基于代谢组学的细胞内源性代谢物研究进展

代谢组学是继基因组学、转录组学和蛋白质组学之后新兴的一种定性和定量分析复杂生物样品中所有小分子代谢物的组学方法,也是目前组学研究领域的热点之一。代谢组学主要考察生物体系受刺激或扰动后内源性代谢物的变化,是系统生物学的有机组成部分。近年来在代谢组学研究中,细胞内源性代谢物的研究已取得了很大进展。本文结合代谢组学的基本含义,综述了细胞样品前处理方法及代谢靶向分析、代谢轮廓分析、代谢组学分析和代谢足迹分析在细胞方面的研究进展。     

气相色谱-质谱联用结合代谢组学方法研究不同极化状态下小胶质细胞的代谢差异

目的 运用代谢组学方法阐明经典激活型(M1型)、选择活化型(M2型)和静息态小胶质细胞的代谢差异。方法 将体外培养小鼠小胶质细胞系(BV2)细胞,分为M1组、M2组和静息态组,用实时荧光定量聚合酶链反应(qRT-PCR)检测特异性mRNA的表达差异以确定细胞极化状态,采用基于气相色谱-质谱联用(GC-MS)技术的代谢组学方法阐明代谢变化。结果 发现M1型与静息态细胞的差异代谢物15个,M2型与静息态细胞的差异代谢物15个。结论 通过代谢组学方法可以找到小胶质细胞极化的差异代谢物,并解释其可能的极化机制,为神经退行性疾病的防治提供了理论依据。     

代谢组学在中药研究和抗肿瘤研究中的应用进展

［摘要］代谢组学是近年来快速发展的“组学”,是代谢控制论提升的学科,是评价细胞和体液的内源性和外源代谢物浓度与功能关系的学科。代谢组学是后基因时代的一门新兴的独立学科。该学科的应用跨越生物技术和医药技术,具有广阔发展前景。代谢组学与药物药效和毒性筛选及评价研究和安全性评价、作用机制研究与合理治疗用药密切相关。从代谢组学技术用于中药研究的可行性、代谢组学的整体观念与中药作用的整体观念的一致性、代谢组学应用于恶性肿瘤的研究等3个方面阐述代谢组学与中药研究及抗肿瘤研究的关系,为代谢组学在中药研究和恶性肿瘤诊断方面的应用及其机制研究提供新的研究方法和思路。     

代谢组学在白血病中的研究进展

代谢组学是对生物系统（细胞、组织、器官、生命体）中所有内源性小分子代谢物进行定性和定量分析的一门新学科,通过研究代谢物和代谢通路改变与生理病理变化之间的规律,揭示疾病的代谢本质。白血病是一种常见的血液系统恶性肿瘤,它的发生发展与代谢密切相关,代谢组学发现的内源性小分子代谢物已经广泛应用于白血病的发病机制、诊断和药效机制研究并取得了显著进展。综述近年来代谢组学在白血病发病机制、诊断和药效机制方面的研究进展,以期为进一步研究提供参考。     

基于代谢组学技术的肺热证候模型评价方法研究

目的建立一套完善的中医证候动物模型的评价方法。方法以肺炎链球菌诱导的肺热证候模型为切入点,在常规模型评价方法的基础上,进一步借助代谢物组学平台,通过对正常组和模型组大鼠尿液的代谢指纹数据及模型组不同时间段尿液的代谢物组进行分析,探讨动物模型的评价方法。结果大鼠在经鼻给予肺炎链球菌后,模型组体温变化和白细胞计数与正常组比较差异有统计学意义（P〈0．01）,肺病理改变明显;代谢组学结果显示,模型组大鼠尿液的代谢轮廓发生改变并且在得分图中代谢物组发生明显偏移,代谢组学研究结果与肺热证候的宏观体征及病理生化指标结果相符。结论代谢组学可用于中医证候动物模型的评价研究。     

