碳包铁纳米粒子作为磁性靶向药物载体的物理性能研究

一种全新的磁靶向纳米药物载体——用直流碳弧法制备的碳包铁纳米粒子,它具有独特的纳米结构,在石墨碳层中完全包裹进铁纳米颗粒,实现在纳米碳包围中的纳米磁性粒子分散状态,其中的纳米碳具有大的比表面积和较高的化疗药物吸附量,达到每毫克吸附160μg的表阿霉素。铁纳米粒子磁场强,靶向效果好,有较佳的磁靶向发热效应,在动物病灶局部放置0.1g碳包铁纳米粒子将能使发热温度平均升到52℃;而将碳包铁纳米粒子均匀与猪肝混合也有明显的产热效果:含量0.4%碳包铁纳米粒子的一组猪肝能将温度升到42℃,而含量0.6%碳包铁纳米粒子的一组猪肝能将温度升到48℃。X射线衍射表明借助于碳的包裹,碳包铁纳米粒子有好的抗氧化性和稳定性。碳包铁纳米粒子有可能作为一种全新的磁性靶向药物载体用于癌症治疗。     

带红色荧光蛋白靶向线粒体载体的构建与表达

选取由核表达的线粒体蛋白细胞色素C氧化亚单位Ⅷ（COX8）的前导序列为靶序列,从人胚胎肺成纤维细胞中扩增出COX8的前导序列,插入到pcDNA3．1／myc—HisA中,并将pDsRED1-n1中的红色荧光蛋白序列RFP克隆至COX8的下游,形成融合蛋白。在脂质体的介导下,将重组载体转染至肿瘤细胞中,在荧光显微镜下观察其在细胞内的表达及分布情况。构建的靶向线粒体的载体以及以红色荧光蛋白为报告基因的靶向线粒体的载体,经酶切、DNA序列测定,结果表明构建正确。将pcDNAmito—RFP转染到HeLa细胞的线粒体中,16h即可见散在荧光,72h达高峰,第10d开始减弱。以上结果表明成功构建了以红色荧光蛋白为报告基因的线粒体靶向的特异表达载体,在靶序列的引导下将红色荧光蛋白输入到线粒体中,为进一步对线粒体疾病的基因治疗研究提供了重要工具。     

蚕豆根端细胞核中微核仁的研究

以蚕豆(Vicia faba)根端分生组织细胞为材料研究了微核仁的超微结构和细胞化学特点。结果表明;微核仁是直径0.3—0.5μm 的卵圆形或球形结构。常规染色时,微核仁与集缩染色质的电子密度相仿,但两者之间在结构上没有任何联系。细胞化学研究指出,微核仁含有 RNA 和蛋白质,其结构成分主要是与核仁颗粒组分十分相似的 RNP 颗粒。报道了植物细胞核中微核仁发生于核仁的过程并对微核仁的本质和功能进行了讨论。     

含RGD修饰的病毒样颗粒递送ICG靶向肿瘤的研究

目的:获取含RGD靶向肽的乙肝核心病毒样颗粒,为药物靶向纳米递送系统提供一种新型载体。方法:将实验室前期构建测序正确的含RGD修饰的乙肝核心病毒重组质粒转化入大肠杆菌BL21(DE3)中,单因素分析及正交试验探究重组蛋白最适表达条件。在最适表达条件下扩培,收集菌体超声破碎后离心,采用凝胶过滤层析、离子交换和蔗糖密度梯度离心进行纯化,利用透射电镜对形成的RGD-HBc VLPs的形态及稳定性进行鉴定。纯化的RGD-HBc VLPs利用其体外自组装的特性,将光敏剂ICG装载到颗粒的内部,通过静脉注射到4T1乳腺癌荷瘤小鼠,探究重组RGD-HBc VLPs作为纳米递送系统的靶向性。结果:RGD-HBc VLPs在温度32℃、IPTG0.5mmol/L、诱导4h时以可溶性蛋白的形式得到高效表达。经蔗糖密度梯度离心纯化后纯度到达95%以上。透射电镜下观察纯化的RGD-HBc VLPs形态、大小均一,直径约为32nm,通过近红外荧光活体成像证实了RGD-HBc作为纳米载体的靶向性。结论:经表达和纯化后,RGD-HBc VLPs具有较高的表达量和大小均一的形态外貌,近红外荧光活体成像证实具有较好的靶向性,这不仅为肿瘤的可视化诊断提供一种快速、精准、方便的方法,而且为今后靶向免疫治疗提供一种新型载体。     

