中国小型猪骨髓间充质干细胞的蛋白质组研究

报道了骨髓间充质干细胞(MSCs)的蛋白质组表达研究。从体外培养的MSCs提取细胞蛋白,经二维电泳分离后用银染方法可检出蛋白点约1600个,选取48个蛋白点进行胶内酶解及质谱分析,经数据库检索成功鉴定了37个蛋白,并对蛋白功能进行初步分析。本实验数据为进一步分析MSCs增殖、分化或凋亡的分子机理提供相关信息。     

血管外膜肌成纤维细胞分化相关蛋白研究

为寻找涉及血管紧张素II (AngII)和转化生长因子β1 (TGF-β1)诱导的血管肌成纤维细胞(MF)分化的蛋白, 本研究采用双向电泳和质谱从整体水平检测了MF分化前后蛋白表达谱的变化, 共找到41个差异表达的蛋白点, 表达水平和/或蛋白位置在Ang II和TGF-β1刺激后都发生明显变化的蛋白点14个, 4个蛋白上调, 6个蛋白下调, 2个蛋白位置发生明显变化, 2个蛋白表达上调,位置也发生变化; 只在Ang II诱导的MF中表达发生变化的蛋白20个, 只在TGF-β1诱导的MF中表达发生变化的蛋白7个. 选取Ang II和TGF-β1共同调节的14个蛋白进行质谱鉴定, 结果除骨架蛋白外, 首次发现MF分化同泛素蛋白酶体系统和嘌呤合成有关; septin 2的下调可能是成纤维细胞分化的标志. 本研究运用蛋白质组学技术发现了新的参与MF分化的蛋白质, 为进一步研究和干预细胞表型转化提供了新的思路和靶点.     

多层面验证共培养微环境诱导法诱导骨髓间充质干细胞心肌样分化

为了多层面探讨共培养微环境诱导法定向诱导骨髓间充质干细胞(MSCs)心肌样分化的可行性,取第3代MSCs与原代心肌细胞(CMs)进行共培养。在显微镜下观察诱导1周后的MSCs形态学变化,用免疫荧光和实时荧光定量聚合酶链式反应(RT-PCR)分别检测诱导的MSCs中心肌肌钙蛋白I(cTnI)、α-肌动蛋白(α-actin)、Nkx-2.5和GATA-4的基因表达变化情况。采用超高效液相色谱-串联质谱(UPLC-MS/MS)分别检测诱导组和对照组的代谢产物。诱导1周后的MSCs形态呈心肌样改变,cTnI、α-actin、Nkx-2.5和GATA-4的基因表达均明显升高,正交偏最小二乘法判别分析(OPLS-DA)模型显示诱导的MSCs代谢物向CMs转变趋势明显。通过多元和单元统计分析筛选差异变量,根据一级质谱和二级质谱比对结果,最终确定12种差异代谢物。与未经诱导的MSCs相比,经诱导的MSCs与CMs中变化趋势相同的差异代谢物有7种,变化趋势不同的差异代谢物有5种。实验结果表明,无论从形态、基因、蛋白质还是代谢层面看,MSCs通过与CMs间接接触共培养后均发生了心肌样改变,但是与CMs仍存在差异。     

维拉帕米作用下急性心梗大鼠比较蛋白质组学研究

以急性心梗大鼠为研究对象, 应用双向凝胶电泳法(2-DE)分析比较了维拉帕米作用下急性心梗大鼠心肌蛋白表达的差异, 从蛋白质水平探讨了维拉帕米心肌保护作用的发生机制. 结果表明, 与假手术组及模型组相比, 维拉帕米给药组心肌组织中有8个蛋白点表达显著上调, 7个蛋白点表达显著下调. 采用质谱(MALDI-TOF-MS)分析结合数据库检索, 共鉴定了其中的15种蛋白质, 可按功能分为如下4类: (1) 能量代谢及线粒体功能相关蛋白; (2) 氧化应激相关蛋白; (3) 细胞骨架蛋白; (4) 其它蛋白. 研究结果表明, 维拉帕米的心肌保护作用与恢复心肌损伤过程中的能量供应及对抗氧化应激等作用有关.     

