【作者】 张晓；

【导师】 张永学； 兰晓莉；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2018， 博士

【摘要】 目的:血管细胞粘附分子-1(VCAM-1)是参与肿瘤演进的重要因子,在多种肿瘤细胞中均有表达。本实验旨在构建99mTc标记靶向VCAM-1单链抗体(scFv)的分子探针,探讨及评估其在不同肿瘤模型中特异性结合的能力以及靶向显像的可行性。方法:通过使用6-肼基烟酸琥珀酰亚胺酯盐酸盐(SHNH),将靶向VCAM-1 的 scFv(VCAM-1scFv)与 99mTc 连接进行标记。VCAM-1 的表达水平可通过不同细胞系(黑色素瘤细胞B16F10及A375m、纤维肉瘤细胞HT1080、卵巢癌细胞SKOV3.ip、乳腺癌细胞MDA-MB-231及肾癌细胞786-0)的免疫荧光染色进行验证。细胞摄取实验、体内单光子发射计算机断层(SPECT)显像和生物分布实验分别用以测定99mTc-6-肼基烟酰胺(HYNIC)-VCAM-1seFv在体内外与VCAM-1的结合能力。与此同时验证肿瘤组织中VCAM-1的表达。结果:99mTc-HYNIC-VCAM-1seFv 的标记率为 81.46±3.61%(n=5),纯化后的放射化学纯度为96.54±1.65%(n=5)。体外摄取实验表明,B16F10和HT1080细胞结合探针能力较强,4小时细胞摄取率分别为6.07±0.55%、5.73±0.41%(n=3),且不同肿瘤细胞对探针摄取能力与免疫荧光的VCAM-1表达水平较为一致。体内SPECT成像及生物分布表明,B16F10和HT1080 肿瘤摄取较高(4.93±0.52%ID/g,4.65±0.39%ID/g,4h),SKOV3.ip肿瘤呈中等摄取(2.99士0.44%ID/g,4h),A375m、MDA-MB-231 和 786-0 肿瘤组织摄取较低(1.33士0.22%ID/g、1.49±0.23%ID/g 及1.47±0.31%ID/g,4h)。B16F10肿瘤与其阻断组肿瘤1h摄取量分别为5.51±0.37%ID/g、2.92±0.26%ID/g(p<0.001),表明 B16F10 肿瘤组织对99mTc-HYNIC-VCAM-1seFv高摄取可被过量的VCAM-1seFv抑制。与此同时,肿瘤组织荧光表达强度与肿瘤组织摄取有一定的相关性(R2=0.875,p<0.001)。结论:99mTc-HYNIC-VCAM-1scFv能够在体内外检测VCAM-1的表达水平,为体内非侵入地评估VCAM-1的表达提供了新的手段。目的:血管细胞粘附分子-1(VascularCellAdhesionMolecule1,VCAM-1)在肿瘤生长及转移中起着至关重要的作用,逐渐成为肿瘤监测和治疗的新靶点。LY2409881是近年来发现的一种新型的IκB激酶β的抑制剂,可以诱导VCAM-1阳性表达的肿瘤细胞凋亡。本研究旨在评估68Ga标记的VCAM-1单链抗体(scFv)检测VCAM-1阳性肿瘤表达的能力,并探讨其监测LY2409881对VCAM-1阳性肿瘤治疗反应的可行性。方法:应用黑色素瘤细胞系B16F10和A375m分别制作VCAM-1过表达和低表达肿瘤模型。以1,4,7-三氮杂环壬烷-1,4,7-三乙酸(NOTA)为放射性标记螯合剂,将VCAM-1 scFv与68Ga进行连接,构建新型示踪剂68Ga-NOTA-VCAM-1scFv。构建B16F10和A375m荷瘤鼠模型,尾静脉注射68Ga-NOTA-VCAM-1seFv后,分别进行microPET/CT成像、生物分布及放射自显影研究,用以探究该分子影像探针在肿瘤模型中的分布和靶向能力。以IKKs抑制剂LY2409881为靶向治疗药物,在体外B16F10细胞中评估其细胞毒性。体内实验中,B16F10荷瘤小鼠腹腔内注射LY2409881(每周两次,100mg/kg),对照组采用等体积的溶剂对照DMSO进行腹腔注射。每隔一天测量小鼠体重及肿瘤体积,同时取5只小鼠每周尾静脉注射68Ga-NOTA-VCAM-1scFv进行microPET/CT成像,动态监测评估治疗疗效。结果:68Ga-NOTA-VCAM-1seFv的标记率为95.00±1.34%(n=5),纯化后的放射化学纯度为97.80±1.65%(n=5)。microPET/CT显像、生物分布及放射自显影实验表明,68Ga-NOTA-VCAM-1seFv对表达VCAM-1p阳性的B16F10肿瘤亲和力较高(3hB16F10和A375m肿瘤摄取分别为5.17±0.68%ID/g、1.87±0.55%ID/g)。随着LY2409881浓度增加及作用时间延长,B16F10细胞活性逐渐降低,半数抑制浓度为17.69μM。体内动物模型实验也显示出该抑制剂对B16F10肿瘤生长具有明显的抑制作用(三周时治疗组肿瘤抑制率为51.76%)。在治疗监测过程中,microPET/CT显像及生物分布实验表明LY2409881治疗组在治疗后第一周肿瘤组织对68Ga-NOTA-VCAM-1seFv摄取减少(从5.02士0.25%ID/g降低至3.68±0.27%ID/g),第二周回到治疗前摄取水平。而在DMSO对照组中,肿瘤对68Ga-NOTA-VCAM-1seFv摄取保持相对稳定水平。结论:LY2409881作为IKK2抑制剂,可以有效地抑制VCAM-1表达及肿瘤生长。68Ga-NOTA-VCAM-1seFv作为显示VCAM-1表达的一种新型分子影像探针,制备简便、标记率高、稳定性好,图像清晰。68Ga-NOTA-VCAM-1scFv的PET显像可以用于VCAM-1阳性肿瘤分子表达的可视化探究,是监测恶性肿瘤LY2409881治疗效果的有效手段,具有良好的临床应用前景。更多还原

