微透析-高效液相色谱法测定大鼠脑脊液中4种氨基酸的含量测定

目的： 通过对大鼠脑透析液中氨基酸的含量测定,为在体氨基酸分析建立方法。方法： 利用微透析技术抽取麻醉下大鼠脑脊液，运用OPA柱前衍生法处理样品，再用高效液相二元梯度洗脱色谱法，BDSC18柱（Hypersil，4.6 mm×250.0 mm，5 μm），荧光检测脑脊液中谷氨酸、天门冬氨酸、γ-氨基丁酸、牛磺酸等4种氨基酸的含量。结果： 该体系可在30 min内对脑脊液中4种氨基酸实现良好的分离，4种氨基酸在0.625-10 μmol/L浓度范围内与色谱峰面积线性关系良好，保留时间分别是天门冬氨酸4.392 min、谷氨酸5.592 min、牛磺酸13.625 min、γ-氨基丁酸14.833 min。结论: 本方法准确、灵敏、方便、重复性好，可用于微透析样品中氨基酸的含量测定。     

应用SELDI-TOF-MS技术对肝损伤模型大鼠血清蛋白质表达谱的比较分析

目的： 应用SELDI-TOF-MS技术对肝损伤模型大鼠血清内蛋白质表达谱进行比较分析，以揭示肝损伤模型大鼠血清促使骨髓间质细胞向肝细胞方向分化的分子基础。方法: 采用2种不同蛋白质芯片CM10和IMAC3先对肝损伤模型大鼠血清进行吸附分馏，后置入蛋白质芯片阅读机，用激光解吸和离子化飞行时间质谱检测被富集到芯片样品点上的蛋白质，对肝损伤模型大鼠血清和正常大鼠血清间的蛋白质表达谱进行比较分析。结果: 肝损伤大鼠血清能诱导骨髓间质细胞向肝细胞方向分化。蛋白质芯片-飞行时间质谱（SELDI-TOF-MS）结果显示，CM10芯片共捕获到9个差异蛋白峰，其中有6个蛋白表达峰的峰值在肝损伤实验血清中明显高于对照组血清，其质荷比（m/z）分别为3 480、7 888、8 275、8 528、15 101和15 787；另有3个蛋白表达峰的峰值在肝损伤实验血清中明显低于对照组血清，其质荷比（m/z）分别为8 833、12 039和12 720。IMAC3芯片共捕获到5个差异蛋白峰，其中有3个蛋白表达峰的峰值在肝损伤实验血清中明显高于对照组血清，其质荷比（m/z）分别为3 481、15 105和15 792；另有2个蛋白表达峰的峰值在肝损伤实验血清中明显低于对照组血清，其质荷比（m/z）分别为9 018和11 593。结论: 肝损伤模型大鼠血清内确实存在差异表达蛋白，这些差异表达蛋白很可能是决定骨髓间质干细胞分化方向的“信号分子”。     

冰片对MnTBAP透过大鼠血脑屏障的影响

目的探讨冰片对锰卟啉(MnTBAP)通过大鼠血脑屏障的影响。方法 SD大鼠分为对照组、131冰片组。对照组以75%乙醇灌胃,冰片组给予冰片0.7g/kg(75%乙醇溶解)灌胃,1h后尾静脉注射I-MnTBAP(1.85MBq/只),分别于注射后5、10、30、60、120、240、1440min取血液和脑脊液,用同位素示踪法测定大鼠血液和脑131-1脊液中I-MnTBAP的放射性计数,计算每克组织摄取注射剂量的百分率(%ID·g)。结果在给药后5¹440131-1 131min时间内,血液中I-MnTBAP的(%ID·g),冰片组和对照组相比无显著差异;而脑脊液中I-MnTBAP的-1(%ID·g),冰片组比对照组明显增加(<0.01)。结论冰片可促进MnTBAP透过血脑屏障,增加其在脑脊液中的浓度,而不改变其血药浓度。     

