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类型 基础研究 预答辩日期 2017-12-05
开始(开题)日期 2015-11-20 论文结束日期 2017-10-19
地点 东南大学电子所楼216 论文选题来源 国家自然科学基金项目     论文字数 6.9 (万字)
题目 磁性氧化铁纳米粒子与细胞互作的基因表达调控
主题词 磁性氧化铁纳米粒子,基因表达谱,免疫,类病毒效应,DMSA
摘要 磁性氧化铁纳米粒子(简称为FeNPs)因良好的生物相容性在生物医学领域具有巨大的应用潜力。当前,一些氧化铁纳米粒子已经被应用为磁性靶向药物载体、补铁剂、磁共振成像(MRI)对比增强剂、细胞标记物等。因此,其相关毒理性效应研究具有重要意义。FeNPs潜在纳米毒性已被逐渐意识,并进行了系列研究,主要体现在细胞水平,如纳米的內化能力和方式,对细胞超微结构、生长活力、氧化应激等方面的影响。随着基因芯片技术的发展与日渐成熟,能检测基因组范围内所有基因表达。纳米作用于细胞后,细胞可迅速地改变它们的转录输出,即基因表达谱,以适应这种变化。在细胞或组织水平筛选鉴别表达受纳米材料影响的所有基因可以为识别任何潜在的纳米毒性及其相关分子机制提供有价值的线索。因此,采用高通量技术评价基因表达谱的变化有助于确定纳米材料潜在的纳米毒性。当前研究表明,我们需要系统地研究纳米颗粒粒径、电荷、形态及表面修饰等多因素的影响,探究其潜在分子机制,并加以开发应用。 静脉注射给药后,纳米材料由于被动靶向性在体内的血液和肝细胞中富集最多最密集。本研究以两种小鼠和相应两种人的血液细胞和肝癌细胞,即小鼠巨噬细胞RAW264.7、小鼠肝癌细胞Hepa1-6、人急性单核细胞THP-1和人肝癌细胞HepG2为研究对象。分别用不同剂量二巯基丁二酸(DMSA)修饰的Fe3O4纳米颗粒(简写为DMSA-FeNPs)处理四种细胞,运用基因芯片技术检测DMSA-FeNPs细胞基因表达谱。通过深入分析数据结合实验验证,从基因水平上探究DMSA-FeNPs与各细胞互作对基因表达的影响、潜在分子机制及可能应用。主要工作如下: 1. 本章运用透射电镜(TEM)、马尔文粒度仪(DLS)、红外光谱检测、普鲁士蓝染色及CCK-8测定细胞活力等技术,表征了DMSA-FeNPs磁性纳米粒子特性,探究了其细胞内化及对细胞活力的影响。TEM和DLS结果表明,DMSA-FeNPs平均粒径约11 nm,水动力尺寸约64 nm,在溶液中具有良好的分散性,且稳定性良好;普鲁士蓝染色检测显示四种细胞均能摄取DMSA-FeNPs,RAW264.7吞噬量最高,而THP-1相对最低。TEM观察可见DMSA-FeNPs以囊泡包覆形式定位于细胞质中。对细胞活力的测定表明,100 μg/mL的DMSA-FeNPs引起部分细胞活力下降,50 μg/mL DMSA-FeNPs未对细胞活力产生明显抑制,反而显著提高了小鼠巨噬细胞RAW264.7的增殖。巨噬细胞是抵御病原体入侵和激活适应性免疫的一类重要免疫细胞,在肿瘤的发生和发展中起到非常重要的作用。这一发现为研究氧化铁磁性纳米粒子激活巨噬细胞进行免疫治疗提供了依据。 2. 评价纳米材料毒性一个可行的策略是先评价小鼠细胞的毒理学效应,再等效评价人细胞效应。本实验室前期已经评估了DMSA-FeNPs对两种小鼠细胞系的毒性效应,本章则在于评价该纳米材料对两种等效的人细胞系(THP-1和HepG2)的毒性影响。取同一批次DMSA-FeNPs对应相同剂量(0、50 μg/mL和100 μg/mL)对细胞处理相同24 h,检测细胞全基因表达谱。分析发现,两种细胞系内分别有几百个DMSA-FeNPs应答基因(FeRGs),且两种细胞中FeRGs大多数是不同的,表明细胞类型特异性效应。通过比较这些FeRGs、注释功能和相关KEGG通路,发现DMSA-FeNPs引起的相同和细胞特异性生物效应。THP-1细胞中,DMSA-FeNPs产生的综合效应是诱导各种反应和抑制蛋白翻译,而HepG2细胞内则表现为促进细胞代谢、生长和迁移。这些效应均体现出纳米剂量依赖性,即高剂量DMSA-FeNPs相对低剂量对细胞的影响更大。通过进一步比较两种人类和两种小鼠相应细胞系中DMSA-FeNPs诱导的共同基因、生物学过程及KEGG通路,发现四种细胞系中Id3基因共同下调。Id3基因是一个氧化还原敏感性信号传导分子,其编码的蛋白还可形成非功能性二聚体与其他转录因子抑制转录,其显著下调表明DMSA-FeNPs可能扰乱了细胞正常的生物学过程,如细胞生长、分化、凋亡及肿瘤发生等。因此,Id3基因可作为DMSA-FeNPs细胞毒性的一个通用和敏感的生物标志物,为探索和鉴定DMSA-FeNPs纳米毒性提供了新的见解。 3. 前面研究中的差异表达基因(DEGs)功能注释(GO)分析发现THP-1细胞中两种剂量下均富集有对病毒的应答反应过程,且C型肝炎病毒通路也被显著富集,而本研究中DMSA-FeNPs和病毒具有相似的粒子结构,暗示THP-1细胞将DMSA-FeNPs类似病毒识别,从而产生类病毒样的细胞免疫效应。本章通过数据深挖,探讨了DMSA-FeNPs对两种免疫细胞(RAW264.7和THP-1细胞)免疫系统的影响,而免疫系统正是细胞用以抗病毒的最重要宿主防御系统。结果表明,DMSA-FeNPs引发了两种细胞类似于病毒的综合免疫反应,主要包括8类先天免疫和3类适应性免疫,并产生大量不同类型的细胞因子。分析还发现几乎一半的DEGs是干扰素刺激基因(ISGs),它们与抗病毒密切相关,如Osa1/2/3/L、Mx1/2编码最典型的抗病毒因子。免疫应答和细胞因子产物均显示剂量依赖性和细胞类型依赖性效应。类病毒免疫效应可能是由于DMSA-FeNPs粒径大小与病毒相近,DMSA表面修饰类似于病毒的蛋白质外壳(称为衣壳)或脂质包膜,DMSA-FeNPs同病毒一样带负电荷,呈类似于许多病毒的单分散球形颗粒形状等因素。实验研究进一步发现,DMSA-FeNPs在体外能激活RAW264.7细胞,显著提升该细胞生长活力,迁移力,攻击力,能在体外有效杀死小鼠肝癌细胞Hepa1-6。因此,本研究首次在基因转录水平上报道了DMSA-FeNPs对细胞的免疫应答系统效应,挖掘DMSA-FeNPs自身在肿瘤免疫治疗中的潜在应用,为其细胞生物效应和潜在的分子机制提供了新见解,促进了具有良好免疫特性的新纳米材料的设计开发,并推动了DMSA-FeNPs自身作为免疫治疗药物的研究与应用。 4. DMSA已经被广泛用于对FeNPs的修饰,可以显著改善FeNPs的稳定性,生物相容性,细胞内化和生物学分布。尽管如此,我们注意到DMSA修饰氧化铁纳米颗粒后,携带大量还原性巯基进入细胞内,其潜在的细胞毒性还有待充分阐明。本章以八个含有大量二硫键的关键词在NCBI数据库中进行搜索,尽可能找到所有富含半胱氨酸蛋白(CRP)编码基因。