【Abstract】 Objectives:Vascular cell adhesion molecule 1(VCAM-1)is overexpressed in a group of cancers.This study aimed to evaluate the apply of 99mTc labeled single chain variable fragment(scFv)of VCAM-1(VCAM-1scFv)as a possible imaging agent in several tumors.Methods:The VCAM-1scFv was labeled with 99mTc using the succinimidyl 6-hydraziniumnicotinate hydrochloride(SHNH).VCAM-1 expression levels were evaluated in a number of cell lines by immunofluorescence staining.In-vitro cell binding assays,single photon emission computed tomography(SPECT)imaging and biodistribution studies with 99mTc-6-hydrazinonicotinamide(HYNIC)-VCAM-1scFv were carried out in a variety of cell lines or tumors.The immunofluorescence study was also performed in the kidneys,livers and different tumor tissues.Results:99mTc-HYNIC-VCAM-1scFv was synthetized with a high radiolabeling yield of 81.46±3.61%(n=5),and a radiochemical purity of 96.54±1.65%(n=5)after purification.In-vitro binding assays showed that different binding affinity of 99mTc-HYNIC-VCAM-1scFv in different tumor cell lines,and high cellular uptake was seen in B16F10 and HT1080 cells(6.07±0.55%,5.73±0.41%,n=3),which were consistent with immunofluorescence staining.In-vivo SPECT imaging demonstrated that B16F10 and HT1080 tumor images could be clearly delineated(4.93±0.52%ID/g,4.65±0.39%ID/g,4h),and a visible signal was observed in SKOV3.ip tumor(2.99±0.44%ID/g,4h)and weak uptake in A375m,MDA-MB-231,and 786-0 tumors(1.33±0.22%ID/g,1.49±0.23%ID/g and 1.47±0.31%ID/g,4h),which were confirmed by biodistribution studies.Moreover,the high uptake in B16F10 tumors could be inhibited by excess VCAM-1ScFv(5.51±0.37%ID/g,2.92±0.26%ID/g,p<0.001).The immunofluorescence intensity of the tumors was correlated well with the in vivo result(R2=0.875,p<0.001),and the kidneys and livers showed relatively low signals.Conclusion:99mTc-HYNIC-VCAM-1scFv,which selectively binds to VCAM-1,can visualize different expression levels of VCAM-1 and provide a qualitative and quantitative method for noninvasive evaluation of VCAM-1 in tumors.Objectives:Vascular cell adhesion molecule-1(VCAM-1)is becoming an attractive candidate for tumor targeting detection and therapy due to its involvement in tumourigenicity and metastasis.LY2409881,an IKK2 inhibitor,can trigger apoptosis of VCAM-1 positive melanoma cells.The aim of this study was to assess the use of the 68Ga-labeled single chain variable fragment(scFv)of VCAM-1 in detecting VCAM-1-positive tumor expression and monitoring the therapeutic effect of LY2409881.Methods:Melanoma cell lines,B16F10 and A375m,were selected as VCAM-1 overexpression and underexpression models,respectively.VCAM-1 scFv was labeled with 68Ga using 1,4,7-triazacyclononane-1,4,7-triacetic acid(NOTA).MicroPET/CT imaging,biodistribution,and autoradiography studies were performed in mice bearing B16F10 and A375m tumors after injection of 68Ga-NOTA-VCAM-1scFv to verify the targeting ability of the tracer.In-vitro cytotoxicity assays of LY2409881 were performed in B16F10 cells,and an in vivo study was performed in B16F10 tumor-bearing mice with intraperitoneal injection of LY2409881,using DMSO injection with same protocol as a control.Treatment monitoring was evaluated with 68Ga-NOTA-VCAM-1scFv microPET/CT imaging weekly.Results:68Ga-NOTA-VCAM-1scFv was successfully synthesized with high labeling efficiency.MicroPET/CT images,biodistribution,and autoradiography studies showed much higher uptake of the tracer in B16F10 tumors than that in A375m tumors.LY2409881 caused dose-and time-dependent growth inhibition and apoptosis in melanoma cellsin vitro,and suppressed B16F10 tumor growth in vivo.In the therapy monitoring group,microPET/CT imaging showed the reduced tumor uptake of 68Ga-NOTA-VCAM-1scFv at the first week of LY2409881 treatment,but increased to the initial uptake level afterward.The tumor uptake of 68Ga-NOTA-VCAM-lScFv in the control group remained relatively steady during the DMSO treatment.Conclusions:LY2409881,an IKK2 inhibitor,inhibits tumor growth and VCAM-1 expression.68Ga-NOTA-VCAM-1scFv,an easily-prepared probe,can be used to visualize VCAM-1 positive tumors and monitor the effect of LY2409881 therapy,suggesting its prospective clinical application.更多还原