靶向超声微泡在肿瘤诊断与治疗领域的研究进展

近年来靶向超声微泡在肿瘤诊断和治疗方面的研究进展十分迅速.实验研究表明,靶向超声微泡用于评价肿瘤血管新生和肿瘤血管内皮分子的变化,具有无创性的优势,并且能够大大提高肿瘤诊断的准确性.同时,靶向超声微泡作为一种新的基因或药物运载的有效工具,通过携带基因和药物对肿瘤组织实现靶向性释放,介导肿瘤细胞坏死、凋亡以及肿瘤微血管的栓塞和阻断,从而起到靶向治疗的作用.     

靶向超声微泡在肿瘤诊断和治疗中的研究进展

近年来肿瘤的发病率有增高趋势,肿瘤的早期、无创、敏感而特异性的诊断和治疗对于降低死亡率及并发症的发生率至关重要。近年研究发现,靶向超声微泡造影剂有望成为一种新的安全有效的载体,从分子水平上介导肿瘤的靶向诊断和治疗。靶向超声微泡是利用微泡表面特有的生物学性质或通过特殊处理将靶向配体与微泡表面连接,使其能够持续、定向地蓄积于靶组织,实现靶向超声分子成像与靶向治疗。     

SonoVue 微泡瞬时均一性对新型绒癌细胞靶向造影剂结合特性的影响

背景与目的:超声微泡造影剂是目前的研究热点,通过其可提高超声扫描对肿瘤血管的检出率,但缺乏组织特异性.本研究目的是探讨在制备靶向造影剂之前SonoVue微泡瞬时均一性对绒癌细胞(choriocarcinoma cell, JAR)靶向造影剂的体外结合特性、结合率及稳定性的影响,为临床研究肿瘤细胞抗原的超声靶向定位显像、提高肿瘤的早期诊断率奠定基础.方法:研究分为微泡不均匀组(n=10),微泡均匀组(n=10),小微泡组(n=10) 3组;主要检测花环形成率、流式细胞计数及花环结合形态等.用均一性不同的SonoVue微泡与兔抗人绒毛膜促性腺激素(human chorionic gonadotrophin, HCG)抗体作用制成靶向造影剂,然后分别与绒癌细胞作用,比较各组的结合率及冲洗前后的结合率.结果:各组中不均一组的靶向造影剂微泡与绒癌细胞的结合率为(60.4±1.5)%,低于微泡均匀组及小微泡组[(84.3±5.5)%和(90.6±6.8)%],差异有统计学意义(P<0.05).冲洗前后比较,微泡均匀组靶向微泡与贴壁生长的绒癌细胞结合率变化最小,冲洗后结合率为(82.4±3.7)%(P>0.05).流式细胞检测结果显示:微泡不均匀组、微泡均匀组、小微泡组悬液中结合了荧光标记的靶向造影剂微泡与绒癌细胞的结合率分别为72.9%、81.03%、88.5%,3组间差异有统计学意义(P<0.05).结论:在体外制备靶向造影剂之前,SonoVu微泡瞬时均一性对绒癌细胞靶向造影剂微泡与绒癌细胞特异性结合能力有明显影响,而SonoVue微泡的均一性与适度振荡有关,SonoVue微泡瞬时不均一性直接影响靶向特异性结合率的数量.通过改善SonoVue微泡的均一性可望提高靶向造影剂结合率及靶向微泡稳定性,改善显影.     

超声纳米微泡造影剂在肿瘤诊疗中的研究进展

随着分子生物学的快速发展,超声纳米微泡造影剂已成为研究热点之一。其作为一种新型的造影剂具有纳米级粒径,在成像方面有多种优势,容易实现对肿瘤的靶向性,在肿瘤诊疗中有一定意义。超声纳米微泡造影剂不仅可以结合超声成像、荧光探针及铁粒子等增强成像效果,还可以包裹肿瘤靶向药物进行药物靶向递送、作为载体和免疫增强剂携载目的基因进行基因转染、促进肿瘤的免疫治疗,为肿瘤的诊断和治疗提供了新方法。本文针对超声纳米微泡造影剂在肿瘤中的研究进展进行综述。     

超声造影剂在肿瘤诊断和治疗中的应用

超声造影剂在临床上的应用,使超声影像对疾病的诊断和鉴别诊断能力明显提高,尤其改善对肿瘤的定性和定位诊断.近年,国内外学者致力于微泡造影剂靶向性显影和靶向性治疗的研究,包括靶向微泡的制备技术、超声生物学技术、微泡相关基因技术等,并取得积极成果.研究显示,新型造影剂和超声的联合应用,可提高对肿瘤诊断的特异性和准确率,并实现有效的肿瘤靶向治疗,做到个性化诊疗,为临床上肿瘤的治疗带来新的希望.     