通过比对筛选出由三个剂量(30、50和100 μg/mL)DMSA-FeNPs处理三个时间点(4、24和48 h)的RAW264.7细胞中的编码富含半胱氨酸蛋白的差异表达基因(CRP-DEGs)。结果显示,每种处理下均有约四分之一的基因编码CRP,表明CRP基因(如锌指蛋白基因)的表达受到显著影响。进一步分析发现,所有CRP-DEGs中约31%的基因编码酶,表明该DMSA-FeNPs极大地影响了酶基因的表达。GO分析表明,DMSA-FeNPs诱导各种应答、免疫活性和细胞凋亡等生物学过程,分子功能则主要与铁离子结合有关。同时,在Hepa1-6、THP-1和HepG2这三种细胞系中也发现类似效应。为了确证CRP基因差异表达源自修饰分子DMSA,本研究采用定量聚合酶链式反应(qPCR)同步检测了DMSA-FeNPs、聚醚酰亚胺(PEI)修饰的Fe3O4磁性纳米粒子(PEI- FeNPs)及纯DMSA分子对部分典型CRP-DEGs表达的影响。结果发现该DMSA-FeNPs对CRP基因表达的影响主要是由DMSA-FeNPs带入细胞内的DMSA造成的。本研究首次报道DMSA作为FeNPs纳米表面修饰分子在基因转录水平上的细胞毒性效应,为DMSA-FeNPs显著影响CRP基因差异表达提供了新的分子机制,为纳米材料表面修饰分子的生物相容性评价提供了新思路。
英文题目 MAGNETIC IRON OXIDE NANOPARTICLES INTERACTION WITH CELLS TO REGULATE GENE EXPRESSION
英文主题词 Magnetic iron oxide nanoparticles, gene expression profile, immunization, virus-like effect, DMSA
英文摘要 Magnetic iron oxide nanoparticles (FeNPs) have great potential applications in biomedical field because of good biocompatibility. At present, some iron oxide nanoparticles have been used as magnetic targeting drug carriers, iron supplements, magnetic resonance imaging (MRI) contrast enhancers, cell markers and so on. Therefore, it is significant to study their nanotoxicity. The potential nanotoxicity of FeNPs has been gradually recognized and studied mainly at the cellular level including internalization and the ways into cell, the effects on cell ultrastructure, growth, and oxidative stress. With the development and maturation of GeneChips technology, gene expression of the genome can be detected completly. Cells can promptly regulate its gene expression profile in response to any changes triggered by nanoparticles. Selecting out all different expression genes induced by a nanomaterial at the cell or tissue levels can provide valuable clues to explore any potential toxicity and investigate the relevant molecular mechanism. Therefore, to evaluate changes of gene expression profiles used high-throughput techniques is much helpful to uncover the potential nanotoxicity of nanomaterials. The current studies show that we should systematically research the effects of size, charge, morphology and surface modification on the nanoparticles, explore their potential molecular mechanisms, and develop and apply them. The nanomaterials are most concentrated in the blood and hepatocytes of the body due to passive targeting after intravenous administration. In this study, two blood cells and two hepatocellular carcinoma cells derived from mouse and the corresponding human were used as the objects of study, respectively, including mouse macrophage RAW264.7, mouse hepatoma Hepa1-6, human acute monocyte THP-1 and human hepatoma HepG2. Four kinds of cells were treated with different doses of dimercaptosuccinic acid (DMSA) modified Fe3O4 nanoparticles (DMSA-FeNPs). Their gene expression profiles were detected by GeneChips. The effect of DMSA-FeNPs on the gene expression profile, the potential molecular mechanisms and possible applications