【作者】 刘伟伟；

【导师】 侯桂华；

【作者基本信息】 山东大学 ， 影像医学与核医学， 2019， 硕士

【摘要】 研究背景目前临床上多数肺癌患者因早期症状较少,确诊时已处于晚期并有远处转移,严重影响患者预后及临床治疗手段的选择。病理类型和分期不同的肺癌患者临床治疗方式不同,且疗效和生存率有明显不同,因此早期诊断对于肺癌患者及时治疗及预后至关重要。目前临床常用诊断肺癌的手段主要包括影像学、血清肿瘤标记物、痰细胞学、肺穿刺和纤维支气管镜检查等,这些检查方法在特异性、实用性等方面存在明显局限性。因此亟需寻找更高选择性、高特异性标志物,为肺癌早期诊断、疗效监测和个体化靶向治疗提供实验基础。近年来分子影像学研究快速发展,尤其是核医学分子影像因其高灵敏、高特异、易于临床转化受到高度重视。放射性核素肿瘤分子显像主要包括放射免疫显像、受体显像和基因显像,可以用于恶性肿瘤的早期诊断和良恶性肿瘤的鉴别诊断,具有较好的临床应用价值。其中肿瘤放射免疫显像技术作为一种将放射性核素与高特异性抗体相结合的一种分子成像示踪技术,适合监测并量化肿瘤组织中靶分子表达水平和分布情况,有助于肿瘤早期诊断、疗效和预后评估,适于临床多种肿瘤的显像。肿瘤靶向分子探针的选择已成为放射免疫成像的先决条件,对肿瘤显像与治疗及临床疗效评估至关重要。目前探索新型核素分子探针成为肿瘤分子显像领域的核心,该类新型探针应满足临床高特异性、高敏感性和高选择性等要求。血管生成可为机体组织和细胞提供氧气和营养物质,参与多种生理病理过程,在实体肿瘤生长中至关重要。肿瘤新生血管具备非典型形态特征,使肿瘤组织血管过度渗透、灌注不良,缺氧微环境可介导肿瘤细胞的扩散、侵袭和转移。肿瘤组织如缺乏血管氧气及能量供应,将无法突破实体瘤临界值大小而进行远处器官转移。目前靶向肿瘤病理性新生血管已经成为临床肿瘤诊断显像基础研究的热点。肿瘤血管新生与多种基因突变和细胞因子及受体的表达密切相关。值得注意的是近年发现的与新生血管生成有关的CD93分子。CD93作为一种跨膜糖蛋白,可在内皮细胞、干细胞及骨髓细胞表达,最主要的表达部位是内皮细胞,而内皮细胞又在肿瘤血管生成过程必不可少。早期研究发现CD93可促进免疫细胞的黏附和穿越。新近文献报道CD93是一种新型血管生成激活剂,主要通过促进内皮细胞粘附,加速肿瘤血管再生,从而影响肿瘤的生长,其高表达可加速肿瘤生长并减少宿主存活。此外,膜表面CD93的蛋白外域在炎症刺激下容易分裂或脱落,形成可溶性CD93,促进血管生成过程,因此CD93可能是肿瘤新生血管发生发展过程中的关键分子,可作为肺癌诊断和预后的生物标志物,有望用于肿瘤早期靶向诊断。研究发现CD93分子在肿瘤组织中的表达量与临床病理类型和患者治疗预后密切相关,早期识别肿瘤组织中CD93表达情况对患者靶向诊断和治疗的选择至关重要。CD93分子在肿瘤血管的内皮细胞高表达,在非增殖的内皮细胞低表达,更凸显CD93分子在肿瘤诊断方面的潜在临床应用价值。然而目前尚未见有关以CD93分子为研究靶点,进行肿瘤早期靶向监测及疗效评价的相关研究报道。探讨放射性核素标记的CD93分子探针是否可用于肺癌靶向分子显像对于研究CD93作为肿瘤分子靶点进行非侵入性诊断、靶向治疗和疗效评估具有重要意义。本论文选择了临床发病率高,早期诊断难的肺癌作为研究对象,利用放射性核素标记抗CD93抗体,通过两部分初步研究了放射性核素CD93分子探针对肺癌靶向性监测的可行性及意义。一是体外细胞水平上研究了三种非小细胞肺癌细胞系CD93表达状况,同时制备新型放射性核素分子探针125I-anti-CD93 mAb并进行鉴定,研究了其对肺癌细胞结合的特异性;二是体内研究,构建肺癌荷瘤小鼠模型,研究125I-anti-CD93 mAb对肺癌肿瘤体内靶向显像的特异性,并与125I-IgG和18F-FDG进行对比分析,评价CD93核素标记物的显像特征及优势。本论文旨在为肺癌靶向诊断寻找新的靶向分子,为早期非侵入性诊断肺癌提供新策略。第一部分CD93放射性核素分子探针制备及鉴定研究方法1.125I-anti-CD93 mAb和125I-IgG制备Iodogen法碘化标记、常规制备放射性核素分子探针125I-anti-CD93 mAb和对照125I-IgG。利用PD-10凝胶柱进行洗脱纯化,测量放射性计数,计算两标记物的标记率和放射性比活度;采用纸层析法测定放射化学纯度,将125I-anti-CD93 mAb和125I-IgG蛋白峰分别与生理盐水、血清混合均匀,利用纸层析法检测24h、48h、72h标记物的体外稳定性。2.RT-PCR和Western Blot检测细胞CD93表达状况常规无菌培养腺癌A549细胞、大细胞癌H460细胞和鳞癌H520细胞三种非小细胞肺癌细胞系。利用RT-PCR检测三种肺癌细胞系CD93基因表达状况;Western Blot分析三种肺癌细胞系CD93蛋白表达状况,分析三种肺癌细胞系之间CD93表达的差异。3.标记物体外细胞结合实验125I-anti-CD93 mAb组:将生长状态良好的三种肺癌细胞系(2×105/孔)接种于24孔板,按浓度梯度(3-100nM)加入标记物,一定时间后,洗涤,测量细胞放射性计数,对比分析各肺癌细胞系对125I-anti-CD93 mAb的亲和解离常数(KD值)和受体结合最大量(Bmax)之间的差异。125I-IgG组:将呈对数生长的A549肺癌细胞(2×10s/孔)接种于24孔板上,按浓度梯度(3-100nM)加入标记物125I-IgG,一定时间后,洗涤,测量A549细胞放射性计数,分析A549肺癌细胞对125I-IgG的亲和解离常数(KD值)和受体结合最大量(Bmax),并与125I-anti-CD93 mAb组A549对比研究。4.标记物体外细胞结合阻断实验将A549肺癌细胞(2×105/孔)接种于24孔板上,同时加入定量125I-anti-CD93 mAb及未标记的anti-CD93 mAb(0、1.42、14.2、28.4nmol/L),一定时间后,洗涤,测量放射性计数,分析anti-CD93 mAb对125I-anti-CD93 mAb与A549结合的阻断情况。研究结果1.成功制备放射性核素探针125I-anti-CD93 mAb,标记率为91.37%,放射性比活度为1096.44MBq/mg,放化纯为96.49%;同种型对照125I-IgG标记率为90.24%,放射性比活度为1082.88MBq/mg,放化纯为94.82%。纸层析法测得125I-anti-CD93 mAb和125I-IgG均具有较好的稳定性,至72h仍维持在90%以上。2.RT-PCR和Western Blot结果均表明,A549细胞、H460细胞高表达CD93分子,H520细胞低表达CD93分子,两者之间差异具有统计学意义。