【作者】 李琳；

【导师】 滕皋军；

【作者基本信息】 东南大学 ， 影像医学与核医学， 2017， 硕士

【摘要】 第一部分GE11多肽对表皮生长因子受体阳性的乳腺癌脑转移瘤的靶向性研究目的探讨GE11多肽对表皮生长因子受体(EGFR)阳性的乳腺癌脑转移瘤靶向结合的可行性,为构建特异性靶向乳腺癌脑转移瘤的分子探针寻找合适的靶向基团。方法应用Western blot及流式细胞学方法检测人三阴性乳腺癌脑转移细胞株(MDA-MB-231-BR)的EGFR的表达,采用细胞免疫荧光染色及流式细胞学方法检测荧光标记的GE11多肽对MDA-MB-231-BR细胞株的体外靶向结合特性,并利用皮下荷瘤鼠进行活体水平靶向性验证。结果Westemblot及流式细胞学检测结果显示EGFR在MDA-MB-231-BR细胞株中表达比例为88.6%,呈明显高表达。流式细胞检测结果显示,10μmol/L浓度GE11多肽与MDA-MB-231-BR细胞结合比例为38.4%,结合比例明显高于相同浓度的对照肽;细胞荧光染色结果显示GE11多肽与细胞共孵育后可检测出更强的荧光信号,并且结合具有剂量及能量依赖性。活体实验结果显示GE11多肽在肿瘤部位有较强的信号,其他组织信号较弱。结论 GE11多肽与EGFR阳性的乳腺癌脑转移瘤具有特异性靶向结合特性,为进一步构建靶向乳腺癌脑转移瘤的分子探针奠定了基础。第二部分靶向EGFR分子成像探针的构建、表征及靶向性验证目的乳腺癌脑转移是乳腺癌患者是最严重的结局之一,具有死亡率高、预后差等特点,尤其是三阴性乳腺癌。目前临床上现存的诊断手段对于早期或者病灶较小的乳腺癌脑转移瘤的诊断存在一定的困难。本研究旨在通过构建一种靶向乳腺癌脑转移瘤EGFR的可跨越血脑屏障的双模态分子成像探针,对乳腺癌脑转移瘤进行实时、无创成像,以期实现对乳腺癌脑转移的早期诊断。方法本实验以PAMAM树枝状分子为载体,GE11为EGFR分子靶向肽,Angiopep2为脑靶向肽,Gd3+-DTPA为MR显像剂,IR783为近红外荧光显像剂,设计并合成一种可跨越血脑屏障的靶向EGFR的磁共振/光学双模态分子成像探针。利用人三阴性乳腺癌脑转移细胞株(MDA-MB-231-BR)、人胶质瘤细胞株(U87)及乳腺导管癌细胞株(BT-474)体外验证该分子探针靶向EGFR的特异性;利用三阴性乳腺癌脑转移细胞株MDA-MB-231-BR构建乳腺癌脑转移瘤荷瘤鼠模型,并通过MR和近红外成像进行分子探针的活体分子成像。结果1)细胞免疫荧光染色实验结果显示,当探针浓度为5μmol/L时,Den-Angio-GE11探针组显微镜下可观察到较多的探针红色荧光信号,并随着探针浓度减低,红色荧光信号强度逐渐减低;Den-Angio-GE11探针与MDA-MB-231-BR及U87细胞株共孵育后红色荧光信号强于BT-474细胞组。2)流式细胞学检测结果显示当探针浓度为5μmol/L时,乳腺癌脑转移细胞株MDA-MB-31-BR 与 Den-Angio-GE11 探针组结合比例为 64.2%,Den-Angio 探针组为 22.7%,Den-PEG 探针组为21.6%,Den-Angio-GE11探针组细胞结合比例显著高于对照探针组(P<0.05);U87细胞与5μmol/L浓度Den-Angio-GE11探针结合比例约为47.5%,BT-474细胞与其结合比例约为0.7%,此结果与三种细胞的EGFR表达水平结果相一致。3)乳腺癌脑转移细胞株MDA-MB-231-BR体外磁共振及近红外成像结果显示Den-Angio-GE11探针组信号强度显著高于对照探针组。4)乳腺癌脑转移瘤荷瘤鼠活体近红外结果显示注射探针2h后,荷瘤鼠活体头部荧光信号达到高峰,且Den-Angio-GE11探针组荷瘤鼠头部荧光信号显著高于对照探针组。5)乳腺癌荷瘤鼠活体磁共振成像结果显示注射分子探针5min后,Den-Angio-GE11探针组脑转移瘤T1WI序列可见增强,对照探针组未见明显强化。结论可跨越血脑屏障的靶向EGFR的磁共振/光学双模态分子成像探针可以实现乳腺癌脑转移瘤荷瘤鼠的活体近红外及磁共振成像。作为一种非创伤性成像技术,该分子探针在乳腺癌脑转移的早期诊断中具有潜在的应用价值。更多还原

