毛兰素抗肿瘤活性研究进展

毛兰素是从石斛中提取的天然产物,近年来发现毛兰素在体外和体内表现出抗癌活性,诱导多种肿瘤细胞生长抑制。目前报道的毛兰素抗肿瘤效应与多个信号通路相关。通过激活细胞凋亡的内部线粒体途径和死亡受体介导的外部凋亡途径诱导凋亡;通过ROS/JNK信号通路诱导细胞周期G2/M期停滞,凋亡和自噬;通过调节促迁移蛋白和迁移抑制蛋白的表达抑制肿瘤细胞的迁移;通过阻断JAK2/STAT3的磷酸化抑制血管生成等。本文针对抗肿瘤活性的研究进行简要综述。     

CRIP1生物学功能的定量蛋白质组学分析

CRIP1（cysteine-rich intestinal protein 1）是含有双锌指结构域的蛋白,在很多肿瘤细胞中高表达,但其在肺癌细胞中的生理学功能尚不明确。本研究应用定量蛋白质组学探究CRIP1过表达对肺癌顺铂耐药细胞（A549/DDP）的影响及作用机制。运用慢病毒载体系统构建了CRIP1过表达的A549/DDP稳转细胞系,利用Western Blotting证实了单克隆细胞系中CRIP1的过表达。发现CRIP1过表达能够加快细胞增殖,增加细胞的耐药性。通过定量蛋白质组学分析,鉴定了CRIP1过表达引起的蛋白质组的变化,并且对上调和下调的蛋白进行聚类分析。结果表明,CRIP1过表达上调了烟酰胺磷酸核糖转移酶（NAMPT）和NAD依赖型氧化还原酶的表达,从而促进细胞的增殖,并且提高了细胞的耐药性。     

蚯蚓对三种芳烃有机磷阻燃剂的毒性响应效应

考察了赤子爱胜蚓(Eisenia foetida)的酶活性对三种芳烃类有机阻燃剂(aryl-OPEs)的毒性响应效应。结果表明:SOD、CAT活性和MDA含量对磷酸三苯酯(TPP),磷酸三甲苯酯(TCP),磷酸-2-乙基己基二苯酯(EHDP)的毒性响应各不相同,其中,SOD活性被TCP和EHDP诱导增加,而在TPP胁迫下,先增加后降低。CAT活性在TPP、TCP和EHDP低浓度下抑制高浓度下诱导。MDA含量在EHDP胁迫下,持续增加,而在TCP和TPP低浓度下抑制高浓度下诱导。     

苦瓜抗HIV活性蛋白及其聚乙二醇修饰研究进展

苦瓜籽含有许多具有抗HIV活性的核糖体失活蛋白,例如α-苦瓜素,β-苦瓜素和MAP30,它们还分别具有堕胎、抗肿瘤活性、抗乙肝病毒活性等。本文从结构、功能、抗HIV分子机制、与其它病毒作用、与其它药物相互作用等方面总结了MAP30蛋白的研究,并根据现有的聚乙二醇修饰方法对MAP30修饰研究提出了展望。     

三维培养结肠癌细胞的微流控芯片荧光成像研究

改良了一种微流控芯片,可用于对结肠癌细胞进行三维培养并实现实时荧光成像。在结肠癌细胞内植入内源性的红色荧光蛋白,使用激光共聚焦显微镜对芯片中三维培养的细胞进行成像。通过细胞内部红色荧光蛋白的表达,可以观测到细胞的生长状态,实现对细胞的实时监测和高分辨率荧光成像。同时,通过免疫荧光染色来表征反映细胞活性的特征蛋白,其荧光强度和蛋白表达呈正相关。研究结果提示,细胞活性相关蛋白的表达受到微环境的影响,其在芯片三维培养中的活性强于二维培养,表明芯片内环境更加接近真实的人体微环境。该方法为进一步探究肿瘤细胞转移机制及相关药物的筛选研究提供了一种新的技术手段及实验平台。     

肝癌细胞外泌体蛋白组成分析

外泌体是细胞分泌的一种大小40-130nm的双层膜结构的微小囊泡。近年来,研究发现外泌体可以携带蛋白质,DNA,RNA,mi RNA和脂类物质等,通过对这些物质的运输,介导细胞与细胞之间,组织与组织之间的相互作用,成为新的信号传递载体。并且最近发现,肿瘤细胞分泌的外泌体可以率先改变靶组织的微环境,进而介导肿瘤细胞的迁移,实现癌症的转移。因此对肿瘤细胞的外泌体蛋白分析非常重要,本文首次分析了肝癌细胞的外泌体蛋白组成,为研究肝癌细胞外泌体的作用和机制,以及肝癌转移打下基础。     