3.标记物体外细胞结合实验显示,A549、H460和H520细胞对125I-anti-CD93 mAb的KD值分别为27.09nmol/L、28.84nmol/L、39.90nmol/L,即A549和H460细胞亲和力明显高于H520细胞;A549细胞和H460细胞的Bmax值是1679cpm/104细胞、1780cpm/104细胞,明显高于H520细胞(756cpm/104细胞)。125I-anti-CD93 mAb与A549肺癌细胞结合率显著高于对照125I-IgG组。4.标记物体外细胞结合阻断实验表明,未标记的anti-CD93 mAb可特异性阻断A549细胞与125I-anti-CD93 mAb的结合,加入量与阻断率呈正比。第二部分CD93放射性核素分子探针对荷肺癌小鼠肿瘤靶向性研究研究方法1.构建三种肺癌荷瘤小鼠模型常规无菌培养肺癌细胞系:A549细胞、H460细胞、H520细胞。选取4-6周雌性BALB/c裸鼠,分别在右前肢肩部皮下注射5 × 1 07/nml肺癌细胞悬液,0.2ml/只,建立三种肺癌荷瘤小鼠模型。全程无菌操作及饲养,记录肿瘤体积变化,待肿瘤直径达0.8-1.0cm时进行实验。2.三种肺癌模型鼠125I-anti-CD93 mAb生物学分布实验三种肺癌荷瘤鼠提前48h饮水中加3%NaI溶液封闭甲状腺,分别经尾静脉注射125I-anti-CD93 mAb,0.37 MBq/只,注射后分别于24h、48h、72h每组随机选取5只小鼠处死,取主要器官、肿瘤及对侧肌肉组织,称重并测定其放射性计数(cpm)和注射标记物(标准源)的放射性,计算%ID/g值。3.三种肺癌模型鼠125I-anti-CD93 mAb全身动态磷屏自显影显像三种肺癌荷瘤小鼠提前48h饮水中加3%NaI溶液封闭甲状腺。荷瘤小鼠模型经尾静脉注射125I-anti-93 mAb,0.37MBq/只,分别在24h、48h、72h麻醉后行模型小鼠全身动态磷屏显像,观察并使用OptiQuantTM图像分析软件对比分析125I-anti-D93 mAb在三种肺癌模型鼠全身放射性分布及在肿瘤部位的浓聚状况。4.A549肺癌荷瘤小鼠125I-anti-D93 mAb和125I-IgG生物学分布实验A549肺癌荷瘤小鼠如前所述方法提前48h封闭甲状腺,并随机分为2组。别经尾静注射125I-anti-D93mAb或125I-IgG,0.37MBq/只,注射后24h、48h、72h随机选取5只小鼠脱椎处死,取主要器官、肿瘤及对侧肌肉组织称重并测定其放射性计数(cpm)和注射标记物(标准源)的放射性,计算%ID/g值。5.A549肺癌荷瘤小鼠三种显像剂全身动态磷屏显像A549肺癌荷瘤小鼠提前48h封闭甲状腺,并随机分为3组。其中两组模型鼠分别经尾静脉注射125I-anti-D93 mAb/125I-IgG,0.373MBq/只,于注射后24h、48h、72h进行全身磷屏动态显像。另一组模型鼠经尾静脉注射18F8FDG,G37MBq/,分别于30min、3h、6h进行全身磷屏显像。使用OptiQuantTM图像分析软件对比分析125I-anti-CD93 mAb、125I-IgG、18FDG全身分布及在肿瘤部位的放射性浓聚状况。6.肺癌组织H&E和CD93免疫组织化学染色随机选取注射标记物后72h完成磷屏显像的肺癌模型鼠,取出肿瘤组织,4%多聚甲醛固定并制备成石蜡切片,进行苏木素-伊红染色,并利用抗CD93抗体进行免疫组织化学染色,显微镜下观察拍照,计算其CD93阳性表达率。研究结果1.肺癌荷瘤小鼠生物学分布结果表明,三组模型鼠125I-antin-CD93 mAb标A物均主要经肝肾代谢,肿瘤的放射性48h时最高。48h时,A549组和H460组放射性和T/NT比值均明显高于H520组,且差异有统计学意义。2.三组肺癌模型鼠全身动态磷屏显像研究表明,均在注射125I-anti-CD93 mAb后24h可见荷瘤鼠轮廓,48h时肿瘤显像最为清晰,且A549组和H460组肿瘤组织放射性摄取明显高于H520组;半定量分析结果表明48h时A549组和H460组放射性活度比(肿瘤区域/对侧区域)显著高于H520组,差异具有统计学意义。3.A549肺癌荷瘤小鼠生物学分布结果提示,两标记物均经肝肾代谢,125I-anti-CD93 mAb组肿瘤聚集明显,125I-anti-CD93 mAb组肿瘤的放射性计数和T/NT比值(肿瘤/对侧肌肉组织)均明显高于125I-IgG组,且具有统计学意义。4.A549肺癌荷瘤小鼠注射标记物后,48h肿瘤显像最为清晰,且125I-anti-CD93 mAb组肿瘤部位放射性浓聚明显高于125I-IgG组,提示125I-anti-CD93 mAb在肺癌肿瘤部位高摄取应该是125I-anti-CD93 mAb与细胞表面CD93受体特异性结合,可排除抗体IgG Fc段非特异性结合的作用。18F-FDG对照组在注射后3h显像最为理想,肿瘤放射性活度比为1.60±0.10,显著低于125I-anti-CD93 mAb注射组(3.34±0.18),表明与非特异性显像剂18F-FDG相比,125I-anti-CD93 mAb靶分子探针对A549肿瘤显像更加清楚。5.肿瘤组织病理学切片显示肿瘤细胞恶性增生,含有较丰富的新生血管,CD93免疫组化染色阳性率高,其中A549组和H460组肿瘤CD93阳性率高于H520组,与体外分析所测得的三种肺癌细胞细胞系CD93表达结果一致。全文小结1.非小细胞肺癌细胞中腺癌A549细胞和大细胞癌H460细胞高表达CD93分子,而鳞癌H520细胞低表达CD93分子。2.放射性核素探针125I-anti-CD93 mAb具有良好的生物学活性;肺癌细胞对125I-anti-CD93 mAb的亲和力与CD93表达密切正相关;Anti-CD93 mAb可特异性阻断A549细胞与125I-anti-CD93 mAb的结合。3.A549肺癌模型小鼠生物学分布结果与全身动态显像结果一致,125I-anti-CD93 mAb组肿瘤放射性计数和T/NT值均明显高于125I-IgG组。4.A549肺癌模型小鼠三种显像剂显像结果对比研究提示,125I-anti-CD93 mAb组肺癌肿瘤部位的放射性活度比明显高于125I-IgG和18F-FDG组。125I-anti-CD93 mAb有望用于肺癌靶向分子显像。5.125I-anti-CD93 mAb可在CD93阳性表达的肺癌荷瘤小鼠肿瘤部位特异性浓聚,浓聚程度和肿瘤CD93表达量明显正相关。研究结果为CD93阳性肿瘤的靶向监测提供了新的思路。创新点1.研究发现非小细胞肺癌中腺癌和大细胞癌细胞高表达CD93分子,鳞癌细胞低表达CD93分子。2.研究发现制备的放射性核素探针125I-anti-CD93 mAb可与肺癌细胞特异性结合,可在荷瘤小鼠CD93阳性肿瘤特异性浓聚。3.研究发现与同种型IgG及非特异性显像剂18F-FDG相比,制备的放射性核素探针125I-anti-CD93 mAb具有较好的肺癌靶向性,因此CD93作为肺癌的新型分子靶点具有潜在的临床应用前景。更多还原