稀土化合物TbCl3对成骨细胞系MC3T3-E1增殖、分化和矿化功能的影响

在细胞和分子水平上,研究了稀土化合物氯化铽(TbCl_3)对成骨细胞MC3T3-E1增殖、分化及矿化功能的影响。结果表明,细胞水平上,浓度为0.000 1、0.001、0.01、0.1、1和10μmol·L-1的TbCl_3均促进MC3T3-E1细胞的增殖、分化及其矿化功能,然而,当浓度升至为100和1 000μmol·L-1时,TbCl_3表现出抑制作用。分子水平上,浓度为0.000 1和0.1μmol·L-1的TbCl_3明显上调成骨分化相关基因骨形成蛋白2(BMP-2),碱性磷酸酶(ALP),骨涎蛋白(BSP),Ⅰ型胶原蛋白(ColⅠ),骨钙素(OCN)和runt相关转录因子2(Runx2)的表达。浓度为1 000μmol·L-1的TbCl_3则抑制上述成骨分化相关基因的表达。浓度为0.000 1、0.1和1μmol·L-1的TbCl_3促进成骨分化相关蛋白Runx2,BMP-2和OCN的表达;结果显示,低浓度的TbCl_3促进MC3T3-E1细胞的成骨分化及矿化功能,而高浓度TbCl_3则呈现出抑制作用。TbCl_3通过调控Runx2的表达刺激早期成骨分化相关基因BMP-2、ColⅠ和晚期成骨分化相关基因ALP、OCN的表达,从而诱导MC3T3-E1成骨分化。     

连接蛋白基因表达与横纹肌肉瘤分化关系研究

报道了用间隙连接蛋白Cx43基因探针斜缝杂交,Cx43和肌专一蛋白免疫荧光染色和罗氏黄荧光传输法观察到人横纹肌肉瘤(RD细胞系)的分化异常,细胞通讯缺陷,Cx43表达下降。单细胞克隆亚系RDL6增殖活跃,分化差,细胞通讯缺陷,Cx43表达很低;另一亚系RDL3增殖慢,有明显肌分化,通讯功能强,Cx43及其mRNA的表达近似正常成肌细胞。Cx43 cDNA+neo基因转染RDL6得到Cx43稳定表达的克隆RDL6/C-4,其通讯功能恢复,增殖抑制,成肌性分化增强。本结果证明RD细胞分化阻断与Cx43降表达有关。Cx43的功能性表达不仅抑制瘤细胞增殖,而且与瘤细胞向正常分化有正相关性。     

Ⅱ型糖尿病大鼠神经视网膜组织中差异蛋白质分析

以高脂饮食联合小剂量链脲菌素(Streptozotocin, STZ ) 注射方法诱导大鼠II型糖尿病 (T2DM) 模型为研究对象, 采用比较蛋白质组学技术鉴定和分析早期糖尿病神经视网膜差异表达蛋白.通过联用液氮内研磨、冰浴超声、高速离心提取全蛋白技术,提取每只大鼠神经视网膜全蛋白质总量约900 μg.采用双向凝胶电泳(2-DE) 分离技术,获得2500个以上可辩认的蛋白质斑点.通过蛋白质组学比对技术筛选出糖尿病状态下神经视网膜差异表达蛋白,用基质辅助激光解吸电离飞行时间串联质谱 (MALDI-TOF- MS/MS) 和肽质量指纹图谱 (PMF) 鉴定出20个差异蛋白点.按照Gene Ontology (GO)分类体系,对差异蛋白归类分析,揭示其亚细胞定位和分子功能.应用Pathway Studio软件对差异表达蛋白的生物学意义进行分析,认为凋亡是早期糖尿病视网膜病变(Diabetic retinopathy,DR) 重要的细胞事件;AnnexinΙ、CRYAB、mtHsp70蛋白是与病理过程相关的关键蛋白,对研究DR致病机制有重要意义.     

