【作者】 伍星；

【导师】 王志刚；

【作者基本信息】 重庆医科大学 ， 生物医学工程， 2009， 博士

【摘要】 恶性肿瘤是严重威胁人类生命健康的重大疾病。早期诊断与早期治疗恶性肿瘤对提高患者的生存率和生活质量具有至关重要的作用。以超声、CT、MRI等为代表的传统医学影像技术能够直观地显示一定体积以上肿瘤的形态学表现。而面对早期或疑似的肿瘤性病变,几乎都依靠普通造影剂进行血池灌注显像来比较组织器官在生理和病理状态下的血流动力学差异,从而做出定性诊断。然而,由于普通造影剂对病变组织缺乏特异性亲和力,不能有效驻留于靶组织,只能在短暂的动脉相中对组织、器官产生一过性增强,因此对疾病诊断的特异性欠佳。如何及时、准确地观测到活体内肿瘤组织在分子及细胞水平的变异信息,一直是医学科研者们亟待攀登的高峰。超声分子影像学为非侵入性地对活体内参与生理和病理过程的分子进行定性或定量可视化提供了一种全新的科学观察方法与手段。其原理是:经静脉注入带有特定配体的靶向超声造影剂,在体内通过配体与受体结合的方式,使之特异性结合并较长时间停留于靶组织,从而在超声检测中观察到靶组织在分子水平的成像,以此来反映病变的分子病理基础。实体肿瘤在生长与转移的过程中均依赖新生血管形成。血管系统在肿瘤发生、发展过程中具有至关重要的作用。研究表明,血管内皮生长因子受体2(Vascular Endothelial Growth Factor Receptor 2,VEGFR2)在肿瘤新生血管内皮细胞表面高度表达,而在正常组织表达高度保守。VEGFR2介导了形成肿瘤新生血管必需的所有内皮细胞功能,其表达强度和表达率已被看作评估肿瘤预后与转移的指标之一。它本身亦成为抗肿瘤血管生成治疗的重要靶点。鉴于肿瘤新生血管内皮高度表达VEGFR2,而VEGFR2的水平可反映肿瘤预后与转移的趋势,以及超声分子显像提供了一种无创性检测活体组织在分子或细胞水平变异的方法,本课题拟制备一种携带抗VEGFR2单抗的靶向超声微泡造影剂,在小鼠H22移植瘤模型上观察其特异性增强显像活体肿瘤的效果与能力;并进一步尝试应用超声分子显像的方法评估肿瘤新生血管VEGFR2表达水平动态变化的可行性与准确性,以期为早期、特异性诊断恶性肿瘤开拓一项新的思路与新的手段,为指导抗肿瘤血管生成治疗与疗效评估提供更加充分、可靠的依据。除了对上述内容进行全面探讨以外,本课题还直接靶向肿瘤细胞开展了血管外超声分子显像的前期探索性研究。因为靶向VEGFR2超声分子显像是针对肿瘤新生血管内皮表面的特异性受体VEGFR2而发生的血池内显像,它虽然可以有力地提高超声对肿瘤的检出率,但我们并无法从中获知直接来自肿瘤细胞自身的信息。而人类对恶性肿瘤的执着探索还希望能够直接针对肿瘤细胞分子显像,以便更全面地认识肿瘤生长特点与规律。血管外超声分子显像如果得以成功实现,必将带来直接展示肿瘤细胞的更直观、更丰富的讯息。根据大量研究表明,叶酸受体在卵巢癌、子宫内膜癌等绝大多数恶性上皮性肿瘤组织高度表达;同时,叶酸与单克隆抗体相比,具有分子量小,到达靶点时间短,血液清除速度快,穿透力强,几乎无免疫源性等优点。因此,我们拟制备一种叶酸靶向超声微泡造影剂,在高度表达叶酸受体的卵巢癌SKOV3细胞株上探讨了该造影剂的主动寻靶能力,以期为血管外靶向肿瘤细胞建立一种理想的分子探针,为将来开展血管外超声分子成像研究奠定扎实的前期实验基础。综上所述,本课题主要包括以下四部分内容。第一部分携带抗VEGFR2单抗靶向超声造影剂的制备及体外性质鉴定目的制备靶向肿瘤新生血管VEGFR2的超声微泡造影剂,并对其基本物理性质与免疫学活性进行鉴定。方法通过生物素-链亲和素桥接构建携带抗VEGFR2单抗的靶向超声微泡造影剂。应用DFY型超声图像定量分析诊断仪(以下简称“DFY软件”)检测造影剂的粒径与浓度。通过免疫荧光法鉴定其活性。结果携带抗VEGFR2单抗超声微泡的基本物理性质与普通微泡相似。该靶向微泡在体外可以与相应的二抗发生特异性结合,具有良好的免疫学活性。结论成功制备了携带抗VEGFR2单抗的靶向超声微泡造影剂。该造影剂具有特异性增强显像肿瘤新生血管的潜能。第二部分靶向VEGFR2超声微泡造影剂增强显像小鼠H22移植瘤目的探讨靶向VEGFR2超声微泡造影剂结合超声造影技术增强显像小鼠H22移植瘤的效果与能力。方法建立小鼠H22移植瘤模型,分别注射靶向VEGFR2微泡或普通对照微泡进行超声造影检查。应用DFY软件量化分析造影图像的视频强度变化,并描绘时间-强度曲线。用荧光素标记微泡进行体内示踪实验,观察两种微泡在体循环7 min后在血管内的分布。免疫组化法检测肿瘤组织内VEGFR2表达情况。结果时间-强度曲线表明靶向微泡造影组较对照组达峰时间提前,峰值更高,持续时间更长。荧光标记微泡的体内示踪实验显示,在体循环7 min后,普通微泡在肿瘤血管内基本被清除,而靶向微泡仍有部分黏附于肿瘤血管内壁。免疫组化证实肿瘤组织内部高度表达VEGFR2。结论靶向VEGFR2超声微泡造影剂结合超声造影技术可较普通微泡造影剂更特异性地显像小鼠H22移植瘤。该靶向造影剂是一种良好的肿瘤新生血管分子探针。第三部分超声分子显像评估小鼠H22移植瘤新生血管VEGFR2动态变化目的探讨用超声分子显像的方法在体无创性评估小鼠H22移植瘤新生血管VEGFR2表达水平动态变化的可行性与准确性。方法分别建立生长1周、2周、3周及4周的小鼠H22移植瘤模型,经尾静脉注射靶向VEGFR2微泡或普通对照微泡,在“充盈-击碎微泡-再充盈”模式下进行超声造影检查。应用DFY软件分别测量肿瘤组织在击碎微泡前、后的视频强度值(VI),以二者之差即视频强度差值(VId)代表粘附于血管内壁的微泡信号。通过Western Blot半定量检测各组肿瘤VEGFR2表达水平。对4个时期肿瘤造影的平均视频强度差值(mean VId)与相应VEGFR2表达水平进行相关性分析。结果在靶向造影组,第2周肿瘤mean VId最高,而第4周最低(P<0.01);4个生长时段肿瘤靶向造影的mean VId与相应VEGFR2表达水平之间呈显著正相关关系(r=0.866,P<0.01)。而在对照组内,mean VId在4个时期肿瘤之间呈小范围波动,以第4周肿瘤mean VId最高(P<0.01);但对照组造影的mean VId与VEGFR2表达水平之间无相关关系(P>0.05)。结论应用超声分子显像方法可以较准确地评估小鼠H22移植瘤新生血管VEGFR2表达水平的动态变化。该方法可能作为一种新型监控手段,为指导抗肿瘤血管生成治疗与疗效评估提供更充分、可靠的依据。第四部分靶向叶酸受体血管外超声分子显像的前期探索性研究目的制备偶联叶酸的靶向超声微泡造影剂,鉴定其基本物理性质,并在体外培养的卵巢癌细胞上考察其主动寻靶能力。方法将DSPE-PEG(2000)Folate溶入微泡成膜材料中,制备叶酸靶向超声微泡造影剂。用DFY软件检测微泡的粒径与浓度。体外培养高度表达叶酸受体的卵巢癌SKOV3细胞。通过光镜和激光共聚焦显微仪分别探究叶酸靶向超声微泡对SKOV3细胞的主动寻靶能力及机制。结果叶酸靶向微泡的基本物理性质与普通微泡相似。体外寻靶试验显示,该靶向微泡可以较多并牢固地结合于SKOV3细胞表面;而在加入游离叶酸干扰后,该结合可以被竞争性阻断。但在普通微泡对照组始终未观察到微泡与SKOV3细胞结合。结论成功制备了偶联叶酸的靶向超声微泡造影剂。该造影剂在体外对高度表达叶酸受体的卵巢癌SKOV3细胞具有极强的特异性亲合力。它有望成为一种理想的血管外超声分子探针,特异性增强显像高表达叶酸受体的肿瘤组织。更多还原

