靶向不同细胞器的硒化氢近红外纳米荧光探针的设计合成及分析应用

发布时间：2018-03-07 17:30

本文选题：Na_2SeO_3　切入点：H_2Se　出处：《山东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：硒,作为人体必需的微量元素之一,与肿瘤预防及抑制肿瘤生长密切相关。硒化物通过体内代谢产生高活性小分子,如H_2Se等,进而导致DNA、蛋白质损伤、谷胱甘肽消耗,最终导致肿瘤细胞死亡。Na_2SeO_3是肿瘤治疗常用药物,被广泛应用于基础研究及临床应用,其作用机制一直是人们关注的热点。众所周知,乏氧是肿瘤组织生存、增殖的主要特征之一。之前的研究均是在常氧条件下进行实验,为此,我们课题组在乏氧条件下重新开展了Na_2SeO_3诱导肿瘤细胞凋亡机制研究,并发现Na_2SeO_3的抗癌效果是通过其代谢过程中产生的重要信号分子H_2Se在细胞内发挥作用实现的,即该过程中H_2Se的升高导致细胞凋亡。但是,H_2Se在细胞内发挥抗癌作用的机制研究必须精准到亚细胞器水平,才能深入探索Na_2SeO_3诱导肿瘤细胞凋亡过程究竟通过什么途径导致细胞凋亡,然而,目前H_2Se在细胞内产生部位及其凋亡作用的具体机制尚不清楚。为了阐述该抗癌机制,必须首先研究H_2Se在亚细胞器中的分布情况。基于此,我们建立纳米靶向检测平台,开发了可以分别靶向不同细胞器的检测H_2Se的近红外纳米荧光定位探针,分别靶向细胞质、溶酶体、线粒体三种重要的细胞器,检测不同细胞器内硒化氢的分布情况,并对其进行可视化研究,这对抗癌药物疗效提高、精准治疗具有非常重要的意义和价值,该平台对Na_2SeO_3乃至其他抗癌药物的抗癌机制研究提供了一种新的途径。与此同时,根据上述结果,我们继续设计开发了可以靶向线粒体同时检测H_2Se和O_2~(·-)的纳米荧光探针,为深入研究和阐释乏氧条件下Na_2SeO_3还原胁迫抗癌机制提供了必备的研究工具。本论文主要研究内容分以下两方面:1、利用介孔二氧化硅易被修饰等特点,我们设计合成了分别靶向细胞质、溶酶体、线粒体三种重要细胞器内检测H_2Se的纳米荧光探针,首次实现了细胞内细胞质、溶酶体、线粒体等细胞器中H_2Se的实时成像与检测。该探针通过介孔二氧化硅装载用于检测硒化氢的近红外分子探针(NIR-H_2Se),进一步通过分别修饰聚乙烯亚胺(PEI)、吗啉(MPP)、三苯基膦(TPP)的靶向基团来分别定位细胞质、溶酶体、线粒体三种重要的细胞器,设计合成了新型靶向不同细胞器的H_2Se近红外纳米荧光探针,分别命名为细胞质定位探针(Cyto-NIR-H_2Se-MSN)、溶酶体定位探针(Lyto-NIR-H_2SeMSN)、线粒体定位探针(Mito-NIR-H_2Se-MSN)。实验结果表明,乏氧条件下Na_2SeO_3诱导肿瘤细胞凋亡过程中,随着Na_2SeO_3诱导时间或浓度的增加,线粒体内H_2Se水平明显升高,而细胞质与溶酶体内的H_2Se含量变化并不明显,同时伴随线粒体膜电位降低,说明此过程在线粒体中产生H_2Se,损伤线粒体后导致线粒体塌陷,这为深入研究Na_2SeO_3诱导肿瘤细胞凋亡过程的分子机制提供重要的理论依据及可靠手段。2、我们设计合成了线粒体内H_2Se和O_2~(·-)的双检测纳米荧光探针,用以Na_2SeO_3诱导肿瘤凋亡过程中高活性还原性分子(H_2Se)水平与高活性氧化性分子超氧阴离子自由基(O_2~(·-))水平检测与可视化分析。基于上一章研究结果我们得知:乏氧条件下Na_2SeO_3诱导肿瘤细胞凋亡过程中为“非氧化胁迫”所致,该过程中H_2Se在线粒体内产生并得到累积,因此接下来我们想要探索该过程中线粒体内氧化还原状态的变化情况,并深入其凋亡通路的研究。我们基于介孔二氧化硅粒子包覆二氢乙锭(DHE)和NIR-H_2Se两种分子探针,并修饰TPP来定位线粒体,设计合成了新型靶向线粒体的双检测定位探针(Mito-N-D-MSN),对Na_2SeO_3诱导肿瘤细胞凋亡过程中线粒体内氧化还原状态进行实时监测,两种分子探针的激发发射分别为λex/λem=488/638 nm和λex/λem=688/735nm,实现了线粒体中O_2~(·-)和H_2Se的同时检测与成像。实验结果表明,乏氧条件下Na_2SeO_3诱导肝癌细胞凋亡过程中,线粒体内只有H_2Se水平逐渐升高,O_2~(·-)水平无明显变化,说明该过程中线粒体内处于还原胁迫状态,结合上一体系结论中线粒体塌陷这一结果,我们推测:乏氧条件下Na_2SeO_3诱导肿瘤细胞凋亡为还原胁迫,且由线粒体凋亡所致,具体分子机制有待进一步研究。
[Abstract]:Selenium, as one of the essential trace elements in human body, and tumor prevention and inhibition of tumor growth are closely related. Selenium compounds can produce high activity by small molecule metabolism, such as H_2Se, leading to DNA, protein damage, glutathione depletion, eventually lead to tumor cell death.Na_2SeO_3 is commonly used drugs for cancer treatment, has been widely used in basic research and clinical application, the mechanism has been the focus of attention. As everyone knows, hypoxia is one of the main features of tumor survival, proliferation. Previous studies were carried out experiments in normoxic conditions, therefore, our research group under hypoxic condition to carry out research on tumor cell apoptosis induced by Na_2SeO_3. And found that the anticancer effect of Na_2SeO_3 is an important signaling molecule of H_2Se produced by the metabolic process in cells play a role in the realization of H_2Se, the process of increasing Guide Apoptosis induced by H_2Se. However, in the cell play mechanism of anticancer effect must be accurate to the subcellular level, to further explore the Na_2SeO_3 induced apoptosis of tumor cells in what way leads to apoptosis, however, the specific mechanism has produced a H_2Se and its apoptosis effect in cells is not clear. In order to explain the anticancer the distribution mechanism, must first study of H_2Se in subcellular organelles. Based on this, we establish a targeted detection platform, developed a target near infrared probe respectively to detect H_2Se nano fluorescence localization of different organelles, are targeting cytoplasmic lysosomes, mitochondria, three important organelles, the distribution of different detection intraorganellar hydrogen selenide, and visualization of their research, the efficacy of anticancer drugs to improve the accurate treatment, has a very important meaning and value, the Provides a new way of anticancer mechanism research platform to the Na_2SeO_3 and