was explored by data in-depth analysis combined with experimental verification. The main works of this study were as follows: 1. The characteristics of DMSA-FeNPs, the cell internalization and viability were detected by transmission electron microscopy (TEM), Malvern particle size analyzer (DLS), infrared spectroscopy, Prussian blue staining, and CCK-8 assay. DMSA-FeNPs had good dispersibility and stability in solution with an average particle size of about 11 nm and the hydrodynamic size of 64 nm observed by TEM and DLS, respectively. Prussian blue staining showed that DMSA-FeNPs were uptaked in four mammalian cell lines. The ability of phagocytosis was the highest for macrophage RAW264.7 cells, while lowest for THP-1 cells. DMSA-FeNPs were transported and located in the cytoplasm with vesicles coating through TEM. DMSA-FeNPs caused the viability of some cells decline when treated with 100 μg/mL, while no obvious effects at dose of 50 μg/mL. Furthermore, the proliferation of RAW264.7 cells with 50 μg/mL treatment was significantly increased. Macrophages are a class of important immune cells that fight against pathogen invasion and activate adaptive immunity, playing a very important role in the development and progression of tumors. This finding provides a basis for studying the immunotherapy of DMSA-FeNPs by activation of macromonocytes. 2. A viable strategy to assess the toxicity of nanomaterials is to evaluate the nanotoxicity with human cells and their mouse equivalents. The toxic effects of DMSA-FeNPs on two mouse cell lines have been detected earlier in our laboratory. The toxicity of DMSA-FeNPs to two equivalent human cell lines (THP-1 cells and HepG2 cells) was appraised at the same two doses (50 and 100 μg/mL) for the same time (24 h). The global profile of each treatment was abtained through GeneChips. Analysis revealed that there were hundreds of DMSA-FeNPs response genes (FeRGs) in the two cell lines, most of which were different, indicating cell type-specific effects. By comparing these FeRGs, annotation functions, and related KEGG pathways, DMSA-FeNPs were found to cause general and cell-specific effects. DMSA-FeNPs induced various responses and inhibited protein translation in THP-1 cells, whereas promoted cell metabolism, growth and migration in HepG2 cells. These effects all showed a dose-dependent, which means that the high dose of DMSA-FeNPs had a greater effect on the cells. The common genes, biological processes and KEGG pathways induced by DMSA-FeNPs were still compared in four cell lines. It is found that Id3 gene was commonly down-regulated in four cell lines.The Id3 gene is a redox-sensitive signal transduction molecule, the protein which encoding can form non-functional dimers with other transcription factors to inhibit transcription. Its significant down-regulation indicated that DMSA-FeNPs might disrupt cellular normal biological processes, such as cell growth, differentiation, apoptosis and tumorigenesis. Therefore, Id3 gene can be used as a universal and sensitive biomarker of DMSA-FeNPs cytotoxicity, which provides new insights into the explotation and identification of nano-toxicity of DMSA-FeNPs. 