急性重型脑外伤后脑蛋白表达变化的蛋白质组研究

目的应用蛋白质组学技术，研究重型人脑外伤后的脑皮层蛋白质组表达变化的情况。方法提取脑挫伤部位皮层的总蛋白。通过双向电泳，分离蛋白。应用胶内酶切、生物质谱，鉴定由图像分析软件所得出的具有表达差异的蛋白质点。结果在急性期8h内，目前已发现138个蛋白质点，表达水平具有显著性差异变化。在已鉴定出的83个蛋白点中，属于64种蛋白质。依其功能可分为：细胞骨架、代谢反应、核酸蛋白合成与更新、信号传导、氧化应激反应、功能未知等几类。在急性期，大多数差异蛋白表达水平呈波动变化。结论蛋白质组技术作为一个有力的大规模、高通量的分析蛋白质混合物工具，可快速的建立脑蛋白表达谱。在急性期，脑蛋白质表达呈剧烈变化主要为参与细胞代谢反应、结构修复等组织代偿反应。     

蛋白质组学在创伤后异位骨化调控蛋白研究中的意义

异位骨化是指不应发生骨化的软组织内出现病理性骨组织形成,多见于骨骼肌肉创伤、骨关节术后、神经系统损伤等。文章总结了运用蛋白质组学技术分析、比较异位骨组织在不同形成阶段的调控蛋白质在种类、数量存在的差异,据此可以筛选出不同时间点差异表达的功能蛋白,并针对性地对其中感兴趣的蛋白进行鉴定和功能验证,这种方式有助于发现新的调控蛋白,了解各种蛋白质之间相互作用的关系。证实蛋白质组学在创伤后异位骨化调控蛋白的研究中有广阔的应用前景,这有助于进一步阐明创伤后异位骨形成机制以及探索新的生物分子调控位点。     

PC12细胞基础状态氧化蛋白质表达谱

目的绘制基础状态下PC12细胞氧化蛋白质的表达谱,期望为以PC12细胞为模型的实验研究提供基本理论依据。方法采用2D凝胶电泳和westernblot技术相结合的方法获得氧化修饰的蛋白点,运用MALDI-TOF质谱鉴定出被氧化的蛋白质。结果PC12细胞总蛋白中共有91个蛋白点发生氧化修饰,运用MALDI-TOF质谱鉴定出19个氧化蛋白质。结论处于基础状态的PC12细胞存在蛋白质的氧化修饰,可以为病理条件下的氧化损伤研究提供可靠的参照系统。     

NOGO-A在创伤性脑损伤后大鼠脑组织含量表达及干预实验研究

目的研究创伤性脑损伤（TBI）后大鼠脑组织内NOGO-A分子含量及大鼠脑超微结构的改变以及二者与RHOA/ROCK信号传导通道的联系。 方法45只健康SD大鼠按照随机数字表法分为对照组、中度创伤组和干预组，每组15只。中度创伤组和干预组大鼠采用击锤、撞杆自由落体原理致伤，制作TBI大鼠模型，干预组大鼠创伤后立即给予静脉注射盐酸法舒地尔注射液。TBI后24 h处死各组大鼠，采用电子显微镜观察各组损伤区脑组织细胞超微结构的变化，采用免疫组化技术观察各组大鼠脑组织中NOGO-A蛋白的含量，并测定NOGO-A蛋白的灰度值。 结果TBI后24 h，中度创伤组及干预组大鼠脑组织损伤区出现病理性损伤，干预组程度较中度创伤组轻。3组大鼠脑组织内NOGO-A蛋白表达量比较，差异有统计学意义（F=176.085，P<0.05），中度创伤组的NOGO-A蛋白含量最高，干预组次之，对照组最低。 结论TBI后24 h大鼠脑内损伤区出现细胞核、线粒体损害及细胞水肿，NOGO-A含量增加。RHOA/ROCK信号通路被阻断后能够改善TBI大鼠脑组织损伤区细胞超微结构损害以及减少NOGO-A的含量。     