携精氨酸-甘氨酸-天冬氨酸肽序列靶向超声微泡结合对比超声评价肿瘤新生血管

目的 探讨携精氨酸-甘氨酸-天冬氨酸(RGD)肽序列靶向超声微泡结合对比超声评价肿瘤新生血管的可行性.方法 应用声振法制备普通脂质微泡(MB),通过共价偶联的方法,将RGD肽序列连接在MB外壳,制备成靶向超声微泡(MBRGD).6只接种人肝癌HepG2细胞14 d的裸鼠(模型组)和6只未接种HepG2细胞的正常裸鼠(对照组),经尾静脉弹丸式注射等量MB和MBRGD,两次注射间隔30 min.注射10 min后,分别行肿瘤和骨骼肌的对比超声检查,测量肿瘤和骨骼肌的第1帧对比超声图像的声强度(VI),并对肿瘤组织和骨骼肌组织进行抗αvβ3-整合免疫组化检测.结果 模型组注射MBRGD和MB后,肿瘤第1帧对比超声图像分别显示显著及轻度的超声显影,VI值分别为17.03±3.58和5.33±1.71,注射MBRGD后的VI值是注射MB的3.18倍(P＜0.05).对照组注射MBRGD和MB后,骨骼肌第1帧对比超声图像均无明显超声显影,VI值分别为3.10±0.48和2.90±0.29,两者差异无统计学意义(P＞0.05).免疫组化检测显示,αvβ3-整合素在肿瘤新生血管内皮高度表达,而骨骼肌组织血管内皮却未见明显表达.结论 应用MBRGD行对比超声检查可有效评价肿瘤的新生血管,将有助于肿瘤性质的早期判断.     

低频超声辐照联合微泡抑制兔VX2肿瘤生长的实验研究

目的:探索低频超声辐照联合静脉注射微泡抑制肿瘤生长的非创伤性治疗肿瘤的新方法。方法:24只新西兰大白兔后腿肌肉内接种VX2肿瘤,瘤体长至1cm左右随机分为对照组、单纯微泡组、单纯超声组和超声微泡组。各组经相应处理后,超声检测肿瘤大小、观察瘤体内血流灌注情况,绘制肿瘤生长曲线,计算肿瘤增长百分率。病理学观察治疗后各组瘤体组织的病理学损伤。结果:治疗后2周结果显示超声微泡组瘤体体积和肿瘤增长百分率均明显小于其余各组(P<0.01),瘤体内血流灌注明显减少,瘤体组织大部分坏死;单纯微泡组、单纯超声组的瘤体体积和肿瘤增长百分率与对照组间均无显著性差异(P>0.05)。结论:20kHz超声辐照联合微泡造影剂SonoVue可有效抑制兔VX2肿瘤的生长。     

低频超声辐照联合微泡抑制兔VX_2肿瘤生长的实验研究

目的：探索低频超声辐照联合静脉注射微泡抑制肿瘤生长的非创伤性治疗肿瘤的新方法。方法：24只新西兰大白兔后腿肌肉内接种VX2肿瘤,瘤体长至1cm左右随机分为对照组、单纯微泡组、单纯超声组和超声微泡组。各组经相应处理后,超声检测肿瘤大小、观察瘤体内血流灌注情况,绘制肿瘤生长曲线,计算肿瘤增长百分率。病理学观察治疗后各组瘤体组织的病理学损伤。结果：治疗后2周结果显示超声微泡组瘤体体积和肿瘤增长百分率均明显小于其余各组（P〈0.01）,瘤体内血流灌注明显减少,瘤体组织大部分坏死;单纯微泡组、单纯超声组的瘤体体积和肿瘤增长百分率与对照组间均无显著性差异（P〉0.05）。结论：20kHz超声辐照联合微泡造影剂SonoVue可有效抑制兔VX2肿瘤的生长。     

妇科恶性肿瘤超声造影表现与微血管密度相关性研究进展

 微血管密度（MVD）是评价肿瘤血管生成的金标准。超声造影近年发展迅速,可通过测定各种灌注参数,间接反映妇科恶性肿瘤新生微血管形成程度,并评估肿瘤的生物学行为以判断预后。对妇科恶性肿瘤超声造影、MVD检测,以及二者之间相关性研究的进展进行了综述。     