【Abstract】 BackgroundAt present,most lung cancer patients have been diagnosed at a late stage and have distant metastasis due to the less early symptoms,which seriously affects the choice of clinical treatment in clinic.Radiotherapy and chemotherapy,commonly used in clinic,which makes the treatment efficiency and patient survival rate significantly lower.The clinical treatment of lung cancer patients with different pathological types and stages is different,so the efficacy and survival rate are significantly different.Early diagnosis is crucial for the treatment and prognosis of lung cancer patients.At present,the commonly used diagnostic methods for lung cancer mainly include imaging examination,serum tumor marker detection,sputum cytology examination,lung puncture and fiberoptic bronchoscopy.These diagnostic methods have limitations in specificity and practicability.Therefore,it is urgent to find high-specific lung cancer-targeted molecular probes for early diagnosis of lung cancer diseases,and provide a basis for efficacy monitoring and individualized targeted therapy.In recent years,molecular imaging developed rapidly and made great achievements,especially nuclear medicine molecular imaging,which with high sensitivity,high specificity and easy clinical transformation.Radionuclide tumor molecular imaging,including receptor imaging,metabolic imaging and gene imaging,plays an important role in the early diagnosis of malignant tumors and the differential diagnosis of benign and malignant tumors,and has good clinical application value.Tumor radioimmunoimaging technology,as a high specificity imaging tracer technology,radionuclide combined with antibody,is suitable for monitoring and quantifying the expression level and distribution of target molecules in tumor tissues,which is helpful for early accurate diagnosis,prognosis and treatment evaluation of tumors.It has been used in imaging of many common tumors in clinic.The lung cancer targeting molecule has become a key factor for radioimmunoimaging establishment and it is basis for tumor imaging and therapeutic and clinical efficacy evaluation.At present,exploring new radionuclide labeled molecular probes has become the core of tumor imaging research.Such molecular probes should meet the requirements of high specificity,high sensitivity and high selectivity,suitable for clinical applicationAngiogenesis providing oxygen and nutrients to all tissues and cells of the body,involving in a variety of physiological and pathological processes,plays a key role in the development of solid tumors.Tumor neovascularization has atypical morphological features,which make the tumor tissue over-infiltrated