血清分离后室温放置24小时内多肽质谱的变化

目的探讨血清分离后室温放置24h内多肽质谱的变化,为确立血清多肽质谱分析最适时间提供参考。方法募集6名志愿者(男性2名,女性4名,平均年龄30岁)分别采集静脉血,离心分离血清后,根据在室温下放置的时间(0h、2h、6h、10h、24h)分装血清冻存并分成5组待测。用弱阳离子磁珠提取多肽,用Clin-TOF平台进行检测,通过BioExplorer软件和SPSS 22.0进行多肽峰的比较。结果5个不同时间组共出峰58个,5个多肽峰差异有统计学意义(P<0.05),在5组峰强度最大的前10个多肽峰中,8个均相同,且峰强无差异(P>0.05);6h组和10h组正态分布的血清多肽峰最多(51个),全峰强度6h组最高(1599499);峰强度大于300的血清多肽峰数为6h组最高(24.7±1.37),峰强累积值为6h组最高(132674);根据正态分布多肽峰个数、全峰强度、峰强大于300的多肽峰数、差异峰4049.9m/z的峰强度这4个因素综合评价,6h组得分最高。结论在室温条件放置24h之内血清多肽变化不足以影响分析,最优的检测时间是采血分离血清后室温放置6h,在这一条件下进行质谱检测,出峰均一性最佳,且峰强度最大,较易获得阳性结果。     

经鼻及皮下给予睾丸酮/丙酸睾丸酮对大鼠相关脑区c-Fos表达效能的影响

目的:探讨经鼻和皮下给予睾丸酮(T)或丙酸睾丸酮(TP)对大鼠中枢神经系统相关脑区激活的效能.方法:利用放射免疫分析法检测大鼠经鼻滴注给予TP后脑脊液和血清睾丸酮浓度的变化；以免疫组织化学观察大鼠各脑区cFos的表达.结果:放射免疫结果显示去势组大鼠脑脊液和血清中睾丸酮含量比正常组降低；去势大鼠皮下注射TP后只增加了血清巾睾丸酮的含量,去势大鼠和正常大鼠经鼻给予TP后脑脊液和血清中睾丸酮的含量均明显增加；去势大鼠经鼻给予TP脑脊液巾睾丸酮的含量高于去势皮下注射TP组,而血清巾睾丸酮的含量低于去势皮下注射TP组.免疫组织化学结果显示正常大鼠鼻腔给予TP或睾丸酮增加大鼠多数脑区的c-Fos免疫反应阳性神经元数目和c-Fos免疫反应强度,而皮下注射TP只增加了少数脑区c-Fos免疫反应阳性神经元数目以及c-Fos免疫反应强度.结论:经鼻给予睾丸酮或TP后能够激活多数脑区c-Fos蛋白的表达,经鼻给予睾丸酮或TP的给药方式有可能成为治疗大雄激素低下导致的巾枢神经系统疾病的一种有效手段.     

血清多肽组谱图的生物信息学

血清多肽组谱图(简称血肽图,serum peptidome profiling)是指通过质谱分析技术获得的血清中多肽组的精确质量数的谱图,是临床蛋白质组学研究领域的一个分支,在生物标志物的发现、疾病早期诊断和个性化治疗等领域有着广阔的应用前景.而且在这些应用中,生物信息学分析是其中一个重要环节.为了给有关的生物医学工作者提供较好的支持,文章就与血肽图相关的生物信息学方法进行综述,内容涉及基线删除、标准化、峰检测、峰比对和模型建立等方面.     

绿茶多酚对脑缺血大鼠血脑屏障通透性的影响

目的探讨绿茶多酚对局灶性脑缺血大鼠血脑屏障的通透性及紧密连接蛋白occludin、claudin-5和蛋白激酶PKCα表达的影响。方法采用线栓法制作大鼠局灶性脑缺血模型,伊文思蓝法检测血脑屏障通透性的变化,免疫组化方法检测缺血脑组织occludin、claudin-5的表达,western blot方法检测脑缺血组织PKCα蛋白的表达。结果大鼠局灶性脑缺血60min、120min的脑组织血脑屏障通透性显著增加,同时点的脑组织BBB紧密连接开放;而绿茶多酚显著降低血脑屏障的通透性(P0.01),使BBB紧密连接处于关闭状态。大鼠局灶性脑缺血60min及120min脑组织紧密连接相关蛋白occludin、claudin-5的蛋白表达水平显著降低,绿茶多酚显著上调这些蛋白表达水平(P0.01)。大鼠局灶性脑缺血30min至120min脑组织中PKCα的蛋白表达水平显著升高,绿茶多酚显著降低PKCα蛋白的表达水平(P0.01)。结论绿茶多酚降低缺血大鼠脑组织血脑屏障通透性可能与上调紧密连接蛋白occludin、claudin-5的表达及下调蛋白激酶PKCα的表达有关。     