超声微泡造影剂增强TRAIL基因转染肝癌细胞的实验研究

目的 探讨采用超声辐照联合微泡造影剂增强重组表达质粒plRES-EGFP-sTRAIL转染肝癌细胞的有效性.方法 构建携带肿瘤坏死因子相关细胞凋亡诱导配体(TRAIL)基因及绿色荧光蛋白(GFP)的真核表达质粒pIBES-EGEP-sTRAIL,转染肝癌细胞HepG2,分为超声辐照联合微泡造影剂转染组、脂质体转染组、单纯微泡造影剂转染组及对照组.荧光显微镜检测GFP以评价转染效率,四甲基偶氮唑盐微量酶反应比色法(MTT)检测细胞生长抑制率,Hoechst33258荧光染色观察细胞形态变化,流式细胞术检测细胞凋亡率,Western blot检测TRAIL凋亡途径中关键凋亡分子caspase-8和caspase-3的蛋白变化.结果 超声辐照联合微泡造影剂可增强pIREs-EGFP-sTRAIL有效转染HepG2细胞,其转染效率与脂质体转染组、单纯微泡造影剂组及对照组比较差异均有统计学意义(P<0.05);携带sTRAIL基因的表达质粒能够激活caspase通路,促进肝癌细胞凋亡,有效抑制肝癌细胞生长.结论 构建的真核表达载体pIRES-EGFP-sTRAIL在肝癌细胞中能有效表达,联合超声微泡造影剂及低强度超声,可显著增强sTRAIL对肝癌细胞的增殖抑制及诱导凋亡作用.     

Ultrasound for the Visualization and Quantification of Tumor Microcirculation

Advances in ultrasound based methods for the non-invasive assessment of the tumor microcirculation are described. Two new ultrasound approaches are highlighted. The first method relies on commercially available ultrasound contrast agents in combination with specialized nonlinear imaging sequences. Nonlinear scattering by microbubble contrast agents provides a unique intravascular signature that can be distinguished from the echoes caused by surrouning tissues. Through destruction-reperfusion experiments it is possible to use microbubble contrast agents as a tracer revealing the kinetics of tumor blood flow. The second ultrasound method for examining the microcirculation involves the use of much higher frequencies. At frequencies on the order of 50MHz, Doppler processing allows the direct assessment of flow dynamics in realtime in arterioles as small as 15µm. Three dimensional Doppler maps of flow patterning are presented. The strengths and weaknesses of these new methods are discussed and the potential for their use in preclinical animal drug studies, clinical drug trials, and prognostic studies is described.     

SonoVue微泡粒径对新型绒癌细胞靶向造影剂结合特性的影响

Objective:To evaluate the impact of the diameter of SonoVue microbubbles on binding characteristics, including the adhesion rate and stability, of a new contrast agent targeted to choriocarcinoma cells (JARs) in vitro, in order to establish a foundation to explore targeted ultrasound imaging for localization of tumor cell antigens and increase the early diagnostic rate for tumors. Methods:The objects were divided into three groups:the large microbubble group (n=15), the middling microbubble group (n=15) and the tiny microbubble group (n=15). The rosette formation rate was counted. JARs were calcu-lated by flow cytometry (FCM). The targeted contrast agent was prepared by mixing SonoVue microbubbles of different diam-eter with rabbit anti-human chorionic gonadotrophin (HCG) antibody. The binding rates of the targeted contrast agent to JARs before and after PBS rinse were analyzed. Results:The binding rate was significantly lower in the large microbubble group [(61.7±1.8)%] than in the middling microbubble group [(82.6±4.5)%] and the tiny microbubble group [(91.3±5.8)%] (P< 0.05). The binding rates of different diameter microbubbles to JARs before and after PBS rinse were different. The middling microbubbles were the most stable ones, with the binding rate of (82.3±4.5)% and (80.4±3.9)% before and after PBS rinse (P > 0.05). The binding rates of the targeted microbubbles labeled with fluorescence to JARs were 68.6%, 81.3% and 89.3% in the large microbubble group, the middling microbubble group and the tiny microbubble group, respectively (P< 0.05). Conclu-sion:The binding capacity of the targeted SonoVue microbubbles to JARs is related to the diameter of the microbubble, which is determined by the shaking method before preparation. Modulating the diameter of SonoVue microbubbles may increase the binding rate and stability of targeted microbubbles to JARs, thus to improve the image of JARs.     

Therapiemonitoring mittels CT und MRT: methodische Grundlagen

New antitumor therapy concepts often include “targeted drugs.” In contrast to chemotherapy or radiation therapy, targeted drugs have antiangiogenic rather than cytotoxic effects. Because tumor shrinkage often takes time, therapy monitoring with conventional morphology-based response evaluation criteria in solid tumors (RECIST) is insufficient, leading to the latest version, RECIST 1.1. Computed tomography (CT) and magnetic resonance imaging (MRI) offer excellent spatial resolution. Recent advances in hardware and software enable monitoring of tumor angiogenesis with CT and MRI. Molecular MR contrast agents, diffusion-weighted MRI, and MR spectroscopy can visualize molecular tumor processes. In this article, the basic principles of these imaging techniques are discussed.     

