额颞叶痴呆的蛋白质组研究

目的 为深入理解额颞叶痴呆分子机制并寻找诊断治疗该疾病的蛋白质标记物,选取5例经病理证实的额颞叶痴呆患者与4例无神经系统疾病老年人尸检脑标本进行蛋白质组学研究.方法 死亡24 h内新鲜取材的尸检额叶、颞叶蛋白质以固相pH梯度等电聚焦为第一向,SDS-PAGE垂直电泳为第二向进行双向电泳（2-DE）,图象分析软件Imagemaster 2D Elite分析电泳图谱,MALDI-TOF质谱或MALDI-TOF/TOF串联质谱鉴定蛋白质.结果 18种蛋白质的表达量在额颢叶痴呆患者与正常老年人显著不同.12个在额颞叶痴呆表达量上调的蛋白质被鉴定为3-磷酸甘油醛脱氢酶、尿嘧啶DNA糖苷水解酶、Cu-Zn超氧化物歧化酶、异柠檬酸脱氢酶亚单位、synaptotagmin I、Peroxiredoxin2、胶质纤维酸性蛋白、P25 alpha、烯酰辅酶A水合酶短链1、吡哆醇-5′-磁酸氧化酶、Mn超氧化物歧化酶、α-烯醇化酶;6个在额颞叶痴呆表达量下调的蛋白质被鉴定为抗氧化蛋白2（酸性钙离子依赖型磷脂酶A）、铁蛋白H链、谷氨酸脱氢酶、肽基脯氨酸顺反异构酶A、血清白蛋白前体、二氢嘧啶酶相关蛋白2.结论 额颞叶痴呆患者脑组织中神绛纤维缠结相关蛋白、氧应激蛋白、凋亡相关蛋白及重要代谢酶的表达发生变化,有助于深入理解额颞叶痴呆分子机制并有望在额颞叶痴呆的早期诊断及新药开发上发挥作用.     

芪苈强心胶囊对心力衰竭大鼠心肌线粒体蛋白质组影响的研究

目的采用蛋白质学方法研究芪苈强心（QLQX）胶囊对心力衰竭大鼠心肌细胞线粒体蛋白表达情况的影响,并探讨其治疗心力衰竭的机制。方法将心肌梗死心力衰竭大鼠模型分为心衰模型组、QLQX（1.0g/kg.d-1）胶囊组、假手术组。灌胃给药,每天一次,连续4周后,差速离心法提取心肌线粒体,双向电泳法分离差异表达的蛋白,凝胶银染后酶切差异蛋白点进行激光解析电离飞行时间（MALDI-TOF）质谱分析,通过Mascot软件在数据库检索。结果共鉴定出11种差异表达的蛋白质,表达上调的有NADH氧化还原酶、ATP合成酶、苹果酸脱氢酶、长链乙酰辅酶A脱氢酶、缩醛酶、肌酸激酶、58 kDa钙调蛋白;表达下调的蛋白有乳酸脱氢酶B、烯醇酶、αB2Crystallin和热休克蛋白27。结论 QLQX胶囊能够部分纠正衰竭心肌线粒体有关能量代谢、氧化应激相关酶的异常表达,可能是其治疗心力衰竭的机制之一。     

怀化侗族高血压人群IL-1Ra、CK-18、α-烯醇化酶蛋白质表达差异

目的分析怀化侗族高血压患者血淋巴细胞蛋白质谱,寻找高血压患者血淋巴细胞差异蛋白质。方法应用MALDI-TOF-MS技术检测130例血淋巴细胞标本(侗族高血压50例,汉族高血压40例,侗族正常人群40例)的蛋白质质谱,用MatrixScience公司的Mascot对蛋白质质谱数据进行数据库查询比对,并搜索鉴定蛋白。结果三组均获得重复性好的血淋巴细胞蛋白质双向凝胶电泳图谱。通过对其中3个差异表达蛋白点分析,并经质谱鉴定。与侗族正常人对照,侗族高血压人群α-烯醇化酶表达上调,CK-18表达差异无统计学意义(P﹥0.05),IL-1Ra表达下调;汉族高血压人群CK-18、α-烯醇化酶两个点表达上调,IL-1Ra表达下调。结论 IL-1Ra和α-烯醇化酶可作为预测高血压发生、发展程度的一个候选生物学标志物。     

生长抑素2型受体基因转染对胰腺癌细胞蛋白表达的影响

目的 探讨生长抑素2型受体(hSSTR2)基因转染对胰腺癌细胞PANC1蛋白表达的影响,以寻找新的胰腺癌敏感治疗靶点.方法 利用前期构建的腺病毒载体Ad.CMV.hSSTR2.GFP将hSSTR2全长cDNA导入胰腺癌细胞PANC1.采用双向荧光差异凝胶电泳(2D-DIGE)技术分离并筛选转染hSSTR2的实验组、空载体对照组以及空白组胰腺癌细胞之间差异表达蛋白,用反射式基质辅助激光解吸附电离串联飞行时间质谱(MALDI-TOF/TOF)技术对差异蛋白进行鉴定.结果 hSSTB2成功地转染了胰腺癌细胞,获得了hSSTR2阴性和阳性表达的PANC1荧光差异蛋白表达图谱,经DeCyder v6.5软件分析,共有18个差异在1.3倍以上的蛋白质点,经质谱鉴定得到13个蛋白质.低表达的蛋白7个,为GMP合酶、磷酸化应激诱导蛋白、谷氨酸脱氢酶、Septin-11、波形蛋白、异柠檬酸脱氢酶α亚基、线粒体内膜易位酶;高表达蛋白6个,为真核延长因子1α1、丙酮酸激酶异构体M2型、烯酰-CoA水合酶、转录调节因子1-β、Mitofilin、HSP105.结论 hSSTR2基因转染胰腺癌细胞PANC1后引起蛋白表达发生变化,这些差异蛋白功能涉及到糖、脂肪、核酸代谢以及细胞生长调节和细胞凋亡等,从而为寻找新的胰腺癌敏感治疗靶点奠定基础.     

