胃癌诊断系列分子影像探针的研发与成像研究

发布时间：2018-07-01 14:38

  本文选题：胃癌诊断 + 分子影像　； 参考：《第四军医大学》2017年博士论文

【摘要】：【背景】胃癌是严重威胁人类生命健康的恶性肿瘤之一,其高病死率与发现时病变已处于晚期密切相关,早期诊断是降低胃癌病死率的关键环节和有效手段,但现有临床诊断手段在诊断灵敏度与特异性上均不能满足早诊的需求。MG7Ab是胃癌特异性单克隆抗体,靶向特异性强、灵敏度高,具有作为胃癌早期诊断分子标志物的应用前景。分子影像作为新兴的成像手段,具有可在活体连续动态观察特定分子改变的特点,逐渐成为基础研究中不可或缺的手段,也极具临床应用前景,其中多模态成像可以使不同的成像模态互补,得到更为精准和信息量更大的影像,为诊断提供有力证据。【目的】以胃癌特异性单克隆抗体MG7Ab为靶向分子,制备系列可用于胃癌诊断成像的分子影像探针:MG7Ab-Cy5.5、64Cu-NODAGA-MG7Ab以及MG7Ab-ICG等,开展体外和在体成像研究,探索并优化成像条件。【方法】1.胃癌特异性单克隆抗体MG7Ab的制备与功能验证:通过杂交瘤-腹水抗体制备系统制备抗体,辛酸-饱和硫酸铵沉淀法及后续免疫学手段纯化MG7Ab,通过Western Blot与免疫荧光染色实验,验证此次所制备抗体的胃癌特异性与亲和力。2.MG7Ab核素显像探针的制备与表征:通过化学耦联法,将螯合剂NODAGA-NHS ester和p-NCS-benzyl-NODAGA与MG7Ab耦联,使用64Cu Cl2进行放射性标记,利用PD-10凝胶柱收集纯化放射性探针,并计算放射性标记效率;通过PBS与FBS孵育,检验探针的溶液稳定性。3.MG7Ab-ICG探针的制备与表征:通过不同化学修饰将ICG与MG7Ab耦联,利用紫外-可见光吸收光谱对探针表征;通过PBS孵育,检测探针的溶液稳定性;利用浓度梯度成像,检测探针的光声成像性质。4.体外细胞学成像研究:在细胞水平,通过细胞摄取实验分别检测各探针的胃癌细胞结合,利用激光共聚焦显微镜明确探针的结合模式,通过竞争性抑制实验验证探针结合的特异性。5.在体成像研究建立荷胃癌细胞的裸鼠模型,分别使用IVIS活体成像、小动物PET/CT成像、光声成像以及近红外成像内镜等系统,通过不同时间点的成像探索最佳成像时间,通过竞争性抑制明确肿瘤成像的特异性,通过生物学分布研究探针在体内的代谢情况。【结果】1.胃癌特异性单克隆抗体MG7Ab的制备与功能:成功通过杂交瘤-腹水抗体制备体系,获得一批含高浓度MG7Ab的腹水,经进一步纯化,共获得干粉态MG7Ab 8.67 mg。选用5种胃癌细胞株和永生化胃粘膜上皮细胞进行Western Blot实验,结果显示以本次制备的MG7Ab为一抗,在胃癌细胞SGC-7901、KATOIII和MKN-28三株细胞株中出现了明显的130 k Da显色条带,胃癌细胞AGS中有弱阳性条带出现,而胃癌细胞MKN-45和永生化胃粘膜上皮细胞GES中,在相应位置无明显条带出现;免疫荧光实验显示,MG7Ab与胃癌SGC-7901和MKN-28细胞株结合均主要位于细胞膜部位,另在细胞浆中可见微弱染色,而在阴性对照GES细胞株中,未见胞膜及胞质有明确染色。2.MG7Ab-Cy5.5探针制备与体外、在体成像:通过耦联反应,成功将Cy5.5-NHS ester与MG7Ab耦联,平均每个抗体上联有2.5个荧光分子。细胞摄取实验显示,MG7Ab-Cy5.5探针可以特异性结合胃癌细胞SGC-7901和MKN-28,而不与永生化胃粘膜上皮细胞GES结合;激光共聚焦显微镜成像表明探针与胃癌细胞结合主要定位在胞膜,并有少量胞质分布;荷瘤裸鼠活体近红外成像显示探针可特异性聚集在肿瘤部位;生物学分布提示探针能特异性聚集于肿瘤部位,并主要经肝脏代谢。3.64Cu-NODAGA-MG7Ab探针制备与体外、在体成像:在不同反应条件下成功将螯合剂NODAGA-NHS ester和p-NCS-benzyl-NODAGA与MG7Ab耦联,制备了两种核素探针前体;在温和反应条件下实现了64Cu核素标记,经PD-10分离纯化,测得两种探针的标记效率分别为85.7%和80.9%;PBS与FBS孵育后,两种探针均有较好的溶液稳定性,在孵育240 min后,两种探针的完整度分别为96.37%、94.60%(PBS中)和95.39%、94.75%(40%FBS中);细胞摄取实验表明两种探针均能特异性结合胃癌细胞SGC-7901,孵育120 min,探针1的摄取率为13.93±0.45%ID,探针2的摄取率为10.62±0.07%ID,而加入MG7Ab竞争性抑制后,细胞摄取率明显下降,在孵育60 min时,探针1的摄取率为12.29±0.27%ID,而加入MG7Ab后,摄取率下降至3.85±0.21%ID;探针2在孵育60 min时摄取率为10.39±0.04%ID,而加入MG7Ab竞争后,摄取率下降至3.26±0.04%ID;细胞亲和力测定得出探针的解离常数KD=1.15±0.17μM;在体成像显示探针1较探针2有更好的代谢行为和更佳的成像对比度,注射后24 h,两种探针在裸鼠肿瘤摄取量分别为2.27±0.54%ID/g和0.25±0.13%ID/g;生物学分布研究显示,MG7Ab可竞争性抑制探针在肿瘤部位的摄取,在注射后24 h,探针组肿瘤摄取为3.44±0.29%ID/g,而竞争抑制组仅为1.01±0.08%ID/g,进一步验证了探针结合的特异性。4.MG7Ab-ICG探针制备与体外、在体成像:通过共价耦联和非共价耦联两种方式制备了MG7Ab-ICG探针,紫外-可见光谱吸收显示,共价耦联探针中,ICG/MG7Ab为5.4,而非共价耦联探针为12.3;光声成像显示探针光声信号与探针浓度具有良好的线性关系,相关性系数R2=0.9984;体外稳定性检测显示,共价耦联探针的稳定性优于非共价耦联探针,其在孵育12 h、24 h和48 h的完整度分别为79.6%、75.9%以及72.2%,而非共价耦联探针为31.7%、26.0%以及25.2%,但最终ICG/MG7Ab在两种探针均在3-4区间;细胞结合实验显示,孵育2 h,探针组细胞信号强度为1.37*106±1.93*105 p/s/cm2/sr,竞争组为1.04*106±1.05*105 p/s/cm2/sr,阴性对照组为1.07*106±1.28*105 p/s/cm2/sr,提示探针可特异性与胃癌细胞结合;在体光声成像显示MG7Ab-ICG探针可显著增强肿瘤区域信号强度,注射2 h,探针组信号为667.3%±102.2%,竞争抑制组为339.8±41.3%,信号强度差别约2倍;近红外内镜可在探针注射15 min区别肿瘤部位的特异性成像信号与非特异成像信号。【结论】成功制备、纯化了一批具有胃癌特异性的单克隆抗体MG7Ab,以其为靶向核心制备了系列可用于胃癌诊断成像的分子影像探针:MG7Ab-Cy5.5、64Cu-NODAGA-MG7Ab以及MG7Ab-ICG等,通过开展体外和在体成像实验,验证了探针成像的肿瘤特异性,探索并优化了成像条件,为提高胃癌诊断的准确率和特异性提供新的分子影像学支持。