缺氧预处理诱导心肌细胞蛋白质组变化的初步研究

缺氧预处理(hypoxia preconditioning, HPC)可模拟缺血预处理(ischemic preconditioning, IPC)对缺血/再灌注心肌的保护作用, 涉及细胞内众多分子事件. 本工作旨在采用双向电泳和质谱分析等蛋白质组分析技术, 发现缺氧预处理后心肌细胞蛋白质整体表达上的变化, 初步分析其与缺氧预处理心肌保护作用的关系. 将原代培养的SD乳鼠心肌细胞分为2组(n＝6): (1)缺氧预处理组(HPC): 将细胞置缺氧仓内短暂缺氧20 min进行缺氧预处理(HPC), 制备心肌细胞蛋白提取物; (2)对照组(control): 细胞置于培养箱内持续常氧孵育至实验结束, 提取蛋白. 采用双向凝胶电泳和图像扫描, 经蛋白样本分离和考马斯亮蓝染色后比较分析, 选取3个差异表达蛋白点进行胶内酶切、肽质量指纹图谱分析和数据库检索. 双向电泳可分离约529±45个蛋白质, 点匹配率约为78%±7.5%. 18种蛋白质在HPC后发生明显表达差异, 其中12种蛋白质表达降低, 6种表达增高. 经质谱分析鉴定出的3种蛋白质分别为myosin light polypeptide 3, nucleoside diphosphate kinase (NDPK)和calreticulin (CRT). 缺氧预处理引起心肌细胞蛋白质组变化, 初步发现其中myosin light polypeptide 3表达下调、nucleoside diphosphate kinase和calreticulin表达增加, 可能通过调节心肌细胞的收缩性、激活G蛋白、调节细胞内Ca^2＋浓度而保护心肌. 本工作通过研究缺氧预处理延迟保护过程中心肌内源性蛋白表达水平的变化, 有助于从细胞水平探讨预处理延迟保护机制.     

间充质干细胞扩增载体材料

间充质干细胞(MSCs)具有高度自我更新能力、多分化潜能、体外易分离和培养的特性,是细胞治疗和组织工程重要的种子细胞来源,但如何大规模地获得具有可再生活性的MSCs一直是限制其临床应用的关键因素,近几年发展起来的贴壁动物细胞动态培养技术为MSCs的大规模体外扩增提供了一条重要的途径。本综述结合动物细胞扩增载体的发展现状,主要介绍了用于间充质干细胞三维动态培养的明胶载体、海藻酸盐载体、壳聚糖载体和其他多糖载体等常规载体及其表面修饰和改性方法,并进一步介绍了以非酶解途径回收扩 增细胞的新型干细胞载体的研究进展。随着新型载体材料的涌现以及人们对干细胞生长和扩增特点的了解,采用三维动态培养技术安全而有效地大规模体外扩增MSCs的必要性将得到进一步的确认。     

中国小型猪心肌梗死模型的比较蛋白质组学研究

利用二维电泳(2DE)分离中国小型猪心肌梗死模型的正常与梗死心肌组织的蛋白提取液, 采用 PDQuest 软件对比分析了两种心肌组织在pH=5─8范围内的2DE谱图. 正常心肌组织检出851个蛋白点, 梗死组织检出1 032个蛋白点. 发现13个蛋白质点只在小型猪的正常心肌组织中表达, 而有14个蛋白质点只在梗死心肌组织中表达. 另外, 还有49个蛋白点在两种组织中表达量上有显著性变化(P<0.05), 选择进行质谱分析其中11个蛋白点, 成功地鉴定出7种蛋白, 蛋白功能分析结果表明, 这些蛋白的差异表达与心肌梗死过程相关.     

Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro

Embryonic stem (ES) cells are totipotent stem cells, which can differentiate into various kinds of cell types, including neurons. They are widely used as a model system for investigating mechanisms of differentiation events during early mouse development. In this study, proteomic techniques were used to approach the protein profile associated with the early-stage differentiation of ES cells into neuronal cells induced by all-trans retinoic acid (ATRA) in vitro. In comparison of the protein profile of parent ES cells with that of ES-derived neural-committed cells, which was induced by ATRA for four days, 24 differentially displayed protein spots were selected from two-dimensional electrophoresis (2-DE) gels for further protein identification by pepide mass fingerprinting (PMF). Nine proteins were known to being involved in the process of neural differentiation and/or neural survival. Of those, alpha-3/alpha-7 tubulin and vimentin were down-regulated, while cytokeratin 8, cytokeratin 18, G1/S-special cyclin D2, follistatin-related protein, NEL protein, platelet-activating factor acetylhydrolase IB alpha-subunit, and thioredoxin peroxidase 2 were upregulated during differentiation of ES cells to neural cells. Additionally, other 12 protein (five upregulated and seven downregulated) spots associated with ES cell differentiation into neuronal cells were not matched to known proteins so far, implicating that they might be novel proteins. The results above indicated that the molecular mechanisms of differentiation of ES cells to neural cells in vitro might be similar to those of other neural systems in vitro and identified that proteomic analysis is an effective strategy to comprehensively unravel the regulatory network of differentiation.     

Biomic study of human myeloid leukemia cells differentiation to macrophages using DNA array,proteomic, and bioinformatic analytical methods

A biomic approach by integrating three independent methods, DNA microarray, proteomics and bioinformatics, is used to study the differentiation of human myeloid leukemia cell line HL-60 into macrophages when induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Analysis of gene expression changes at the RNA level using cDNA against an array of 6033 human genes showed that 5950 (98.6%) of the genes were expressed in the HL-60 cells. A total of 624 genes (10.5%) were found to be regulated during HL-60 cell differentiation. Most of these genes have not been previously associated with HL-60 cells and include genes encoded for secreted proteins as well as genes involved in cell adhesion, signaling transduction, and metabolism. Protein analysis using two-dimensional gel electrophoresis showed a total of 682 distinct protein spots; 136 spots (19.9%) exhibited quantitative changes between HL-60 control and macrophages. These differentially expressed proteins were identified by mass spectrometry. We developed a bioinformatics program, the Bulk Gene Search System (BGSS, http://www.sinica.edu.tw:8900/perl/genequery.pl) to search for the functions of genes and proteins identified by cDNA microarrays and proteomics. The identified regulated proteins and genes were classified into seven groups according to subcellular locations and functions. This powerful holistic biomic approach using cDNA microarray, proteomics coupled to bioinformatics can provide in-depth information on the impact and importance of the regulated genes and proteins for HL-60 differentiation.     

Regulation of protein biosynthesis in neonatal rat cardiomyocytes by adrenoceptor-stimulation: investigations with high resolution two-dimensional polyacrylamide gel electrophoresis.

The role of alpha- and beta-adrenoceptor-mediated regulation of protein patterns in cultured neonatal rat cardiomyocytes was studied with high resolution two-dimensional polyacrylamide gel electrophoresis. Spontaneously beating neonatal rat cardiomyocytes were cultured for 72 h at 37 degrees C in serum-free medium, supplemented with either the catecholamine norepinephrine (0.1 microM), or with norepinephrine (0.1 microM) plus the alpha 1-adrenoceptor blocker prazosin (1 microM), or with neither of these substances. In transmission light, 105 protein spots could be seen in the lysates from control cells, 244 spots were identified in lysates from norepinephrine-treated cells, and 114 protein species were counted in the case of lysates from cardiomyocytes which had been cultured in the presence of norepinephrine plus prazosin. This experimental approach allows a clear classification of alpha- and non-alpha (probably beta)-adrenoceptor-mediated catecholamine effects on protein synthesis in cardiomyocytes. In comparison with previous experiments (Chang et al., Electrophoresis, this issue, pp. 748-754), less protein species were identified in the untreated control cardiomyocytes, as well as in the norepinephrine treated cells. The only actual modification in the experimental setup in these two series was the addition of dimethyl sulfoxide--a substance known for its repressive effect on oncoprotein expression--to the culture medium, as solvent for prazosin.     

