甲氨蝶呤对大鼠尿液蛋白质组的影响

甲氨蝶呤是常见的免疫抑制剂,曾被大剂量用于治疗癌症,近年来其被小剂量用于治疗类风湿性关节炎。文中尝试研究甲氨蝶呤对大鼠尿蛋白质组的影响。大鼠口服甲氨蝶呤构造用药模型,再收集大鼠10 h内的尿液,并且采用液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)分析大鼠的尿蛋白。总共鉴定到31个差异蛋白,其中7个蛋白与甲氨蝶呤药物作用和类风湿性关节炎症状有关。部分大鼠的生物学过程反映了甲氨蝶呤对机体谷胱甘肽代谢以及对JAK/STAT信号通路的影响。结果显示,尿蛋白具有反映甲氨蝶呤对大鼠机体影响的能力。对单个大鼠个体差异蛋白的分析体现出不同个体对该药物的反应有较大差异。     

细菌性脑膜炎大鼠模型的尿液蛋白质组学变化

与脑脊液和血液不同,尿液不受到稳态机制的调节,更倾向于积累和反应机体生理和病理状态下的变化。生物标志物的本质特征是变化,因此尿液是寻找疾病早期标志物的更好来源。在世界范围内,细菌性脑膜炎依然是引起新生儿和儿童疾病的主要原因。为了降低死亡率和致残率,需要用无创的方法寻找细菌性脑膜炎的相关线索。本研究中,使用大鼠脑室内注射大肠杆菌模型模拟细菌性脑膜炎,在第1天和第3天的大鼠尿液中寻找尿液蛋白谱的差异变化,为进一步寻找大肠杆菌性脑膜炎的早期生物标记物研究进行初步的探索。通过膜上酶切和胶内酶切将尿蛋白切成肽段并通过液相色谱串联质谱技术(LC-MS/MS)分析肽段信息。第1天的尿液通过胶内酶切方法鉴定到17个差异蛋白,通过膜上酶切鉴定到20个差异蛋白;第3天的尿液通过膜上酶切方法鉴定到5个差异蛋白。这些差异蛋白为寻找细菌性脑膜炎早期生物标志物的初步探索奠定了基础。     

原料乳冷藏过程中微生物细胞的宏蛋白组学分析

原料乳在投入生产前通常需4°C冷藏,在这个过程中微生物的污染将造成冷藏原料乳的变质。【目的】本文旨在分子水平上探究原料乳冷藏过程中微生物表达的差异蛋白质的动态变化,为原料乳的冷藏提供理论支撑。【方法】利用Label-free技术研究原料乳4°C冷藏6 d期间微生物菌体蛋白质的物种来源、功能及参与通路,筛选差异蛋白质并对主要差异蛋白质参与的通路进行富集,探究其变化规律。【结果】冷藏过程中共鉴定出341个微生物菌体蛋白质,其中冷藏3 d后产生的蛋白质占所有检出蛋白质的60.12%。COG及KEGG分析表明,蛋白质所体现的功能及参与的通路随时间而变化。冷藏4 d,参与糖酵解/糖异生、ABC转运蛋白、氨基糖和核苷酸糖代谢等通路的蛋白数量显著增加。随冷藏时间延长,相邻时间点差异蛋白质数目逐渐增多,且富集在不同的通路中。【结论】冷藏过程中原料乳中的微生物所产生的蛋白质参与的通路及其所体现的功能复杂,4 d时的变化最为明显,或可作为原料乳质量控制的关键节点。     

大鼠急性低氧模型尿液蛋白质组的变化

本研究旨在探究急性低氧对大鼠尿液蛋白质组造成的影响。在该项研究中,大鼠被放置于模拟海拔5 000 m高原环境的低氧舱内24 h。在低氧后0、12、24 h收集尿液样本,并使用液相色谱-串联质谱技术（LC-MS/MS）对尿蛋白进行分析。与低氧0 h相比,低氧12 h组共鉴定到144个差异蛋白,低氧24 h组共鉴定到129个差异蛋白。功能分析显示,差异蛋白参与了一系列与低氧应激有关的生物学通路,如抗氧化应激、糖酵解、补体和凝血级联反应等。研究结果表明,尿液蛋白质组可以反映急性低氧刺激后的显著变化。这些发现可能提供了一种判断机体缺氧状态的方法,有助于辅助检测缺氧状态。     