3. Previous study found that the biological processes of response to the virus and the pathway of hepatitis C virus were significantly enriched in THP-1 cells treated with DMSA-FeNPs. The particles had similar particle structure with virus, suggesting that DMSA-FeNPs were recognized like virus, resulting in virus-like cellular immune effects. The effects of DMSA-FeNPs on the immune system of two immune cells (RAW264.7 and THP-1 cells) were investigated through data mining in depth, which is the most important host defense system for cell antivirus. The results show that DMSA-FeNPs triggered the immune response like viral in the two kinds of cells, mainly including 8 innate immunity pathways and 3 adaptive immunity pathways, and producing a large number of different cytokines. Thereamong, almost half of the DEGs were found to be interferon-stimulating genes (ISGs), which are closely related to anti-virus. For example, proteins encoded by Osa1/2/3/L, Mx1/2 are the typical antiviral factors. Many cytokines were induced production. Both the immune response and cytokine products showed dose-dependent and cell-type-dependent effects. The virus-like immunoactivation effect may result from the size of the DMSA-FeNPs, DMSA coating, negative charge, or monodisperse spherical particles similar to that of the virus. Experimental study found that DMSA-FeNPs could activate RAW264.7 cells. DMSA-FeNPs significantly promoted cell viability, mobility and the attack power. RAW264.7 cells surrounded and effectively killed Hepa1-6 cells in vitro. Therefore, this study first reported the systematic immune response of DMSA-FeNPs at the level of gene transcription and the potential application of DMSA-FeNPs itself in tumor immunotherapy, which sheds new insights into its cellular biological effects and potential molecular mechanisms and promotes the design and development of new nanomaterials with good immune properties or useful nanoimmunoassay. Furthermore, the study is developing application of DMSA-FeNPs itself as immunotherapy drug in clinic. 4. DMSA has been widely used in the modification of FeNPs, which can significantly improve the stability, biocompatibility, intracellularization and biological distribution of FeNPs. Nevertheless, a large number of reductive thiol groups were carried into cells once DMSA modified on the surface of FeNPs. The potential cytotoxic remains to be fully elucidated. The study searched the NCBI database with eight keywords containing large amounts of disulfide bonds to find all genes that encoding cysteine-rich protein (CRP) as much as possible. At three time points (4, 24 and 48 h) treated with