海洋弧菌鉴定方法学评价

近年来，由海洋细菌导致的感染越来越常见，弧菌是海洋细菌中常见的类群之一，可引起人类腹泻、菌血症及外伤感染，严重时可导致死亡。目前，对海洋弧菌尚缺乏快速可靠的鉴定方法。16S rRNA 序列分析目前被公认为细菌鉴定的金标准。然而由于海洋细菌进化缓慢，且基因具有高同源性，16S rRNA 测序并不能对海洋弧菌进行可靠鉴定。编码 RNA 聚合酶β亚基的基因 rpoB 为序列较长的单拷贝基因，可以克服16S rRNA 的高度保守性。至今为止，许多研究已经证明 rpoB 基因可用于各种细菌的分类，包括芽孢杆菌、军团菌、分枝杆菌、葡萄球菌等［1］。基质辅助激光解吸/电离飞行时间质谱（ matrix-assisted laser desorption / ionization time-of-flight mass spectrometry，MALDI-TOF/ TOF MS）技术是近年发展起来的一种全新的用于微生物快速鉴定的技术，具有快速、稳定、准确、重复性好的特点。为促进海洋弧菌的快速可靠鉴定，本研究利用16S rRNA 测序、rpoB 测序和MALDI- TOF/ TOF MS 3种技术对128株海洋弧菌同时进行鉴定，对比分析检测结果并进行方法学评估。

高脂血症性胰腺炎患者代谢组学研究

核磁共振波谱(nuclear magnetic resonance,NMR)、气相色谱-飞行时间质谱(gas chromatography time-of-flight mass spectrometry,GC-TOF MS)等是当前代谢组学的主要分析手段.

DNA芯片与蛋白质质谱技术在生殖内分泌研究中的应用

今天的生命科学已经进入了以基因组学(genomics)、RNA组学(transcriptomics)及蛋白质组学(proteomics)为代表的组学(omics)时代.高通量技术的广泛使用是组学时代生命科学研究的一个显著特征.这一特征的出现,是科技进步的必然结果.首先,生命体是复杂度很高的巨系统,在任何一个层次上的生命活动,都是由众多参数所决定的,只有应用系统生物学的方法,才可能理解生命活动的规律,而只有高通量技术,才能为系统生物学研究提供所需的大量数据.其次,成熟的电子、自动化和计算机技术,使得多种高通量技术的出现成为现实.DNA芯片技术和蛋白质质谱技术就是在这样的条件下产生的.

Objective To analyze the protein expression in the rat hippocampus by the proteomic approach.Methods Proteins from hippocampal tissue homogenates of the rat were separated by two-dimensional gel electrophoresis(2-DE),and stained with colloidal Coomassie blue to produce a high-resolution map of the rat hippocampus proteome.Selected proteins from this map were digested with trypsin,and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The mass spectrometric data were used to identify the proteins through searches of the NCBI protein sequence database.Results 37 prominent proteins with various functional characteristics were identified.The identified brain protein classes covered metabolism enzymes,cytoskeleton proteins,heat shock proteins,antioxidant proteins,signalling proteins,proteasome-related proteins,neuron-specific proteins and glial-associated proteins.Furthermore,3 hypothetical proteins,unknown proteins so far only proposed from their nucleic acid structure,were identified.Conclusion This study provides the first unbiased characterization of proteins of the rat hippocampus and will be used for future studies of differential protein expression in rat models of neurological disorders.

Yizhijiannao granules have been shown to improve cognitive function in Alzheimer’s disease patients. The present study sought to explore the mechanisms involved in the cognitive enhanc-ing effects ofYizhijiannao granule. Senescence-accelerated mouse prone 8 mice with learning and memory disorders were intragastrically treated withYizhijiannao granule for 8 weeks. Mice intragastrically treated with double distilled water for 8 weeks were considered as the control group. 2D gel electrophoresis was used to isolate total protein from the temporal lobe of senes-cence-accelerated mouse prone 8 mice, and differential protein spots were obtained by mass spectrometry. Thirty-seven differential protein spots were found in the temporal lobe area of both groups. Ten protein spots were identiifed: high mobility group box 1, dimethylarginine dimethylaminohydrolase-1, neuroglobin, hemoglobin beta adult major chain, peroxiredoxin-6, coiflin-1, lfotillin 1, peptidylprolyl isomerase A, voltage-dependent anion channel-2 and chap-eronin containing TCP1, and subunit 2. Among other functions, these proteins are separately involved in the regulation of amyloid beta production, oxidative stress, neuroinflammation, regulation of tau phosphorylation, and regulation of neuronal apoptosis. Our results revealed thatYizhijiannao granule can regulate the expression of various proteins in the temporal lobe of senescence-accelerated mouse prone 8 mice, and may be therapeutically beneifcial for the treat-ment of Alzheimer’s disease.

表面增强激光解离和激光离子化蛋白芯片技术与二维凝胶电泳质谱技术的比较

人类基因计划组揭开了人类基因框架的面纱,自此,生命科学研究步入了后基因时代,其核心便是蛋白质组研究.蛋白质是生理功能的执行者,是生命现象的直接体现者.