【Abstract】 Part I Characterization of GE11 peptide with affinity for EGFR-overexpressing human breast cancer brain metastasesObjective To investigate GE11 peptide targeting property of human epidermal growth factor receptor(EGFR)positive breast cancer brain metastases,and find suitable target groups for the construction of molecular nanoprobe specific targeting breast cancer brain metastases.Methods The EGFR expression level of triple negative breast cancer cell lines(MDA-MB-231-BR)was detected using Western blot and flow cytometry.GE11 peptide targeting property was tested using cell immunofluorescence staining and flow cytometry detection in MDA-MB-231-BR cell and in the subcutaneous tumor model.Results Western blot and flow cytometry examination results showed that EGFR cell binding percent in MDA-MB-231-BR cell was 88.6%,significantly higher expressed than other cells.Cell flow cytometry test results indicated that the cell binding percent with 10μmol/L concentration GE11 peptide was 38.4%,significantly higher than control peptide with same concentration.Cell immunofluorescence staining results showed GE11 peptides can be detected stronger signal after incubation with cells,and its combination with the energy and dose dependence.Experiment results show that the GE11 peptide has stronger signal in tumor site,other organizations signal is weak.Conclusion GE11 peptide can specific target with EGFR positive breast cancer brain metastases,laid the foundation of building targeting molecular nanoprobe for breast cancer brain metastases.Part II Construction,characterization and targeting property verification of EGFR-targeted molecular imaging nanoprobes Objective Breast cancer brain metastases is one of the most serious outcomes with high mortality and poor prognosis,especially the triple negative breast cancer.It is difficult to diagnosis of brain metastases in the early stage or smaller lesions with the existing clinical diagnostic methods.This study aims to synthesis an EGFR-targeted and blood-brain-barrier permeable dual-modality molecular imaging nanoprobes as the real time and noninvasive imaging for accurate detection of early breast cancer brain metastases.Methods Designing and synthesising of an EGFR-targeted molecular imaging nanoprobes used PAMAM dendrimers as the carrier,GE11 peptide as EGFR targeting peptide,Angiopep2 for brain targeting peptide,Gd3+-DTPA as MR imaging agent,IR783 for near-infrared fluorescence imaging agents.Using triple negative breast cancer brain metastasis cell line(MDA-MB-231-BR),human glioma cell line(U87)and breast ductal cancer cell(BT-474)to verify the specificity of molecule nanoprobe targeting EGFR in vivo.Using triple negative breast cancer brain metastasis cell line(MDA-MB-231-BR)to establish a mouse model of breast cancer brain metastases and image with magnetic resonance imaging and near infrared imaging in vivo.Results(1)Cell immunofluorescence staining results showed that Den-Angio-GE11 nanoprobe group can be detected stronger signal after incubation with cells,and its combination with the energy and dose dependence;MD-MB-231-BR and U87 cell lines can be detected stronger signal than the BT-474 cell after incubation with Den-Angio-GE11 nanoprobe.(2)Flow cytometry examination results showed that Den-Angio-GE11 nanoprobe binding percent in MDA-MB-231-BR cell was 64.2%with the concentration of 5μmol/L and was significantly higher expressed than other nanoprobes,Den-Angio was 22.7%,Den-PEG was 21.6%(P<0.05).The binding percent of U87 cell was 47.5%after incubation with Den-Angio-GE11 nanoprobe,BT-474 cell was 0.7%.(3)The magnetic resonance and near infrared imaging of MDA-MB-231-BR cells results showed that Den-Angio-GE11 group signal intensity was significantly higher than control nanoprobes.(4)After injecting nanoprobes 2h,the near infrared imaging of breast cancer brain metastases-bearing mouse model showed that Den-Angio-GE11 group’s fluorescence signal was significantly higher than control nanoprobes.(5)After injecting Den-Angio-GE11 nanoprobe 5min,the T1-weighted magnetic resonance imaging of metastases-bearing mouse model showed the signal enhancement in the brain metastases,none of control nanoprobe groups.Conclusion The EGFR-targeted molecular imaging nanoprobe can realize the metastases lesions of breast cancer brain metastases-bearing mouse model with magnetic resonance imaging and near infrared imaging in vivo.As a real time and noninvasive imaging,this nanoprobe has potential in the early diagnosis of breast cancer brain metastases.更多还原