鸡软骨肽化学结构特征及抗氧化、抗炎活性成分研究

目的:明确鸡软骨肽的主要化学结构特征,及其抗氧化、抗炎的物质基础。方法:对鸡软骨肽进行水解,采用异硫氰酸苯酯(PITC)-柱前衍生高效液相色谱法检测鸡软骨肽水解后所得氨基酸的组成;借助LC-MS/MS以及物种蛋白质数据库,确定鸡软骨肽中主要水解多肽的氨基酸序列;利用固相合成方法制备已知氨基酸序列的多肽,采用DPPH法、水杨酸法、FRAP方法测定合成肽段的抗氧化作用,采用H_2O_2致C518大鼠膝关节软骨细胞损伤模型测定合成肽段对氧化损伤的保护作用,采用LPS诱导的C518大鼠膝关节软骨细胞炎症模型测定合成肽段的抗炎作用。结果:确定18种氨基酸在鸡软骨肽中的占比情况,其中占比最高的是甘氨酸(G)29.5%,其次为脯氨酸(P)11.63%、丙氨酸(A)10.61%、谷氨酸(E)9.2%;从水解鸡软骨肽中鉴定出的11条分子量为794～1 860 Da的肽段,并筛选出多条抗氧化、抗炎作用显著的活性肽段,其中综合功效最佳的肽段为G4(MEGNAEESTLFCFAVRG)。结论:LC-MS/MS结合物种蛋白质数据库是一种快速、有效研究鸡软骨肽化学结构特征的新方法,本实验借助该方法初步确定了鸡软骨肽的化学结构特征以及抗氧化、抗炎活性成分,将为深入开展鸡软骨肽的应用研究提供基础。     

超声辅助酶切法用于溶液内蛋白质样品酶切特点分析

目的分析超声辅助酶切法对溶液内蛋白质样品酶切的特点。方法分别应用超声辅助酶切法与传统酶切法对牛血清白蛋白进行溶液内酶切,通过飞行时间质谱进行分析,对数据库检索后得到的多肽进行比较分析。结果超声辅助酶切法的酶切时间可缩短到10 min,酶切后多肽的回收率比传统酶切法低8%,但飞行时间质谱鉴定评分(MASCOT评分)比传统方法高约1倍,肽段的覆盖率高10%,匹配的多肽数多40%以上。对超声辅助酶切法鉴定出的多肽进一步分析表明,这些多肽的误切率较传统方法高22%,比传统方法多鉴定出的多肽分子质量主要在2～3 ku之间,且大部分为含有误切位点的多肽。结论超声辅助酶切可明显缩短蛋白质酶切时间,得到更好的多肽鉴定,但部分多肽酶切不完全,样品损失会更多。     

TNF结合肽和TNFR封闭肽对TNBS诱导的大鼠溃疡性结肠炎的治疗作用

目的 检测TNF-α结合肽和TNFR封闭肽对TNBS诱导的大鼠溃疡性结肠炎的治疗作用.方法 以TNBS诱导的大鼠溃疡性结肠炎为模型,联合给予TNF-α结合肽和TNFR封闭肽[2.5 mg/(k·次)]处理,同时设各种对照;观察大鼠的症状、体重、结肠组织病理改变等;采用一般的生物化学检测方法检测大鼠血清和结肠组织中NO含量、活性氧以及结肠组织中MP0活性等炎性介质;采用RT-PCR技术检测大鼠腹腔MφIL-1β和IL-8等mRNA水平的变化;采用免疫组织化学SP法检测大鼠结肠组织中TNF-α蛋白质表达水平的改变.结果 TNF结合肽和TNFR封闭肽联合多肽组大鼠的体重、结肠组织形态学评分、组织病理学评分及血清和结肠组织中NO含量、MP0活性等指标均显著低于模型组及无关肽组(P＜0.01);其腹腔Mφ的IL-1β和IL-8 mRNA水平和结肠组织中TNF-α蛋白的表达水平及阳性细胞率也显著低于显著低于模型组及无关肽组(P＜0.01).结论 TNF结合肽和TNFR封闭肽联用可显著减轻TNBS诱导大鼠实验性溃疡性结肠炎的病理损伤,改善症状,有效抑制大鼠血清和结肠组织中NO含量以及结肠组织中MP0活性的活性,抑制大鼠腹腔巨噬细胞中Ⅱ-1β、IL-8的mRNA转录和结肠组织中TNF-α蛋白的表达,对TNBS诱导的大鼠溃疡性结肠炎具有治疗作用.本研究为进一步研制和开发拮抗TNF-α的新型肽类药物提供了重要的实验依据.     

