外泌体作为是细胞旁分泌的重要介质,在促血管形成方面有重要作用.在我们前期研究中,已经成功从嗅黏膜间充质干细胞(olfactory mucosa mesenchymal stem cells,OM-MSCs)分离、鉴定了其外泌体,然而,OM-MSCs源外泌体对血管生成的影响尚不清楚.本研究旨在探讨OM-MSCs来源外泌体对内皮细胞血管生成能力的影响.采用PKH67荧光标记OM-MSCs源外泌体,与人脑微血管内皮细胞(human brain microvessel endothelial cells,HBMECs)共培养,观察OM-MSCs外泌体能否进入HBMECs.采用CCK-8法、Transwell迁移实验和小管实验,观察OM-MSCs外泌体对HBMECs增殖、迁移及管状结构形成的影响.采用基质胶塞实验及CD31免疫荧光,观察OM-MSCs外泌体在体内对血管生成的影响.上述研究均以等量PBS作为对照.结果 提示,OM-MSCs外泌体可被HBMECs摄取.CCK-8法检测显示,在处理1、2、3、4、5d各时间点,实验组细胞增殖均优于对照组(1.32±0.14 vs.0.98±0.04,1.36±0.14 vs.1.04±0.06,1.75±0.18 vs.1.33±0.11,2.16±0.11 vs.1.50±0.19,2.71±0.11 vs.1.81±0.20,P＜0.01).Transwell实验结果显示,实验组跨膜迁移细胞吸光度值较对照组显著增多(1.12±0.05 vs.0.02±0.02,P＜0.05).在体外小管实验中,从节点、交叉点、网眼数、血管分支数和总长度5个方面,实验组均高于空白对照组(374.33±127.74vs.193.33±44.79,104.56±33.07 vs.54.33±11.65,20.11±11.20 vs.7.56±3.64,81.67±19.07 vs.57.00±13.02,11466.22±2781.03 vs.8544.00±1848.61,P＜0.05);在体内实验中,实验组成血管及CD31阳性率(％)亦显著高于对照组(85.00±5.57 vs.8.00±2.08,P＜0.05).本研究表明:OM-MSCs外泌体可促进HBMECs增殖、迁移及管样结构形成,提示OM-MSCs外泌体可促进血管新生.

目前磷酸化肽段鉴定主要依赖于质谱技术,但磷酸化肽段的低丰度性以及来自非磷酸化肽段的干扰等因素,影响质谱的分析与鉴定.因此质谱分析前磷酸化肽段的富集,是深入研究磷酸化蛋白质组学的先决条件.该文介绍了磷酸化蛋白质组学中传统的以及新建立的一些磷酸化肽段分离富集方法的原理及优缺点,这些方法包括固相金属离子亲和色谱法(IMAC)、金属氧化亲和色谱法(MOAC)、强阳/阴离子交换色谱法(SCX/SAX)、亲水相互作用色谱法(HILIC)、静电排斥亲水相互作用色谱法(ERLIC)、化学衍生法、MALDI靶盘富集法以及多种富集方法相结合.

Objective The role of secreted protein SC1 in synaptogenesis after neural stem cells ( NSCs ) induced by olfactory ensheathing cells (OECs) is discussed.Methods Mouse NSCs line C17.2 was induced by mouse OECs conditioned medium, then the presynapses were labeled with primary antibody bassoon and the expression of bassoon was identified with Western blot.Results The synapse number and expression of bassoon in OECs group were significantly higher than those of RA group.After mouse SC1 recombinant protein added into retinoic acid (RA) group, the synapse number and the expression of bassoon were close to those of OECs group. Conclusion OECs secreted protein SC1 can promote synaptogenesis after C17.2 NSCs induced by OECs conditioned medium.

动物肝是具有极强再生能力的器官,研究并阐明肝再生的机制可为肝移植等与肝损伤相关的疾病治疗提供理论依据.质膜包括“脂筏(lipid rafts)”和“质膜微囊(caveolae)”的微区,具有参与胞吞胞饮、信号转导、运输胆固醇等重要功能.肝再生过程中,肝质膜微区脂筏蛋白质受到内部调控的影响会发生改变.捕获脂筏微区信号蛋白分布的变化,对于理解和阐明肝再生过程中信号通路途径有重要意义.本研究应用成熟的大鼠2/3肝切除模型结合蔗糖密度梯度离心法,提取假手术组与肝再生组大鼠肝细胞质膜,并进一步纯化获得质膜微区蛋白质.通过SDS-PAGE分离以及ESI-Q-TOF质谱鉴定,对获得的质膜微区蛋白质进行差异分析.结果显示,有30个微区蛋白质差异表达,其中13个上调、17个下调.生物信息学分析表明,所鉴定到的蛋白质主要参与细胞增殖、程序性死亡、细胞凋亡等调控,同时涉及到与肝再生密切相关的血管生成等信号通路.本文为质膜微区蛋白质的研究提供了方法上的参考以及相关基础数据,为后续临床肝再生的研究奠定了一定的基础.