and poorly perfused,and the hypoxic microenvironment can mediate the spread,invasion and metastasis of tumor cells.Tumor tissue,lacking oxygen and energy supply of blood vessels,will not be able to break through the critical tumor size for distant organ metastasis.Tumor neovascular imaging and treatment have become the hotspot of basic research in clinical tumors.Tumor angiogenesis involves mutations in many genes and expression of different cytokines and receptors.Recent studies have found that CD93 is a transmembrane glycoprotein,expressed in endothelial cells,stem cells and bone marrow cells.The most important expression is on the surface of endothelial cells,and endothelial cells are essential for neovascularization of tumors.Early studies have shown that CD93 was an immune molecule involved in the adhesion and migration of immune cells.The factors affecting angiogenesis include activators and inhibitors.Recent literature reports that CD93 is a new type of angiogenesis activator.It mainly affects the growth of tumors by promoting endothelial cell adhesion and accelerating tumor angiogenesis.High expression of CD93 can promote the growth of tumors and reduce the survival of hosts.In addition,the extracellular domain of CD93 on the membrane surface is prone to cleavage or shedding under inflammatory stimuli,forming soluble CD93 and promoting angiogenesis.Therefore,it is postulated that CD93 may be a key molecule in tumor neovascularization and can be used as a molecular marker for the diagnosis and prognosis of lung cancer,and it is expected to be used for early targeted diagnosis of tumors.Studies have shown that the expression of CD93 in cancer tissues is closely related to clinical pathological types and prognosis of patients.Early identification of CD93 expression in lung cancer tissues is very important for patients to choose targeted therapy.However,there is no relevant research report on the early detection and evaluation of tumors using CD93 as a molecular target.The high expression of CD93 molecule on endothelial cells of tumor vessels and low expression in non-proliferating endothelial cells highlight the potential clinical value of CD93 molecule in cancer diagnosis.To explore whether radionuclide labeled CD93 molecular probe can be used for targeted imaging of tumors is of great significance for evaluation of CD93 as a non-invasive diagnosis of tumor molecular targets.In this paper,we selected lung cancer with high clinical incidence and difficult early diagnosis as the research object.We used radionuclide labeled anti-CD93 antibody to preliminarily study the targeted monitoring of lung cancer with new CD93 molecular probe in two parts.Firstly,the expression of CD93 in non-small cell lung cancer cell line was studied in vitro,and a novel radionuclide molecular probe 125I-anti-CD93 mAb was