应激心肌细胞蛋白质组双向凝胶电泳分析

采用双向凝胶电泳技术和计算机辅助的图像分析方法 ,对去甲肾上腺素诱导的应激心肌细胞与正常心肌细胞蛋白质进行分离和比较分析 .正常心肌细胞可分离 12 32± 5 6个蛋白点 ,蛋白点匹配率为 83 3%± 1 0 %.有 11种蛋白质在NE应激后发生了明显和稳定的质和量的改变 (P <0 0 5 ) ,其中 6种 (Mr pI :4 9 7kD 7 8,38 3kD 5 9,37 1kD 6 6 ,2 9 3kD 7 4 ,18 7kD 6 1,18 5kD 7 7)在应激后表达降低 ,4种 (Mr pI:4 7 6kD 5 5 ,31 9kD 4 4 ,2 6 6kD 4 6 ,33 2kD 8 1)在应激后表达增高 ,1种 (Mr pI:19 4kD 6 9)只在应激后发生表达 .这些差异表达的蛋白质可能参与了心血管应激反应乃至应激损伤发生的过程 .     

樟子松突变丛生枝蛋白质的双向电泳分析

目的：研究樟子松突变丛生枝的发生机理。方法：对其正常枝和突变丛生枝进行蛋白质双向电泳,通过ImageMaster2-DE计算机分析系统对结果进行定性、定量分析。结果：共有59个蛋白点在表达的质和量上有变化,其中35个蛋白点在突变丛生枝2-DE图谱中表达量均高于正常枝的蛋白点,20个蛋白点在突变丛生枝2-DE图谱中表达量均低于正常枝的蛋白点,4个蛋白点在突变丛生枝图谱上发生缺失。结论：差异表达的蛋白点可能与突变丛生枝的发生有关。     

大豆雄性不育系与其保持系不同器官蛋白质比较

采用双向凝胶电泳技术对大豆质核互作雄性不育系NJCMS2A及其保持系NJCMS2B的种子、叶片和花药等不同器官蛋白质进行比较分析.结果显示,不育系NJCMS2A与其保持系NJCMS2B的花药2-DE图谱间存在较多差异表达蛋白点,种子2-DE图谱间仅有少量差异表达蛋白点,而叶片2-DE图谱间基本没有差异表达蛋白点.结果表明,不育基因表达具有时空性和器官特异性,与育性有关的蛋白主要在花药中表达.     

乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化

应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27．1kD、p,7．3),TE2(17．5kD、pI8．2)2个特异蛋白,45d存在TE3(11．4kD、pI7．6),TE4(13．2kD、pI9．9)2个特异蛋白,52d存在TE5(22．6kD、pI7．2),TE6(18．6kD、pI8．3),TE,(23．5kD、pI3．6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失．对胚胎的分化发育具有重要作用。     

ALA-PDT对红斑狼疮患者外周血CD40阳性淋巴细胞的影响

探讨了δ氨基酮戊酸—光动力疗法 ( ALA PDT)对红斑狼疮患者外周血中 CD4 0阳性淋巴细胞的影响及其作用方式。方法 :采用免疫荧光双标记—流式细胞仪检测了 1 2例活动期红斑狼疮患者外周血淋巴细胞在光动力疗法前后 CD4 0、CD95的表达。结果 :1 ALA PDT后 CD4 0阳性淋巴细胞下降至 9.2 8± 1 .2 2 ,较 ALA PDT前的 1 3.36± 0 .89,有显著性的差异 ( P<0 .0 5)。2 CD95 /CD4 0 淋巴细胞在 ALA PDT后立即上升至 1 3.2 3± 2 .1 0 ,较 ALA PDT前的 7.84± 1 .93,有非常显著的差异 ( P<0 .0 1 )。 ALA PDT后继续培养 1 8小时检测 CD95 /CD4 0 淋巴细胞下降至 7.68± 1 .4 6,较 ALA PDT后即刻检测有显著性差异( 1 3.2 3± 2 .1 0 ,P<0 .0 1 )。结论 :ALA PDT能降低 CD4 0阳性淋巴细胞百分比 ,即可能抑制活动期红斑狼疮患者外周血中 B细胞的活性。这种作用可能与 Fas介导的凋亡有关     