Hela细胞增殖抑制模型结合离心超滤质谱筛选中药抗癌药物

采用基于MTT比色法的Hela细胞增殖抑制模型评价中药提取物的抗癌活性,并利用离心超滤质谱方法对活性最强的中药提取物中抗癌活性成分进行筛选和鉴定。结果表明,长春花提取物对Hela细胞增殖的抑制活性最强,与阳性对照药阿霉素相当,且抑制活性与给药浓度呈正相关。延胡索提取物在3个不同给药浓度下也可以显著抑制Hela细胞的增殖。三尖杉、黄连和喜树果提取物在高浓度时能够显著抑制Hela细胞的增殖,而刺五加提取物则没有明显抑制作用。利用离心超滤质谱从长春花提取物中筛选得到2种存活素基因启动子片段结合剂和6种钙调蛋白结合剂,经多级串联质谱分析鉴定,分别为蛇根碱、鸡骨常山碱、它波宁、环氧长春碱、长春新碱和21’-氧代环氧长春碱。本研究可为中药抗癌药物的筛选提供一种简便、快速的方法。     

三种苯基有机磷阻燃剂对蚯蚓的氧化胁迫效应

以赤子爱胜蚓(Eisenia foetida)为受试生物,通过滤纸接触法研究磷酸三甲苯酯(TCP)、磷酸-2-乙基己基二苯酯(EHDP)和磷酸三苯酯(TPP)三种污染物对蚯蚓的毒性效应。结果显示:随着TCP暴露浓度的增加,蚯蚓体内总超氧化物歧化酶(SOD)呈现先上升后逐渐下降的趋势,低浓度(0.0001 mg/cm2)时被显著诱导,高浓度(0.1 mg/cm2)时被显著抑制。EHDP和TPP随浓度增加,SOD活性均被刺激。TCP、EHDP、TPP胁迫下,蚯蚓体内过氧化氢酶(CAT)活性随浓度增加呈现上升趋势,依次在0.01 mg/cm2、0.001 mg/cm2、0.1 mg/cm2时,与对照相比,表现出显著差异。TCP胁迫下,蚯蚓体内丙二醛(MDA)呈现先下降后上升的趋势,而EHDP和TPP随浓度增加,MDA均被诱导,而TPP0.001 mg/cm2,MDA被持续诱导且作用更为显著。     

亲和超滤质谱技术在中药活性成分筛选中的研究进展

亲和超滤质谱技术是20世纪90年代中期发展起来的一种快速、简单、有效的药物小分子发现模式。该技术利用配体与受体之间特异性结合,通过超滤装置快速筛选活性小分子化合物,再结合液相色谱 质谱联用技术（LC-MS）,鉴定活性成分结构。亲和超滤质谱技术集药物活性成分筛选、结构鉴定于一体,尤其适用于从复杂体系中筛选潜在的活性小分子化合物。近年来,针对中药发挥药理作用具有多组分、多靶点的重要特点,亲和超滤质谱技术已被广泛用于从中药提取物中筛选与特定蛋白靶点相结合的小分子活性物质,对阐明中药药效的物质基础和以活性成分作为先导化合物的新药开发具有重要意义,是对传统药物发现方法的有利补充。本工作综述了该技术在中药活性成分筛选中的原理、特点、应用进展,以及对未来的展望。     

脂质质谱成像揭示三氯生促进三维肝癌肿瘤细胞球增长的相关机理

三氯生(triclosan, TCS)作为一种广谱抗菌剂被广泛应用于多种日常消费品中。当前研究表明，TCS暴露可以促进肝癌肿瘤增长，但涉及的相关脂质代谢机理尚不完全清楚。基于此，本研究建立了TCS暴露的肝癌肿瘤细胞球模型，利用质谱成像技术分析对照组和暴露组间脂质小分子在肿瘤细胞球内丰度和分布的差异。结果表明，6μmol/L TCS暴露可以显著促进肝癌肿瘤细胞球的增长，并引起脂质代谢的变化。27种脂质代谢小分子(含19种甘油磷脂、3种甘油酯和5种鞘脂)的丰度在对照组和暴露组的肿瘤细胞球内发生了显著性的变化。在暴露组的19种甘油磷脂中，16种在肿瘤细胞球外围增殖区发生了显著性上调；在3种甘油酯中，1种在外围增殖区发生了显著性上调，2种在内部凋亡区发生了显著性下调；5种鞘脂在肿瘤细胞球内部凋亡区均发生了显著性下调。可见，TCS可能是通过促进肿瘤细胞球外围细胞的增殖以及抑制内层细胞的凋亡来促进三维肿瘤细胞球的增长。该结果可为进一步探讨环境污染物对肿瘤发展影响的分子机制提供参考。     