prepared and identified,and we studied its binding specificity to lung cancer cells.Secondly,lung cancer-bearing mouse model was established to study the specificity of 125I-anti-CD93 mAb in targeting imaging of lung cancer tumors in vivo,and compared with the isotype imaging agent 125I-IgG and the non-specific imaging agent 18F-FDG.This paper aims to explore new target molecule for early diagnosis of lung cancer and provide a new way for early non-invasive diagnosis of lung cancer.Part One Preparation and identification of CD93 radionuclide molecular probeMethods1.Preparation of 125I-anti-CD93 mAb and 125I-IgGThe nuclide probes 125I-anti-CD93 mAb and 125I-IgG were labeled with Iodogen method,and PD-10 gel column was used for elution and purification.Radioactivity was measured to calculate the labeled rate and specific activity of the two tracers.Radiochemical purity was determined by paper chromatography.125I-anti-CD93 mAb and 125I-IgG protein peaks were mixed with saline and serum,respectively.The stability was measured by paper chromatography at room temperature at 24h,48h and 72h.2.Detection of the expression of CD93 in cells by RT-PCR and Western BlotThree kinds of lung cancer cell lines were routinely cultured:adenocarcinoma A549 cells,large cell carcinoma H460 cells,and squamous carcinoma H520 cells.RT-PCR was used to detect the expression of CD93 mRNA in three cell lines,Western Blot was used to detect the expression of CD93 protein in three cell lines,and the difference of CD93 expression among three lung cancer cell lines was analyzed.3.Binding assay of probes in vitro125I-anti-CD93 mAb group:three kinds of lung cancer cells(2×105/well)were inoculated into 24-well plate.Labeled antibody was added according to the concentration gradient(3-100nM).After a certain period of time,washed and the radioactivity counts of the cells were measured,and the differences of affinity(KD value)of each lung cancer cell line to 125I-anti-CD93 mAb were compared and analyzed.125I-IgG group:A549 lung cancer cells(2×105/well)with good growth condition were inoculated into 24-well plates.125I-IgG were added according to the concentration gradient(3-100nM).After a certain period of time,washed and the radioactivity counts of the cells were measured to analyze the affinity(KD value)of A549 lung cancer cell line to125I-IgG,and compared with 125I-anti-CD93 mAb A549 group.4.Binding blocking assay of probe in vitroA549 lung cancer cells(2×105/well)with good growth condition were inoculated into 24-well plates.Quantitative 125I-anti-CD93 mAb and anti-CD93 mAb were added(0,1.42,14.2,28.4nmol/L),after a certain time,washed and measured the radioactivity,and analyzed the blocking of the binding of between 125I-anti-CD93 mAb and A549 by non-labeled anti-CD93 mAb.Results1.The radionuclide probe 125I-anti-CD93 mAb was