磁共振纳米铁颗粒成像技术在肿瘤及转移淋巴结中的基础实验研究

【作者】 雷晶；

【导师】 金征宇； 许海燕； 宋伟； 薛华丹；

【作者基本信息】 中国协和医科大学 ， 影像医学与核医学， 2010， 博士

【摘要】 目的：研究不同浓度超小超顺磁性氧化铁颗粒(USPIO)在兔胭窝炎性淋巴结中的增强磁共振特征,建立USPIO淋巴结显像的常规注射及扫描方案。材料与方法：12只健康新西兰兔于足垫处注射完全弗氏佐剂,建立胭窝淋巴结的炎性增生模型。静脉注射不同剂量(45/90/135μmol Fe/kg)的USPIO,注射前后行磁共振扫描,观察不同浓度组及90μmol Fe/kg浓度组在不同时间点淋巴结强化特征,测量各组淋巴结T1信号强度、T2信号强度、T2*值及T2值变化并计算T2强化率。结果：胭窝炎症淋巴结在USPIO增强后24小时,90和135μmol Fe/kg浓度组T2加权像序列强化效果及强化率差异无显著性,但均显著优于45μmol Fe/kg浓度组(P<0.05)：90μmo1 Fe/kg组在6-24h T1信号强度、T2信号强度、T2值及T2强化率差异无显著性(P>0.05),90μmol Fe/kg组淋巴结信号强度降低在18h达峰值,于18-24h均能获得较好的图像。结论：90μmol Fe/kg可以较好地强化淋巴结,注射USPIO后18-24h采集淋巴结图像可以达到满意的强化效果。目的：研究不同性质淋巴结超小超顺磁性氧化铁颗粒(USPIO)的增强磁共振特征,分析其与淋巴结病理超微结构的关系；在此基础上探讨USPIO增强MR在诊断淋巴结转移方面的作用及价值。材料与方法：36只健康新西兰兔随机分为炎症组及肿瘤转移组,兔足垫注射完全弗氏佐剂,用于建立胭窝淋巴结的炎性增生模型；兔后小腿肌肉接种VX2瘤株,用于建立胭窝肿瘤淋巴结转移模型。两组建模后动物均经静脉注射90μmolFe/kg的USPIO,于注射前及注射后24h分别行磁共振成像(MRI)扫描观察淋巴结信号强度及T2值变化并计算强化率,扫描后取出胭窝淋巴结行HE染色、普鲁士蓝染色及电镜切片,观察淋巴结超微结构的变化、铁颗粒在淋巴结内的分布特征,巨噬细胞吞噬铁颗粒的情况,分析病理显微结构与淋巴结强化的关系。确定淋巴结USPIO增强MR影像诊断标准,评价其诊断的准确性。结果：在观测的72枚淋巴结中,病理证实共有46个炎性增生性淋巴结和26个转移性淋巴结,其中4枚仅见小片状包膜下转移灶。USPIO增强MR诊断淋巴结的敏感性为84.6%,特异性为87.0%,阳性预测值为78.6%,阴性预测值为90.9%。平扫时炎性增生性淋巴结和肿瘤转移性淋巴结的T2信号强度差异无显著性,USPIO强化后,炎性增生组淋巴结中心T2信号强度明显降低,而肿瘤转移组淋巴结T2信号降低不明显,二者淋巴结强化率分别为57.22%和29.45%,差异具有显著性(P<0.01)。HE染色、普鲁士蓝染色及电镜切片显示淋巴结内的铁颗粒主要分布于淋巴结髓索结构内,皮质及副皮质区相对较少,与MRI图像相对应；电镜检查显示炎性增生性淋巴结内铁颗粒主要存在于巨噬细胞的胞饮泡内,而转移性淋巴结巨噬细胞胞饮泡内则被大量的细胞碎屑所取代。结论：淋巴结USPIO增强磁共振成像是一种良好的鉴别淋巴结性质的方法,良恶性淋巴结USPIO强化特征与淋巴结的超微结构特别是巨噬细胞在结内的分布及其功能状态有较密切关系,可能影响USPIO对淋巴结性质的诊断准确性。目的：研究DMSA-USPIO纳米颗粒表征、体外磁学性能及其在健康裸鼠体内的分布特征。方法：FT-IR方法检测DMSA-USPIO表面羧基情况。配置相同浓度梯度的DMSA-USPIO溶液及Ferumoxtran-10溶液,比较其体外磁共振T2及T2*信号强度及T2／T2*值；3只健康BALB/C nu-nu裸鼠尾静脉注射DMSA-USPIO,注射前后行磁共振扫描,观察其各脏器在MRI多种序列中的影像特征及在不同时间点肝、肾、肌肉和脑组织的强化特点和T2值变化,并进行病理组织学检查,普鲁士蓝染色观察各器官铁颗粒分布情况。结果：FT-IR波谱图显示DMSA-USPIO具有-OH和-C=O特征性吸收强峰。DMSA-USPIO浓度与R2／R2*值呈线性正比关系,在相同浓度下,DMSA-USPIO的横向弛豫时间明显短于Ferumoxtran-10的横向弛豫时间。DMSA-USPIO注射后肝脏信号强度比注射前减低,肾脏、肌肉与脑信号强度无明显改变；肝脏DMSA-USPIO注射前与注射24小时后T2值比较差异有显著性(p<0.05),强化率为26.8%。病理切片普鲁士蓝染色可见肝、脾、淋巴结组织内存在蓝染铁颗粒,肾、脑、肺、心肌及肠道未见明显铁颗粒沉积。结论：DMSA-USPIO颗粒有着优良的磁共振成像性能,表面被覆有丰富的可修饰集团,可作为靶标分子的拼接载体用于磁共振成像；肝脏可作为评价DMSA-USPIO纳米氧化铁颗粒裸鼠体内强化效果的目标脏器之一,用于后续磁共振增强研究。目的：构建血管内皮生长因子受体3(VEGFR3)抗体与超微超顺磁性氧化铁粒子(USPIO)的磁共振分子探针VEGFR3-USPIO,探讨其对人乳腺癌MDA-MB-231细胞的体外靶向结合能力及体外磁共振靶向成像能力。方法：利用细胞免疫荧光方法检测MDA-MB-231细胞VEGFR3的表达情况及表达部位；利用Western Blot方法检测VEGFR3分子量并进行半定量分析；用碳二亚胺(1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)将VEGFR3抗体和USPIO进行偶联；检测相同浓度梯度下VEGFR3-USPIO、USPIO溶液磁共振T2弛豫时间的变化及差异；将VEGFR3-USPIO、USPIO分别与MDA-MB-231细胞孵育,行普鲁士蓝染色观察铁颗粒结合及存在情况,并行体外磁共振成像测定T2值变化情况。结果：MDA-MB-231细胞VEGFR3受体表达阳性,VEGFR3表达部位在细胞膜及胞质内,VEGFR3肽段与Marker相比大致位于分子质量为73KDa处；体外磁共振测量结果显示,VEGFR3-USPIO与USPIO相比,在各浓度下,T2及T2*弛豫时间均无明显差异,且R2及R2*弛豫时间与溶液浓度存在线性正比关系；铁颗粒与细胞孵育后,普鲁士蓝染色可见VEGFR3-USPIO组铁颗粒存留较多,且集中分布于细胞膜周围,而USPIO组铁颗粒存留稀少,且散在分布于细胞间隙。VEGFR3-USPIO-细胞孵育组的T2弛豫时间低于USPIO-细胞孵育组,两者的T2驰豫时间变化率分别为61.0%和43.5%。结论：VEGFR3-USPIO颗粒在体外细胞水平对表达VEGFR3的人乳腺癌MDA-MB-231细胞具有较好的靶向结合能力和靶向成像能力。目的：利用临床型MRI仪探讨VEGFR3-USPIO颗粒的健康裸鼠体内分布特征及其对荷瘤裸鼠在体瘤块的靶向成像能力。