家兔^3H—cAMP颈内动脉注射后脑脊液放射活性的检测

在许多类型的发热过程中,血浆中cAMP含量都明显升高。但是否能通过血脑屏障进入中枢,从而介导发热,仍是一个有争议的问题。本实验经家兔颈内动脉注入~3H-cAMP,并用抗体结合法证明在开始注入后5、15及30分钟,有大量~3H-cAMP出现于脑脊液中;并以注射后5分钟含量最高,但此含量仍明显低于同时血浆中~3H-cAMP含量。此结果提示,至少在浓度较高的情况下,家兔血浆中的cAMP能有限制地通过血脑屏障而进入脑组织。     

Peptides and the senescent blood-brain barrier

Peptides can influence numerous systems known to be altered by aging. They can affect the passage of non-peptide substances across the blood-brain barrier (BBB) and themselves can cross by both saturable and non-saturable transport mechanisms. The passage of peptides across the BBB is influenced by numerous physiological and pathological events, including aging. Taken together, the evidence suggests that alterations in peptide/BBB interactions may play a significant role in senescence.     

Delivery of gold nanoparticles to the brain by conjugation with a peptide that recognizes the transferrin receptor

The treatment of Alzheimer's disease and many other brain-related disorders is limited because of the presence of the blood-brain barrier, which highly regulate the crossing of drugs. Metal nanoparticles have unique features that could contribute to the development of new therapies for these diseases. Nanoparticles have the capacity to carry several molecules of a drug; furthermore, their unique physico-chemical properties allow, for example, photothermal therapy to produce molecular surgery to destroy tumor cells and toxic structures. Recently, we demonstrated that gold nanoparticles conjugated to the peptide CLPFFD are useful to destroy the toxic aggregates of β-amyloid, similar to the ones found in the brains of patients with Alzheimer's disease. However, nanoparticles, like many other compounds, have null or very low capacity to cross the blood-brain barrier. In order to devise a strategy to improve drug delivery to the brain, here we introduced the peptide sequence THRPPMWSPVWP into the gold nanoparticle-CLPFFD conjugate. This peptide sequence interacts with the transferrin receptor present in the microvascular endothelial cells of the blood-brain barrier, thus causing an increase in the permeability of the conjugate in brain, as shown by experiments in?vitro and in?vivo. Our results are highly relevant for the therapeutic applications of gold nanoparticles for molecular surgery in the treatment of neurodegenerative diseases such as Alzheimer's disease.     

Low prevalence of TT virus in the cerebrospinal fluid of viremic patients with central nervous system disorders

TT virus (TTV) is a widespread infectious agent of humans identified in 1998. In infected individuals, TTV induces persistent viremia but its life cycle and pathogenic potential are still poorly understood. In the present study, the presence of TTV DNA in 32 consecutive paired serum and cerebrospinal fluid (CSF) samples from patients with neurological (mainly multiple sclerosis) disorders was investigated by means of a sensitive quantitative real-time PCR assay. Of the 24 patients who were found to carry TTV DNA in serum, 3 also had detectable TTV DNA in their CSF. Two TTV positive CSF samples had markers indicative of blood contamination or a disrupted blood-brain barrier and contained considerably lower TTV loads as compared with the corresponding serum samples, thus suggesting that the virus they contained was of plasma origin. These findings indicated that in general TTV does not permeate effectively an intact blood-brain barrier. Furthermore, the CNS does not represent a common site of TTV replication and persistence. However, at least one exception was observed: the third TTV positive CSF sample (obtained from a patient with subacute dementia of unknown origin) showed no markers suggestive of disrupted blood-brain barrier or blood contamination and had a TTV DNA concentration similar to that found in the patient's serum. In addition, the TTV isolates detected in the two body fluids were distinct genetically. The detection of TTV DNA in CSF is of considerable interest but the clinical significance remains unknown.     

Failure of dithiothreitol and pronase to reveal a false-positive cryptococcal antigen determination in cerebrospinal fluid

A patient with squamous cell carcinoma of the lung and a serum rheumatoid factor (RF) of 1:1,280 had a positive cerebrospinal fluid (CSF) latex agglutination test (LAT) for cryptococcal antigen, in culture-negative, India-ink-negative CSF. Pretreatment of the sample of CSF with 2-mercaptoethanol (2-ME) ablated the antigen titer and established the presence of a false-positive LAT, whereas CSF pretreated with dithiothreitol (DTT) and pronase continued to yield a false-positive result. The differing ability of pronase, DTT, and 2-ME to eliminate interfering substances from CSF has not been previously described. Moreover, because RF is unlikely to cross the blood-brain barrier, the authors postulated that malignant disease was responsible for the patient's false-positive LAT in CSF. Hence, the authors report the case to emphasize that false-positive LAT results in CSF are unlikely to be produced by RF and to underscore the benefit of using enzymatic and sulfhydryl-reducing agents when the validity of the initial test results are in doubt. Such a procedure will optimize the chances of accurately identifying false-positive LAT results in CSF.     