腹腔注射百草枯构建小鼠肺纤维化模型

目的:探讨百草枯一次性腹腔注射致小鼠肺纤维化的病理改变及半数致死剂量(LD50),进而制备肺纤维化病理改变稳定的百草枯中毒小鼠肺纤维化模型。方法:60只正常雌性C57BL/6J小鼠被随机分为6组,10只/组,分别为正常对照组及百草枯给药30、40、50、60和80 mg/kg组,所有小鼠于造模后28 d处死,取其左肺用于病理观察(HE染色),并计算LD50及各组肺纤维化Ashcroft评级。结果:至观察期28 d,小鼠一次性腹腔注射百草枯溶液的LD50为55.1923 mg/kg;各染毒组均出现不同程度的肺纤维化改变,且注射剂量越高,肺纤维化病变越严重,但早期死亡率亦越高。结论:一次性腹腔注射百草枯可制备小鼠肺纤维化模型,40和50 mg/kg为较合适的造模剂量。     

不同年龄大鼠尿液代谢组学研究

旨在探究健康大鼠的尿液代谢物在生长、发育和衰老过程中的年龄相关性变化.采集大鼠出生后3周、5周、7周、9周、12周、56周和111周共7个年龄点的尿液样本,利用GC/TOF-MS平台进行代谢组学检测.结果显示,所有尿液样本在主成分分析与偏最小二乘法判别分析中出现组内聚类和组间分离.氨基酸类、有机酸类和碳水化合物类代谢物...     

三种不同方法检查尿液有形成分结果的比较研究

目的:探讨利用三种不同方法检查尿液有形成分的符合率及其影响因素.方法:采用AX-4280全自动干化学分析仪、IQ200全自动尿液分析仪和人工镜检法联合检测尿液,并对结果进行比较和分析.结果:尿液各种有形成分的检测结果均存在不同程度的假阳性,人工镜检法与AX-4280的符合率为:红细胞71.1%;白细胞76.4%.人工镜检法与IQ200全自动尿液分析仪人工修饰前和人工修饰后结果的符合率分别为:红细胞81.3%、91.5%;白细胞85.9%、93.8%;透明管型81.4%、89.7%,未分类管型52.6%、77.4%:结晶84.1%、92.4%;细菌65.1%、88.2%;精子72.0%、91.5%.以人工镜检为标准,IQ200全自动尿液分析仪人工修饰前和人工修饰后各检测项目的假阳性率分别为:红细胞23.0%、9.3%,白细胞16.4%、6.6%,透明管型22.9%、11.4%,未分类管型90.2%、29.2%.结晶19.004、8.2%,细菌53.7%、11.0%,精子38.9%、9.3%.结论:三种检查方法的联合应用更能提高尿液有形成分检查结果的准确性,为临床诊治提供依据.     

腹腔注射重组腺病毒诱导的免疫反应

为研究重组腺病毒接种实验动物后的免疫反应性,利用ＲＴ－Ｐ　Ｃ　Ｒ方法,从ＨＡＶ的ＲＮＡ中克隆了结构蛋白基因插入穿梭质粒ｐＸＣＸ２Ｎｏｔ　Ｉ,通过磷酸钙－ＤＮＡ共　沉淀技术,将复制缺陷型腺病毒载体与线形化的ｐＸＣＸ２－ＣＭＶ－ＨＡＶ共转染２９３细胞。一系列检测方法证明产生了重组腺病毒ｒＡｄＨＡＶ。纯化后的ｒＡｄＨＡＶ滴度为１×１０９ＴＣＩＤ５０／ｍＬ　,腹腔注射免疫昆明种小白鼠后,可诱导产生抗ＨＡＶ　ＩｇＧ和ＨＡＶ中和抗体。复制缺陷型腺病毒　可作为发展基因工程病毒疫苗载体的有效系统。     

腹腔注射重组腺病毒诱导的免疫反应

为研究重组腺病毒接种实验动物后的免疫反应性,利用RT-PCR方法,从HAV的RNA中克隆了结构蛋白基因插入穿梭质粒pXCX2Not I,通过磷酸钙-DNA共沉淀技术,将复制缺陷型腺病毒载体与线形化的pXCX2 CMV-HAV共转染293细胞.一系列检测方法证明产生了重组腺病毒rAdHAV.纯化后的rAdHAV滴度为1×109TCID50/mL,腹腔注射免疫昆明种小白鼠后,可诱导产生抗HAV IgG和HAV中和抗体.复制缺陷型腺病毒可作为发展基因工程病毒疫苗载体的有效系统.     