材料与方法：3只健康BALB/C nu-nu裸鼠尾静脉注射VEGFR3-USPIO,注射前后行磁共振扫描,观察其各脏器在MRI多种序列中的影像特征及在不同时间点肝、肾、肌肉和脑组织的强化特点和T2值变化,并进行病理组织学检查,普鲁士蓝染色观察各器官铁颗粒分布情况；建立人乳腺癌MDA-MB-231裸鼠皮下瘤模型6只,随机分为二组,每组3只,分别经尾静脉注射VEGFR3-USPIO及空白USPIO造影剂,注射前与注射后24小时行磁共振扫描,测量在体肿瘤及肝脏的T2值强化程度,并进行病理组织学检查。结果：VEGFR3-USPIO注射后健康裸鼠肝脏T2值比注射前明显减低,而肾脏、肌肉与脑的T2值无明显改变。肝脏VEGFR3-USPIO注射前与注射24小时后T2值比较差异有显著性(p<0.05),强化率为25.7%。普鲁士蓝染色显示VEGFR3-USPIO强化24小时后可见肝、脾、淋巴结组织内蓝染铁颗粒,其他脏器未见明显铁颗粒沉积。T2WI图像上显示VEGFR3-USPIO组肿瘤T2值明显减低,强度率为28.6%；而USPIO组肿瘤T2值未见明显改变。二者肝的T2值降低均接近30%左右。离体标本普鲁士蓝染色观察,VEGFR3-USPIO组肿瘤及肿瘤间质可见蓝染铁颗粒,而USPIO组铁颗粒主要位于聚集在肿瘤周围的巨噬细胞中。结论：VEGFR3-USPIO与USPIO在健康裸鼠体内的分布相似,主要位于肝脏、脾脏血窦及淋巴结中；肝脏可作为评价VEGFR3-USPIO裸鼠强化效果的目标脏器；VEGFR3-USPIO有望成为一种高效、特异性的MRI靶向成像分子探针,用于肿瘤早期诊断及无创实时的评价治疗效果成像。更多还原

【Abstract】 Purpose To study magnetic resonance enhancement features of inflammatory lymph nodes using different doses of ultrasmall superparamagnetic iron oxide (USPIO) particles in order to establish a standardized protocol for USPIO-enhanced MR imaging of lymph nodes. Materials and Methods A total of 12 healthy New Zealand rabbits were injected complete Freund’s adjuvant in foot pad to establish popliteal inflammatory lymph node model. Different doses (45/90/135μmol Fe/kg) of USPIO were injected intravenously. Magnetic resonance scans were performed before and after USPIO injection to observe the enhancement features of different groups. T1SI, T2SI, T2*value and T2 value were measured and T2 enhancement ratio was calculated at different time points to draw time-dependent curves.Results Twenty-four hours after USPIO injection, there was no statistical difference in T2SI and enhancement ratio between 90 and 135μmol Fe/kg dose groups, but both were superior to 45μmol Fe/kg group (P<0.05); There were no statistical differences in T1SI, T2SI, T2 value or enhance ratio from 6 to 24 hours after USPIO injection in 90μmol Fe/kg group (P>0.05), and signal reduction of lymph nodes peaked 18 hours after injection. Better images were acquired with a post-contrast delay of 18-24 hours. Conclusion Lymph nodes can be enhanced well with a dose of 90μmol Fe/kg. Postcontrast delay of 18-24 hours is appropriate for acquiring satisfactory enhancement images. Purpose To assess the characteristics of lymph nodes in USPIO enhanced Magnetic resonance (MR) imaging in rabbit tumor and inflammatory models, and explore its relevance with histologic ultrastructural findings; To discuss the accuracy and clinical value of USPIO enhanced MR imaging in the diagnosis of lymph node.Materials and Methods 36 New Zealand white rabbits were randomly divided into inflammatory group and tumor group. Complete Freund’s adjuvant was injected into the footpads to set up inflammatory model. VX2 tumor cell suspension was implanted into thighs to set up metastatic lymph node model. MR scans of the popliteal fossa were performed before and 24 hours after USPIO (90μmol Fe/kg) administration. T2 values of each lymph node were measured and enhancement rate was calculated as well. HE staining, Prussian blue staining, and electronic microscopy were performed