three doses (30, 50 and 100 μg/mL) of DMSA-FeNPs in RAW264.7 cells, DEGs encoding cysteine-rich proteins (CRP-DEGs) were screened through comparision DEGs with genes coding CRP. The results demonstrate that about quarter of DEGs encoded CRP in each treatment, indicating that DMSA-FeNPs significantly affected the expression of CRP gene (such as gene encoding zinc finger protein). Furthermore, about 31% of all CRP-DEGs encoded enzymes, suggesting that the DMSA-FeNPs significantly affected the expression of the enzyme genes. GO analysis showed that DMSA-FeNPs induced various biological processes, such as response, immunological activity and apoptosis, while the molecular function was mainly related to iron ion binding. The similar effects were found in the three other cell lines, Hepa1-6, THP-1 and HepG2. To confirm the different expression of CRP genes resulted from the modified molecule DMSA, the study simultaneously detected the expression of some typical CRP-DEGs in the cells treated by DMSA-FeNPs, Fe3O4 nanoparticles coating with polyethyleneimine (PEI), and pure DMSA through quantitative polymerase chain reaction (qPCR). The results display that the effect of DMSA-FeNPs on CRP gene expression was mainly caused by DMSA into the cells. This study first reported that the cytotoxic effect of DMSA as surface modifier of DMSA-FeNPs at the gene transcription level, shedding a new molecular mechanism for the significant effects of DMSA-FeNPs on the differential expression of CRP gene. The study also provides a new insight for the biocompatibility evaluation of surface modification molecules of nanomaterials.
学术讨论
主办单位时间地点报告人报告主题
东南大学生物电子学国家重点实验室 2013.11.1 电子所楼216 会议室 张玲 纳米制备实验方案设计
东南大学生物电子学国家重点实验室 2014.5.12 电子所楼216 会议室 张玲 两种人细胞基因表达谱数据分析
东南大学生物电子学国家重点实验室 2015.11.23 电子所楼216 会议室 张玲 细胞活力检测实验
东南大学生物电子学国家重点实验室 2016.1.22 电子所楼216 会议室 张玲 伤愈实验
东南大学生物电子学国家重点实验室 2016.12.26 电子所楼216 会议室 张玲 细胞混合培养实验
东南大学生物电子学国家重点实验室 2017.2.27 电子所楼216 会议室 张玲 Transwell实验
东南大学生物电子学国家重点实验室 2017.3.13 电子所楼216 会议室 张玲 细胞流式分析
东南大学生物电子学国家重点实验室 2017.4.24 电子所楼216 会议室 张玲 类病毒论文修改
     
学术会议
会议名称时间地点本人报告本人报告题目
3rd internantional conference on advanced composite materials and manufacturing 2015.5.30-2015.5.31 北京 Preparation of CB-load Polyacrylamide nanoparticles and their cellular internalization
5th Euro Biosensors & Bioelectronics Conference 2016.6.30-2016.7.2 西班牙 DMSA-coated Iron Oxide Nanoparticle Greatly Affect the Expression of Genes Coding Cysteine-rich Proteins by its DMSA Coating
     
代表作
论文名称
DMSA-Coated Iron Oxide Nanoparticles Greatly Affect the Expression of Genes Coding Cysteine-Rich Pro
Effects of an 11-nm DMSA-coated iron nanoparticle on the gene expression profile of two human cell l
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
张云 正高 教授 博导 南京军区军事医学研究所 医学
马飞 正高 教授 博导 南京师范大学 生物学
周昕 正高 教授 博导 扬州大学 纳米生物医学
肖鹏峰 正高 教授 博导 东南大学 生物医学工程
万遂人 正高 教授 博导 东南大学 生物医学工程
      
答辩秘书信息
姓名职称工作单位备注
李敏俐 副高 副教授 东南大学 生物医学工程