关参数,并行肿瘤组织血管生成拟态MMP2免疫组化的检测。结果成功制备携抗MMP2单抗的MBt;造影结果表明MBt组较MBc组达峰时间提前、持续时间更长(P0.01),两组峰值强度之间的差异无统计学意义(P0.05),MBt组峰值强度有二次高峰现象出现;免疫组化证明肿瘤组织血管生成拟态中MMP2高表达。结论携MMP2靶向造影剂在体内能与特异度表达MMP2受体的血管拟态特异度结合,能较好地评估卵巢癌血管拟态状况。     

造影多普勒超声显像评价兔肌肉VX2肿瘤的血流灌注

目的 :评价造影多普勒超声显像对兔肌肉 VX2 肿瘤 (兔鳞癌 )血流灌注的作用。方法 :将 8只双侧股外侧肌肉种植 VX2 肿瘤的新西兰白兔经耳缘静脉注射氟碳微泡造影剂后 ,分别应用彩色多普勒、能量多普勒超声显像观察兔肌肉 VX2 肿瘤血流灌注情况并定量分析。结果 :造影后 ,增强了肌肉 VX2 肿瘤血管显示率及血流信号强度 ,造影前后比较 ,彩色多普勒血流显像视频密度增加 69.88,能量多普勒血流显像视频密度增加 94.49,二者比较 P<0 .0 1 ,延长了肿瘤血流显影时间。结论 :造影多普勒超声显像提高了兔肌肉 VX2 肿瘤血流灌注的显示 ,增强其血流信号。造影能量多普勒血流显像优于彩色多普勒血流显像     

造影多普勒超声显像检测兔肝VX2肿瘤血液供给与肿瘤血管生成相关性的实验研究

目的探讨造影多普勒超声检测兔肝VX2肿瘤血流及其与肿瘤血管生成的关系。方法选用肝VX2肿瘤模型兔8只,注射自制声学造影剂后,应用彩色及能量多普勒观察肿瘤病灶周边和内部血流信号的变化、分布和特征;采用免疫组化技术检测肿瘤血管内皮生长因子(VEGF)表达,并进行半定量分级。结果造影后肿瘤内部血流的显示率明显增加,尤以能量多普勒成像(PDI)变化最为明显;免疫组化显示血管内膜生长因子(VEGF)弥漫性表达于癌巢中,表达阳性率52%～85%。统计结果表明,造影能量多普勒显像检测的血管阳性反应面积百分率分级与VEGF表达程度显著相关(r=0.764,P<0.05)。结论应用多普勒超声造影显像技术,有助于评估肿瘤血管生成。     

靶向超声造影评价裸鼠肾癌新生血管的实验研究

目的 探讨以血管内皮生长因子受体2 (VEGFR2)为靶点的靶向超声造影评价裸鼠肾癌新生血管的应用价值.方法 建立人肾透明细胞癌皮下移植瘤裸鼠模型,制备携VEGFR2抗体的靶向造影剂(MBV),每只裸鼠分别随机注射普通造影剂(MBC)和MBV并行超声造影检查,比较两种造影剂的视频强度,并对肿瘤组织行VEGFR2免疫组化检测.结果 超声造影示MBV组视频强度(6.50±1.43)Db,MBC组视频强度(2.59±0.99)Db,两者差异有统计学意义(P<0.01),且MBV组持续强化时间较MBC组长,免疫组化结果证实肿瘤血管内皮VEGFR2高表达.结论 以VEGFR2为靶点的靶向造影剂可特异性增强肾癌显影,对评估肿瘤新生血管有重要价值.

Abstract：

Objective To investigate the value of contrast enhanced ultrasound(CEUS) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in murine tumor models.Methods Established human renal cell carcinomas(RCC) subcutaneous xenograft tumor model in nude mice,microbubbles targeted to VEGFR2(MBV) was prepared,control microbubbles(MBC) and MBV were injected respectively in each mouse,the intensity of each microbubble was compared,the expressions of VEGFR2 in tumors were tested by immunohistochemistry.Results CEUS imaging showed the intensity of MBV and MBC was (6.50±1.43)dB,(2.59±0.99)dB,respectively.There was significantly higher intensity when using MBV compared with MBC (P<0.01).The time to wash out was longer in MBV contrast group compared with MBC group.Immunohistochemistry showed VEGFR2 was highly expressed in novel vessel walls.Conclusions Contrast microbubbles targeted to VEGFR2 can specially enhance the images of RCC and has tremendous significance in the assessment of angiogenesis.     

