本文選題：NGR-釓 + 腫瘤細(xì)胞��； 參考：《遵義醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：CD13是腫瘤細(xì)胞膜及腫瘤新生血管內(nèi)皮細(xì)胞膜上的受體，對(duì)腫瘤細(xì)胞的生長(zhǎng)、繁殖、侵襲及轉(zhuǎn)移起著重要作用。本研究以腫瘤細(xì)胞膜上CD13受體為靶點(diǎn)，合成能與其特異性結(jié)合的配體NGR短肽，并以釓及熒光作為MR的成像部分，構(gòu)建熒光-NGR-釓的新型多模態(tài)MR靶向?qū)Ρ葎�，并探討該MR對(duì)比劑在體外的生物活性及其體外靶向性能，為活體內(nèi)靶向成像奠定實(shí)驗(yàn)基礎(chǔ)。 方法：通過免疫組化實(shí)驗(yàn)檢驗(yàn)?zāi)[瘤細(xì)胞膜上CD13表達(dá)情況。分為實(shí)驗(yàn)組（HePG2人肝癌細(xì)胞和HT1080人肉瘤細(xì)胞）和對(duì)照組（HT29人結(jié)腸癌細(xì)胞）.用化學(xué)合成方法偶聯(lián)對(duì)比劑。首先合成熒光-NGR，再合成熒光-NGR-釓對(duì)比劑，并進(jìn)行相關(guān)理化數(shù)據(jù)測(cè)試最后細(xì)胞熒光實(shí)驗(yàn)驗(yàn)證熒光-NGR體外靶向性能：a.實(shí)驗(yàn)組：實(shí)驗(yàn)組（HePG2，HT1080細(xì)胞）和對(duì)照組(HT29細(xì)胞)爬片上加等量熒光-NGR，觀察細(xì)胞爬片熒光強(qiáng)度。b.通過拮抗實(shí)驗(yàn)進(jìn)一步從反面驗(yàn)證NGR對(duì)CD13的體外靶向性。分為兩批實(shí)驗(yàn)：第一批HT1080細(xì)胞拮抗組，提前在HT1080腫瘤細(xì)胞爬片上滴加非熒光NGR，封閉CD13靶點(diǎn)之后再滴加與同一次實(shí)驗(yàn)組等劑量熒光-NGR，觀察每組細(xì)胞爬片熒光強(qiáng)度；第二批HePG2拮抗組，提前在HePG2腫瘤細(xì)胞爬片上滴加非熒光NGR，之后再滴加熒光-NGR，劑量和實(shí)驗(yàn)組（同一次實(shí)驗(yàn)）是一致的，觀察每組細(xì)胞爬片熒光強(qiáng)度，及細(xì)胞熒光實(shí)驗(yàn)驗(yàn)證熒光-NGR-釓對(duì)比劑體外靶向性能。a.對(duì)比觀察熒光-NGR-釓對(duì)CD13高表達(dá)和CD13低表達(dá)細(xì)胞的結(jié)合能力：實(shí)驗(yàn)組用HT1080細(xì)胞，對(duì)照組用HT29細(xì)胞，同時(shí)在HT1080和HT29細(xì)胞爬片上分別滴加熒光-NGR-釓，觀察兩種細(xì)胞爬片上的熒光強(qiáng)度。b.通過拮抗組實(shí)驗(yàn)反證熒光-NGR-釓在體外的生物活性。實(shí)驗(yàn)分為HT1080細(xì)胞實(shí)驗(yàn)組和拮抗組，實(shí)驗(yàn)組細(xì)胞爬片上分別滴加遞減劑量熒光-NGR-釓，拮抗組HT1080細(xì)胞爬片或腔式蓋玻片上事先滴加遞增劑量非熒光NGR，然后再加入與同次實(shí)驗(yàn)組等量的熒光-NGR-釓劑量，比較實(shí)驗(yàn)組和拮抗組熒光的強(qiáng)度。 結(jié)果：實(shí)驗(yàn)組HePG2和HT1080細(xì)胞免疫組化實(shí)驗(yàn)呈CD13高表達(dá)，對(duì)照組HT29細(xì)胞呈CD13低表達(dá)；.熒光-NGR與熒光-NGR-釓體外偶聯(lián)成功；實(shí)驗(yàn)組HePG2和HT1080細(xì)胞熒光-NGR實(shí)驗(yàn)均可看到大量熒光標(biāo)記物，對(duì)照組僅看到微弱熒光標(biāo)記物在HT29細(xì)胞上。實(shí)驗(yàn)組熒光強(qiáng)度高于對(duì)照組及拮抗組，半定量對(duì)比分析，差異有顯著統(tǒng)計(jì)學(xué)意義（P0.005）；熒光-NGR-釓實(shí)驗(yàn)實(shí)驗(yàn)組（HT1080細(xì)胞）熒光強(qiáng)度高于對(duì)照組（HT29細(xì)胞）及拮抗組（HT1080細(xì)胞），半定量對(duì)比分析，差異有顯著統(tǒng)計(jì)學(xué)意義（P0.005）。 結(jié)論：熒光-NGR-釓偶聯(lián)成功，，在體外對(duì)HePG2和HT1080腫瘤細(xì)胞膜上的CD13具有特異性靶向性。
[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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R445.2;R73-3

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本文編號(hào)：1913072