Using 4-chloropheny1aldehyde as the starting material, tebuconazole was synthesized by condensation, hydrogenation, epoxidation, and ring-opening. A new catalyst was used to avoid production of the byproduct, 4,4- dimethyl-3-(4H-1,2,4-triazole-4-ylmethyl)-1-(4-chlorophenyl)-3-pentanol. Overall yield was over 65%, and purity was over 98.0% without requiring recrystallization. Economic and technical indices of this synthetic method are more favorable than those previously reported, and the method has already been commercialized.

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a very promising method applied in proteomics for the characterization of protein-protein interactions. BIA/MS is an approach combining the surface plasmon resonance(SPR) technology with mass spectrometry for the real-time analysis and identification of interaction molecules that interact specifically with a known immobilized ligand. In this study, by using the Biacore-X instrument, about 0.1 pmol beta(2)-Microglobulin was immobilized on sensor chip, the interacting protein in solution was delivered to the biosensor chip surface. Then, a microrecovery method of the principle of "sandwich" structure was used to elute the bonding microglobulin protein from the sensor chip, and the protein identification was then achieved after tryptic digestion by matrix-assisted laser desorption/ionization-time of flight mass fingerprint mapping and data-base search. The strategy was successfully applied to the model protein microglobulin interacting with its antibody, a unambiguous identification was obtained at 10(-15) mol level.

OBJECTIVE To synthesize 1-(2,4-dichloropheny1)-2-(1-imidazoly1)ethanol. METHOD 1-(2,4-dichloropheny1)-2-(1-imidazoly1)ethanol, the important intermediate of miconazole, was synthesized by epoxidation of 2,4-dichloropheny1aldehyde with sulphonium ylide and by reacting of 2,4-dichloropheny1oxirane with imidazole.RESULTS The overall yield was 54% and the purity was over 99.0%.CONCLUSION This synthetic method is feasible and suitable for industry.

采用纳升喷雾(Nano)技术和碰撞诱导解离(CIDcollisioninducddissociation)方法,在电喷雾四极杆飞行时间质谱(ESI-Q-TOFelectrspectrometryionization-quadrupole-timeofflight)上,对两种序列部分未知的天然多肽进行从头测序(denovosequence),结果证明质谱的denovosequence可以方便有效的解决传统的Edman降解法测序中常见的实际问题,如末位残基的丢失,赖氨酸和亮氨酸难鉴定等,此方法的建立是对Edman降解测序法很好的补充.

利用反相高效液相色谱(RP-HPLC)和电喷雾串联质谱(ESI-MS/MS)联用技术直接对模式蛋白分子(牛血清白蛋白,BSA)的胰蛋白酶酶解产物进行分离和测定.获得的一系列BSA酶解片段的一级(MS)和二级(MS/MS)质谱数据经分析软件处理后,分别在不同处理和不同参数条件下,用3种不同的方法通过网上蛋白质数据库进行蛋白质搜寻鉴定.结果显示,3种搜寻法都能正确地鉴定该蛋白质,其中以利用MS数据的肽质量指纹谱搜寻法(PMF法)较为快捷方便,但鉴定结果易受数据处理和数据库搜寻鉴定时参数设置等因素的影响;利用未解析MS/MS数据(raw MS/MS data)的搜寻法可在较宽的搜寻参数变化范围内获得明确的鉴定结果;而借助从头测序(de novo sequencing)结果的序列搜寻法(sequence query)则显示出更高的专一性,利用较少酶解片段数据就能得到稳定和明确的鉴定结果,搜寻参数变化的影响很小.就酶解条件、数据处理和搜寻参数设置对蛋白质鉴定结果的影响展开详细的讨论,为蛋白质组学研究中的数据处理和库搜寻鉴定积累了可借鉴的资料.

双向凝胶电泳(2-DE)具有高分辨率、高通量等特点,已被广泛地用于蛋白质组的分离.但是它在分离疏水性蛋白质和碱性蛋白质时却遇到了极大的挑战.然而,疏水性与碱性蛋白质在全蛋白质中占相当大的比例,且具有很重要的生物学意义.因而,近年来,越来越多的研究者将目标瞄准这些蛋白质,并且取得了一些令人鼓舞的进展:用亚细胞预分离技术,顺序提取法等方法来富集疏水性蛋白质,用一些新的有效的增溶剂如硫脲,ASB-14等来改善疏水性蛋白质的溶解,应用这些技术2-DE可分辨出总平均疏水值达0.80的蛋白质;在碱性蛋白质分离方面,通过等电聚焦预处理,使用窄pH梯度胶条等大大地改善了碱性蛋白质在2-DE中的分离,能分辨出等电点达11.7的蛋白质.现对2-DE技术中疏水性和碱性蛋白质分离的研究进展进行综述.