Application of high resolution two-dimensional polyacrylamide gel electrophoresis of polypeptides from cultured neonatal rat cardiomyocytes: regulation of protein synthesis by catecholamines.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to cultured neonatal rat heart muscle cells, incubated for 72 h at 37 degrees C in serum-free medium, either in the absence or in the presence of 0.1 microM norepinephrine. After silver staining, about 340 and 550 protein spots could be seen in cardiomyocytes, cultured either in the absence or presence of norepinephrine. Of these spots, 141 could be further characterized according to isoelectric point and molecular weight, with 71 protein spots being present under both conditions. In cells cultivated in presence of norepinephrine, 58 new protein spots appeared, whereas 12 spots disappeared, and 22 spots increased (whereas 3 spots decreased) in intensity. In comparison with 2-D PAGE of rat cardiomyocytes, the protein pattern of the intact heart of neonatal rats is incongruent. 2-D PAGE of polypeptides of cultured neonatal rat cardiomyocytes may be a suitable tool to study the regulation of protein synthesis by various stimuli with relevance to cardiac growth adaptation, inotropy and heart failure.     

Profiling and semiquantitative analysis of the cell surface proteome in human mesenchymal stem cells

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n?=?1,001) were identified from cells cultured either with (n?=?857) or without (n?=?667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus

Cell adhesion and orientation are important for both natural and engineered tissues to fully achieve physiologic functions. Based on diverse cellular responses induced by electrical stimulus on 2D substrate, we applied non-invasive electrical stimulus to regulate cell adhesion and orientation of bone marrow-derived mesenchymal stem cells (MSCs) and fibroblasts in a reconstituted 3D collagen-based scaffold. While fibroblasts were induced to reorient perpendicularly in response to direct current electrical stimulus, rat MSCs showed only slight changes in cell reorientation. Multiphoton microscopy revealed that rat MSCs exhibited much stronger 3D adhesion, which appears to resist cell reorientation. Only in response to a large electrical stimulus (e.g., 10 V/cm), collagen fibers around rat MSCs became disconnected and loosely reorganized. In contrast, the collagen fibers surrounding the fibroblasts were entangled in a random network and became preferentially aligned in the direction of the electrical stimulus. When incubated with integrin antibodies, both fibroblasts and rat MSCs failed to respond to electrical stimulus, providing evidence that integrin-dependent molecular mechanisms are involved in 3D cell adhesion and orientation. Elucidation of physical regulation of 3D cell adhesion and orientation may offer a novel approach in controlling cell growth and differentiation and could be useful for stem cell-based therapeutic application and engineering tissue constructs.     

苯肾上腺素诱导乳鼠心肌细胞蛋白质表达变化的蛋白质组分析

α₁-肾上腺素受体(α₁-Adrenergic receptor, α₁-AR)是G蛋白偶联受体(G-protein coupled receptor, GPCR), 也是内源性儿茶酚胺、 去甲肾上腺素和肾上腺素最重要的靶受体之一. α₁-AR广泛分布于机体的各种器官、 组织和细胞中, 并介导多种生理效应, 如血管收缩、 蛋白质合成及心脏变力变时作用等^[1,2]. 很多研究已经证实, α₁-AR及其信号转导通路与许多心血管疾病存在密切关系^[3,4]. 蛋白质组学可提供一种发现在疾病情况下异常表达蛋白质的方法, 为疾病的早期诊断和愈后判断提供指南, 并为针对性疾病治疗提供科学依据