靶向肿瘤细胞CD13的荧光-NGR-钆磁共振对比剂的体外实验研究

发布时间：2018-05-20 03:43

本文选题：NGR-钆 + 肿瘤细胞　；参考：《遵义医学院》2014年硕士论文

【摘要】：目的：CD13是肿瘤细胞膜及肿瘤新生血管内皮细胞膜上的受体，对肿瘤细胞的生长、繁殖、侵袭及转移起着重要作用。本研究以肿瘤细胞膜上CD13受体为靶点，合成能与其特异性结合的配体NGR短肽，并以钆及荧光作为MR的成像部分，构建荧光-NGR-钆的新型多模态MR靶向对比剂，并探讨该MR对比剂在体外的生物活性及其体外靶向性能，为活体内靶向成像奠定实验基础。方法：通过免疫组化实验检验肿瘤细胞膜上CD13表达情况。分为实验组（HePG2人肝癌细胞和HT1080人肉瘤细胞）和对照组（HT29人结肠癌细胞）.用化学合成方法偶联对比剂。首先合成荧光-NGR，再合成荧光-NGR-钆对比剂，并进行相关理化数据测试最后细胞荧光实验验证荧光-NGR体外靶向性能：a.实验组：实验组（HePG2，HT1080细胞）和对照组(HT29细胞)爬片上加等量荧光-NGR，观察细胞爬片荧光强度。b.通过拮抗实验进一步从反面验证NGR对CD13的体外靶向性。分为两批实验：第一批HT1080细胞拮抗组，提前在HT1080肿瘤细胞爬片上滴加非荧光NGR，封闭CD13靶点之后再滴加与同一次实验组等剂量荧光-NGR，观察每组细胞爬片荧光强度；第二批HePG2拮抗组，提前在HePG2肿瘤细胞爬片上滴加非荧光NGR，之后再滴加荧光-NGR，剂量和实验组（同一次实验）是一致的，观察每组细胞爬片荧光强度，及细胞荧光实验验证荧光-NGR-钆对比剂体外靶向性能。a.对比观察荧光-NGR-钆对CD13高表达和CD13低表达细胞的结合能力：实验组用HT1080细胞，对照组用HT29细胞，同时在HT1080和HT29细胞爬片上分别滴加荧光-NGR-钆，观察两种细胞爬片上的荧光强度。b.通过拮抗组实验反证荧光-NGR-钆在体外的生物活性。实验分为HT1080细胞实验组和拮抗组，实验组细胞爬片上分别滴加递减剂量荧光-NGR-钆，拮抗组HT1080细胞爬片或腔式盖玻片上事先滴加递增剂量非荧光NGR，然后再加入与同次实验组等量的荧光-NGR-钆剂量，比较实验组和拮抗组荧光的强度。结果：实验组HePG2和HT1080细胞免疫组化实验呈CD13高表达，对照组HT29细胞呈CD13低表达；.荧光-NGR与荧光-NGR-钆体外偶联成功；实验组HePG2和HT1080细胞荧光-NGR实验均可看到大量荧光标记物，对照组仅看到微弱荧光标记物在HT29细胞上。实验组荧光强度高于对照组及拮抗组，半定量对比分析，差异有显著统计学意义（P0.005）；荧光-NGR-钆实验实验组（HT1080细胞）荧光强度高于对照组（HT29细胞）及拮抗组（HT1080细胞），半定量对比分析，差异有显著统计学意义（P0.005）。结论：荧光-NGR-钆偶联成功，，在体外对HePG2和HT1080肿瘤细胞膜上的CD13具有特异性靶向性。
[Abstract]:Objective CD13 is a receptor on tumor cell membrane and tumor neovascularization endothelial cell membrane, which plays an important role in the growth, proliferation, invasion and metastasis of tumor cells. In this study, the ligand NGR short peptide, which can specifically bind to CD13 receptor on tumor cell membrane, was synthesized, and gadolinium and fluorescence were used as Mr imaging part to construct a novel multimode Mr targeting contrast agent with fluorescence-NGR- gadolinium. The bioactivity of Mr contrast agent in vitro and its in vitro targeting performance were discussed, which laid an experimental foundation for in vivo target imaging. Methods: the expression of CD13 on tumor cell membrane was detected by immunohistochemistry. Two groups were divided into two groups: the experimental group (HePG2 human hepatoma cell and HT1080 human tumor cell) and the control group (HT29 human colon cancer cell line). Coupling contrast agent by chemical synthesis method. Fluorescence-NGR was synthesized at first, then fluorescence-NGR-gadolinium contrast agent was synthesized. In the experimental group, the same amount of fluorescence (NGR) was added to the climbing slices of the experimental group (HePG2 + HT1080 cells) and the control group (the HT29 cells), and the fluorescence intensity of the cell climbing slices was observed. The in vitro targeting of NGR to CD13 was further verified by antagonistic experiments. In the first batch of HT1080 cell antagonistic group, non-fluorescent NGR was added on the HT1080 tumor cell climbing slice in advance. After blocking the target of CD13, the same dose of fluorescence was added to the same experimental group, and the fluorescence intensity of each group was observed. In the second group of HePG2 antagonists, non-fluorescent NGRs were added to the HePG2 tumor cells in advance, and then fluorescent NGR was added later. The dose was the same as that in the experimental group (the same experiment). The fluorescence intensity of the climbing slices of each group was observed. The in vitro targeting performance of fluorescent-NGR-gadolinium contrast agent was verified by cell fluorescence assay. The binding ability of fluorescent-NGR- gadolinium to high expression of CD13 and low expression of CD13 was observed: HT1080 cells were used in experimental group, HT29 cells were used in control group, and fluorescence-NGR- gadolinium was added to HT1080 and HT29 cell climbing slices respectively. The bioactivity of fluorescence-NGR-gadolinium in vitro was confirmed by antagonistic experiment. The experiment was divided into two groups: HT1080 cell experimental group and antagonistic group. In the antagonistic group, the fluorescence intensity of the experimental group and the antagonistic group were compared by adding the increasing dose of non-fluorescent NGR to the climbing slice or cavity glass slide, and then adding the same dose of fluorescence-NGR- gadolinium as the same experimental group. Results: the expression of CD13 was high in HePG2 and HT1080 cells in the experimental group, and the low expression of CD13 in the HT29 cells in the control group. HePG2 and HT1080 cells in the experimental group could see a large number of fluorescent labeled substances, while in the control group, only the weak fluorescent labeled substance could be seen on the HT29 cells. The fluorescence intensity of experimental group was higher than that of control group and antagonistic group. The fluorescence intensity of HT1080 cells in the fluorescent NGR- gadolinium group was higher than that in the control group (HT29 cells) and in the antagonist group. The semi-quantitative comparison analysis showed that the difference was statistically significant (P 0.005). Conclusion: fluorescence-NGR-gadolinium coupling is successful and has a specific targeting of CD13 on the cell membrane of HePG2 and HT1080 tumors in vitro.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R445.2;R73-3

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