to observe the pathological microstructure changes and the distribution of the iron particle in lymph node. Relationship between lymph nodes USPIO enhancement imaging and its microstructures were further analyzed. Diagnoses of popliteal lymph nodes were evaluated based on dedicated criteria and the accuracy was also discussed. Results There were 46 inflammatory and 26 metastatic lymph nodes, including four with subcapsular metastatic foci. Sensitivity, specificity and positive and negative predictive values of the diagnosis of nodal metastasis were 84.6%,87.0%,78.6% and 90.9%, respectively. Plain scan showed no obvious difference of T2 signal intensity between the inflammatory and metastasis lymph nodes. After USPIO administration, there was distinctly T2 signal reduction at the center in inflammatory lymph nodes, and metastatic lymph nodes showed faint T2 signal reduction. Enhancement rate of benign and malignant lymph nodes were 57.22% and 29.45%, respectively (P< 0.01). HE staining and Prussian blue staining indicated USPIO particles located mainly in the macrophages at inflammatory lymphatic medulla, while paracortical area and cortical area contained relatively much less USPIO particles due to less macrophages distribution. MRI findings were correlated with the pathological results. Electronic microscopy also verified that the majority of USPIO particles were located in the numerous cytophagic bubbles of macrophages in inflammatory nodes, while in metastatic nodes, they contained predominantly cellular residues.Conclusion USPIO-enhanced MR is a particularly promising technique that improves the sensitivity and specificity of metastatic lymph node. USPIO enhancement pattern of different lymph nodes is closely related to distribution and functional status of the intra-node macrophages. It may affect the accuracy of the lymph node property diagnosis based on USPIO enhanced image. Purpose:To investigate granulometric characteristics of DMSA modified Ultrasmall Superparamagnetic Iron Oxide (USPIO) nanoparticles, the magnetic property in vitro and distribution features in healthy nude mice.Materials and Methods:FT-IR method was used to detect carboxyl groups on the surface of DMSA-USPIO. MR signal intensity of modified USPIO solutions with different concentrations was evaluated, and was compared with that of Ferumoxtran-10 solutions. USPIO nanoparticles were injected into 3 healthy BALB/C nu-nu nude mice through tail veins. MR imaging was performed before and after USPIO administration to study the MR enhancement features and T2 signal intensity changes on different time points in liver, kidney, muscle and brain. Mice were sacrificed right after MR imaging scan, tissues were collected for Prussian blue staining to investigate iron distribution in different organs.Results:DMSA-USPIO had absorption peaks at CO-and OH-groups. R2 and R2* values had a linear correlation with USPIO concentrations in both particles. At the same