荧光磁性纳米粒子标记间充质干细胞靶向胃癌研究

目的:探索一种高效的早期胃癌诊断体系.方法:荧光磁性纳米粒子与间充质干细胞共培养,不同时间点检测干细胞存活率.利用荧光显微镜、普鲁士蓝染色、透射电子显微镜等方法观测干细胞被标记情况;建立裸鼠的胃癌模型,并将标记后的干细胞尾静脉注射入裸鼠体内,14d后用动物成像仪和核磁共振成像仪检测.结果:荧光磁性纳米粒子在低于 100μg/mL浓度下对间充质干细胞没有毒性作用,荧光磁性纳米粒子与干细胞共培养6h后,荧光显微镜、普鲁士蓝染色、透射电子显微镜检测结果显示粒子被内吞后定位于细胞质中,动物实验显示通过检测肿瘤部位的荧光信号和核磁信号,被标记的干细胞能够准确定位到胃癌的发生部位.结论:这种双模式检测体系为胃癌的早期诊断与治疗提供一种新的方法.     

基于鞣质的碳量子点荧光探针测定西咪替丁的研究

本研究以鞣花酸为原料,在碱性条件下,采用便捷的超声法制备了碳量子点(CQDs),并用紫外光谱法和荧光光谱法研究了该CQDs与西咪替丁之间的相互作用,研究发现以该CQDs为荧光探针可以测定西咪替丁(CMTD)的含量。研究结果：鞣花酸(0.089 4 mol/L)与氢氧化钠溶液(0.100 0 mol/L)以1:3的比例混合后,在50℃下超声4 h得出的荧光碳量子点与西咪替丁结合,可以使碳量子点的荧光信号减弱。其浓度在1×10^-8～1×10^-7mol/L范围内与碳量子点荧光信号强度的减少值ΔF成良好的线性关系,相关系数r为0.996 42。以此提出用鞣质的碳量子点荧光探针测定西咪替丁的研究,该方法简便高效,可应用于样品中西咪替丁含量的测定,S_RSD范围为0.86%～1.06%;回收率范围为98.18%～103.2%。     

正常造血细胞的核仁组成区计数定量研究

本文应用银染技术对正常造血细胞的核仁组成区进行了计数定量研究。结果显示,在粒系和红系中,随细胞的成熟,细胞中簇状ＡｇＮＯＲ的数量逐渐减少．而点状ＡｇＮＯＲ数量逐渐增多,无分裂能力的成熟细胞仅有少数点状ＡｇＮＯＲ。淋巴细胞中为一完全集结的银染颗粒,而巨核细胞内为各自分离的点状银染颗粒。本结果为正常造血细胞的核仁组成区提供了基础数值。     

响应面法优化微波辅助提取山苍子核仁油的研究

通过微波辅助提取技术结合响应面法优化山苍子核仁油提取条件,以期建立更高产率的提取方法。该研究在单因素设计基础上,选取液料比、微波功率、萃取时间、萃取温度4个主要因素,分析这4个因素对山苍子核仁油提取率的影响。结果表明:通过建立多元回归拟合分析,得出山苍子核仁油提取最佳工艺条件为液料比1∶16,萃取温度为69℃,微波功率为337 W,萃取时间为63 min,在此条件下山苍子核仁油提取率为37.42%,与环己烷溶剂回流法相比较提取率提高了30.11%。气质联用仪分析结果显示,山苍子核仁油主要成分有16种占总成分的88.21%,鉴定出10种脂肪酸占总成分的78.24%,饱和脂肪酸有4种占总成分的43.23%,不饱和脂肪酸有6种占总成分的35.01%,脂肪酸中含量最高的为月桂酸(31.36%)。该研究结果表明该方法严谨、可靠,采用微波辅助提取山苍子核仁油是可行的。     

综述——新型纳米生物材料的研发：稀土上转换荧光纳米材料的制备与生物应用

近几年,稀土上转换荧光纳米材料作为新型的荧光探针受到研究者的广泛关注,其优势在于光化学稳定性好、发射谱带窄、荧光寿命长、Stokes位移大等．同时,它利用近红外激光器作为激发光源,组织穿透能力好、对生物组织的损伤小、几乎没有背景荧光,使其应用于生物活体荧光成像成为可能．本文主要综述了最近稀土上转换荧光纳米材料在制备与生物应用方面的研究进展．     