Proteins as biomarkers of oxidative/nitrosative stress in diseases: the contribution of redox proteomics

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) contribute to the pathogenesis and/or progression of several human diseases. Proteins are important molecular signposts of oxidative/nitrosative damage. However, it is generally unresolved whether the presence of oxidatively/nitrosatively modified proteins has a causal role or simply reflects secondary epiphenomena. Only direct identification and characterization of the modified protein(s) in a given pathophysiological condition can decipher the potential roles played by ROS/RNS-induced protein modifications. During the last few years, mass spectrometry (MS)-based technologies have contributed in a significant way to foster a better understanding of disease processes. The study of oxidative/nitrosative modifications, investigated by redox proteomics, is contributing to establish a relationship between pathological hallmarks of disease and protein structural and functional abnormalities. MS-based technologies promise a contribution in a new era of molecular medicine, especially in the discovery of diagnostic biomarkers of oxidative/nitrosative stress, enabling early detection of diseases. Indeed, identification and characterization of oxidatively/nitrosatively modified proteins in human diseases has just begun.     

Proteomics analysis of tumor microenvironment: Implications of metabolic and oxidative stresses in tumorigenesis

Tumorigenesis is always concomitant with microenvironmental alterations. The tumor microenvironment is a heterogeneous and complex milieu, which exerts a variety of stresses on tumor cells for proliferation, survival, or death. Recently, accumulated evidence revealed that metabolic and oxidative stresses both play significant roles in tumor development and progression that converge on a common autophagic pathway. Tumor cells display increased metabolic autonomy, and the hallmark is the exploitation of aerobic glycolysis (termed Warburg effect), which increased glucose consumption and decreased oxidative phosphorylation to support growth and proliferation. This characteristic renders cancer cells more aggressive; they devour tremendous amounts of nutrients from microenvironment to result in an ever‐growing appetite for new tumor vessel formation and the release of more “waste,” including key determinants of cell fate like lactate and reactive oxygen species (ROS). The intracellular ROS level of cancer cells can also be modulated by a variety of stimuli in the tumor microenvironment, such as pro‐growth and pro‐inflammatory factors. The intracellular redox state serves as a double‐edged sword in tumor development and progression: ROS overproduction results in cytotoxic effects and might lead to apoptotic cell death, whereas certain level of ROS can act as a second‐messenger for regulation of such cellular processes as cell survival, proliferation, and metastasis. The molecular mechanisms for cancer cell responses to metabolic and oxidative stresses are complex and are likely to involve multiple molecules or signaling pathways. In addition, the expression and modification of these proteins after metabolic or oxidative stress challenge are diverse in different cancer cells and endow them with different functions. Therefore, MS‐based high‐throughput platforms, such as proteomics, are indispensable in the global analysis of cancer cell responses to metabolic and oxidative stress. Herein, we highlight recent advances in the understanding of the metabolic and oxidative stresses associated with tumor progression with proteomics‐based systems biology approaches. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:267–311, 2013     

药物免修饰的药靶鉴定蛋白质组方法研究进展

药物靶蛋白的筛选是现代药物研发过程必不可少的步骤。传统的药物靶点筛选通常使用假设驱动的生物学实验进行药物靶点的逐个验证，近年来高通量、高分辨质谱技术的进步以及液相色谱-质谱联用技术的发展，促进了蛋白质组学技术在药物靶蛋白筛选领域的广泛应用。化学蛋白质组学技术在药物-蛋白相互作用研究领域已取得巨大突破，因其需要在药物小分子结构上进行化学衍生等问题，最近新兴的药物免修饰的靶蛋白筛选技术引起广泛关注。本文综述了近15年来药物免修饰（非化学修饰）的药物靶蛋白筛选方法的研究进展，包括细胞热位移分析（CETSA）、热蛋白质组分析（TPP）、蛋白质氧化速率稳定性分析（SPROX）、基于有机溶剂沉淀蛋白稳定性方法（SIP）、药物亲和相应靶点稳定性分析（DARTS）、脉冲酶解法（PP）、限制性酶解法（LiP）等，并比较了这些方法的优势、局限性以及应用场景。     

