摘要
近年来,荧光纳米材料优良的荧光性质使其在细胞及亚细胞层面的生物标记研究中得到了广泛的关注,为生物分析及生物医学领域的研究提供了新的技术手段和方法。本研究小组利用二氧化硅荧光纳米颗粒开展了一系列生物标记研究工作,但二氧化硅纳米颗粒在亚细胞标记中的研究还未有报道,本论文在实验室已有工作基础上,开展了二氧化硅荧光纳米颗粒用于线粒体标记的相关研究,研究发现纳米颗粒粒径较大会影响亚细胞运输的进行,因此选择小粒径荧光材料金纳米簇作为研究对象,考察其作为细胞荧光标记物的可行性。本论文主要从以下三个方面开展工作：
1.信号肽功能化二氧化硅纳米颗粒的离体线粒体靶向作用研究
以N-(p-马来酰亚胺基苯基)异氰酸酯(N-(p-Maleimidophenyl)isocyanate,PMPI)为交联剂,将线粒体信号肽分子共价修饰到二氧化硅荧光纳米颗粒表面,构建线粒体信号肽功能化二氧化硅荧光纳米颗粒。采用荧光分光光度计、Zeta电位仪以及透射电子显微镜对修饰前后的二氧化硅纳米颗粒进行表征。在此基础上以分离纯化的细胞核作为对照,采用流式细胞术考察了信号肽功能化二氧化硅荧光纳米颗粒与分离纯化的线粒体间的相互作用。结果表明：信号肽成功修饰在二氧化硅纳米颗粒表面,并且纳米颗粒粒径在信号肽分子修饰前后没有发生明显变化；同时,线粒体信号肽修饰到二氧化硅纳米颗粒表面后依然保持良好的生物活性,能够介导二氧化硅纳米颗粒特异性识别及结合分离纯化的线粒体,从而为线粒体监测以及线粒体功能调控研究提供了新的思路。
2.信号肽功能化二氧化硅纳米颗粒的活细胞线粒体靶向研究
在上一章研究工作基础上,以Hela细胞为研究对象,考察了线粒体信号肽功能化二氧化硅荧光纳米颗粒在活细胞内的线粒体靶向情况。将信号肽功能化二氧化硅荧光纳米颗粒和Hela细胞共同培育,并利用Lyso Tracker Green和Mito Tracker Deep Red对细胞内溶酶体及线粒体进行染色,通过激光共聚焦显微镜对纳米颗粒在Hela细胞内的分布情况进行分析。结果表明：功能化二氧化硅荧光纳米颗粒能够通过细胞内吞作用进入细胞,但是未能实现线粒体靶向而是滞留于溶酶体。氯喹处理能够破坏溶酶体膜的完整性,但在氯喹处理后二氧化硅荧光纳米颗粒并未进入细胞质而是粘附于溶酶体膜上,仍滞留于溶酶体中。本章工作为纳米颗粒用于亚细胞标记提供了具有参考价值的信息,荧光纳米颗粒在亚细胞标记领域的进一步应用有赖于发展分散性好、粒径小、非特异吸附小的纳米材料。
3.牛血清白蛋白介导合成的金纳米簇用于细胞荧光成像研究
在上一章研究工作中发现纳米颗粒粒径较大不利于亚细胞运输的进行,发展小粒径荧光物质对实现亚细胞标记十分必要。近年来金纳米簇因其荧光特性得到很大改善且具有粒径小、无毒性等优点而成为纳米材料领域科学家们关注的热点并在荧光标记研究中得到了一定应用。本章以牛血清白蛋白介导合成了金纳米簇,并利用荧光分光光度计,纳米粒度及zeta电位仪和非变性聚丙烯酰胺蛋白质电泳进行了表征；在此基础上进一步考察了金纳米簇与Hela细胞间的相互作用,同时将其与生物素相交联考察其用作荧光标记物的可行性。结果表明：牛血清白蛋白介导合成的金纳米簇荧光信号较强且在不同pH值溶液中荧光稳定性好；与细胞共同孵育时可以通过内吞作用进入活细胞内,在优化培育时间和金纳米簇浓度的条件下可实现较好的活细胞荧光标记效果,并且在经过细胞固定化处理后仍可保持其标记形态；此外生物素化的金纳米簇可以识别及标记链霉亲和素包被的微珠。以上研究对于金纳米簇作为一种新型荧光标记物在活细胞荧光成像领域中的进一步应用具有一定的指导意义。
In recent years, fluorescent nanoparticles have been widely used in fluorescence labeling at cellular and subcellular level in cell biology research as it has excellent fluorescence characteristics, which have provided new technological methods for the biological analysis and the biomedical research. Based on the fluorescent silica nanoparticles'platform in our laboratory, we first carried out the study of using fluorescent silica nanoparticles for subcellular labeling. The results showed that the big diameter of nanoparticles was a block for subcellular targeting. Then, gold clusters with small size were also investigated to be used as a potential fluorescence marker for cell labeling.