[Abstract]:[background] gastric cancer is one of the malignant tumors that seriously threaten human life and health. The high mortality rate is closely related to the disease in the late stage. Early diagnosis is the key link and effective means to reduce the mortality of gastric cancer. However, the diagnostic sensitivity and specificity of the existing diagnostic methods can not meet the needs of early diagnosis.MG7Ab. It is a specific monoclonal antibody for gastric cancer with high specificity and high sensitivity. It has the potential to be used as a molecular marker for early diagnosis of gastric cancer. As a new imaging means, molecular imaging has the characteristics of continuous dynamic observation of specific molecules in living bodies, and has gradually become an indispensable means in basic research, and it is also highly clinical. With the prospect, multi-modal imaging can make different imaging modalities complementary, get more accurate and more information, and provide strong evidence for diagnosis. [Objective] to prepare a series of molecular imaging probes for gastric cancer diagnosis imaging with the specific monoclonal antibody MG7Ab of gastric cancer as the target molecule, and to prepare a series of molecular imaging probes for diagnosis of gastric cancer: MG7Ab-Cy5.5,64Cu-NODAGA-MG7Ab And MG7Ab-ICG and so on, carry out in vitro and in vivo imaging research, explore and optimize imaging conditions. [Methods] preparation and functional verification of 1. gastric cancer specific monoclonal antibody MG7Ab: preparation of antibodies by hybridoma ascites antibody preparation, octane saturated ammonium sulfate precipitation and sequel immunological methods to purify MG7Ab by Western Blot and immunity The immunofluorescence staining experiments were conducted to verify the preparation and characterization of the.2.MG7Ab nuclear imaging probe for the specificity and affinity of the prepared antibodies. By coupling the chelating agent NODAGA-NHS ester and p-NCS-benzyl-NODAGA and MG7Ab by chemical coupling, the radioactivity markers were labeled with 64Cu Cl2, and the radioactive probe was collected and purified by the PD-10 gel column, and the radioprobe was collected and purified by the PD-10 gel column. The efficiency of radioactivity labeling was calculated; the preparation and characterization of the solution stability.3.MG7Ab-ICG probe of the probe by PBS and FBS were prepared and characterized: the probe was characterized by the coupling of ICG with MG7Ab by different chemical modifications and the UV visible light absorption spectrum was used to characterize the probe; the solution stability of the probe was detected by PBS incubation; the probe was detected by the concentration gradient imaging. .4. in vitro cytological imaging study of photoacoustic imaging: at the cell level, cell uptake was detected by cell uptake experiments, and the binding mode of gastric cancer cells was detected by the laser confocal microscopy, and the specific.5. of the probe was verified by competitive inhibition experiments to establish nude mice bearing gastric cancer cells in vivo. Model, using IVIS living body imaging, small