Targeted Contrast-Enhanced Ultrasound Imaging of Angiogenesis in an Orthotopic Mouse Tumor Model of Renal Carcinoma

Previous studies have reported that microbubbles bearing targeting ligands to molecular markers of angiogenesis can be successfully detected by ultrasound imaging in various animal models of solid cancer. In the present study, we sought to investigate the activity of microbubbles targeted to vascular endothelial growth factor receptor 2 (VEGFR2) in an orthotopic model of renal cell carcinoma (RCC). Microbubbles conjugated to an anti-VEGFR2 antibody (MB_V) were compared with microbubbles conjugated to an isotype control antibody (MB_C) or naked microbubbles (MB_N). An orthotopic mouse model of human RCC was established by surgically implanting an established tumor within the renal capsule in mice. Tumor growth and blood flow were verified by B-mode and color Doppler ultrasound imaging. VEGFR2 expression within the tumor and renal parenchyma was detected by immunohistochemistry. The duration of contrast enhancement of MB_V was much longer than those of MB_N and MB_C when assessed over 10 min. The baseline-subtracted contrast intensity within the tumor was higher for MB_V than for MB_C and MB_N (p < 0.01). Additionally, the contrast intensity for MB_V was significantly higher in the tumor region than in normal parenchyma (p < 0.01). Microbubbles targeting VEGFR2 exhibit suitable properties for imaging angiogenesis in orthotopic models of renal cell carcinoma, with potential applications in life science research and clinical medicine.     

Annexin V微泡对凋亡组织寻靶的试验研究

目的制备Annexin V靶向微泡,评价其理化性质,并进行初步体外寻靶试验探究。方法生物素-亲和素桥接法制备Annexin V靶向微泡,粒径分析仪、Malvern电位仪评价其理化性质,免疫荧光染色法评估靶向配体结合状况,用荧光显微镜观察其形态、大小及分布状态。建立人甲状腺未分化癌裸鼠模型,对其进行微波消融,诱导肿瘤组织凋亡。随机分组,A组为普通微泡,B组为Annexin V抗体预饱和+靶向微泡,C组为Annexin V靶向微泡,比较3组微泡与消融后凋亡肿瘤组织的结合情况。结果成功制备Annexin V靶向微泡,微泡形态大小一致,理化性质稳定;A组和B组,基本未见微泡与肿瘤组织切片稳定结合,而C组见Annexin V微泡与组织稳定结合。结论采用生物素-亲和素桥接法制成Annexin V靶向微泡;且在体外能与凋亡肿瘤组织稳定结合,即成功寻靶,从而实现识别检测凋亡组织的功能。     

Fast microbubble dwell-time based ultrasonic molecular imaging approach for quantification and monitoring of angiogenesis in cancer

PURPOSE: To develop and test a fast ultrasonic molecular imaging technique for quantification and monitoring of angiogenesis in cancer. MATERIALS AND METHODS: A new software algorithm measuring the dwell time of contrast microbubbles in near real-time (henceforth, fast method) was developed and integrated in a clinical ultrasound system. In vivo quantification and monitoring of tumor angiogenesis during anti-VEGF antibody therapy was performed in human colon cancer xenografts in mice (n=20) using the new fast method following administration of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast microbubbles. Imaging results were compared with a traditional destruction/replenishment approach (henceforth, traditional method) in an intra-animal comparison. RESULTS: There was excellent correlation (R(2)=0.93; P<0.001) between the fast method and the traditional method in terms of VEGFR2-targeted in vivo ultrasonic molecular imaging with significantly higher (P=0.002) imaging signal in colon cancer xenografts using VEGFR2-targeted compared to control non-targeted contrast microbubbles. The new fast method was highly reproducible (ICC=0.87). Following anti-angiogenic therapy, ultrasonic molecular imaging signal decreased by an average of 41±10%, whereas imaging signal increased by an average of 54±8% in non-treated tumors over a 72-hour period. Decreased VEGFR2 expression levels following anti-VEGF therapy were confirmed on ex vivo immunofluorescent staining. CONCLUSIONS: Fast ultrasonic molecular imaging based on dwell time microbubble signal measurements correlates well with the traditional measurement method, and allows reliable in vivo monitoring of anti-angiogenic therapy in human colon cancer xenografts. The improved work-flow afforded by the new quantification approach may facilitate clinical translation of ultrasonic molecular imaging.     

双靶向超声微泡造影剂评价小鼠缺血再灌注心肌

目的 制备同时携载P选择素和ICAM-1抗体的靶向超声微泡造影剂,以评估小鼠缺血再灌注损伤心肌声学造影显像效果.方法 采用生物素-亲和素方法制备靶向微泡,于激光共聚焦显微镜下观察微泡形态,流式细胞仪检测连接效率.将24只健康昆明小鼠随机分成3组:结合双抗体选择素微泡组(MBd组)10只、结合P选择素单抗组(MBp组)10只,空白微泡组(MBc组)4只.结扎冠状动脉左前降支近左主干分支,制作心肌缺血再灌注模型,60 min后经尾静脉分别注射MBd、MBp及MBc,采集心肌对比造影图像.所有图像均采用Sonomath超声影像分析仪处理.结果 MBd组和MBp组对缺氧内皮细胞的黏附力以及对缺血再灌注区心肌显像增强程度显著高于MBc组(P均＜0.05),MBd组对缺血再灌注区心肌显像延迟时间高于MBp组(P＜0.05).结论 双靶向微泡联合超声造影是检测和评估小鼠缺血再灌注心肌的无创性手段.     