溴氰菊酯胁迫下小菜蛾幼虫体内差异表达蛋白的分析

小菜蛾Plutella xylostella L.是世界性十字花科蔬菜的主要害虫, 已对多种杀虫剂产生抗性, 其中以对拟除虫菊酯类杀虫剂的抗性发展最快。溴氰菊酯是拟除虫菊酯杀虫剂中杀虫毒力最强的品种。我们前期的研究发现, 小菜蛾溴氰菊酯敏感品系（DS）和抗性品系（DR）成虫期的蛋白质双向电泳(2-DE)图谱存在显著差异。本研究通过双向电泳技术从小菜蛾4龄幼虫中分离出89个有明显差异的蛋白点, 从中选出30个进行串联质谱(MALDI-TOF-MS)实验, 并利用蛋白质数据库检索这些在抗性品系中表达而在敏感品系中不表达或者不同品系中差异表达的蛋白质的归属、 性质和功能, 最终成功鉴定出10个蛋白。对其中的3个基因进行了荧光定量PCR验证, 发现这些蛋白质在mRNA水平的表达与在蛋白水平的表达是一致的。这些在溴氰菊酯胁迫下差异表达的蛋白为研究溴氰菊酯的作用靶标和作用机理, 以及筛选与其抗性相关的蛋白质提供了依据。     

家蚕蛾触角蛋白的双向电泳分析

为探讨家蚕Bombyx mori蛾触角发生发育、结构与功能的分子基础和调控机制, 我们采用双向电泳结合基质辅助质量飞行时间质谱(MALDI-TOF/MS)技术初步探讨了家蚕蛾触角蛋白及其雌雄表达差异。采用ImageMast 6.0软件分析电泳图谱, 在家蚕蛾触角中检测到约550个蛋白点, 主要集中在分子量14～70 kD, 等电点4～8之间。从雌雄蛾触角电泳图谱中分别检测到419和489个蛋白点, 其中雌雄匹配蛋白点有326对, 匹配率为71.81%。雌雄间表达差异点有34个, 雌雄分别所特有的特异蛋白点分别为9和20个。对特异和差异点进行MALDI-TOF/MS鉴定获得其中5种蛋白--成虫原基生长因子(imaginal disk growth factor)、表皮蛋白RR-1基序15(cuticular protein RR-1 motif 15)、硫醇过氧化还原酶(thiol peroxiredoxin)、空泡ATP酶B亚基(vacuolar ATPase B subunit)以及gasp前体(gasp precursor), 它们在雄蛾触角中表达量都比雌蛾高。这为家蚕触角蛋白的进一步研究提供了基本信息。     

肾阳虚证候的人血清比较蛋白质组学分析

利用双向电泳（2-DE）优化分离了除去高丰度蛋白（白蛋白和IgG）的老年体虚肾阳虚患者（以健康组为参照）的血清样本,对比分析pH 4～7范围的2-DE谱图,肾阳虚患者和参照组的平均蛋白点分别为(393±32)和(455±19)个. 其中肾阳虚证候表达量上调2倍（P<0.05）以上的蛋白点有26个,下调2倍以上的有33个. 用质谱获得上述差异蛋白的肽质量指纹图谱,并经数据库检索共鉴定出了49种差异蛋白质,其中有10种在肾阳虚血清中特异表达,有6种在健康组血清中特异表达. 蛋白功能分析发现,其中33种蛋白质的差异表达与肾阳虚证密切相关. 蛋白质TCRβ及transthyretin的蛋白印迹实验验证了2-DE 的结果. 该研究结果为阐明中医肾阳虚证的机理提供了一条新途径.     

水稻红莲型细胞质雄性不育系小孢子不同发育时期花药总蛋白质比较分析

采用双向凝胶电泳对水稻红莲型细胞质雄性不育的不育系小孢子发育单核期和二核期花药总蛋白进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱,且单核期和二核期花药总蛋白质在双向电泳胶上分布的图谱十分相似。PDQuest 2DE图像分析软件在等电点(pI)3.0~10.0、分子量(M.W.)9.0~98.0 kD之间可识别约1 800个蛋白质点。比较分析发现单核期和二核期花药中共有241个差异表达的蛋白质点,其中仅在单核期中表达的点数为125,仅在二核期中表达的为13点;表现为表达量差异的105点,其中在二核期表达下调的点数为70点,表达上调的为33点。还对蛋白质点集中的区域(pI 4.5~8.0,M.W.25.0~70.0 kD)中的41个差异蛋白质点进行了分子量和等电点分析。     