Strategies for drug delivery through the blood-brain barrier

Strategies for drug delivery through the blood-brain barrier include neurosurgical-based, pharmacologic-based, and physiologic-based approaches. Neurosurgical-based techniques involve the implantation of intraventricular catheters that allow for chronic drug delivery. However, this approach is limited by the much faster rates of bulk flow of CSF through the ventricular compartments as compared to the slow rates of drug diffusion down into brain parenchyma. Pharmacologic-based strategies, e.g., synthesis of lipid soluble pro-drugs, or physiologic-based strategies, e.g., the development of chimeric nutrients or chimeric peptides allow for drug delivery through the brain capillary wall, i.e., the blood-brain barrier, and allow for drug delivery directly into brain parenchyma.     

Autoimmune epilepsy: some epilepsy patients harbor autoantibodies to glutamate receptors and dsDNA on both sides of the blood-brain barrier, which may kill neurons and decrease in brain fluids after hemispherotomy

PURPOSE: Elucidating the potential contribution of specific autoantibodies (Ab's) to the etiology and/or pathology of some human epilepsies. METHODS: Six epilepsy patients with Rasmussen's encephalitis (RE) and 71 patients with other epilepsies were tested for Ab's to the "B" peptide (amino acids 372-395) of the glutamate/AMPA subtype 3 receptor (GluR3B peptide), double-stranded DNA (dsDNA), and additional autoimmune disease-associated autoantigens, and for the ability of their serum and cerebrospinal-fluid (CSF) to kill neurons. RESULTS: Elevated anti-GluR3B Ab' s were found in serum and CSF of most RE patients, and in serum of 17/71 (24%) patients with other epilepsies. In two RE patients, anti-GluR3B Ab's decreased drastically in CSF following functional-hemispherotomy, in association with seizure cessation and neurological improvement. Serum and CSF of two RE patients, and serum of 12/71 (17%) patients with other epilepsies, contained elevated anti-dsDNA Ab's, the hallmark of systemic-lupus-erythematosus. The sera (but not the CSF) of some RE patients contained also clinically elevated levels of "classical" autoimmune Ab's to glutamic-acid-decarboxylase, cardiolipin, beta2-glycoprotein-I and nuclear-antigens SS-A and RNP-70. Sera and CSF of some RE patients caused substantial death of hippocampal neurons. CONCLUSIONS: Some epilepsy patients harbor Ab's to GluR3 and dsDNA on both sides of the blood-brain barrier, and additional autoimmune Ab's only in serum. Since all these Ab's may be detrimental to the nervous system and/or peripheral organs, we recommend testing for their presence in epilepsy, and silencing their activity in Ab-positive patients.     

Brainpeps: the blood–brain barrier peptide database

Peptides are able to cross the blood-brain barrier (BBB) through various mechanisms, opening new diagnostic and therapeutic avenues. However, their BBB transport data are scattered in the literature over different disciplines, using different methodologies reporting different influx or efflux aspects. Therefore, a comprehensive BBB peptide database (Brainpeps) was constructed to collect the BBB data available in the literature. Brainpeps currently contains BBB transport information with positive as well as negative results. The database is a useful tool to prioritize peptide choices for evaluating different BBB responses or studying quantitative structure-property (BBB behaviour) relationships of peptides. Because a multitude of methods have been used to assess the BBB behaviour of compounds, we classified these methods and their responses. Moreover, the relationships between the different BBB transport methods have been clarified and visualized.     

Effects of melanotan II, a melanocortin agonist, on grooming and exploration in rats after repeated restraint/immobilization

We wanted to verify the magnetic resonance imaging (MRI) abnormalities that occur in the central nervous system (CNS) of cobalamin-deficient (Cbl-D) rats. The rats were made Cbl-D by means of total gastrectomy or feeding a Cbl-D diet. MR images of the cervical tract of the vertebral canal were recorded using a vertical spectrometer, and the volume of cerebrospinal fluid (CSF) in this part of the vertebral canal was calculated. The findings of the present study demonstrate that: (i) there was a significant decrease in cervical tract CSF volume regardless of the way in which the vitamin deficiency was induced; (ii) this volume normalized in the totally gastrectomized rats after chronic Cbl treatment; (iii) no blood-brain or blood-CSF barrier lesions were found in Cbl-D rats, using either MRI with a paramagnetic contrast agent or calculating the albumin CSF/serum concentration quotient. Cbl deficiency decreases CSF volume in the cervical tract of the vertebral canal of the rat, without apparently impairing the blood-brain barrier.