successfully prepared.The labeled rate was 91.37%,the specific radioactivity was 1096.44MBq/mg,and the radiochemical purity was 96.49%.The labeled rate of 125I-IgG was 90,24%,the specific radioactivity was 1082.88MBq/mg,and the radiochemical purity was 94.82%.Paper chromatography showed that both 125I-anti-CD93 mAb and 125I-IgG had good stability in saline solution and serum,and keep above 90%until 72h.2.Both RT-PCR and Western Blot showed that CD93 was high expressed in A549 cells and H460 cells,and low expressed in H520 cells.There was a significant difference between them.3.The cell-binding assay showed that the KD of 125I-anti-CD93 mAb in A549 cells,H460 cells and H520 cells was 27.09nmol/L,28.84nmol/L and 39.90nmol/L,respectively.The affinity of A549 cells and H460 cells was significantly higher than that of H520 cells.The Bmax values of A549 cells and H460 cells were 1679cpm/104 cells and 1780cpm/104 cells,which were significantly higher than that of H520 cells(756cpm/104 cells).Comparative analysis with the 125I-IgG group indicated that the 125I-anti-CD93 mAb specifically binds to A549 lung cancer cells.4.The cell binding blocking experiments showed that the non-labeled anti-CD93 mAb specifically blocked the binding of A549 cells to 125I-anti-CD93 mAb,and the amount of addition was proportional to the blocking rate.Part Two Study of CD93 radionuclide probe on tumor targeting in lung cancer-bearing miceMethods1.Establishment of lung cancer-bearing mice modelThree kinds of lung cancer cell lines:A549,H460 and H520 were cultured routinely.Female BALB/c nude mice,aged 4-6 weeks,were injected subcutaneously with 5×107ml cell suspension on the shoulder of the right forelimb,0.2ml/each,to establish lung cancer-bearing mice models.The whole process was aseptically operated and the tumor volume change was recorded.The experiment was performed when the maximum diameter of the tumor reached 0.8-1.0cm.2.Biodistribution of 125I-anti-CD93 mAb in three kinds of lung cancer-bearing miceThree kinds of lung cancer-bearing mice,thyroid blocked with 3%NaI solution 48h before,were injected with 125I-anti-CD93 mAb,0.37MBq/each by tail vein.And five mice were randomly selected from each group at 24h,48h and 72h after injection.Sacrificed,collected main organs,tissues,tumors and opposite muscle tissues to weigh and measure radioactivity(cpm).To calculate the percentage of radioactivity per gram of tissue or organ to standard source(%ID/g)and target to non-target radioactivity ratio(T/NT ratio).3.Dynamic phosphor-autoradiography of 125I-anti-CD93 mAb in three kinds of lung cancer modelThree kinds of lung cancer model mice were injected with 125I-anti-CD93 mAb,0.37MBq/mice,through tail vein separately after thyroid blocked with 3%NaI solution 48h earlier.Dynamic in vivo whole-body phosphor autoradiography was aquired at 