快速老化小鼠额叶皮质的蛋白组学研究

目的:探讨快速老化过程中差异蛋白质及其与学习记忆的关系.方法:以13月龄和8月龄快速老化小鼠模型的快速老化亚系SAMP8和抗快速老化亚系SAMR1的额叶为研究对象,经双向电泳技术和考马斯亮兰G250染色,分别获取13月龄和8月龄SAMP8和同龄SAMR1额叶的2-DE考染图谱,用PDQuest 7.40图像分析软件,建立不同月龄蛋白质组的匹配差异图谱,分析图谱中SAMP8与SAMR1蛋白质的表达变化.结果:8月龄SAMP8额叶检测到579个蛋白点,同龄SAMR1额叶检测到612个蛋白点;13月龄SAMP8额叶检测到705个蛋白点,同龄SAMR1额叶检测到621个蛋白点.PDQuest7.40软件匹配差异分析显示,8月龄SAMP8与同龄SAMR1比较,SAMP8表达缺失33个蛋白,两者共有显著差异表达蛋白35个.13月龄SAMP8与同龄SAMR1比较,SAMR1中表达缺失84个蛋白,两者均有但表达有显著变化的蛋白质36个.13月龄和8月龄的SAMP8互相比较,13月龄组新增蛋白126个,二者共有差异表达蛋白58个.13月龄和8月龄的SAMR1比较,13月龄组新增9个蛋白,二者共有差异表达蛋白33个.结论:两组不同月龄小鼠额叶SAMP8和SAMR1存在差异表达蛋白质,进一步研究有助于了解衰老的发生机制,并为研发调节学习记忆蛋白的新药提供依据.     

全反式维甲酸及依托泊苷对急性早幼粒细胞白血病细胞磷脂酰丝氨酸暴露及促凝血活性的影响

目的：探讨磷脂酰丝氨酸（PS）暴露在急性早幼粒细胞白血病（APL）细胞促凝血活性中的作用及不同药物对其产生的影响。方法：实验共分为4组：新采集APL细胞组、APL细胞单纯培养组、APL细胞全反式维甲酸（ATRA）处理组及APL细胞依托泊苷（VP16）处理组。提取10名初发APL患者的骨髓APL细胞进行实验,提取10名健康成人外周血单个核细胞作为凝血实验的正常对照。分别用1μmol·L-1ATRA和1μmol·L-1VP16处理APL细胞24 h,利用共聚焦显微镜及流式细胞术检测各组细胞PS暴露情况。利用凝血实验检测各组细胞总的促凝活性及细胞表面磷脂的促凝血活性。利用PS特异结合蛋白乳粘素对各组细胞进行凝血抑制实验。结果：新采集的APL细胞存在一定量的PS外翻,并且与外周血单个核细胞相比,存在更高的促凝血活性（P〈0.05）,ATRA对APL细胞的PS外翻及促凝活性有抑制作用（P〈0.05）,VP16则对其有显著的促进作用（P〈0.001）。乳粘素可以拮抗APL细胞至少70%的促FXa和FIIa生成活性。结论：PS暴露在APL细胞促凝血过程中发挥着重要作用。分化治疗药物ATRA和化疗药物VP16分别通过减少和增加APL细胞表面PS的暴露来减轻和加重凝血紊乱。乳粘素通过与PS特异结合可以有效地阻断暴露的PS的促凝活性,是一种潜在的治疗APL凝血紊乱的抗凝剂。     

The effect of temperature on midgut and brain protein profiles in Morimus funereus larvae (Coleoptera: Cerambycidae)

The 7-days shift of M. funereus larvae, from nature to a constant temperature of 23 degrees C led to changes in midgut and brain protein quality and quantity. The changes in midgut protein profiles are characterized by an intensified protein band Mr of 29 kD, the absence of protein Mr of 22 kD and less intense bands Mr of 8.5-2.5 kD. Electrophoretic patterns of brain proteins showed less intense Mr of 66-2.5 kD protein bands.     

Analysis of proteins in normal and regenerating rat liver using two-dimensional electrophoresis

The major proteins of homogenate, cytosol, nuclei and nuclear membrane extract from normal and regenerating rat liver were studied by two-dimensional electrophoresis with a view of detecting proteins involved in DNA replication regulation. Essential quantitative differences in three out of 200 polypeptides separated as spots and dyed with Coomassie R-250 on two-dimensional maps were revealed. The content of the p38 nuclear protein (Mr congruent to 38 kD, pI congruent to 4) increases 6-8-fold in the S-phase. The level of another nuclear protein, p50 (Mr congruent to 50 kD, pI congruent to 6.5) decreases 2-3-fold. The cytoplasmic protein p35 (Mr congruent to 35 kD, pI congruent to 8) also decreases 2-3-fold. Moreover, the p40 protein (Mr congruent to 40 kD, pI congruent to 6) whose content in the nuclei sharply rises up to 20 times after sham operation was revealed.     

