CD22靶向纳米材料设计、合成与肿瘤靶向性能评价

发布时间：2018-07-03 09:57

本文选题：CD22 + 靶向识别　；参考：《大连医科大学》2017年硕士论文

【摘要】：目的:基于受体-配体(receptor-ligand)间相互特异性识别原理,借助化学生物学偶联的手段,设计、合成具有CD22靶向功能的荧光纳米材料,表征荧光纳米材料的材料性质,并通过细胞靶向实验实现CD22高表达的细胞系的示踪,为CD22的生物学诊断提供有力工具。方法:1.基于receptor-ligand相互识别原理,借助N-乙酰神经氨酸(NANA)对CD22的识别作用,对其进行衍生化处理,得到不稳定的O-酰基异基酯(NANA-EDC),用于氨基量子点(NH2-QDs)的生物偶联;将NANA-EDC与NH2-QDs进行偶联,得到NANA-QDs纳米复合物。2.对NANA-QDs纳米复合物的材料性质进行表征,通过测定NANA-QDs纳米复合物中配体NANA的偶联度,粒径分布,Zeta电位,量子产率,光谱学性质,生物毒性等性质,明确NANA-QDs纳米复合物的结构,荧光性能和安全性。3.借助体外细胞实验对NANA-QDs纳米复合物的靶向性能进行评价,通过模式CD22高表达细胞株Daudi表面荧光分布、竞争性结合实验、纳米粒子细胞内吞实验、免疫荧光共定位等实验确定NANA-QDs特异性和亲和性。4.将NANA-QDs纳米复合物用于多种肿瘤细胞的CD22表达水平检测,并通过传统方法蛋白质免疫印迹(Western blotting analysis)、流式细胞术、细胞免疫荧光对结果可靠性进行验证。结果:经TEM及DLS测试,结果表明NANA-QDs纳米复合物的平均粒径分布为10nm左右且分布均匀;平均偶联度为1分子QD纳米粒子表面可偶联294个NANA分子,配体分子浓度较高,有利于下一步细胞靶向实验;偶联后,量子产率稳定,保持在70%左右,且荧光光谱谱峰对称,荧光性能良好;在细胞靶向性测试中,NANA-QDs纳米粒子在Daudi细胞表面产生强烈的红色荧光信号,初期荧光信号具有类似细胞膜结构分布,而阴性对照中荧光微弱且无特异性分布;通过与游离NANA的竞争性结合及免疫荧光共定位实验表明,NANA-QDs纳米复合物与Daudi细胞的结合是NANA上环状结构对CD22的特异性识别产生;纳米粒子细胞动力学实验表明,随着时间延长,荧光分布逐渐由Daudi细胞表面进入细胞内部,表明NANA-QDs可通过细胞的CD22内吞作用进入细胞;以上一系列实验表明NANA-QDs对细胞表面CD22具有特异性识别及靶向作用。将NANA-QDs纳米粒子用于淋巴瘤、肝癌、肺癌及乳腺癌六种肿瘤细胞CD22表达水平的测定,表明CD22除在淋巴瘤细胞特异性高表达外,在肝癌细胞、肺癌及乳腺癌细胞均呈低表达,在A549细胞中CD22的表达随传代次数的增加呈现上调趋势;利用细胞免疫荧光、流式细胞术和Westerning blotting等传统鉴定手段验证了上述实验结果的可靠性。结论:本研究利用NANA环状结构特异性识别CD22受体的原理,制备了NANA-QDs纳米荧光材料,其粒径分布均匀,荧光性能良好,低生物毒性,对细胞表面跨膜糖蛋白CD22具有较强的靶向性,为CD22的生物学诊断提供了有力工具。
[Abstract]:Aim: to design and synthesize fluorescent nanomaterials with CD22 targeting function based on the principle of mutual specificity recognition between receptor and ligand (receptor-ligand), and to characterize the properties of fluorescent nanomaterials. The high expression of CD22 cell line can be traced by cell targeting experiment, which provides a powerful tool for the biological diagnosis of CD22. Method 1: 1. Based on the principle of receptor-ligand mutual recognition, the unsteady O-acyl isoester (NA-EDC) was obtained by using the recognition of N-acetylneuraminic acid (NANA-EDC) on CD22, which was used to couple amino quantum dots (NH2-QDs), and NANA-EDC was coupled with NH2-QDs. NANA-QDs nanocomposites. The properties of NANA-QDs nanocomposites were characterized. The structure of NANA-QDs nanocomposites was determined by determining the coupling degree, particle size distribution, quantum yield, spectroscopic properties and biotoxicity of NANA-QDs nanocomposites. Fluorescence properties and safety. The targeting properties of NA-QDs nanocomposites were evaluated by cell experiments in vitro. The surface fluorescence distribution, competitive binding assay and endocytosis of NA-QDs nanocomposites were determined by the model CD22 high expression cell line Daudi. The specificity and affinity of NANA-QDs were determined by immunofluorescence co-localization. NANA-QDs nanocomplexes were used to detect the expression of CD22 in various tumor cells, and the reliability of the results was verified by Western blotting analysis), flow cytometry (FCM). Results: the results of TEM and DLS showed that the average particle size distribution of NANA-QDs nanocomposites was about 10nm and the average coupling degree was 1 molecular QD nanoparticles. After coupling, the quantum yield is stable, about 70%, and the fluorescence spectrum peak is symmetrical, and the fluorescence performance is good. In the cell targeting test, NANA-QDs nanoparticles produced strong red fluorescence signals on the surface of Daudi cells. The initial fluorescence signals were similar to the cell membrane structure distribution, but the fluorescence in negative controls was weak and non-specific. The results of competitive binding with NANA and immunofluorescence co-localization showed that the binding of NANA-QDs nanoparticles to Daudi cells was due to the specific recognition of CD22 by the ring structure on NANA. The fluorescence distribution gradually entered the cells from the surface of Daudi cells, indicating that NANA-QDs could enter the cells through the endocytosis of CD22, and the above series of experiments showed that NA-QDs had a specific recognition and targeting effect on the CD22 on the cell surface. NANA-QDs nanoparticles were used to detect the expression of CD22 in lymphoma, liver cancer, lung cancer and breast cancer cells. The results showed that CD22 expression was low in hepatoma cells, lung cancer cells and breast cancer cells, except for the specific high expression of CD22 in lymphoma cells. The expression of CD22 in A549 cells was up-regulated with the increase of passage times, and the reliability of the above results was verified by traditional identification methods such as cellular immunofluorescence, flow cytometry and Westerning blotting. Conclusion: in this study, NANA-QDs nanofluorescent materials were prepared by the principle of NANA ring structure specific recognition of CD22 receptor. The NANA-QDs nanophore materials have the advantages of uniform particle size distribution, good fluorescence performance, low biotoxicity and strong targeting to transmembrane glycoprotein CD22 on cell surface. It provides a powerful tool for the biological diagnosis of CD 22.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.1;R73

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