基于磁性纳米材料的肿瘤靶向治疗研究进展

磁性纳米材料具有独特的磁学性质，可响应外磁场，产生力、热等效应。如在静磁场下将药物磁靶向递送至肿瘤部位；低频交变磁场下可将纳米药物主动渗透至病灶部位，实现瘤内均一分布；中频交变磁场作用下磁滞损耗产生热和增强的活性氧，用于肿瘤治疗。磁性纳米材料同时具有尺寸依赖的磁学性质以及表面多功能化等特点，可将磁靶向、分子靶向以及磁热疗联合。此外，磁性纳米材料具有磁共振成像性能以及纳米酶催化特性，使其在肿瘤诊疗一体化治疗方面获得了广泛应用。近年来，纳米给药系统不断被优化，基于磁性纳米材料的肿瘤靶向治疗也得到了长足的发展。鉴于此，本文围绕提高靶向肿瘤治疗效果，从磁靶向药物治疗、被动靶向磁热疗和主动分子靶向磁热疗、纳米酶特性以及诊疗一体化应用等几方面出发，综述了基于磁性纳米材料的肿瘤靶向治疗研究进展。     

核仁和核仁蛋白

核仁是位于细胞核内的非膜结构。电子显微镜下的核仁从形态上可以分为三层结构包括纤维中心区(FC)、高密度纤维区(DFC)和颗粒区(GC)。核仁内的蛋白有核糖体蛋白和非核糖体蛋白两种。利用蛋白质组学方法已经鉴定了350多种核仁蛋白,其中包括80多种核糖体蛋白。核仁是核糖体合成的场所,核仁中的非核糖体蛋白对核糖体的生物合成起关键调控作用。核仁不仅是细胞内通讯和核糖体：RNA加工的中心,而且在细胞周期、细胞增殖和衰老中起重要调控作用;核仁也是tRNA、mRNA和其它类型小分子RNA加工的场所。因此核仁是一个多功能的细胞生命活动中心。     

荧光铜纳米簇介导的生物传感器的研究进展

荧光铜纳米簇(Fluorescent copper nanoclusters,CuNCs)是以脱氧核糖核酸链(Deoxyribonucleic acid,DNA)为模板,以二价铜离子(Cu2+)、抗坏血酸等为反应物生成的铜晶体,纳米级大小,其具有荧光性,可以作为生物传感器输出信号的一种方式。荧光铜纳米簇的生成快速、简便、安全,因此近年来涌现出很多关于荧光铜纳米簇原理和应用方面的研究。从支持传感器工作的介导物质以及信号输出方式两方面对荧光铜纳米簇进行分类,详细阐述了每一类别传感器工作的原理,并对比同类型传感器的优缺点,最后对荧光铜纳米簇介导的生物传感器目前存在的不足及今后的发展趋势进行了展望。以便读者对荧光铜纳米簇生物传感器发展历程和方向,对荧光铜纳米簇生物传感器的实用性和多形性有所了解,在未来的研究发展中得到启示,使荧光铜纳米簇成为一种更加实用和便捷的生物传感工具。     

肿瘤细胞的标记及其活体荧光成像

以绿色荧光蛋白(GFP)作为标记基因转入人类肺癌细胞系（ASTC-a-1）,经800 mg/L G418筛选,获得5株高表达细胞系.利用流式细胞仪对GFP表达的稳定性进行了初步研究,结果表明本实验中有些细胞株间GFP表达稳定性有显著差异（P＜0.01）.将稳定表达的细胞系（3＃）植入裸鼠皮下,成瘤后用氩离子激光器产生的488 nm蓝光经扩束后直接激发,瘤体部位发出强烈的绿色荧光.用530 nm长通滤色片滤除激发光,数码相机记录荧光的分布情况.实验尝试利用激光作为激发光源,检测GFP标记的肿瘤细胞在裸鼠中的定位,期望建立一种新的肿瘤早期检测技术并改进肿瘤转移研究的手段,实验取得了阶段性进展.     

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.¹ In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.² Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo ³ and intracellular sodium probes ⁴ do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.^5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.^7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. ¹⁰ This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.⁶ They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.¹¹ Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.³ Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.     

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. ¹ and ² for further information on VNPs).From a materials scientist''s point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods ³. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level⁴, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs ^5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics ^8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect¹². The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.     

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T₁ and T₂-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.     

洋葱细胞核仁内DNA的结构变化及排布过程(英文)

以洋葱 (AlliumcepaL .)细胞为研究材料 ,应用DNA细胞化学特异染色方法 (NAMA_Ur)及常规电子显微镜技术 ,观察了洋葱细胞核仁FC(纤维中心 )内DNA的超微结构 ,发现FC内DNA存在着一个介于集缩到解集缩之间的变化过程 ,并揭示了DNA在核仁内的连续排布过程 ,即核仁外DNA经过核仁通道进入到FC后 ,继续沿FC的边缘或DFC(致密纤维成分 )与FC的交界处环绕FC而排布 ,再经FC之间的核仁通道 ,延伸到另外的FC区域