基于荧光硅纳米颗粒的基因载体的构建及其在活细胞内的生物成像基础研究

发布时间：2018-03-20 00:24

本文选题：荧光硅纳米颗粒　切入点：基因载体　出处：《苏州大学》2016年硕士论文　论文类型：学位论文

【摘要】：随着纳米科学技术的飞速发展,各种各样的纳米材料被用于构建非病毒基因载体。其中,具有荧光性能的基因载体因为能够示踪细胞内基因运输的相关过程而倍受瞩目。在基因运输的过程中,长时程实时的示踪成像可以让人们更加综合地理解基因载体的胞内行为,进而有利于更加合理地设计高性能的基因载体。近年来,零维荧光硅纳米颗粒受到了人们的广泛关注。因其具备优越的光学性质和良好的生物安全性,被认为是生物成像应用中潜在的理想荧光探针。在本文中,我们首先构建了一种基于荧光硅纳米颗粒的基因载体,并成功将其应用于基因运输和荧光生物成像。在DNA的负载和保护试验中,基于荧光硅纳米颗粒的基因载体不仅具有相对较高的DNA负载率,而且能够有效地保护DNA,避免DNA酶的降解。在基因转染测试中,基于荧光硅纳米颗粒的基因载体在人宫颈癌(HeLa)细胞的基因转染效率约为35%,比相同条件下聚乙烯亚胺(Polyetherimide, PEI)的转染效率要高。基于荧光硅纳米颗粒的基因载体同时也具备良好的生物安全性,在与HeLa细胞共孵育培养时间长达24和48小时的时间内,均未产生明显的细胞毒性,HeLa细胞仍然维持90%以上的存活率。其次,通过追踪荧光硅纳米颗粒强而稳定的荧光,我们长时程实时地研究了基因载体的胞内行为(比如：细胞内吞、胞内转运等),进而揭示了基因载体与溶酶体的“复融合”(re-fusion)现象。我们选择了另外一种典型的人乳腺癌(MCF-7)细胞作为对照组来研究载体的基因转染和胞内行为。基于荧光硅纳米颗粒的载体在MCF-7细胞中的基因转染效率约为16%,比相同实验条件下HeLa细胞的转染效率小的多。在活细胞长时程实时的示踪成像中,MCF-7细胞中同样存在基因载体与溶酶体的“复融合”现象。在相同的时间内(15分钟的实时示踪成像),基因载体与溶酶体的“复融合”在MCF-7细胞中发生了3次,而在HeLa细胞中仅发生了1次。最后,通过进一步分析此两种细胞系的基因转染效率和胞内基因载体与溶酶体“复融合”现象之间的关系可知,“复融合”现象在基因转染效率低的MCF-7细胞中比在基因转染效率高的HeLa细胞中发生的次数更多。综上可知,基于荧光硅纳米颗粒的基因载体不仅具有强而稳定的荧光和良好的生物安全性,而且具有相对较高的DNA负载和基因转染能力。利用其优异的光学性质,本文进一步研究了基因载体的胞内命运。这些结果表明基于荧光硅纳米颗粒的基因载体有望作为具有荧光性能的新型基因载体应用于活细胞内长时程实时的示踪成像,并为人们提供更加有价值的信息来深入地了解和研究基因载体的胞内行为。
[Abstract]:With the rapid development of nanotechnology, a variety of nanomaterials have been used to construct non-viral gene vectors. Gene vectors with fluorescence properties have attracted much attention because of their ability to trace the related processes of gene transport within cells. In the process of gene transport, long-term real-time tracer imaging can provide a more comprehensive understanding of the intracellular behavior of gene vectors. In recent years, zero-dimensional fluorescent silicon nanoparticles have attracted wide attention because of their superior optical properties and good biosafety. In this paper, we first constructed a gene vector based on fluorescent silicon nanoparticles. In the loading and protection experiments of DNA, the gene carrier based on fluorescent silicon nanoparticles not only has a relatively high DNA loading rate, but also has been successfully applied to gene transport and fluorescence imaging. And it can effectively protect the DNA from the degradation of DNA enzyme. The transfection efficiency of the gene vector based on fluorescent silicon nanoparticles in human cervical cancer cell line HeLa was about 35, which was higher than that of PEI under the same conditions. The gene vector based on fluorescent silicon nanoparticles was also found to be more efficient than that of PEI under the same conditions. With good biological safety, After incubation with HeLa cells for 24 and 48 hours, no significant cytotoxicity was observed. The survival rate of HeLa cells remained above 90%. Secondly, the strong and stable fluorescence of fluorescent silicon nanoparticles was traced. We have studied the intracellular behavior of gene vectors (E. G. endocytosis) over a long period of time. We selected another typical human breast cancer cell line MCF-7 as control group to study the gene transfection and intracellular behavior of the vector. The transfection efficiency of fluorescent silicon nanoparticles in MCF-7 cells was about 16, which was much lower than that of HeLa cells under the same experimental conditions. The gene was also present in MCF-7 cells in live cell long time and real time tracer imaging. The phenomenon of "refusion" of vector and lysosome. In the same time, the "fusion" of gene vector and lysosome took place three times in MCF-7 cells after 15 minutes of real-time tracer imaging. But only once in HeLa cells. Finally, Through further analysis of the gene transfection efficiency of the two cell lines and the relationship between the intracellular gene vector and lysosomal "refusion" phenomenon, it can be seen that the "refusion" phenomenon in MCF-7 cells with low gene transfection efficiency is more than that in the gene transfer cell line. HeLa cells with high dyeing efficiency occur more frequently. The gene vector based on fluorescent silicon nanoparticles not only has strong and stable fluorescence and good biological safety, but also has relatively high DNA loading and gene transfection ability. In this paper, the intracellular fate of gene vectors has been further studied. These results indicate that the novel gene vectors based on fluorescent silicon nanoparticles are expected to be used as novel gene vectors with fluorescent properties for real-time tracer imaging in living cells for a long time. And provide more valuable information for people to understand and study the intracellular behavior of gene vectors.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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