过敏性鼻炎患者血液代谢组学研究

目的：采用代谢组学方法分析过敏性鼻炎患者血清中小分子代谢物的变化。方法：采用气相色谱-质谱联用技术（GC-MS）检测20例过敏性鼻炎患者（病例）和同期20例健康者（对照组）血清中代谢物,采用正交偏最小二乘法（OPLS）的方法,观察病例组和对照组的代谢谱变化,并寻找有差异的小分子代谢物。结果：对照组和病例组的代谢谱能明显区分,鉴定了23个差异代谢物,并对差异代谢物进行了ROC曲线绘制,计算ROC曲线下的面积,得出曲线下面积大于0.5的物质有甘油酸、丝氨酸、苏氨酸、甲硫氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、色氨酸和十九烷酸。结合代谢途径分析,发现过敏性鼻炎患者体内存在丙氨酸、天冬氨酸和谷氨酸代谢,甘氨酸、丝氨酸和苏氨酸代谢,半胱氨酸和甲硫氨酸代谢和色氨酸代谢异常。结论：基于GC/MS的代谢组学技术能全面客观地反映过敏性鼻炎患者体内血液中的代谢物变化,小分子代谢物分析及代谢途径探讨将为过敏性鼻炎的诊断和发病机制研究提供一个可借鉴的思路和手段。     

福多司坦干预炎症微环境下肺癌细胞的代谢组学研究

本研究借助GC-MS代谢组学技术研究福多司坦干预炎症微环境下肺癌A549细胞的作用机制。代谢组学结果鉴定出苹果酸、异亮氨酸、乳糖、肌醇半乳糖苷二水合物、肌酸酐、葡萄糖醛酸、油酸、磷酸、S-(羧甲基)-L-半胱氨酸、塔格糖和尿苷共11个内源性代谢物可作为炎症微环境状态下肺癌A549细胞的药效生物标志物。通路富集分析显示主要包括异亮氨酸、缬氨酸、亮氨酸、甘氨酸、丝氨酸和苏氨酸等氨基酸代谢途径与半乳糖、磷酸戊糖等碳水化合物代谢途径发生明显扰动。福多司坦对炎症状态下肺癌A549细胞有明显降低炎症因子水平与纠正肺癌炎症微环境的作用,潜在机制为对多种氨基酸代谢物及氨基酸等代谢途径的干预作用。该研究揭示了福多司坦有可能通过改善肺癌炎症微环境来扼制炎症的持续反应从而防止肺癌的进一步恶化。     

代谢组学技术在临床诊断中的研究进展

代谢组学是系统生物学的重要组成部分。研究显示,代谢组学在临床诊断过程中有着不可比拟的作用。当机体受到外源性或内源性刺激时,体内小分子代谢物的种类及含量会发生显著改变,代谢组学技术通过对体内复杂代谢物的动态变化进行分析,分析代谢物变化与机体病理或生理变化的直接相关性。随着分析技术的不断发展,核磁共振、色谱-质谱联用等先进技术被广泛应用于代谢组学的研究中。目前临床运用代谢组学技术可以全面分析患者体液中代谢物的动态变化,发现体液中与疾病密切相关的特征性生物标志物,从而实现疾病的早期临床诊断。本文主要对代谢组学技术及其在临床诊断过程中的广泛应用作一综述。     

基于非靶向代谢组学的促乳腺癌曲妥珠单抗耐药 代谢产物分析

摘 要 目的：探究人类表皮生长因子受体2（HER2）阳性乳腺癌曲妥珠单抗（trastuzumab）耐药细胞（HCC1954）和敏感细胞（SK-BR-3）之间的代谢物差异。方法：利用超高效液相色谱-串联质谱（UHPLC-MS/MS）非靶向代谢组学方法进行代谢物的鉴定与定量,结合多元统计方法主成分分析（PCA）和正交偏最小二乘-判别分析（OPLS-DA）以筛选差异代谢物,并对差异代谢物进行KEGG通路富集分析。结果：在正负离子模式下鉴定筛选出15种显著性变化差异代谢物,在曲妥珠单抗耐药细胞中丙酰肉碱和谷胱甘肽等7种代谢物上调,肉豆蔻酸和D-葡萄糖酸等8种代谢物下调。KEGG通路富集分析发现,9条代谢通路具有统计学意义（P<0.05）,其中氮代谢和谷氨酰胺与谷氨酸代谢最为显著,P值为0.001 3,还发现谷氨酸和谷氨酰胺均在这两条通路中发挥作用。结论：本研究初步揭示了HER2阳性乳腺癌曲妥珠单抗耐药和敏感细胞之间的代谢物差异,为HER2阳性乳腺癌细胞曲妥珠单抗诊断标志物进一步研究提供了代谢组学的数据支持。     