【Abstract】 Malignant tumor is a serious threat to the life and health of human beings. Early diagnosis and early treatment is always the best principle to rescue patients’life.Traditional medical imaging technologies including ultrasound, CT, MRI, etc provide doctors the intuitive figures of tumor beyond certain volumes. As to the early tumorigenesis or suspicious neoplasms, blood perfusion imaging with common contrast agents was used to support diagnosis for all the various technologies.However, after decades’researches, people found that only the above methods may not provide timely and accurate information that the clinic needed. Because the common contrast agents are not specific to diseased tissue, they only bring about contrast-enhanced images during the transient arterial phase. But after the short enhancement, nothing special left in the abnormal organs. In this case, early or small tumorigenesis would be missed by the traditional means. Therefore, scientific researchers are making every effort to look for novel approaches that can detect specific molecular abnormalities of early tumorigenesis timely and sensitively.Ultrasound molecular imaging provides an encouraging tool for specific diagnosis of early disease at the molecular level. Molecular imaging with contrast-enhanced ultrasound relies on the detection of targeted microbubbles which retained in diseased tissue where they produce an acoustic signal because of their resonant properties in the ultrasound field. Targeting is accomplished either through manipulating the chemical properties of the microbubble shell or through conjugation of disease-specific ligands for the targeted molecule to the microbubble surface.In the recent years, researchers have demonstrated that angiogenesis is promoted early with cancer cells in tumorigenesis and it is a critical determinant of tumor growth, invasion and metastatic potential. One of the major regulators of tumor angiogenesis is vascular endothelial growth factor receptor 2 (VEGFR2). This molecule is overexpressed on tumor vascular endothelial cells. Upregulation of VEGF/VEGFR2 has been associated with tumor progression and poor prognosis in several tumors.Therefore, noninvasive imaging strategies for the detection and quantification of VEGFR2 may be particularly helpful for detection of early neoplasm and guidance of antiangiogenic treatments. In the current study, we hypothesized that VEGFR2-targeted microbubbles with contrast-enhanced ultrasound could particularly depict the novel vessels of mice xenografted H22 tumor, which may benefit timely detection of neoplasm as well as noninvasive assessment of angiogenesis progression.On the other side, as VEGFR2-targeted ultrasound molecular