大鼠肝移植术后腹腔细菌感染模型的建立与评价

目的建立大鼠肝移植术后腹腔感染的模型。方法构建DA大鼠到LEW大鼠的肝移植模型,采用腹腔内细菌注射的方法建立感染模型,通过对大鼠肝功、血气、血细胞计数等各项指标的检测对模型进行综合评价。结果肝移植术后5 d注射细菌,大鼠死亡率高,不利后续研究;术后3 d注射细菌,并选定5×105cfu/mL为最终注射浓度,感染后大鼠的7d存活率累计可达到37.5%左右,随之感染的加重,大鼠状态逐渐变差,直肠温度不断升高,WBC计数也随之增加,pH下降,大鼠出现代谢性酸中毒,肝功能损害进行性加重,肝实质的损害重于胆道的损伤,大约在感染5 d左右相继死亡,多器官病理分析表明,大鼠死亡原因为肝损害,不并发肺脏、肾脏损害。结论采用的腹腔内大肠埃希菌注射建立肝移植术后腹腔细菌感染的模型是比较成功的,可用于相关领域的研究。     

Human urine proteome analysis by three separation approaches

The urinary proteome is known to be a valuable field of study related to organ functions. There have been several extensive urine proteome studies. However, the overlapping rate among different studies is relatively low. Whether the low overlapping rate was caused by different sample sources, preparation, separation and identification methods is unknown. Moreover, low molecular mass (<10 kDa) proteins have not been studied extensively. In this report, male and female pooled urine samples were collected from healthy volunteers. The urinary proteins were acetone precipitated, separated and identified by three approaches, 1-DE plus 1-D LC/MS/MS, direct 1-D LC/MS/MS and 2-D LC/MS/MS. 1-D tricine gels were used to separate low molecular mass proteins. The tandem mass spectra of positive identifications were quality controlled both by manual validation and using advanced mass spectrum scanner software. A total of 226 urinary proteins were identified; 171 proteins were identified by proteomics approach for the first time, including 4 male-specific proteins. Twelve low molecular mass proteins were identified. Most urinary proteins had a molecular mass between 30 and 60 kDa and a pI between 4 and 10. The apparent molecular masses of many proteins were different from theoretical ones, which indicated their post-translational modification and degradation. The effects of sample preparation, separation and identification methods on the overlapping rate of different experiments are discussed.     

Physiological conditions can be reflected in human urine proteome and metabolome

Biomarkers are the measurable changes associated with physiological or pathophysiological processes. Urine, unlike blood, lacks mechanisms for maintaining homeostasis: it is therefore an ideal source of biomarkers that can reflect systemic changes. Urinary proteome and metabolome have been studied for their diagnostic capabilities, ability to monitor disease and prognostic utility. In this review, the effects of common physiological conditions such as gender, age, diet, daily rhythms, exercise, hormone status, lifestyle and extreme environments on human urine are discussed. These effects should be considered when biomarker studies of diseases are conducted. More importantly, if physiological changes can be reflected in urine, we have reason to expect that urine will become widely used to detect small and early changes in pathological and/or pharmacological conditions.     

Comparative testis proteome of cattleyak from different developmental stages

Cattleyak (hybrid of cattle and yak) exhibit higher capability in adaptability and production than cattle and yak, while the infertility of F1 males greatly restricts the effective utilization of this hybrid and little progress has been made on investigating the mechanisms of the cattleyak infertility. Cattleyak individuals at three development stages (10, 12 and 14-month old) were sampled in this work and the isobaric tag for relative and absolute quantification method was employed to identify differences between their testicular proteomes. The proteomic analysis identified 318 proteins differentially expressed with significance at 12-month stage and 327 at 14-month compared with 10-month stage, respectively. Compared with the testicular proteome from 10-month cattleyak, the gene ontology (GO) annotations of the differentially expressed proteins at 12 months did not indicate significant differences from those at 14 months, which confirmed the histological observation that germ cell reduction was more obvious and spermatogenic arrest may become more serious in 12-month-old cattleyak. On the other hand, 56 differentially expressed proteins were coexpressed at 12 and 14-month stage compared with 10-month stage, in which 32 proteins were upregulated and 24 downregulated. GO analysis revealed that most of the differently expressed proteins were involved in the molecular function of catalytic activity, transporter activity, oxidoreductase activity and protein binding. Further analysis indicated that the differently expressed proteins including testis-expressed protein 101 precursor, RNA-binding motif protein, X chromosome, putative RNA-binding protein 3, heparin-binding proteins, tudor domain-containing protein 1, glutathione S-transferases (GSTA2, GSTP1), heat shock-related 70 kDa protein 2, estradiol 17-β-dehydrogenase11, 2,4-dienoyl-CoA reductase and peroxiredoxin-2 were possibly associated with testis development and spermatogenesis, which could be selected as candidate proteins in future study to examine the mechanisms of cattleyak infertility.     