concentration, T2 and T2* relaxation time of DMSA-USPIO nanoparticles was obviously shorter than that of Ferumoxtran-10. After USPIO administration, T2 signal intensity of liver reduced significantly, whereas no obvious T2 signal intensity changes was found in kidney, muscle and brain. T2 values of liver showed significant differences between pre-and 24 hours post-USPIO administration (p<0.05), with an enhancing ratio of 26.8%. Prussian blue staining showed iron nanoparticles scattering in liver, spleen and lymph nodes, and there were no visible iron deposition in other tissues including kidney, brain, lung, myocardiac wall and bowel wall.Conclusion:DMSA-USPIO nanoparticles with superb magnetic features, which were encapsulated with rich modifiable surface groups, can be used as conjugation carrier of the target molecular for MR imaging. The significant T2 value enhancement in the liver might be recommended as one of indexes for evaluation of USPIO enhancement effectiveness. Purpose:To establish a magnetic resonance molecular conjugator of vascular endothelial growth factor receptor 3 (VEGFR3) antibody and ultrasmall superparamagnetic iron oxide particles (USPIO). And to investigate in vitro targeted combination specificity and MRI targeted imaging feasibility of VEGFR3-USPIO to human breast cancer MDA-MB-231 cells.Materials and Methods:Expression level and expression site of VEGFR3 on MDA-MB-231 cells was detected using immunofluorescence technique. Molicular weight measurement and semi-quantitative analysis were performed using Western Blot test. VEGFR3 anti-body and USPIO were coupled using carbodiimide (1-ethyl-(3-dimethylamino propyl) carbodiimide hydrochloride (EDC). T2 relaxation time differences of VEGFR3-USPIO conjugator and USPIO solutions were evaluated at same concentration grads. After incubating the MDA-MB-231 cells with VEGFR3-USPIO and USPIO respectively, existing of iron particles was observed by Prussian blue staining, changes of T2 relaxation time of each in vitro samples were measured using a clinical MRI scanner.Results:VEGFR3 receptor expression was positive both on the membrane and in the cytoplasm of MDA-MB-231 cells. Molecular weight of VEGFR3 peptide is about 73Kda according to the result of Western blot test. In vitro MR scan proved that there was no significant T2 or T2* relaxition time difference between VEGFR3-USPIO and USPIO at every different concentrations, and there was linear correlation between R2 and R2* relaxation time and the particle concentration. After incubation, Prussian blue staining showed more iron particles left in the VEGFR3-USPIO group. Distribution of iron particles was mainly around the cell membrane in the VEGFR3-USPIO group, while scattered iron particles were found in the inter-cell space. T2 relaxation time of VEGFR3-USPIO-cell incubation group was lower than USPIO-cell incubation group, T2 relaxation time change rate were 61.0% and 43.5% respectively.Conclusion:VEGFR3-USPIO conjugator