右美托咪啶预处理脂多糖诱导的巨噬细胞代谢物研究

摘要：目的:应用代谢组学方法,研究右美托咪啶(DEX)预处理LPS(脂多糖)诱导的巨噬细胞炎症模型代谢物的变化,探讨其抗炎机制可能性代谢途径。方法:DEX预处理LPS诱导的巨噬细胞炎症模型,利用超高效液相色谱-三重四级杆-线性离子阱质谱(UPLC-Q-TRAP-MS/MS)采集细胞代谢物信息,并采用多元变量统计方法:主成分分析法、正交偏最小二乘法判别分析法筛选细胞差异代谢物。结果:与对照组相比,DEX干预后筛选出甜菜碱、胆碱、乳酸、谷氨酰胺、蛋氨酸、S-腺苷L-甲硫氨酸、磷脂酰甘油、磷脂酰胆碱、瓜氨酸、脯氨酸、二磷酸腺苷(ADP)、环磷酸腺苷(c AMP)、丙酮酸等22种差异代谢物。结论:代谢组学方法显示DEX预处理炎症细胞特征性代谢物分子,为DEX抗炎生物机制奠定基础。     

Cell-penetrating peptides

The established view in cellular biology dictates that the cellular internalization of hydrophilic macromolecules can only be achieved through the classical endocytosis pathway. However, in the past five years several peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway. These peptides have been used successfully for the intracellular delivery of macromolecules with molecular weights several times greater than their own. Cellular delivery using these cell-penetrating peptides offers several advantages over conventional techniques because it is efficient for a range of cell types, can be applied to cells en masse and has a potential therapeutic application.     

In vitro antiproliferative and cytotoxic activities of novel kojic acid derivatives: 5-benzyloxy-2-selenocyanatomethyl- and 5-methoxy-2-selenocyanatomethyl-4-pyranone

Some kojic acid (KA) derivatives and selenium containing compounds possess antiproliferative properties. The present study tested novel selenocyanatomethyl derivatives of KA for growth inhibitory and LDH cytotoxic activities. Human skin carcinoma (A431) and human breast carcinoma (MCF7) cells were treated with 5-benzyloxy-2-selenocyanatomethyl-4-pyranone (P763) and 5-methoxy-2-selenocyanatomethyl-4-pyranone (P764) in selected concentrations for 24, 48 and 72 h. Cell viability tests aimed at intracellular injury of mitochondria (MTT) and lysosomes (neutral red uptake, NR) revealed (a) higher growth inhibitory activity of the benzyloxy-selenocyanatomethyl derivative of KA (P763) compared with the methoxy derivative (P764) in both cell lines, (b) an intensified effect with time of exposure (MTT test only). The results demonstrate that NR cell survival/viability assay is more sensitive than the MTT test to detect subcellular changes induced by test compounds. Cell membrane integrity determined by LDH leakage confirmed an exaggerated cytotoxic effect of P763 but similar sensitivity of both cell lines to membrane injury. The ED50 values for all three tests used indicate that injury of intracellular mitochondria and lysosomes precedes the loss of membrane integrity. Cell growth inhibitory activities of new selenium containing kojic acid derivatives are preferentially aimed at the intracellular compartment rather than the plasma membrane and enlarge the group of antiproliferative active compounds.     

Application of metabonomics in a compound ranking study in early drug development revealing drug-induced excretion of choline into urine

Selecting drug candidates based on toxicity is an important step in early drug development. In this case study, it is shown how metabonomics is applied to a ranking study, in which drug candidates with equal pharmacological activities are selected based on least toxic side effects. The metabonomic analyses were carried out on an animal study that followed an established protocol for pilot toxicology/ranking studies in rats, however, not specifically modified for a metabonomic assessment. It is shown how conditions not specificially adopted for metabonomics investigations can significantly influence the metabolic profiles recorded by NMR. Furthermore, it is shown how the multivariate analysis of the NMR spectra identified an extreme excretion of an endogenous metabolite into urine induced by two out of the five drug candidates. The subsequent structure elucidation by two-dimensional NMR experiments and a subsequent validation by spiking experiments identified the metabolite as choline. The discussion of the mechanistic background for the excretion of choline, which is usually well-conserved in the body, results in two hypotheses of either a massive degradation of cell membranes or an inhibition of the choline oxidation. Although the validation of these hypotheses needs a follow-up study, the finding of a increased excretion of the important metabolite choline warrants exclusion of these two compounds as viable drug candidates from a metabonomics point of view.     