animal PET/CT imaging, photoacoustic imaging and near infrared imaging endoscopy, the optimal imaging time was explored by imaging at different time points, the specificity of tumor imaging was determined by competitive inhibition, and the metabolic status of the probe in the body was studied by biological distribution. [results] 1. gastric cancer was specific. The preparation and function of sex monoclonal antibody MG7Ab: successfully using hybridoma ascites antibody preparation system to obtain a batch of ascites containing high concentration of MG7Ab. After further purification, a total of 5 kinds of gastric cancer cell lines and immortalized gastric mucosa epithelial cells were selected for Western Blot experiment of dry powder MG7Ab 8.67 mg.. The results showed that the MG7Ab was prepared by this method. For one resistance, there were obvious 130 K Da bands in the three cells of gastric cancer cells SGC-7901, KATOIII and MKN-28, and there were weak positive bands in gastric cancer cell AGS, while MKN-45 in gastric cancer cells and GES of immortalized gastric mucosa epithelial cells had no obvious bands in the corresponding position, and the immunofluorescence experiment showed MG7Ab and SGC-7901 and M of gastric cancer. The combination of KN-28 cell lines is mainly located in the membrane part of the cell, and the weak staining is visible in the cytoplasm. In the negative control GES cell line, there is no clear staining.2.MG7Ab-Cy5.5 probe in cell membrane and cytoplasm, and in vivo imaging: by coupling reaction, Cy5.5-NHS ester is coupled to MG7Ab, and the average of each antibody is 2.5. The cell uptake experiments showed that the MG7Ab-Cy5.5 probe could specifically bind the gastric cancer cells SGC-7901 and MKN-28, but not with the immortalized gastric epithelial cells GES. The laser confocal microscope imaging showed that the probe and gastric cancer cells were located mainly in the cell membrane, with a small amount of cytoplasm distribution, and the near infrared imaging of the tumor bearing nude mice was near infrared imaging. It shows that the probe can be specifically clustered at the tumor site. Biological distribution suggests that the probe can be specifically clustered at the tumor site and is mainly prepared and in vitro by the liver metabolism.3.64Cu-NODAGA-MG7Ab probe. In vivo imaging, two kinds of nuclei are prepared by coupling the chelating agent NODAGA-NHS ester and p-NCS-benzyl-NODAGA with MG7Ab under different reaction conditions. 64Cu nuclide labelling was realized under mild reaction conditions. The labeling efficiency of the two probes was 85.7% and 80.9% respectively by PD-10 separation and purification. After incubating with PBS and FBS, the two probes had better solution stability. After incubating 240 min, the integrity of two probes was 96.37%, 94.60% (PBS) and 95.39%, 94.75% (40%). In FBS), the cell uptake experiments showed that the two probes were able to specifically bind the gastric cancer cell SGC-7901 and incubate 120 min, the uptake rate of probe 1 was 13.93 + 0.45%ID, and the uptake rate of probe 2 was 10.62 + 