imaging is limited in tumor blood pools, a preliminary study of extravascular ultrasound molecular imaging was carried out in the aim of understanding tumors more comprehensively from the tumor cells themselves.As we have known, FR is highly overexpressed in most cancer cells of endothelial origin, especially ovarian carcinoma; while folic acid is a naturally existed substance of small molecule and no immunogenicity, we believe that folic acid and FR are potentials in tumor-targeted imaging beyond blood pools. Therefore, a preliminary exploration in this field was tried in the last part.Overall, the whole study was composed of the following four parts.PartⅠPreparation of VEGFR2-targeted Microbubbles and Characterization in VitroObjective To prepare VEGFR2-targeted ultrasound microbubbles and to characterize the agent’s physical properties as well as immunocompetence. Methods Microbubbles targeted to VEGFR2 (MBv) were prepared by biotin-streptavidin interaction. The diameter and concentration of MBv were analyzed by DFY software. The immunocompetence was validated using immunofluorescence. Results The basic physical properties of MBv were similar to that of the common agents. MBv could conjugate with corresponding secondary antibody specifically in vitro, showing its active immunocompetence. Conclusion MBv were successfully synthesized and demonstrated good immunocompetence. This agent is potential for targeted imaging of tumor angiogenesis in vivo.PartⅡUltrasonic Imaging of Mice Xenografted H22 Tumor with Microbubbles Targeted to VEGFR2Objective To investigate the efficiency of contrast-enhanced ultrasound with microbubbles targeted to VEGFR2 (MBv) for imaging tumor angiogenesis in vivo. Methods Contrast-enhanced ultrasound imaging with MBv or control microbubbles (MBc) was performed in 20 mice bearing xenografted H22 tumors. Sonograms were analyzed by video intensity (VI) by DFY software, and time-intensity curve (TIC) was depicted according to VI change. Internal tracer experiment with DiI-labled microbubbles was carried out to show the distribution difference between MBv and MBc when they circulated for 7 min in vivo. Finally, tumor samples were harvested for analysis of VEGFR2 expressions by immunohistochemistry. Results TIC showed that the time to peak was earlier, peak intensity was higher, and the time to wash out was prolonged in the MBv contrast group compared to that of the MBc control. Internal tracer experiment demonstrated that several MBv were still shown attaching to the tumor vascular endothelium after circulating for 7 min, whereas few or no MBc left in the tumor vessels. Immunohistochemistry demonstrated that VEGFR2 was highly expressed inside the tumor tissues. Conclusion Contrast-enhanced ultrasound with MBv could enhance mice xenografted H22 tumor much more significant than the control. MBv would be a promising molecular probe for imaging tumor angiogenesis in the near future.PartⅢAssessment