A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus.

By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.     

ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL’s hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1 expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.     

ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL's hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1 expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.     

双向电泳联合MALDI-TOF-MS技术在筛选食管癌铂类耐药患者中的应用研究

目的:采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术检测食管癌(EC)患者化疗敏感性血清蛋白质谱,指导临床EC患者化疗方案的选择。方法:收集2013年1月至2014年6月我院收治的EC术后复发并行含铂化疗的患者40例,按照化疗疗效分为敏感组和耐药组。将两组蛋白质双向凝胶电泳图谱用Image Master 2D platinum 5.0软件分析并匹配,筛选出两组差异斑点,并采用MALDI-TOF-MS技术检测EC患者化疗敏感性血清蛋白质谱。结果:两组差异斑点共有68个,以耐药组为参照,敏感组患者血清蛋白下调的斑点有42个,上调的斑点有26个。从这68个差异斑点中筛选出8个斑点进行质谱鉴定,通过数据库检索分析和鉴定最终确定上述斑点所对应的蛋白质名称分别为Kininogen-1,Alpha-2-HS-glycoprotein,Fibrinogen beta chain,Clusterin,Retinol-binding protein 4,Beta-2-glycoprotein 1,Ig kappa chain C region,Complement C4-A。结论:基于铂类化疗的EC敏感患者和耐药患者间存在差异表达蛋白。     

Identification of a type 6 protein ser/thr phosphatase regulated by interleukin-2 stimulation

We have identified a 36 kD phosphoprotein that forms a complex with spliceosomal small nuclear ribonucleoproteins in lymphocyte extracts. This 36 kD protein is differentially phosphorylated in transformed human lymphoid cell lines and is regulated by IL-2 in peripheral blood T cells. We purified the 36 kD protein from human lymphocytes by employing a combination of immuno-affinity chromatography and preparative two-dimensional gel electrophoresis. Internal amino acid sequence analysis of the purified protein yielded two peptides that had perfect matches with sequences in the human protein serine/threonine phosphatase 6 (PP6). Using degenerate primers corresponding to the peptides, we obtained from a human T lymphocyte cDNA library a DNA fragment whose sequence is homologous to an EST cDNA clone (R05547). The predicted amino acid sequence of this clone showed over 98% sequence identity to human PP6. The identification of an IL-2 regulated type 6 protein serine/threonine phosphatase in lymphocytes was further substantiated by immunoblotting with anti-peptide antibodies. These findings suggest that PP6 is a component of a signaling pathway regulating cell cycle progression in response to IL-2 receptor stimulation.     

Proteomic analysis of sensitive and multi drug resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Multidrug-resistant tuberculosis (MDR-TB) is caused by bacteria that are resistant to the most effective anti TB drugs (Isoniazid and Rifampicin) with or without resistance to other drugs. Novel intervention strategies to eliminate this disease based on finding proteins can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profile of MDR-TB with sensitive isolates. Two-dimensional gel electrophoresis (2DE) along with mass spectrometry is a powerful and effective tool to identification and characterization of Mycobacterium tuberculosis. Two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for diagnosis and comparison of proteins. We identified 14 protein spots in MDR-TB isolates that 2DE analysis showed these spots absent in M. tuberculosis sensitive isolates (Rv1876, Rv0379, Rv0147, Rv2031c, Rv3597c, Rv1886c, MT0493, Rv0440, Rv3614c, Rv1626, Rv0443, Rv0475, Rv3057 and unknown protein. The results showed 22 protein spots which were up regulated (or expressed) by the MDR-TB isolates, (Rv1240, Rv3028c, Rv2971, Rv2114c, Rv3311, Rv3699, Rv1023, Rv1308, Rv3774, Rv0831c, Rv2890c, Rv1392, Rv0719, Rv0054, Rv3418c, Rv0462, Rv2215, Rv2986c, Rv3248c and Rv1908c)). Two up regulated protein spots were identified in sensitive isolate (Rv1133c and Rv0685). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug resistant and sensitive of M. tuberculosis.