0.07%ID, and the uptake rate of cells decreased significantly after the competitive inhibition of MG7Ab, and the uptake rate of probe 1 was 12.29 + 0.27%ID when incubating 60 min. After entering MG7Ab, the uptake rate decreased to 3.85 + 0.21%ID, and the uptake rate of probe 2 at 60 min was 10.39 + 0.04%ID, and the uptake rate decreased to 3.26 + 0.04%ID after MG7Ab competition, and the dissociation constant of the probe was determined by KD=1.15 + 0.17 mu M, and in vivo imaging probe 1 was better than the probe 2 for better metabolic behavior and better imaging pair. The uptake of the two probes in nude mice was 2.27 + 0.54%ID/g and 0.25 + 0.13%ID/g, respectively. The biological distribution study showed that the uptake of the MG7Ab competitive inhibition probe at the tumor site was 24 h after the injection, and the tumor uptake was 3.44 + 0.29%ID/g in the probe group, while the competition inhibition group was only 1.01 + 0.08%ID/g, which was further verified. The probe binding specific.4.MG7Ab-ICG probe was prepared and in vitro, in vivo imaging: MG7Ab-ICG probes were prepared by covalent coupling and non covalent coupling two methods. UV visible absorption showed that ICG/MG7Ab was 5.4 in covalent coupling probe and 12.3 for non covalent coupling probe; photoacoustic imaging showed the probe photoacoustic signal and probe concentration. With good linear relationship and correlation coefficient R2=0.9984, the stability test in vitro showed that the stability of covalent coupling probe was better than non covalent coupling probe, and the integrity of 12 h, 24 h and 48 h were 79.6%, 75.9% and 72.2% respectively, while the non covalent coupling probes were 31.7%, 26% and 25.2%, but finally ICG/MG7Ab was in two probes. In the 3-4 interval, the cell binding experiment showed that the cell signal intensity of the incubated 2 h was 1.37*106 + 1.93*105 p/s/cm2/sr, the competition group was 1.04*106 + 1.05*105 p/s/cm2/sr, and the negative control group was 1.07*106 + 1.28*105 p/s/cm2/sr, suggesting that the probe was specific to the gastric cancer cells, and the bulk photoacoustic imaging showed that the MG7Ab-ICG probe could be significantly enhanced. The signal intensity of the tumor was 2 h, the probe group signal was 667.3% + 102.2%, the competition inhibition group was 339.8 + 41.3% and the signal intensity difference was about 2 times. The specific imaging signal and the nonspecific imaging signal could be distinguished by the near infrared endoscopy with the probe injection of 15 min. A series of molecular imaging probes, such as MG7Ab-Cy5.5,64Cu-NODAGA-MG7Ab and MG7Ab-ICG, were prepared by cloning antibody MG7Ab, which were used as the target core for the diagnosis of gastric cancer. In vitro and in vivo imaging experiments, the specificity of the probe imaging was verified. The imaging conditions were explored and optimized to improve the accuracy of diagnosis of gastric cancer and the accuracy of the diagnosis of gastric cancer. Specificity provides new molecular imaging support.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2
，

本文编号：2088105