DNA加合物测试新进展

1加速器质谱法(Accelerator mass spectrometry,AMS)在加合物的鉴定和量化方面的应用DNA加合物是外来活性化合物在体内的中间代谢产物对DNA碱基共价修饰所形成的化合物,目前已有多种方法用于检测实验动物和一些接触人群体内的加合物含量.加合物检测剂量-反应关系存在的问题是:动物模型怎样外推到人体,加合物水平是否能反映低剂量水平致癌物暴露情况?目前,多数研究方法在反映人体实际接触情况下DNA加合物定量方面存在明显不足.

结直肠癌分类树模型建立的初步探讨

本研究采用表面增强激光解析电离飞行时间质谱(surface-enhanced laser desorption/ionization time-of-flight mass spectrometry,SELDI-TOF-MS)技术对结直肠癌Dukes B、C、D期患者、结直肠良性疾病患者和正常人血清中的蛋白质进行对比分析,建立用于在普通人群中诊断Dukes B、C、D期结直肠癌的分类树模型,并探讨其临床意义.

应用蛋白芯片技术探索乙型肝炎相关肝癌诊断新方法

表面加强激光解析电离飞行时间质谱(surface-enhanced laser desorption-ionization time-of-flight mass spectrometry,SELDI-TOF-MS)技术可将蛋白质沉析在芯片表面,离子化后用飞行时间质谱进行检测[1].这一技术操作简单,可直接检测体液,具有快速、高通量、准确等优点.

Objective:To establish a positive feedback circuit triggered by Reg4 and investigates its signiifcance in peritoneal metastasis of gastric cancer.Methods: Reg4 expression was detected in gastric cancer samples by immunohistochemistry staining. Reg4 overexpressed and knocked down cells were used to investigate its biological function. Activation of EGFR pathway and promotion of Reg4 were evaluated upon EGFR ligand stimulation. Mass spectrometry was performed to identify Reg4 receptor. ADAM phosphorylation and EGFR ligand shedding was detected after upstream manipulation. Possible downstream effectors were investigated by PCR array.Results:Expression of Reg4 was higher in gastric cancer tissues. Reg4 enhanced prometastatic abilities of gastric cancer cells. EGFR ligand was able to induce Reg4 expression through sequential EGFR pathway activation. Reg4 receptor was identified by mass spectrometry. ADAM phosphorylation and EGFR ligand shedding were enhanced after Reg4 overexpression. PCR array showed several promising downstream effectors.Conclusion:Reg4 is able to trigger a positive feedback regulatory circuit that promotes peritoneal metastasis of gastric cancer.

AIM:Atherosclerosis primarily involved systemic arteries .Luminal surface , a monolayer of endothelial cells , of artery directly exposes to blood and is susceptible to active substances in the blood .Exosomes contain significantly amount of proteins and RNAs .Ex-osomes can be good and bad for cells , depending on their component .Thus, exosomes may contribute to atherosclerosis by affecting endothelial cells .This study analyzed the relationship of exosome proteins and atherosclerosis .METHODS: Fifty-six patients and healthy subjects were recruited and divided into two comparisons:healthy subjects vs atherosclerosis ( HS vs AS) , and hypertension vs hypertension plus atherosclerosis ( HT vs HT+AS) .Serum exosomes were decoded by protein mass spectrometry .The protein profile and function were analyzed by gene ontology ( GO) .RESULTS:It was found that five child terms repeatedly appeared in “response to stimulus” and “immune system process” of BP of the two categories ( HS vs AS and AS vs HT+AS):“positive regulation of innate immune response”,“immune response-activating signal transduction”,”activation of innate immune response”,“innate immune re-sponse-activating signal transduction” and “innate immune response activating cell surface receptor signaling pathway ”.Two child terms repeatedly showed in “binding” of MF of the two categories:“antigen binding” and “enzyme binding”.Two proteins, PSMA6 and PSMA7, were repeatedly shown in the two categories .CONCLUSION:GO analysis was utilized for structure hierarchy “tree” to illustrate these proteins involved in various terms in BP , CC and MF.The PPI analysis supplied proteins which may play potentially im-portant roles in AS process .Innate immune system and blood coagulation pathway contribute to AS formation .The proteins, PSMA6, PSMA7 and Annexin A2, may can be the new target proteins for prevention and treatment of AS .

血流感染实验室诊断新技术

血流感染引起的败血症临床常见,且发病危急,后果严重.可导致血流感染的细菌种类很多,针对不同细菌采用不同的抗生素进行治疗, 是败血症能否治愈的关键.如何实现血流感染致病菌的快速有效检测及其药敏结果的准确回报,是长期困扰临床的问题.随着分子生物学技术的广泛应用,对各种致病菌的检测也从常规的单纯分离培养鉴定及药敏方法过渡到与聚合酶链反应(polymerase chain reaction,PCR)技术及其扩展试验、基因芯片、飞行时间质谱(time-of-flight mass spectrometry,TOF MS)技术等现代手段相结合,大大提高了检出率,并且缩短了细菌鉴定结果的报告时间.本文就以上几种方法的优缺点进行浅析.

