【作者】 周伟；

【导师】 詹维伟；

【作者基本信息】 上海交通大学 ， 影像医学与核医学， 2020， 博士

【摘要】 背景及目的热消融已普遍应用于肿瘤微创治疗,但消融不彻底可能会加速肿瘤的进展。由于肿瘤边界模糊或不规则,消融边界难以准确估计,因此术后准确的超声造影评估成为治疗的关键。纳米靶向超声造影剂的应用可对肿瘤组织精准靶向定位。本研究主要是评估非致死热刺激后乳腺癌细胞增殖与侵袭能力变化;通过构建新型的VEGFR2靶向铁掺杂二氧化硅（Si O₂）空心纳米球超声造影剂,评估其理化性质和安全性;并应用于乳腺癌移植瘤微波消融术后疗效的判断,以评估其应用价值。方法利用细胞分子生物学、动物模型等技术建立乳腺癌体外热消融模型,观察非致死温度刺激下细胞增殖与侵袭能力改变;利用非标记的定量蛋白质组学技术,获得与非致死温度刺激有关的蛋白质组信息。利用醋酸亚铁刻蚀纳米Si O₂,表面连接VEGFR2单克隆抗体进行靶向修饰,制得VEGFR2-PEG-HSNs-Fe;利用纳米粒度电位分析仪和激光共聚焦显微镜等观察其主要理化性质,并以定性和定量方式评估体外靶向能力;利用流式细胞仪、生物学行为观察、血生化、免疫指标及器官病理检测等考察其体外、内生物安全性;利用超声评估其体外、内显像效果;对小鼠乳腺癌细胞4T1移植瘤微波消融后,通过注射不同造影剂进行消融范围的评估。结果45℃为4T1乳腺癌细胞的非致死性温度,可增强细胞增殖及侵袭能力;Heat shock protein 1B、NOB1与CRIP1在45℃刺激后表达升高,而BCLAF1在45℃刺激后表达降低。制备的VEGFR2-PEG-HSNs-Fe粒径为276.64±30.31 nm,表面电位为-13.46±2.83m V。体外实验:随着造影剂浓度增大,超声信号强度增强。VEGFR2-PEG-HSNs-Fe可与人脐静脉内皮细胞靶向结合,结合率为74.80±2.21%。随造影剂浓度增加,细胞存活率逐渐减小,但保持在80%左右或以上。体内实验:注射VEGFR2-PEG-HSNs-Fe后小鼠无异常生物学行为,血细胞、生化和免疫指标均正常,主要脏器组织结构未见异常。注射VEGFR2-PEG-HSNs-Fe后肿瘤区超声信号显示增强,覆盖整个肿瘤。消融后注射VEGFR2-PEG-HSNs-Fe评估,可以发现残留灶。不完全消融的病灶表现为肿瘤边缘残留造影剂灌注,再次消融后造影评估,呈无灌注表现,最终均能达到完全消融。与常规造影剂比较,利用VEGFR2-PEG-HSNs-Fe超声造影评估,消融手术所需时间更短。结论不完全消融时,非致死性的热刺激可以促进乳腺癌的增殖和侵袭。纳米靶向超声造影剂VEGFR2-PEG-HSNs-Fe具有良好的生物安全性及特异性显像能力,对于肿瘤消融术后疗效评估具有一定价值,可以避免不完全消融。更多还原

【Abstract】 ObjectivesThermal ablation has been widely used in minimally invasive treatment of tumors,however,incomplete ablation might accelerate tumor progression.Due to the blur or irregular boundary of tumor,it is difficult to accurately estimate the boundary of the ablation zone.Therefore,accurate evaluation by contrast enhanced ultrasound（CEUS）plays a key role in the ablation.The application of nano-targeted ultrasound contrast agent（UCA）could target and locate tumor tissues accurately.The purpose of this study was to evaluate the changes of proliferation and invasion ability of breast cancer cells with sublethal heat.A novel UCA VEGFR2-targeting iron-doped silica（Si O₂）hollow nanosphere was made to evaluate its physicochemical properties and safety.It was applied in microwave ablation for breast cancer xenograft to investigate its value in the evaluation of effectiveness after ablation.MethodsIn vitro thermal ablation models of breast cancer were established by using cell and molecular biology and animal models to observe the changes of cell proliferation and invasion ability under the stimulation of sublethal heat.Proteomics information related to sublethal heat stimuli was obtained by using label free proteomics.VEGFR2-PEG-HSNs-Fe was prepared by using nano-Si O₂,which was regarded as a substrate and etched by Ferrous acetate,and then modified with targeted VEGFR2antibody.The nanometer particle size potential analyzer and laser confocal microscope were used to observe its main physicochemical properties,and we also evaluated its specific targeting ability in vitro qualitatively and quantitatively.The biological safety was investigated in vitro and in vivo by flow cytometry,biological behavior observation,blood biochemical index,serum immune index and pathological examination of organs.Ultrasound was used to evaluate the quality of imaging.After the xenograft tumor of 4T1cells was treated by microwave ablation,the extent of perfusion defect was evaluated by injecting different UCAs.ResultsThe temperature 45℃ was a sublethal temperature of 4T1 cells,which could enhance the ability of proliferation and invasion.Heat shock protein 1B,NOB1 and CRIP1 increased after stimulation at 45℃,while BCLAF1 decreased.The average particle size of VEGFR2-PEG-HSNs-Fe was 276.64±30.31 nm,and the surface potential was-13.46±2.83 m V.In vitro,the intensity of ultrasound signal increased with UCA concentration.VEGFR2-PEG-HSNs-Fe can combine with human umbilical vein endothelial cells with a binding rate of 74.80±2.21%.With the increase of UCA concentration,the cell survival rate decreased gradually,but kept at about 80%or above.In vivo,after injecting VEGFR2-PEG-HSNs-Fe,no abnormal biological behaviors were observed in the mice.The main blood cells,biochemical and immune indicators were within normal range,and the structure of main organs was normal.Enhanced ultrasound signal was detected in the tumor after injecting VEGFR2-PEG-HSNs-Fe,covering the whole tumor.After ablation,tumor residue was detected by injecting VEGFR2-PEG-HSNs-Fe.The lesions,which were incompletely ablated,showed contrast agent perfusion at the periphery of the tumor.After complementary ablations,there was no perfusion in the ablated zone,and complete ablation was achieved in all cases.Compared with conventional UCA,less time was required for ablation by using VEGFR2-PEG-HSNs-Fe.ConclusionDuring incomplete ablation,the stimulation of sublethal heat could promote the proliferation and invasion of breast cancer.Nano-targeted UCA VEGFR2-PEG-HSNs-Fe has good biosafety and ability of specific imaging,which might be helpful to evaluate the efficacy of tumor ablation and avoid incomplete ablation.更多还原