The extracellular proteome of Bacillus licheniformis grown in different media and under different nutrient starvation conditions

The now finished genome sequence of Bacillus licheniformis DSM 13 allows the prediction of the genes involved in protein secretion into the extracellular environment as well as the prediction of the proteins which are translocated. From the sequence 296 proteins were predicted to contain an N-terminal signal peptide directing most of them to the Sec system, the main transport system in Gram-positive bacteria. Using 2-DE the extracellular proteome of B. licheniformis grown in different media was studied. From the approximately 200 spots visible on the gels, 89 were identified that either contain an N-terminal signal sequence or are known to be secreted by other mechanisms than the Sec pathway. The extracellular proteome of B. licheniformis includes proteins from different functional classes, like enzymes for the degradation of various macromolecules, proteins involved in cell wall turnover, flagellum- and phage-related proteins and some proteins of yet unknown function. Protein secretion is highest during stationary growth phase. Furthermore, cells grown in complex medium secrete considerably higher protein amounts than cells grown in minimal medium. Limitation of phosphate, carbon and nitrogen sources results in the secretion of specific proteins that may be involved in counteracting the starvation.     

Collection fluid helps preservation in voided urine cytology

Objectives:  Degenerative change caused by delay in processing contributes to false-negative and false-positive diagnosis of urothelial carcinoma in cytology. The aim of the study was to see if the use of a collection fluid for urine samples made a significant difference to urine cytology diagnosis, and if one was better suited for routine use in the hospital laboratory. Three cell collection fluids were evaluated by analysing the preservation and degeneration of cells in urine samples, as was the routine preparation which did not use a collection fluid.
Methods:  In the design study 50 voided urine specimens were taken at random from the hospital haematuria clinic. Three commercially available collection fluids cytolyt^TM, cytospin^® and cytoRich^®Blue and the hospital's routine conventional preparation of urine were compared. The degree of degeneration, and so preservation, was assessed by a table of chosen criteria; then ranked and analysed by Friedman's nonparametric test, at P  = 0.05. A second table showing the cell content of each slide was also made.
Results:  These showed no significant diagnostic difference between the collection fluids, but there was a significant difference between the collection fluids and the routine preparation. Minor differences that do not affect diagnosis, such as crystals and ghost red blood cells, were noted in cytospin^® and cytoRich^®Blue.
Conclusion:  It is recommended that a collection fluid is used. This choice should be made after health and safety issues and cost are considered.     

尿液蛋白富集方法的比较

人尿液中蛋白含量低,在进行质谱分析时易被高丰度蛋白掩盖。因此,发展高效和高选择性的富集方法,是实现尿蛋白标记物深度覆盖的必要前提。探究不同实验方法对尿液蛋白富集和尿蛋白质组的影响尤为重要。本研究采用超滤法、硝酸纤维素膜富集法和饱和硫酸铵沉淀法,等体积各处理5例健康志愿者和膀胱癌患者10 mL尿液样本,富集尿液蛋白,SDS-PAGE分离尿蛋白,比较不同方法纯化的效率;通过质谱分析,比较不同纯化方法的肽段鉴定效果,确定针对尿液蛋白质组蛋白的最佳富集方法。相对于超滤和硝酸纤维素膜富集法,饱和硫酸铵沉淀法成功地应用于健康人尿蛋白的富集和质谱检测,在保证回收蛋白质量的前提下,可减少高丰度白蛋白的干扰,富集更多低丰度蛋白,提高了质谱鉴定的灵敏度。综上所述,饱和硫酸铵提取尿蛋白的效果较好,该方法具有大规模处理尿液、提高蛋白质组学筛选临床诊断标记物研究的应用潜力。     

Comparison of protein enrichment strategies for proteome analysis of plasma

Efforts to discover protein biomarkers in plasma are hampered by the high abundance of few proteins, which interfere with the detection of low‐abundant proteins. Different commercially available protein‐partitioning products were tested for their ability to lower the detection limit of proteins in 2‐D gels. Immuno‐depletion using polyclonal antibodies raised against the proteins of highest abundance (Seppro IgY14 System) was compared with a two‐step immuno‐depletion strategy, where depletion with the Seppro IgY14 column was followed by depletion with the Seppro IgY‐SuperMix system. The third strategy tested was protein pre‐fractionation using the ProteoMiner kit, where proteins compete for binding sites on bead‐bound peptide hexamers with different binding properties. The pre‐fractionated protein samples were analyzed using 2‐DE, which revealed stunning differences in protein patterns. However, detectable protein spots in the different plasma fractions contained exclusively high‐abundant proteins normally present in plasma at concentrations between 1 μg and 40 mg/mL.