基质辅助激光解吸电离飞行时间质谱在微生物检测中的应用进展

目前，临床微生物实验室鉴定细菌主要依赖于传统的生物化学、分子生物学和形态学等方法［1］。基于单菌落的生化特征鉴定耗时长，不能满足临床对检测结果时效性的要求；基于分子生物学方法进行微生物鉴定大大地提高了灵敏度和时效性，但对工作人员技术要求高，检测成本高，仅针对某些特定细菌，难以满足临床常规要求［2］。基质辅助激光解吸电离飞行时间质谱（matrix assisted laser desorption ／ionizationtime -of -flight mass spectrometry，MALDI -TOF MS）以其操作简便、自动化、快速、高通量等优势受到青睐，成为了一种新的微生物鉴定方法。由于仪器昂贵，一次性投入成本高，MALDI -TOF MS 在国外临床微生物实验室的应用尚未普及，而在国内临床微生物检验中的应用刚刚起步。MALDI -TOF MS 以其固有的优势进入临床微生物诊断，将彻底改变微生物实验室的面貌。因此，了解并引进该技术用于快速低成本鉴定临床分离菌株是国内临床微生物工作者的迫切需要。本文就该技术和两种常用质谱系统在临床微生物实验室用于鉴定各种常见或重要细菌和真菌纯菌落，以及应用该技术直接检测临床标本中的细菌和真菌等方面作一综述，以便国内临床微生物工作者初步了解MALDI -TOF MS 在临床微生物诊断的应用和价值，并且基于实验室情况选择合适的质谱系统。

Bioinformatics analysis of exosome proteome derived from hepatic carcinoma HepG2 cells

Objective:The aim of this study was to analyze the exosome proteome derived from hepatic carcinoma HepG2 cells and normal hepatocytes HL-7702,and look for the key factors in carcinogenesis of hepatocellular carcinoma (HCC).Methods:HepG2 and HL-7702 cells were cultured,and exosome samples were obtained from the culture supernatant and verified.LTQ-Orbitrap Elite mass spectrometry was applied to analyze and identify the exosome proteome derived from the hepatic carcinoma cells and normal liver cells,which was for seeking hepatic carcinoma cells specifically expressed proteins.Furthermore,gene ontology (GO),protein-protein interaction (PPI) network,and pathway enrichment were constructed to analyze hepatic carcinoma cells specifically expressed proteins.Results:The exosomes of HL-7702 expressed 3,366 proteins,and the exosomes of HepG2 expressed 2,874 proteins,of which 1,224 were expressed specifically in HepG2 exosomes.102 target proteins were selected and going on bioinformatics analysis under the condition that the unique peptide was ≥1 and PSMs ≥10.GO analysis results showed that the target expression protein of HepG2 was concentrated on metabolism,proliferation,and localization adhesion function.76 target proteins were chosen and formed a PPI network;combining with pathway analysis,ACTN1,FN1,RAC1 and GSN were found to be involved in important signaling pathways of HCC.The 26 target proteins which not in PPI network were analyzed pathway involved in,respectively.AKR1C2 and C5 were found to be involved in differently important signaling pathways of HCC.Conclusion:Exosomes proteomics provides the sources of specific protein derived from hepatic carcinoma cell;when it combines with bioinformatics analysis,they are able to reveal the important molecules that are related to carcinogenesis of HCC and provide new ideas for investigation of HCC biomarkers.

ICP-MS技术在药物分析中的应用

ICP-MS(Inductively Coupled Plasma Mass Spectrometry)即电感耦合等离子体质谱,是以独特的接口技术将ICP的高温(8 000K)电离特性与四极杆质谱仪灵敏快速扫描的优点相结合而形成的一种新型的元素和同位素分析技术.在分析能力上,可取代传统的如电感耦合等离子体光谱技术(ICP-AES)、石墨炉原子吸收(GF-AAS)、火焰原子吸收光谱法(F-AAS)等无机分析技术.

蛋白指纹技术在肿瘤诊断中的应用与进展

蛋白指纹技术(protein fingerprinting)是表面增强激光解吸离子化飞行时间质谱技术(surface-enhanced laser desorption/ionization time-of-flight mass spectrometry,缩写SELDI-TOF-MS,简称SELDI-TOF或SELDI)的俗称,它指的就像每个人的指纹不同于他人一样,每种疾病在发生发展过程中所形成的特定蛋白组或其降解产物也不一样,所以对此各种特异性蛋白指纹图谱的识别和判断,就可达到对各类疾病进行准确诊断的目的.

维拉帕米在大鼠肝微粒体中代谢产物的体外鉴定

AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MSn), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eight metabolites, including two novel metabolites (M4 and M8), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (M5-M7), and N, O-didemethyl-verapamil (M8). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.