Metabonomics Techniques and Applications to Pharmaceutical Research & Development

In this review, the background to the approach known as metabonomics is provided, giving a brief historical perspective and summarizing the analytical and statistical techniques used. Some of the major applications of metabonomics relevant to pharmaceutical Research & Development are then reviewed including the study of various influences on metabolism, such as diet, lifestyle, and other environmental factors. The applications of metabonomics in drug safety studies are explained with special reference to the aims and achievements of the Consortium for Metabonomic Toxicology. Next, the role that metabonomics might have in disease diagnosis and therapy monitoring is provided with some examples, and the concept of pharmacometabonomics as a way of predicting an individual's response to treatment is highlighted. Some discussion is given on the strengths and weaknesses, opportunities of, and threats to metabonomics.     

代谢组学在肝肾损伤研究中的应用

代谢组学是近年来新发展起来的一门组学,已成为生物医学研究领域的新热点.由于其广泛的应用前景,目前已是系统生物学的重要组成部分.肝损伤及肾损伤是临床常见的危害人类健康的疾病,但目前的指标缺乏特异性,且灵敏度不高,造成临床上难以对肝肾损伤做出准确而及时的预警和监测.代谢组学能从代谢的角度整体分析疾病,快速、安全地对疾病的严重程度进行判别,在肝肾损伤的早期诊断等方面的研究已初显优势.本文主要综述了代谢组学技术在肝肾损伤研究中的应用现状.     

Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca concentration

Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24 h. The 50% inhibiting concentration (IC50) was 17.5 ± 3.2 μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca²⁺ concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures.     

肼引发损伤相关小分子代谢物的研究进展

肼是化学合成工业及制药领域的重要中间产物,也是肼类药物的共同代谢产物。肼具有肝、肾、中枢神经等多器官毒性,严重威胁人类健康。随着生命科学技术的发展,代谢组学能够全面、整体地获得机体受到外源性刺激后体内内源性小分子代谢物的扰动信息。代谢组学已用于发现与肼损伤相关的具有特殊生物功能的可能生物标志物。该文总结了近年来基于代谢组学方法对肼引发损伤相关的小分子代谢物的研究进展,以期为肼损伤的机制研究、早期发现及治疗提供研究思路。     

Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca(2+) concentration

Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24h. The 50% inhibiting concentration (IC50) was 17.5±3.2μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca(2+) concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures.     

Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides

The cellular membrane constitutes an effective barrier that offers protection for the complex, yet highly ordered, intracellular environment that defines the cell. Passage of molecules across this barrier is highly regulated and highly restricted, with molecular size being the most significant criteria. Over the last 15 years, a class of small cationic peptides has been discovered that can defy the rules of membrane passage and can gain access to the intracellular environment. Importantly, cellular entrance is also permitted for covalently coupled cargo. The cationic nature of these peptides is crucial for their ability to bind and traverse the anionic cellular membrane. Cell penetrating peptides (CPPs) have been used for the delivery of a wide range of macromolecules including peptides, proteins and antisense oligonucleotides. With the recent advancement and understanding of RNA interference (RNAi), CPPs offer an attractive means for the cellular uptake of double-stranded siRNAs to induce a RNAi response. This review focuses on the potential use of CPPs to deliver siRNA into cells and the implications of this technology for both gene function determination and therapeutic potential.     

Mouse Melanoma Cell Migration is Dependent on Production of Reactive Oxygen Species under Normoxia Condition

Cell migration plays a role in many physiological and pathological processes. Reactive oxygen species (ROS) produced in mammalian cells influence intracellular signaling processes which in turn regulate various biological activities. Here, we investigated whether melanoma cell migration could be controlled by ROS production under normoxia condition. Cell migration was measured by wound healing assay after scratching confluent monolayer of B16F10 mouse melanoma cells. Cell migration was enhanced over 12 h after scratching cells. In addition, we found that ROS production was increased by scratching cells. ERK phosphorylation was also increased by scratching cells but it was decreased by the treatment with ROS scavengers, N-acetylcysteine (NAC). Tumor cell migration was inhibited by the treatment with PD98059, ERK inhibitor, NAC or DPI, well-known ROS scavengers. Tumor cell growth as judged by succinate dehydrogenase activity was inhibited by NAC treatment. When mice were intraperitoneally administered with NAC, the intracellular ROS production was reduced in peripheral blood mononuclear cells. In addition, B16F10 tumor growth was significantly inhibited by in vivo treatment with NAC. Collectively, these findings suggest that tumor cell migration and growth could be controlled by ROS production and its downstream signaling pathways, in vitro and in vivo.