颅脑创伤后亚低温脑保护的蛋白质组研究

目的 本研究应用差异蛋白质组学技术,探讨亚低温和常温条件下,创伤性颅脑损伤大鼠海马组织蛋白质的表达变化.方法 采用侧方液压冲击装置,建立大鼠中度脑损伤模型,亚低温组(n=3)于伤后维持体温(33±0.5)℃持续3h,常温组(n=3)始终维持体温(37±0.5)℃.取大鼠海马组织,通过差异荧光双向凝胶电泳、分离蛋白,获得二维的蛋白质分离图谱,然后通过胶内酶切、抽提酶解肽段、基质辅助激光解吸飞行时间质谱分析差异的蛋白质点,鉴定出变化的蛋白质.结果 通过DeCyder5.0(GE Healthcare)软件分析报告发现差异1.5倍以上的蛋白点17个(P＜0.05).通过对这些蛋白考染点进行质谱鉴定,鉴定出14个蛋白质,2个为同一种蛋白,实际差异蛋白数为13个.分别是细胞骨架蛋白、介导能量代谢的酶类、参与核酸合成、氧化应激反应的蛋白质、神经突触功能蛋白、细胞内信号传递蛋白及未知蛋白.结论 脑损伤后亚低温及常温条件下,大鼠海马组织蛋白质存在表达差异,鉴定出的差异蛋白质可能与亚低温保护效应的潜在作用机制有关.     

蛋白质组学技术鉴定的PC12细胞的细胞骨架蛋白

目的:使用蛋白质组学的技术手段,鉴定大鼠肾上腺皮质细胞瘤细胞系PC12细胞内的细胞骨架蛋白。方法:提取PC12细胞的蛋白质,建立固相pH梯度双向电泳图谱,应用图像扫描仪及ImageMaster 2D Elite分析软件获得蛋白质点的数字化和匹配性信息,挑选匹配良好的高峰度蛋白点,进行基质辅助激光解吸／电离飞行时间质谱(MALDI- TOF-MS)分析,鉴定。结果:用二维电泳技术分离,并用MALDI-TOF-MS成功鉴定出5个PC12细胞的细胞骨架蛋白。结论:PC12细胞蛋白质组中部分细胞骨架蛋白胶图位点的建立,为今后探讨这一类蛋白在神经系统疾病中的作用奠定了基础,并提供了新的侯选治疗靶点。     

诺帝诱导人胶质瘤细胞系CHG-5分化的蛋白质表达图谱的建立及差异蛋白质组分析

目的建立诺帝诱导人胶质瘤细胞系CHG-5分化的蛋白质表达图谱并比较其表达差异。方法分别用100μmol/L、200μmol/L诺帝诱导CHG-5细胞分化,观察处理后24h、48h、72h细胞的形态改变;分别提取200μmol/L诺帝处理后的CHG-5细胞和对照组细胞总蛋白,进行双向电泳,所获蛋白表达图谱用PDquest 7.1软件比较其蛋白质表达差异,选取部分高表达的差异蛋白进行质谱分析。结果100pμmol/L和200μmol/L诺帝处理后均可明显诱导CHG-5细胞分化,以72h时细胞分化最为明显。双向电泳发现诱导分化后细胞出现了18个差异蛋白点,其中9个蛋白点表达上调,9个蛋白点表达下调,并获得差异蛋白的分子量、等电点等信息。对其中部分高表达的差异蛋白点成功地进行了质谱鉴定。结论诺帝诱导人胶质瘤细胞系CHG-5分化时蛋白质组改变涉及到细胞增殖、凋亡、基因转录、蛋白表达调控等各个方面。     