New magnetic resonance imaging techniques for the detection of breast cancer

Summary The importance of contrast agents in enhancing diagnoses from magnetic resonance images has been established in numerous cases. However, the development of a potent tissue-specific contrast agent, as a sensitive probe for early detection and investigation of the physiological characteristics of a tumor, has not yet been realized in MR imaging (MRI). In nuclear scintigraphy the technique has been demonstrated; however, the poor spacial resolution inherent to the modality and the substantial dose of radioactivity administered to the patient has hindered its widespread use. This article will review the different classes of contrast agents in MRI, with special focus on the strategies involved in the development of targeted tissue-specific MRI contrast agents for the early detection of breast cancer. The features of a new class of contrast agents for targeted MR imaging will be described. Gadolinium-containing melanin polymers (GMP's) have been synthesized as MR contrast agents in our laboratory. These GMP's demonstrate significantly higher relaxivities than any other paramagnetic contrast agents reported; consequently, they are extremely effective contrast enhancing, imaging agents by themselves. The successful coupling of these potent GMP's to a monoclonal antibody specific for breast carcinoma, the 323/A3 monoclonal antibody, suggests thatin vivo tissue-specific MR imaging, at the receptor level, will become feasible in the near future.     

Monitoring response to anticancer therapy by targeting microbubbles to tumor vasculature.

PURPOSE: New strategies to detect tumor angiogenesis and monitor response of tumor vasculature to therapy are needed. Contrast ultrasound imaging using microbubbles targeted to tumor endothelium offers a noninvasive method for monitoring and quantifying vascular effects of antitumor therapy. We investigated the use of targeted microbubbles to follow vascular response of therapy in a mouse model of pancreatic adenocarcinoma. EXPERIMENTAL DESIGN: Microbubbles conjugated to monoclonal antibodies were used to image and quantify vascular effects of two different antitumor therapies in s.c. and orthotopic pancreatic tumors in mice. Tumor-bearing mice were treated with anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and/or gemcitabine, and the localization of microbubbles to endoglin (CD105), VEGF receptor 2 (VEGFR2), or VEGF-activated blood vessels (the VEGF-VEGFR complex) was monitored by contrast ultrasound. RESULTS: Targeted microbubbles showed significant enhancement of tumor vasculature when compared with untargeted or control IgG-targeted microbubbles. Video intensity from targeted microbubbles correlated with the level of expression of the target (CD105, VEGFR2, or the VEGF-VEGFR complex) and with microvessel density in tumors under antiangiogenic or cytotoxic therapy. CONCLUSIONS: We conclude that targeted microbubbles represent a novel and attractive tool for noninvasive, vascular-targeted molecular imaging of tumor angiogenesis and for monitoring vascular effects specific to antitumor therapy in vivo.     

Non-invasive assessment of tumor neovasculature: techniques and clinical applications

The ability to induce and sustain angiogenesis is regarded as one of the hallmarks of cancer growth and metastasis. Marked advances in understanding vascular physiology and mechanisms of angiogenesis have led to the development of vascularly-targeted anti-cancer therapies. Two broad subclasses of agents are in clinical use currently: those with anti-angiogenic activity (i.e. those that target angiogenic pathways) and those that have direct cytotoxic activity against proliferating endothelial cells (i.e. vascular disruptive agents (VDAs)). This article reviews the various imaging modalities, including magnetic resonance imaging, computed tomography, photon emission imaging (positron emission tomography and single photon emission computed tomography), ultrasound and optical (near-infrared absorption and scattering) techniques that have been used in an attempt to assess the status of tumor neovasculature in vivo. In each case, we describe the basic imaging methodology, methods for image quantification, technical standardization and reproducibility, and current and potential clinical applications for monitoring the status of tumor neovasculature before and during treatment with vascular targeted agents.     

Preclinical MRI Experience in Imaging Angiogenesis

Magnetic resonance imaging (MRI) provides a range of non-invasive measures for visualization of tumor angiogenesis in the clinic as well as in experimental tumor models. MRI methods were developed for assessment of spatial and temporal changes in perfusion, blood volume fraction, vascular permeability, vascular function, vascular maturation, vessel diameter and tortuosity. Molecular targeted contrast agents were used for mapping specific markers of neovasculature. These approaches were applied for analysis of a number of regulatory mechanisms controlling tumor angiogenesis and for preclinical evaluation of tumor response to antiangiogenic agents.