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题名: L-抗坏血酸转运蛋白SVCTs荧光探针的合成及在活细胞成像分析中的应用
作者: 宋媛媛
学位类别: 硕士
答辩日期: 2015-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 尹俊发 ; 汪海林
关键词: L-抗坏血酸,磺基罗丹明B氨基己酸抗坏血酸酯,抗坏血酸转运蛋白,活细胞成像,荧光探针,L-ascorbic acid, RB-A-Vc, sodium vitamin C transporters(SVCTs), living cells imaging, fluorescent probe
其他题名: A small-molecule fluorescent probe for visualization of sodium vitamin C transproters (SVCTs) in living cells
学位专业: 分析化学
中文摘要:     维生素C是人体必需的一种营养物质。由于人体中不能合成维生素C,需要借助于细胞内的维生素C转运蛋白SVCT1和SVCT2将其转运到体内并发挥作用。抗坏血酸转运蛋白(SVCTs)对于维持体内维生素C的稳态具有重要作用,其表达水平的改变与包括肿瘤在内的疾病有关。所以检测SVCTs在细胞内表达水平的变化以及亚细胞的分布,对于相关疾病的诊断及毒性效应机制的研究具有重要意义。
    本文中,我们合成了一种新型的小分子荧光小分子探针(RB-A-Vc),并将其应用到活细胞中内源SVCTs的成像分析。本文共分为四章:
第一章:综述了SVCTs转运蛋白的生理功能及与疾病的关联,介绍了现有的成像分析方法及其局限性,从而引出本研究的研究目标和内容。
第二章:以带负电的磺基罗丹明B磺酰氯作为荧光标记基团,氨基己酸作为连接臂,合成了磺基罗丹明B-氨基己酸-抗坏血酸酯(RB-A-Vc),采用正相硅胶柱色谱对其进行了纯化制备,考察了其化学稳定性。
第三章:将新合成的荧光探针RB-A-Vc应用到固定化细胞(HEK-293T和HepG2)中SVCTs的成像,发现其荧光分布在线粒体和细胞膜周围,与免疫荧光方法得到的结果相吻合。免疫荧光方法和Western blot验证了RB-A-Vc与SVCTs结合的特异性。实验将其应用到活细胞中进行SVCTs的荧光成像及亚细胞定位,发现此荧光探针可以被转运蛋白转运进入细胞中并主要分布在线粒体周围和细胞膜上,与固定化得到的结果相一致。最后对RB-A-Vc进行了剂量效应的考察,得到其最佳使用浓度约为100 μM。
第四章:将RB-A-Vc用于活细胞中SVCTs表达量的变化检测,将细胞中的SVCTs经PMA的上调表达和siRNA的下调表达处理后用RB-A-Vc进行成像分析,结果表明此荧光探针可以指示细胞中SVCTs表达量的变化。用酶标仪和流式细胞仪对其表达量的变化进行了定量分析,进一步验证了RB-A-Vc可以用于指示细胞内SVCTs表达水平的变化。最后将合成的小分子荧光探针及发展方法应用于实际样品的检测,检测了细胞中DNA受到卤代苯醌引起的氧化损伤后SVCTs表达水平的变化。
第五章:对全文进行了总结,并对小分子荧光探针RB-A-Vc用于活细胞中SVCTs实时成像监测进行了展望。新合成的小分子荧光探针RB-A-Vc可以通过转运蛋白的作用进入到细胞内实现活细胞中SVCTs的成像并可以监测活细胞内总体SVCTs表达水平的改变及其分布。
英文摘要:     Ascorbic acid (AA, or vitamin C) is an essential nutrient for mammals. It plays a key role in antioxidant defense against harmful free radicals. Essentially, human beings do not synthesize viatamin C by themselves, and rely on the uptake of AA through the sodium vitamin C transporters (SVCTs) including SVCT1 and SVCT2.
SVCTs are thus crucial for maintaining intracellular ascorbate levels. Deficiency of SVCTs brings haemorrhage in lower brainstem areasand increased oxidative stress in several organs of mice, even causes mice to die after birth, with respiratory failure and cortical brain haemorrhage. Changes in expression or function of the SVCTs may be associated with human diseases including Huntington's disease, breast and prostate cancers, and colorectal adenoma. Therefore, intracellular SVCTs monitoring and detection can provide valuable information for biological diagnosis, adaptive therapy and drug discovery. In this regard, live-cell imaging of SVCTs is meaningful because it can provide high spatiotemporal resolution for the analysis of SVCTs dynamics, reflects the ascorbate transporters expression levels, subcellular distribution and spatiotemporal dynamics. Up to now, the options for live cell labelling of SVCTs were very limited. The antibodies against SVCT1 and/or SVCT2 in immunofluorescence assays are not membrane permeable. Thus they can only be used for indicating SVCTs in fixed and permeabilized cells. Another important issue is, although antibodies for SVCT1 and SVCT2 are provided, there is lack of a reporter for monitoring the whole level and changes of SVCTs. In fact, the previous studies revealed that the biological and toxicological functions of AA transporters are commonly associated with both SVCT1 and SVCT2. To the best of our knowledge, up to date, there are no fluorescent probes capable of selective detection of the total SVCTs in living cells. The development of easy-to-use probes, which can pass the cell membrane and selectively bind and illuminate the total SVCTs, was thus required.‘
    Herein, we synthesized a fluorophore-conjuated vitamin C (RBA-Vc), and demonstrated its application in selectively visualizing SVCTs in living cells. And the probe RB-A-Vc was purified and identified by liquid chromatography-mass spectrometry and characterized by fluorescent spectrometry. Before proceeding to the living cell visualization experiments, we first examined the applicability of the synthesized RB-A-Vc for SVCTs imaging in the fixed cells. Then to gain further insight into fluorescent sensing and imaging of SVCTs in living cells, we applied RB-A-Vc for directly staining SVCTs in living HEK-293T and HepG2 cells. After the validation of RB-A-Vc’s function in SVCTs labelling and localization, we applied it to indicate the changes of SVCTs expression in living cells. At last, we detected changes in the expression of SVCTs in the cells exposed to polyhalogenated quinines.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34371
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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宋媛媛. L-抗坏血酸转运蛋白SVCTs荧光探针的合成及在活细胞成像分析中的应用[D]. 北京. 中国科学院研究生院. 2015.
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