蛋白质组学技术分离及鉴定人脑氧化蛋白质***☆◆

背景：氧化修饰蛋白质在脑老化及阿尔茨海默病等老年变性神经病的发病机制中扮演着重要角色。蛋白质组学技术可以发现氧化修饰蛋白质,为相关疾病的药物治疗选择新的靶标。 目的：建立以双向电泳结合Western blot及生物质谱技术分离、鉴定人脑氧化修饰蛋白质的方法。 方法：取3个无神经系统疾患的男性脑组织蛋白,第一向等电聚焦电泳后,固相pH梯度胶条同二硝基苯肼反应,然后行第二向SDS-PAGE电泳。2张相同凝胶中的1张行考马斯亮蓝染色,另1张凝胶上免疫荧光显色。同PVDF膜上显色点相对应的考染凝胶上的蛋白质点即为氧化修饰蛋白质。凝胶蛋白胶内酶解,基质辅助激光解析飞行时间串联质谱鉴定氧化修饰蛋白质。 结果与结论：从提取的脑组织中鉴定出β-肌动蛋白、天冬氨酸氨基转移酶、果糖二磷酸醛缩酶、磷酸甘油酸激酶、1,3-磷酸甘油醛脱氢酶、碳酰还原酶1、磷酸丙糖异构酶、转酮醇酶、丙酮酸激酶、血清白蛋白前体、二氢嘧啶酶相关蛋白质2、热休克蛋白60为氧化修饰蛋白质。说明实验成功建立了用蛋白质组学技术分离及鉴定人脑氧化蛋白质的方法。     

Aβ_(25-35)诱导PC12细胞损伤的蛋白质组学研究

目的 识别AD细胞模型中蛋白质组改变,在蛋白质水平上揭示Aβ的作用机制.方法 利用双向差异凝胶电泳技术(2D-DIGE)和质谱(MS)技术.探索20μmol/L浓度Aβ_(25-35)肽段作用48h后PC12细胞蛋白质组的改变.结果 2D-DIGE图像上出现约2000个蛋白点.与对照组比较,Aβ_(25-35)作用下共有29个蛋白的表达有显著差异,其中25个蛋白表达量上调及4个蛋白表达量下调超过30%.质谱鉴定出7个蛋白点,分为3类:(1)具有分子伴侣活性的蛋白:葡萄糖调节蛋白75(glucose-regulated protein75,GRP75)、热休克同源蛋白71(heat shock cognate 71 kDa protein,HSC71)、calreticulin表达量均上调.(2)细胞骨架蛋白:β-微管蛋白(tubulin beta chain 15,TBETA-15)和低分子量神经细丝蛋白(neurofilament light polypeptide,NF-L)表达量亦上调.(3)与能量代谢有关的酶:肌酸激酶B(creatine kinase-B,CKB)和醛缩酶A(aldolaseA)蛋白表达量均下调.结论 本实验首次将DIGE和MS方法应用于Aβ_(25-35)神经毒性作用机制研究,在蛋白质组水平上揭示了Aβ_(25-35)毒性作用的早期机制.     

深低温脑保护的差异蛋白质组学研究

目的应用差异蛋白质组学技术，研究深低温和常温停循环后大鼠海马组织蛋白质的变化。方法采用深低温停循环模型，取大鼠海马组织，通过双向电泳、分离蛋白，然后通过胶内酶切、生物质谱分析差异的蛋白质点，鉴定出变化的蛋白质。结果通过Image Master软件分析报告发现有差异的Ratio值大于1．5的蛋白考染点14个。通过对这些蛋白考染点进行质谱鉴定，鉴定出28个蛋白质，其中4个为同一种蛋白，实际的蛋白数为24个。它们是细胞骨架蛋白、介导代谢的酶类、参与核酸合成、参与氧化应激反应的蛋白质及未知蛋白。结论鉴定出的差异蛋白质可能与深低温脑保护作用有关，某些蛋白质在低温脑保护中的作用尚未报道。     

Two-dimensional electrophoretogram of acute brain injury-associated proteins ***☆ Comparison between injured and normal cerebral cortex