1. Study on the target interaction between mitochondria and signal peptide functionalized silica nanoparticles
The mitochondrial signal peptide functionalized fluorescent silica nanoparticles were prepared by conjugated the mitochondrial signal peptide to the nanoparticles through N-(p-Maleimidophenyl)isocyanate (PMPI). The fluorescence spectrophotometer, zeta sizer and TEM have been used to characterize the fluorescent silica nanoparticles. By using the isolated nucleus as a control, the target interaction between the mitochondrial signal peptides modified fluorescent silica nanoparticles and the isolated mitochondria has been investigated by flow cytometry. The results showed that the signal peptides were successfully conjugated to the surface of fluorescent silica nanoparticles, and the size of the fluorescent silica nanoparticles did not change obviously before and after modification. It was revealed that the bioactivity of the signal peptide was well remained after the conjugation with the silica nanoparticles, and the peptides could facilitate the specific recognition and binding between the silica nanoparticles and the isolated mitochondria, which put forwards a new idea for the monitoring and function controlling of the mitochondria.
2. Study on the signal peptide functionalized silica nanoparticles targeting of mitochondria in living cells
Based on the above research work, we investigated the mitochondrial targeting ability of signal peptide functionalized silica nanoparticles in living Hela cells. The functionalized fluorescent silica nanoparticles were incubated with Hela cell, and then the subcellular distribution was studied by cofocal microscopy in the situation that the lysosome and mitochondrion were costained by LysoTracker Green and MitoTracker Deep Red. It was found that the functionalized fluorescent silica nanoparticles can enter cells through endocytosis. However, its further movement was prevented by the lysosome. Although the cells were further treated with chloroquine in order to release the nanoparticles into the cytoplasm, experimental results showed that most of nanoparticles were adsorbed in the lysosomal membrane and retained in the lysosome and could not able to reach the mitochondria. The results indicated that the nanoparticles with smaller size, better dispersion and low specific absorption should be prepared to make better use of fluorescent nanoparticles for subcellular labeling.
3. Study on the live cells fluorescence imaging by bovine serum albumin-mediated synthesized gold nanoclusters
As discussed aboved, it is necessary to develope nanoparticles with smaller size for cell labeling. In recent years, gold nanoclusters have attracted the attention of scientists in the field of nano-materials because of its improved fluorescent properties and the advantages of small size and nontoxic. In this chapter, the gold nanocluster was synthesized by bovine serum albumin directing, and was characterized by fluorescence spectrophotometer, zeta sizer and non-denaturing polyacrylamide gel electrophoresis. And further study was carried out to investigate the interaction between gold nanoclusters and Hela cells, and the gold nanoclusters were conjugated with biotin to investigate the feasibility of being used as a fluorescent marker.The results showed that the gold nanocluster's fluorescence signal was strong and stable at different pH. It demonstrated that the gold naoclusters could be successfully uptaken by living cells. The good effects of the fluorescent labeling of living cells can be achieved by using optimal incubation time and gold nanoclusters'concentration. Moreover, the fixation treatment did not change the form of labeling. Furthermore, the gold-nanoclusters can also be used as fluorescent markers to label the biotin and did not affect the interaction between biotin and avidin. These results provide some guidance to the further application of fluorescent gold nanoclusters in the field of live cells labeling.
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