PMF方法由于具有高灵敏度、高通量和容易自动化等优点,在蛋白质组学鉴定中占有重要的地位.然而,许多样品(比如:小分子蛋白,混合物等)仅仅通过PMF方法不能明确鉴定.在这种情况下,在测定PMF的同一个样品上,选择一个酶解片段峰进行PSD测序,并把这些序列信息输入MS-Tag软件进行搜索,结合PMF方法,表观分子量等电点等参数,能够对胶上的点进行明确的鉴定.本文先用PSD方法对胶上的三个标准蛋白进行鉴定,都得到了非常准确的结果,同时鉴定了胶上的几个未知点.

A reversed phase HPLC was applied for determing the active ingredients of 30% Mefenacet-bensulfron-acetochlor mixed powder.Taking acetonitrile,methanol and water of 30∶40∶30 as the mobile phase at UV-230?nm ,SD and CV of the method for mefenacet, bensalfron and acetochlor are 0.038,0.15%,0.015,0.59% and 0.016,0.63%. The recovery is 98.1%～102.28%.

High performance liquid chromatography-tandem mass spectrometry was used to investigate a nonapeptide H2N-Tyr-Val-Asn-Val-Asn-Met-Gly-Leu-Lys-CONH2(Mr1035.6) and its main by-products from solid phase synthesis. The target sequence was assembled in stepwise fashion from the C-terminal to N-terminal using Fmoc chemistry on a Fmoc-PAL-PEG*PS resin manually. The product was deprotected and cleaved from the resin by treatment with reagent R (90%TFA, 5%thioanisole, 3%ethanedithiol, 2%anisole, V/V/V/V) for 2.5 hours at room temperature (20-25℃). Reverse-phase HPLC showed that the crude product contained a major component, two minor components (one early eluting and the other late eluting) and several trace-level components. The major component and the two minor components were analyzed by high performance liquid chromatography-tandem mass spectrometry, respectively. The major component was confirmed as the desired nonapeptide. The molecular weight of the early-eluting component was 16 more than that of the desired nonapeptide, and the by-product was proven to consist of two kinds of nonapeptide derivatives, one with Tyr1 oxidized and the other with Met6 oxidized. The late-eluting by-product was confirmed as resulting from 12 increase in mass of Tyr1 residue, which has not been reported early. The possible origins of those by-products were discussed.

The proteins of thermo-sensitive genetic sterile rice anther were separated by two-dimensional electrophoresis with immobilized pH(3-10) gradients as the first dimension and SDS-PAGE as the second. Mean standard deviations of about 1.45±0.23 mm for IPG-IEF and 1615±0.17 mm for SDS-PAGE were obtained. The silver-stained protein spots were analyzed and identified by employing an improved procedure, including (1) in-gel reduction, alkylation and enzymatic digestion; (2) extraction and desalting by using the Ziptip TM; (3)direct MALDI-TOF mass spectrometry analysis and protein database searching. 60 protein spots out of about 950 detectable spots on the 2D-gels were identified. The 2D protein maps of the rice anther during the sterile and fertile stages were compared. While the anther changed from sterile to fertile stages, about 20 protein sports showed variations that were significant and reproducible. Five up-regulated proteins and three down-regulated proteins were identified.

从雷氏大疣蛛(Macrothele raveni)粗毒中,结合阳离子交换和反相高效液相色谱分离到一种多肽神经毒素,命名为大疣蛛毒素-VI(Raventoxin-VI).经MALDI-TOF质谱分析,它的分子量为(5 371.6±0.5) Da.利用Edman降解气相蛋白质测序仪测得其氨基酸序列是NH2-NIIKGRVVKLCGGCAQKCCDREPRCDPCRTCVENVGT-GGGYLSSNKKCNGS-COOH,其中8个Cys形成4对二硫键.Raventoxin-Ⅵ能阻断小鼠膈神经膈肌标本神经肌肉接头传递,脑室注射能使小鼠瘫软.从一级结构上没有发现与该毒素有较高相似性的其他蜘蛛毒素.

dichlorophenyl)-1,2-epoxyhexane(1) was synthesized according to the following procedures. Compound (1) was prepared by reaction of 2,4-dichlorophenylpentanone(2) with (CH 3) 3S +CH 3SO - 4 which as sulphonium yield under base condition. The yield is up to 95% and the purity is over 95%. The reaction is mild and easy to be controlled.