BACKGROUND： To this date, specific molecular markers for early diagnosis and prognosis monitoring of craniocerebral injury in clinical medicine do not exist. Therefore, differential detection of specific proteins might play an important role in diagnosis and treatment of this type of brain injury.
OBJECTIVE： To compare differential cerebral cortical protein expression of craniocerebral injury patients and normal subjects through the use of proteomics.
DESIGN： Contrast observation.
SETTING： Department of Neurosurgery, Xiangya Hospital of Central South University.
PARTICIPANTS： Ten patients （6 males and 4 females, 20 58 years old）, with severe craniocerebral injury, were selected at the Department of Neurosurgery, Xiangya Hospital of Central South University, from June 2004 to December 2006. All patients were diagnosed with CT test and Glasgow test （scores 〈 8）. Surgery was performed 4-12 hours after craniocerebral injury, and injured cortical tissues of the frontal and temporal lobes were resected for sampling. At the same time, control cortical tissues were collected from frontal and temporal lobes of 2 epileptic patients who underwent hippocampus-nucleus amygdala resection, and 2 lateral ventricular tumor patients who underwent tumor resection. The participants and their relatives provided confirmed consent, and this study received confirrned consent from the local ethics committee.
METHODS： Ten samples from injured patients and 4 normal samples were compared through the use of proteomics. Total protein was separated by using two-dimensional electrophoresis with immobilized pH gradients, and the differential protein expressions were compared using image analysis after blue-sliver staining. Differential protein spot expressions were analyzed with a matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI/TOF MS） and electrospray ionization-quadrupole time of flight mass spectrometry （ESI-Qq TOF MS）.
MAIN OUTCOME MEASURES：① Two-dimensional el     

人脑低级别星形细胞瘤及瘤周脑组织的差异蛋白表达

Differential protein expression between various pathological grades of glioma has been shown in studies of glioma proteomics. However, very little data is available regarding normal brain tissues and glioma differential protein expression, because normal human brain tissues are difficult to harvest. The present study selected samples from low-grade astrocytomas and peritumoral brain tissues to analyze differential protein expression by two-dimensional (2D) electrophoresis and mass spectrometry techniques. Results revealing 36 protein spots by 2D electrophoresis, including 23 spots revealing increased expression and 13 spots revealing decreased expression. However, 25 differential proteins were identified by mass spectrometry, including 16 proteins with increased expression and 9 with decreased expression. Western blot analysis confirmed the mass spectrometry results, i.e., heat shock protein 70 (HSP70) and human transthyretin (TTR) expressions were increased, but glial fibrillary acidic protein (GFAP) was decreased, in astrocytomas. The present study constructed a 2D electrophoresis pattern between low-grade astrocytomas in the human brain and peritumoral tissues. Results demonstrated that a majority of differential proteins, such as HSP70, TTR, and GFAP, participate in malignant progression of gliomas.     

Relationship of calpain-mediated proteolysis to the expression of axonal and synaptic plasticity markers following traumatic brain injury in mice

The role of neuronal plasticity and repair on the final functional outcome following traumatic brain injury (TBI) remains poorly understood. Moreover, the relationship of the magnitude of post-traumatic secondary injury and neurodegeneration to the potential for neuronal repair has not been explored. To address these questions, we employed Western immunoblotting techniques to examine how injury severity affects the spatial and temporal expression of markers of axonal growth (growth-associated protein GAP-43) and synaptogenesis (pre-synaptic vesicular protein synaptophysin) following either moderate (0.5 mm, 3.5 M/s) or severe (1.0 mm, 3.5 M/s) lateral controlled cortical impact traumatic brain injury (CCI-TBI) in young adult male CF-1 mice. Moderate CCI increased GAP-43 levels at 24 and 48 h post-insult in the ipsilateral hippocampus relative to sham, non-injured animals. This increase in axonal plasticity occurred prior to maximal hippocampal neurodegeneration, as revealed by de Olmos silver staining, at 72 h. However, moderate CCI-TBI did not elevate GAP-43 expression in the ipsilateral cortex where neurodegeneration was extensive by 6 h post-TBI. In contrast to moderate injury, severe CCI-TBI failed to increase hippocampal GAP-43 levels and instead resulted in depressed GAP-43 expression in the ipsilateral hippocampus and cortex at 48 h post-insult. In regards to injury-induced changes in synaptogenesis, we found that moderate CCI-TBI elevated synaptophysin levels in the ipsilateral hippocampus at 24, 48, 72 h and 21 days, but this effect was not present after severe injury. Together, these data highlights the adult brain's ability for axonal and synaptic plasticity following a focal cortical injury, but that severe injuries may diminish these endogenous repair mechanisms. The differential effects of moderate versus severe TBI on the post-traumatic plasticity response may be related to the calpain-mediated proteolytic activity occurring after a severe injury preventing increased expression of proteins required for plasticity. Supporting this hypothesis is the fact that GAP-43 is a substrate for calpain along with our data demonstrating that calpain-mediated degradation of the cytoskeletal protein, alpha-spectrin, is approximately 10 times greater in ipsilateral hippocampal tissue following severe compared to moderate CCI-TBI. Thus, TBI severity has a differential effect on the injury-induced neurorestorative response with calpain activation being one putative factor contributing to neuroregenerative failure following severe CCI-TBI. If true, then calpain inhibition may lead to both neuroprotective effects and an enhancement of neuronal plasticity/repair mechanisms post-TBI.     