Chemotherapy Augmentation Using Low-Intensity Ultrasound Combined with Microbubbles with Different Mechanical Indexes in a Pancreatic Cancer Model

The aim of the study was to explore the optimal mechanical indexes (MIs) for low-intensity ultrasound (LIUS) combined with microbubbles to enhance tumor blood perfusion and improve drug concentration in pancreatic cancer-bearing nude mice. Fifty-four nude mice bearing bilateral pancreatic tumors on the hind legs were randomly divided into three groups (the MI was set at 0.3, 0.7 and 1.1 in groups A, B and C, respectively). Five nude mice in each group were intravenously injected with the fluorescent dye DiR iodide (DiIC18(7),1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide); for each mouse, one tumor was treated with LIUS combined with microbubbles, and the contralateral tumor was exposed to sham ultrasound. In vivo fluorescence imaging was performed to detect the enrichment of intratumoral DiR iodide. Twelve mice in each group were intravenously injected with doxorubicin (DOX) and underwent ultrasound therapy as described above. Tumor blood perfusion changes were quantitatively evaluated with pre- and post-treatment contrast-enhanced ultrasound (CEUS, MI = 0.08). One hour after the post-treatment CEUS, nude mice were sacrificed to determine the DOX concentration in tumor tissue; one mouse in each group was sacrificed after ultrasound treatment for tumor hematoxylin–eosin staining examination. CEUS quantitative analysis and in vivo fluorescence images confirmed that LIUS at MI = 0.3 combined with microbubbles was able to enhance tumor blood flow and increase regional fluorescence dye DiR iodide concentration. The DOX concentration on the therapeutic side was significantly higher than that on the control side after ultrasound-stimulated (MI = 0.3) microbubble cavitation (USMC) treatment (1.45 ± 0.53 μg/g vs. 1.07 ± 0.46 μg/g, t = –5.163, p = 0.001). However, in groups B and C, there were no significant differences in DOX concentration between the therapeutic and control sides (Z = –0.297, –0.357, p = 0.766, 0.721). No hemorrhage or other tissue damage was observed in hematoxylin–eosin-stained tumor specimens of both sides in all groups. LIUS at MI = 0.3 combined with microbubbles was able to enhance tumor blood perfusion and improve local drug concentration in nude mice bearing pancreatic cancer.     

Enhanced ultrasound imaging and anti-tumor in vivo properties of Span–polyethylene glycol with folic acid–carbon nanotube–paclitaxel multifunctional microbubbles

With Span and polyethylene glycol (PEG) as the membrane material, the as-prepared folate–carbon nanotube–paclitaxel (FA–CNT–PTX) complex was added to the reaction system under sound vibration cavitation and Span–PEG with FA–CNT–PTX microbubbles was obtained. The maximum tolerating dose of the obtained composite microbubbles on Kunming mice was determined by acute toxicity test. Utilizing the breast cancer tumor model in the nude mice to assess the anti-tumor activity in vivo, the inhibition effect of the composite microbubbles on tumor growth was analyzed by recording the weight and tumor volume of the nude mice. HE staining observations, the immunohistochemistry method, and TUNEL were, respectively, used to examine the inhibition effect of the composite microbubbles on breast cancer tumors in the nude mice. The ultrasound imaging effects and the changes in the peak intensities of the composite microbubbles were inspected using a Doppler color ultrasound imaging system. The experimental results showed that the maximum tolerated dose of the composite microbubbles was 3500 mg kg⁻¹, indicating that the composite microbubbles had low toxicity and good biocompatibility. The composite microbubbles could reach the breast cancer tumor via a targeting factor, and then hindered the tumor growth by inhibiting the proliferation of tumor cells and inducing apoptosis of the tumor cells. The composite microbubbles contributed toward enhancing the ultrasound signal and improved the resolution of the ultrasound images and extended the imaging time. Also, the addition of CNTs in the composite microbubbles could enhance the ultrasound contrast. Simultaneously, the peak intensity at the tumor was significantly reduced after the treatment.

With Span and polyethylene glycol (PEG) as the membrane material, the as-prepared folate–carbon nanotube–paclitaxel (FA–CNT–PTX) complex was added to the reaction system under sound vibration cavitation and Span–PEG with FA–CNT–PTX microbubbles was obtained.