other anticancer drugs. At the same time, according to the above results, we continue to design and development a mitochondria targeted simultaneous detection of H_2Se and O_2~ (-) fluorescent probes, for further research and interpretation under the condition of lack of oxygen reduction of Na_2SeO_3 provides a research tool the necessary stress anti-cancer mechanism. This is the main content is divided into the following two aspects: 1, use the characteristic of mesoporous silica is easy to be modified, we designed and synthesized were targeting cytoplasmic lysosomes, mitochondria, fluorescent probes three important organelles for the detection of H_2Se for the first time in cell cytoplasm, lysosomes, real-time imaging detection of H_2Se and mitochondria in the probe. The mesoporous silica loaded for near infrared molecular probes for the detection of hydrogen selenide (NIR-H_2Se), further through Modification of polyethylene imine (PEI), morpholine (MPP), three phenyl phosphine (TPP) of the target group to lysosomes, mitochondria were located in cytoplasm, three important organelles, the design and synthesis of novel targeted different organelles of H_2Se near infrared fluorescent probes, which were named as cytoplasmic localization probe (Cyto-NIR-H_2Se-MSN) the positioning probe (Lyto-NIR-H_2SeMSN), lysosome, mitochondria localization probe (Mito-NIR-H_2Se-MSN). The experimental results show that under hypoxic conditions Na_2SeO_3 induced apoptosis of tumor cells in the process, with the increase of concentration or induced by Na_2SeO_3, H_2Se level in mitochondria increased significantly, while the cytoplasm and H_2Se content of the lysosomes is not obvious, accompanied by mitochondrial membrane to illustrate the potential reduction and process of producing the H_2Se in the mitochondria, leading to mitochondrial damage to mitochondria after the collapse, for further research on Na_2SeO_3 induced apoptosis of tumor cells The molecular mechanism of the apoptosis process provides an important theoretical basis and reliable means of.2, we designed and synthesized in the mitochondria of H_2Se and O_2~ (-) - detection of fluorescent probes, with induced reduction of high molecular activity of tumor apoptosis by Na_2SeO_3 (H_2Se) level and high activity of superoxide anion free radical oxidation of molecular (O_2~ (-)) analysis of the level of detection and visualization. The last chapter based on the results we know: Na_2SeO_3 under hypoxic conditions induced apoptosis of tumor cells is caused by non oxidative stress, the H_2Se generated in the process in mitochondria and accumulation, so we want to explore the changes of mitochondrial redox state during the study, and further the apoptosis pathway. We based on mesoporous silica particles coated with two hydrogen ethidium (DHE) and NIR-H_2Se two molecular probes, and modified TPP to locate the grain line The body, the design and synthesis of novel targeted double probe detection and localization of mitochondria (Mito-N-D-MSN), on Na_2SeO_3 induced apoptosis of tumor cells in the mitochondrial redox state monitoring, excitation and emission were ex/ em=488/638 nm and lambda lambda lambda lambda ex/ em=688/735nm two molecular probes, the mitochondrial O_2~ (-) at the same time and H_2Se detection and imaging. The experimental results show that under hypoxic conditions Na_2SeO_3 induced hepatocarcinoma cell apoptosis process, only mitochondrial H_2Se levels increased gradually, O_2~ (-) level had no obvious change, that the process of reduction in mitochondrial stress state, with a conclusion of mitochondrial system collapse this result we hypothesized that, under hypoxic conditions Na_2SeO_3 induced apoptosis of tumor cells was reduced by stress, and mitochondrial apoptosis caused by specific molecular mechanism needs further research.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3;O657.3

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