应用蛋白质组学技术探讨亚低温脑保护机制

目的 利用双向电泳及质谱鉴定在内的蛋白质组学技术探讨哑低温脑保护的作用机制.方法 选择同龄、同家系雄性SD大鼠制作中度液压冲击性脑损伤模型,以最大化消除个体间差异;动物分为对照组和治疗组,治疗组大鼠伤侧腩组织接受亚低温治疗仪局部降温处理4 h,12 h内缓慢复温至正常脑温;获取低温处理4 h的脑组织进行双向电泳和质谱鉴定,剩余大鼠进行神经功能缺陷评分比较.结果 共筛选出21个差异蛋白质点,16个得到认证,这些蛋白质参与炎性反应与氧化应激、细胞增殖与老化、神经胶质再牛及细胞代谢等多个病理生理过程;亚低温治疗大鼠平均神经功能缺陷评分明显好于对照组(P＜0.05).结论 亚低温脑保护机制复杂,同时涉及多个分子病理通路,以双向电泳及质谱鉴定为基础的蛋白质组学技术可能成为研究该复杂机制的最有效工具之一.     

Proteomic comparison between human young and old brains by two-dimensional gel electrophoresis and identification of proteins

To investigate molecular mechanisms of human brain aging, brain proteins were isolated from postmortem human young and old brains and profiled by two-dimensional gel electrophoresis (2-DE). With the help of special software, five down-regulated protein spots in two-dimensional gel electrophoresis gels of old brains were found compared with young brains, four of which was identified as a protein similar to peroxiredoxin 2 (accession-numbered as gi | 13631440), two of stathmin (phosphoprotein p19) and apolipoprotein A-I precursor (apo-AI) by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight common proteins, whose expressions were not altered between young and old brains, were also identified. The possible relevance of changes was analyzed. This study shows that the contribution of proteomics could be valuable in experimental gerontology field.     

Acutely increased cyclophilin a expression after brain injury: a role in blood-brain barrier function and tissue preservation

Blood-brain barrier (BBB) compromise is a significant pathologic event that manifests early following traumatic brain injury (TBI). Because many signaling cascades are initiated immediately after the traumatic event, we were interested in examining acute differential protein expression that may be involved in BBB function. At acute time points postinjury, altered protein expression may result from altered translation efficiency or turnover rate rather than from a genomic response. The application of tandem 2-D gel electrophoresis and mass spectrometry analysis is a powerful approach for directly screening differential protein expression following TBI. Using comparative 2-D gel analysis, we selected candidate protein spots with apparent altered expression and identified them by mass spectrometry. Cyclophilin A was selected for further analysis because it has been implicated in endothelial cell activation and inflammation, and studies have suggested cyclosporine A, an inhibitor of all cyclophilin isoforms, might be beneficial after TBI. We examined if altered expression of cyclophilin A in the brain vasculature might play a role in BBB function. We found significantly increased cyclophilin A levels in isolated brain microvessels 30 min following injury. Postinjury administration of cyclosporine A significantly attenuated BBB permeability measured 24 hr postinjury, suggesting cyclophilin activity after TBI may be detrimental. However, direct injection of purified recombinant cyclophilin A attenuated both BBB permeability and tissue damage in a stab wound model of injury. These findings suggest that increased expression of cyclophilin A may play a protective role after TBI, whereas other cyclophilin isoforms may be detrimental.     

Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration

The reinforcing effects and long-term consequences of cocaine self-administration have been associated with brain regions of the mesolimbic dopamine pathway, namely the nucleus accumbens (NAc). Studies of cocaine-induced biochemical adaptations in rodent models have advanced our knowledge; however, unbiased detailed assessments of intracellular alterations in the primate brain are scarce, yet essential, to develop a comprehensive understanding of cocaine addiction. To this end, two-dimensional difference in gel electrophoresis (2D-DIGE) was used to compare changes in cytosolic protein abundance in the NAc between rhesus monkeys self-administering cocaine and controls. Following image normalization, spots with significantly differential image intensities (P<0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser-desorption ionization time-of-flight time-of-flight (MALDI-TOF-TOF). In total, 1098 spots were subjected to statistical analysis with 22 spots found to be differentially abundant of which 18 proteins were positively identified by mass spectrometry. In addition, approximately 1000 protein spots were constitutively expressed of which 21 proteins were positively identified by mass spectrometry. Increased levels of proteins in the cocaine-exposed monkeys include glial fibrillary acidic protein, syntaxin-binding protein 3, protein kinase C isoform, adenylate kinase isoenzyme 5 and mitochondrial-related proteins, whereas decreased levels of proteins included beta-soluble N-ethylmaleimide-sensitive factor attachment protein and neural and non-neural enolase. Using a complimentary proteomics approach, the differential expression of phosphorylated proteins in the cytosolic fraction of these subjects was examined. Two-dimensional gel electrophoresis (2DGE) was followed by gel staining with Pro-Q Diamond phosphoprotein gel stain, enabling differentiation of approximately 150 phosphoprotein spots between the groups. Following excision and trypsin digestions, MALDI-TOF-TOF was used to confirm the identity of 15 cocaine-altered phosphoproteins. Significant increased levels were detected for gamma-aminobutyric acid type A receptor-associated protein 1, 14-3-3 gamma-protein, glutathione S-transferase and brain-type aldolase, whereas significant decreases were observed for beta-actin, Rab GDP-dissociation inhibitor, guanine deaminase, peroxiredoxin 2 isoform b and several mitochondrial proteins. Results from these studies indicate coordinated dysregulation of proteins related to cell structure, signaling, metabolism and mitochondrial function. These data extend and compliment previous studies of cocaine-induced biochemical alterations in human postmortem brain tissue, using an animal model that closely recapitulates the human condition and provide new insight into the molecular basis of the disease and potential targets for pharmacotherapeutic intervention.     

颅脑损伤程度对大鼠脑血流及氧代谢的影响

目的 对颅脑损伤影响脑血流及氧代谢进行前瞻性研究。方法 30只Wistar大白鼠分成3组：颅脑损伤1组（TBI1）、2组（TBI2）及3组（TBI3）各10只，分别为轻、中、重型颅脑损伤。用脑阻抗（REG）测定脑血流量，颈内静脉血氧饱和度（SjVO2)反映全脑氧代谢情况。结果 TBI、TBI2及TBI3组影响脑血流和氧代谢程度依次为TBI3＞TBI2＞TBI1，健侧脑组织含水量各组无明显差异，伤侧脑组织含水量TBI3组最多，其次为TBI2，明显高于TBI1组（P＜0．01）。结论 颅脑损伤后脑血流和氧代谢变化取决于损伤程度，脑血流和氧代谢各参数的监测对正确认识脑组织病理生理变化，指导临床治疗，判断预后有重要价值。     

急性重型颅脑创伤患者脑组织、血清蛋白质组分析

目的评价急性重度颅脑创伤患者脑组织、血清同时筛选损伤标记物的意义；对比出两种组织之间相同的差异蛋白质，以直接利用血清中的蛋白质来评估颅脑损伤。方法应用表面加强激光解析电离时间飞行质谱技术比较创伤组和对照组皮层组织之间、血清之间蛋白质的差异表达，筛选出血清、脑组织之间相同的差异蛋白质。结果每例患者脑组织、血清之间比较出13—39个相同差异蛋白质峰。创伤组脑组织、血清之间比较出8个相同差异蛋白质峰：m／z3178、m／z3412、m／z4201、m／z4692、m／z8605、m／z8676、m／z9387、m／z9607。结论脑组织、血清同时筛选差异蛋白质，发挥出两种组织各自对比的优势，克服彼此的缺点，为直接利用血清中的差异性蛋白质评估颅脑损伤奠定了实验基础。