of VEGFR2 Dynamic Change in Mice Xenografted H22 Tumor Neovessels by Ultrasound Molecular ImagingObjective To explore the feasibility of ultrasound molecular imaging for assessment of VEGFR2 dynamic change in mice xenografted H22 tumor neovessels. Methods Tumor-bearing mice were allowed to live for different stages of 1 week, 2 weeks, 3 weeks and 4 weeks respectively. Contrast-enhanced ultrasound imaging with VEGFR2-targeted microbubbles (MBv) or control microbubbles (MBc) was performed in each mouse in a“perfusion-distruction–reperfusion”mode. Sonograms were analyzed by video intensity (VI) using DFY software, and the mean VI difference (mean VId) before and after microbubble destruction was calculated and regarded as signals from adherent microbubbles inside tumor neovessels. VEGFR2 expression in the tumor tissues were quasi-quantitatively determined by Western Blotting. The correlation between mean VId and VEGFR2 expression was analyzed by SPSS software. Results In the MBv group, mean VId was the highest in 2-week tumor, but lowest in 4-week tumor (P<0.01). There was a strong correlation between mean VId and tumor VEGFR2 expressions in the MBv group (r=0.866, P<0.01). In the control group, there was a narrower fluctuation of mean VId among the 4-stage tumors (P<0.01). However, no correlation was found between mean VId and tumor VEGFR2 expressions in the control group (P>0.05). Conclusion Ultrasound molecular imaging could assess VEGFR2 dynamic change in mice xenografted H22 tumor neovessels accurately. It may serve as a novel guidance for antiangiogenesis therapy.PartⅣPreliminary study of extravascular ultrasound molecular imaging targeted to folate receptorObjective To prepare folate-targeted microbubbles (MBF) as ultrasound contrast agent for potential molecular imaging beyond blood pools. The physical properties of the agent were characterized, and its affinity to folate receptor (FR) was investigated on ovarian cancer cells in vitro. Methods MBF was synthesized by incorporating DSPE-PEG(2000)Folate into the lipid membrane of microbubbles. The diameter and concentration of MBF were determined by DFY software. Ovarian cancer SKOV3 cells of FR overexpression was cultured in vitro. The affinity of MBF to FR was validated by targeting-conjugation test on SKOV3 cells with the observation of light microscope and confocal imaging microscope respectively. Results The mean diameter and distribution of MBF were similar to that of the common microbubbles. In the targeting-conjugation test in vitro, it was shown that a number of MBF conjugated with SKOV3 cells tightly; but after free folic acid influence, the conjugation would be inhibited. There was always no conjugation in the control group. Conclusion MBF were prepared successfully and demonstrated highly affinitive to ovarian cancer SKOV3 cells. MBF is potential to be an ideal ultrasound contrast agent for extravacular molecular imaging of cancers overexpressin FR.更多还原