had satisfied in vitro targeted combination specificity and MRI targeted imaging feasibility according to a VEGFR3 expression positive cell MDA-MB-231. Purpose:To investigate the distribution feature of VEGFR3 antibody-conjugated Ultrasmall Superparamagnetic Iron Oxide nanoparticles (VEGFR3-USPIO) in healthy nude mice and assess its targeted imaging viability in the tumor bearing nude mice using a clinical type MRI scanner.Materials and Methods:VEGFR3-USPIO molecular probes were injected into 3 healthy BALB/C nu-nu nude mice via tail veins. MR imaging including multiple sequences was performed pre-and post-VEGFR3-USPIO administration in order to study the MR enhancement features and T2 signal intensity changes on different time points in liver, kidney, muscle and brain. Mice were sacrificed right after MR imaging scan, tissues were collected for Prussian blue staining. Iron particles distribution in different organs was investigated. Subcutaneous human mammary tumor (MDA-MB-231) models were established in 6 BALB/C nu-nu female nude mice. They were then randomly divided into two groups. VEGFR3-USPIO and DMSA-USPIO were injected into tumor bearing nude mice via tail veins. MR images were acquired before and 24 hours after contrast agent administration. T2 value enhancement of the tumor and the liver was measured and tissues were collected for histological examination.Results:After VEGFR3-USPIO administration in healthy nude mice, T2 value of liver got lower, whereas no obvious T2 value change was found in the kidney, muscle and brain. T2 values of the liver showed significant differences between before and 24 hours after VEGFR3-USPIO administration (p<0.05), with an enhancing ratio of 25.8%. Prussian blue staining showed iron oxide nanoparticles scattering in liver, spleen and lymph nodes, and there were no visible iron deposition in other tissues including kidney, brain, lung, myocardia wall and bowel wall. On T2WI image, T2 value of tumor tissue showed a dramatic decrease in VEGFR3-USPIO group, with enhancing ratio of 28.6%. However, in the USPIO group, T2 value of tumor tissue had no obvious changes. In both groups, T2 value of the liver showed equally 30% reduction. Prussian blue staining in VEGFR3-USPIO group showed that blue iron nanoparticles scattered around the tumor cells or inside their stoma. While in USPIO group, iron nanoparticles mostly deposited surround the macrophages which gathered around the tumor tissue.Conclusion:Similar to DMSA-USPIO nanoparticle, VEGFR3-USPIO mainly distributed in the liver, spleen and lymph nodes. The significant T2 value enhancement in the liver might be recommended as one of indexes for evaluation of VEGFR3-USPIO enhancement effectiveness. VEGFR3-USPIO is a potentially superb MRI molecular targeted imaging probe with high efficiency and high specificity. It is possible to be used as an imaging tool for early tumor detection and non-invasive real-time evaluation of anti-tumor therapy.更多还原