24h,48h and 72h after injection to compare the accumulation of 125I-anti-CD93 mAb in the tumors of three kinds of lung cancer models.4.Biodistribution of 125I-anti-CD93 mAb and 125I-IgG in A549 lung cancer-bearing miceA549 lung cancer-bearing mice were randomly divided into two groups and were injected with 125I-anti-CD93 mAb or 125I-IgG through tail vein after thyroid blocked separately.Animals were sacrificed at 24h,48h and 72h after injection to collect blood,tumor,opposite normal muscle and other main organs,to study the biodistribution and calculate T/NT ratio in two groups of mice.5.Dynamic whole-body phosphorescent screen imaging with three imaging tracers in A549 tumor-bearing miceA549 lung cancer-bearing mice was randomly divided into three groups with thyroid blocked 48 hours earlier,model mice were injected with 125I-anti-CD93 mAb/125I-IgG,separately,0.37MBq/each to dynamic image at 24h,48h and 72h after injection.Another group of model mice was injected with ’8F-FDG,37MBq/each through the tail vein,to study dynamic imaging at 30min,3h and 6h after injection.The radioactivity of 125I-anti-CD93 mAb,125I-IgG,and 18F-FDG at the tumor site was compared and analyzed using OptiQuantTM image analysis software.6.H&E staining and CD93 immunohistochemical staining of lung tumor tissueTumor tissues from the three groups of animals which have been used for dynamic whole-body phosphor-autoradiography were harvested,fixed into 4%paraformaldehyde and prepared paraffin sections.Hematoxylin-eosin staining was used routinely.Anti-CD93 antibody was used for immunohistochemical staining.The positive expression was observed and photographed under the microscope.Results1.The biodistribution of three kinds of lung cancer-bearing mice showed that the 125I-anti-CD93 mAb tracer were mainly metabolized by liver and kidney.The radioactivity count of tumors was the highest at 48h,and the radioactivity counts and the T/NT ratio of A549 and H460 groups at 48h were higher than the H520 group,and the difference between the two groups was statistically significant.2.The whole body dynamic phosphor screen imaging in lung cancer-bearing mice showed that the mice were weakly visible at 24h after injection of 125I-anti-CD93 mAb in the three groups,and the tumor imaging was the clearest at 48h.The radioactivity accumulation on tumor tissues in A549 group and H460 group,were significantly higher than that in H520 group.The semi-quantitative analysis showed that the ratio of radioactivity in A549 group and H460 group was higher than that in H520 group at 48h,the difference was statistically significant.3.The biodistribution in A549 lung cancer bearing mice showed that both tracers were metabolized by liver and kidney.Tumor radioactivity accumulation was obviously in 125I-anti-CD93 mAb group compared with 125I-IgG group.The radioactivity