Exosomes: Key tools for cancer liquid biopsy

Precision medicine is based on the identification of biomarkers of tumor development and progression. Liquid biopsy is at the forefront of the ability to gather diagnostic and prognostic information on tumors, as it can be noninvasively performed prior or during treatment. Liquid biopsy mostly utilizes circulating tumor cells, or free DNA, but also exosomes. The latter are nanovesicles secreted by most cell types, found in any body fluid that deliver proteins, nucleic acids and lipids to nearby and distant cells with a unique homing ability. Exosomes function in signalling between the tumor microenvironment and the rest of the body, promoting metastasis, immune remodelling and drug resistance. Exosomes are emerging as a key tool in precision medicine for cancer liquid biopsy, as they efficiently preserve their biomarker cargo. Moreover, exosomes strongly resemble the parental cell, which can help in assessing the oxidative and metabolic state of the donor cell. In this respect, exosomes represent one of the most promising new tools to fight cancer. This review will discuss the clinical applications of profiling exosomal proteins and lipids by high-throughput proteomics and metabolomics, and nucleic acids by next generation sequencing, as well as how this may allow cancer diagnosis, therapy response monitoring and recurrence detection.     

Integrating centrosome structure with protein composition and function in animal cells

The centrosome found in animal cells is a complex and dynamic organelle that functions as the major microtubule organizing center. Structural studies over the past several decades have defined the primary structural features of the centrosome but recent studies are now beginning to reveal structural detail previously unknown. Concurrent with these studies has been an explosion in the identification of the proteins that reside within the centrosome. Our growing understanding of how protein composition integrates with centrosome structure and hence with function is the focus of this review.     

Oxidative stabilization of C/C tow pregs studied by light microscopy

Tow pregs obtained from polyacrylonitrile-based carbon fibres and a commercial binder coal-tar pitch were stabilized by oxidation at 548, 588 and 623 K for periods ranging between 1 and 15 h. The microstructural changes involved in the stabilization of tow pregs on the carbonized laminates were studied by light microscopy. Under the conditions studied, the oxidative stabilization of tow pregs produced a significant increase in carbon yield. At the same time, the development of porosity on carbonization was avoided and the size of the optical texture was reduced. However, the extreme oxidation of pitch components at the edges of the carbonized laminate adversely affected pitch binding capability and the sinterizability of the laminates.     

Biochemical applications of mass spectrometry in pharmaceutical drug discovery

Biochemical applications of mass spectrometry (MS) are important in the pharmaceutical industry. They comprise compositional analyses of biomolecules, especially proteins, and methods that measure molecular functions such as ligand binding. In early drug discovery, MS is used to characterize essential reagents and in structural biology. A number of MS-based methods have been proposed for use in high-throughput screening (HTS), but are unlikely to supplant established radiometric and fluorometric methods for this purpose. These methods, which include pulsed-ultrafiltration MS, frontal affinity chromatography-MS, and size-exclusion chromatography-MS, may ultimately be most successful in the post-screening lead development phase. In full development, MS is used heavily in the search for biomarkers that can be used to gauge disease progression and drug action. This review gives equal attention to the technical aspects of MS-based methods and to selective pressures present in the industrial environment that influence their chances of gaining wide application.     

基于灵敏度分析的机械系统损伤识别方法

提出了一种基于振动灵敏度分析的机械系统损伤识别方法。该方法同时考虑了固有频率灵敏度和固有模态灵敏度 ,既可用于损伤定位 ,也能用于确定损伤大小 ,且对单个损伤和多个损伤情况都适用。为了提高识别的精度 ,考虑了二阶灵敏度。针对工程实际的需要 ,分析了不完备模态和模态测量误差对该方法识别精度的影响。算例表明 :本方法合理可靠 ,具有足够的精度。     

质谱在金属抗癌药物与蛋白质相互作用研究中的应用

研究细胞毒性金属抗肿瘤药物与蛋白质的相互作用,以及这种相互作用对药物的细胞摄入、转运、代谢和生物利用度的影响,对金属抗癌药物的结构设计和优化,提高药物的抗癌活性,降低毒副作用具有重要意义。基于软电离技术的电喷雾质谱和基质辅助激光解析电离质谱能够在分析检测过程中很好的保留金属抗癌药物与蛋白质的共价(配位)结合,获得药物与蛋白质结合位点的信息。同时,质谱分析还具有灵敏度高,所需样品量少,耗时短以及适用于分析复杂生物样品等优点,已成为研究金属抗癌药物与蛋白质相互作用最强有力的工具,在为药物发现提供大量化学、生物信息的同时,也极大地促进了质谱技术自身的发展。本文将结合我们在金属抗癌药物相互作用组学研究中取得的最新进展,系统地总结、评述Bottom-up和Top-down质谱分析方法在铂、钌类金属抗癌药物与蛋白质相互作用研究中的发展动态,并分析这一前沿交叉领域未来的发展趋势。