本文選題：Na_2SeO_3　切入點：H_2Se　出處：《山東師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：硒,作為人體必需的微量元素之一,與腫瘤預(yù)防及抑制腫瘤生長密切相關(guān)。硒化物通過體內(nèi)代謝產(chǎn)生高活性小分子,如H_2Se等,進(jìn)而導(dǎo)致DNA、蛋白質(zhì)損傷、谷胱甘肽消耗,最終導(dǎo)致腫瘤細(xì)胞死亡。Na_2SeO_3是腫瘤治療常用藥物,被廣泛應(yīng)用于基礎(chǔ)研究及臨床應(yīng)用,其作用機制一直是人們關(guān)注的熱點。眾所周知,乏氧是腫瘤組織生存、增殖的主要特征之一。之前的研究均是在常氧條件下進(jìn)行實驗,為此,我們課題組在乏氧條件下重新開展了Na_2SeO_3誘導(dǎo)腫瘤細(xì)胞凋亡機制研究,并發(fā)現(xiàn)Na_2SeO_3的抗癌效果是通過其代謝過程中產(chǎn)生的重要信號分子H_2Se在細(xì)胞內(nèi)發(fā)揮作用實現(xiàn)的,即該過程中H_2Se的升高導(dǎo)致細(xì)胞凋亡。但是,H_2Se在細(xì)胞內(nèi)發(fā)揮抗癌作用的機制研究必須精準(zhǔn)到亞細(xì)胞器水平,才能深入探索Na_2SeO_3誘導(dǎo)腫瘤細(xì)胞凋亡過程究竟通過什么途徑導(dǎo)致細(xì)胞凋亡,然而,目前H_2Se在細(xì)胞內(nèi)產(chǎn)生部位及其凋亡作用的具體機制尚不清楚。為了闡述該抗癌機制,必須首先研究H_2Se在亞細(xì)胞器中的分布情況。基于此,我們建立納米靶向檢測平臺,開發(fā)了可以分別靶向不同細(xì)胞器的檢測H_2Se的近紅外納米熒光定位探針,分別靶向細(xì)胞質(zhì)、溶酶體、線粒體三種重要的細(xì)胞器,檢測不同細(xì)胞器內(nèi)硒化氫的分布情況,并對其進(jìn)行可視化研究,這對抗癌藥物療效提高、精準(zhǔn)治療具有非常重要的意義和價值,該平臺對Na_2SeO_3乃至其他抗癌藥物的抗癌機制研究提供了一種新的途徑。與此同時,根據(jù)上述結(jié)果,我們繼續(xù)設(shè)計開發(fā)了可以靶向線粒體同時檢測H_2Se和O_2~(·-)的納米熒光探針,為深入研究和闡釋乏氧條件下Na_2SeO_3還原脅迫抗癌機制提供了必備的研究工具。本論文主要研究內(nèi)容分以下兩方面:1、利用介孔二氧化硅易被修飾等特點,我們設(shè)計合成了分別靶向細(xì)胞質(zhì)、溶酶體、線粒體三種重要細(xì)胞器內(nèi)檢測H_2Se的納米熒光探針,首次實現(xiàn)了細(xì)胞內(nèi)細(xì)胞質(zhì)、溶酶體、線粒體等細(xì)胞器中H_2Se的實時成像與檢測。該探針通過介孔二氧化硅裝載用于檢測硒化氫的近紅外分子探針(NIR-H_2Se),進(jìn)一步通過分別修飾聚乙烯亞胺(PEI)、嗎啉(MPP)、三苯基膦(TPP)的靶向基團(tuán)來分別定位細(xì)胞質(zhì)、溶酶體、線粒體三種重要的細(xì)胞器,設(shè)計合成了新型靶向不同細(xì)胞器的H_2Se近紅外納米熒光探針,分別命名為細(xì)胞質(zhì)定位探針(Cyto-NIR-H_2Se-MSN)、溶酶體定位探針(Lyto-NIR-H_2SeMSN)、線粒體定位探針(Mito-NIR-H_2Se-MSN)。實驗結(jié)果表明,乏氧條件下Na_2SeO_3誘導(dǎo)腫瘤細(xì)胞凋亡過程中,隨著Na_2SeO_3誘導(dǎo)時間或濃度的增加,線粒體內(nèi)H_2Se水平明顯升高,而細(xì)胞質(zhì)與溶酶體內(nèi)的H_2Se含量變化并不明顯,同時伴隨線粒體膜電位降低,說明此過程在線粒體中產(chǎn)生H_2Se,損傷線粒體后導(dǎo)致線粒體塌陷,這為深入研究Na_2SeO_3誘導(dǎo)腫瘤細(xì)胞凋亡過程的分子機制提供重要的理論依據(jù)及可靠手段。2、我們設(shè)計合成了線粒體內(nèi)H_2Se和O_2~(·-)的雙檢測納米熒光探針,用以Na_2SeO_3誘導(dǎo)腫瘤凋亡過程中高活性還原性分子(H_2Se)水平與高活性氧化性分子超氧陰離子自由基(O_2~(·-))水平檢測與可視化分析。基于上一章研究結(jié)果我們得知:乏氧條件下Na_2SeO_3誘導(dǎo)腫瘤細(xì)胞凋亡過程中為“非氧化脅迫”所致,該過程中H_2Se在線粒體內(nèi)產(chǎn)生并得到累積,因此接下來我們想要探索該過程中線粒體內(nèi)氧化還原狀態(tài)的變化情況,并深入其凋亡通路的研究。我們基于介孔二氧化硅粒子包覆二氫乙錠(DHE)和NIR-H_2Se兩種分子探針,并修飾TPP來定位線粒體,設(shè)計合成了新型靶向線粒體的雙檢測定位探針(Mito-N-D-MSN),對Na_2SeO_3誘導(dǎo)腫瘤細(xì)胞凋亡過程中線粒體內(nèi)氧化還原狀態(tài)進(jìn)行實時監(jiān)測,兩種分子探針的激發(fā)發(fā)射分別為λex/λem=488/638 nm和λex/λem=688/735nm,實現(xiàn)了線粒體中O_2~(·-)和H_2Se的同時檢測與成像。實驗結(jié)果表明,乏氧條件下Na_2SeO_3誘導(dǎo)肝癌細(xì)胞凋亡過程中,線粒體內(nèi)只有H_2Se水平逐漸升高,O_2~(·-)水平無明顯變化,說明該過程中線粒體內(nèi)處于還原脅迫狀態(tài),結(jié)合上一體系結(jié)論中線粒體塌陷這一結(jié)果,我們推測:乏氧條件下Na_2SeO_3誘導(dǎo)腫瘤細(xì)胞凋亡為還原脅迫,且由線粒體凋亡所致,具體分子機制有待進(jìn)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.

【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R73-3;O657.3

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相關(guān)期刊論文 前2條

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本文編號：1580257