and T/NT ratio of 125I-anti-CD93 mAb group were significantly higher than those in 125I-IgG group.4.After injecting the probe into A549 lung cancer bearing mice,the tumour imaging was the most clear at 48 hours,and the radioactivity of 125I-anti-CD93 mAb group was significantly higher than that of 125I-IgG group.This indicated that the radioactivity of 125I-anti-CD93 mAb in the tumour location was the specific binding between 125I-anti-CD93 probe to CD93,rather than the non-specific binding of the homologous IgG Fc fragment.The 18F-FDG control group showed the best imaging at 3 hours after injection.The radioactivity ratio of tumors was 1.60±0.10,which was significantly lower than that of the CD93 probe injection group(3.34±0.18).This indicated that 125I-anti-CD93 mAb was clearer imaging than the non-specific imaging agent 18F-FDG in imaging A549 tumors.5.Pathological analysis of tumor tissues showed that malignant proliferation of tumor with neovascularization,and the positive rate of CD93 immunohistochemical staining were high among three kinds of tumors.The CD93 positive rate of CD93 in A549 group and H460 group were higher than that in H520 group,which was consist with the expression of CD93 analyzed in vitro with lung cancer cells.Conclusion1.The expression of CD93 was significantly different in three kinds of lung cancer cells.In non-small cell lung cancer,the expression of CD93 was higher in lung adenocarcinoma and large cell carcinoma cells,but lower in squamous cell carcinoma cells.2.The radionuclide tracer 125I-anti-CD93 mAb has good biological activity;125I-anti-CD93 mAb can specifically bind to lung cancer cells,and the affinity of lung cancer cells to 125I-anti-CD93 mAb is positively correlated with the expression of CD93.3.The bioldistribution of 125I-anti-CD93 mAb in A549 lung cancer model mice was consist with the results of whole body phosphorus screen dynamic imaging,which indicated that 125I-anti-CD93 mAb could be specifically accumulated on the tumor location in CD93 positive lung cancer bearing mice,and accumulation degree was positively correlated with the expression of CD93.The results provided a new possible molecular target for early targeted monitoring of CD93 positive tumors.innovation1.It was found that CD93 molecule was high expressed in lung adenocarcinoma and large cell carcinoma cells of non-small cell lung cancer,while CD93 molecule was low expressed in squamous cell carcinoma cells.2.The radionuclide labeled probe 125I-anti-CD93 mAb has good targeting ability in lung cancer both in vivo and in vitro.3.The radionuclide labeled probe 125I-anti-CD93 mAb has apparently lung cancer targeting ability compared with the isotype IgG and non-specific imaging agents.CD93 may be used as a new molecular target for lung cancer and has potential clinical application value.更多还原