荧光差异双向凝胶电泳法筛选大鼠脑缺血相关蛋白

目的 鉴定局灶性脑缺血相关蛋白并筛选早期神经保护蛋白.方法 应用先进的差异蛋白质组学荧光差异双向凝胶电泳（2D DIGE）技术,比较大鼠大脑中动脉闭塞（MCAO）6 h病灶侧大脑皮层和正常大鼠相应部位蛋白质变化;采用DeCyder-DIA软件、单因素方差分析ANOVA,选择两组间蛋白表达量差异具有统计学意义（P＜0.05）且AR＞1.4的蛋白点;基质辅助激光解析/电离-飞行时间（MALDI-TOF）质谱鉴定差异蛋白.结果 脑缺血6 h组与正常对照组比较13个蛋白点符合统计学要求;经质谱分析仅鉴定出一个缺血组明显减少的蛋白点为α-微管蛋白.结论 作为结构蛋白之一的α-微管蛋白在脑缺血早期即发生明显变化,是缺血性脑血管病早期相关蛋白.     

人结肠癌羟基喜树碱多药耐药细胞的蛋白质组学研究

目的对结肠癌羟基喜树碱多药耐药细胞（SW1116／HCPT）及其亲代细胞（SW1116）进行蛋白质组学比较研究，探讨肿瘤细胞的多药耐药机制。方法培养羟基喜树碱多药耐药细胞和亲代细胞，提取蛋白质，以固相pH梯度等电聚焦为第一向，十二烷基磺酸钠-聚丙烯酰胺凝胶垂直电泳为第二向进行双向电泳，图像分析软件分析电泳图谱，基质辅助激光解吸电离飞行时间质谱或基质辅助激光解吸电离飞行时间／飞行时间串联质谱鉴定蛋白质。结果发现9个蛋白质点在SW1116／HCPT细胞中表达量发生改变，4个蛋白质点表达量增加，5个蛋白质点表达量减少。质谱鉴定了6个蛋白质点分别为：琥珀酸脱氢酶复合物（亚单位A）、3-磷酸甘油醛脱氢酶、泛素融合降解1相似蛋白、胞核氯离子通道蛋白、β-微管蛋白和ORF蛋白。结论该研究有助于深入理解结肠癌羟基喜树碱多药耐药机制，并有望在新型耐药逆转剂的开发上发挥作用。     

人肺腺癌细胞系A549线粒体差异蛋白质组学研究

目的 研究人肺腺癌细胞系A549与人正常支气管上皮细胞系16HBE线粒体蛋白质组的差异表达.方法 传代培养细胞系A549及16HBE,用线粒体提取试剂盒获取细胞线粒体蛋白质,进行双向凝胶电泳,运用液相色谱串联质谱分析技术筛选出A549和16HBE细胞系线粒体间表达水平显著差异的蛋白,所得结果通过Data Analysis软件标峰,用MASCOT进行结果搜索和数据分析.结果 双向电泳结果显示A549、16HBE细胞系线粒体存在差异的蛋白质点共41个,3倍以上差异的16个,A549细胞系中表达上调的15个,表达下调的26个,其中3倍以上差异表达上调的7个,表达下调的9个.运用液相色谱串联质谱技术鉴定出A549细胞系线粒体表达上调的蛋白质2个:AAA+ ATP酶家族结构域蛋白3B、tRNA鸟嘌呤糖基转移酶,表达下调的蛋白质7个:热休克蛋白75、复合物Ⅲ亚基1、复合物Ⅲ亚基2、鸟氨酸氨基转移酶、异柠檬酸脱氢酶亚基α、SLP-2、抗增殖蛋白.结论 应用亚细胞蛋白质组学方法,鉴定出肺腺癌细胞系线粒体差异表达蛋白,为阐明肺腺癌发生的分子机制、筛选早期诊断标志物提供了有益的线索.     

不同转移潜能肝癌细胞株的差异蛋白质组的二维液相色谱分析

目的:对不同转移潜能肝癌细胞株MHCC97- H(高转移)和MHCC97-L(低转移)差异表达的蛋白质进行二维液相色谱分离和MALDI-TOF质谱鉴定.方法:将肝癌细胞株MHCC97-H和MHCC97- L细胞裂解样品按蛋白质PI进行一维的色谱聚焦分离,然后每个PI组分再按疏水性经二维反相无孔硅胶HPLC分离,利用ProteoVue软件将UV光吸收图谱转换成PI对疏水性的胶图,再利用DeltaVue软件比较升高或降低的差异蛋白.收集差异蛋白峰进行胰酶酶解,然后进行MALDI-TOF质谱鉴定.结果:2D图谱显示共有72个差异蛋白条带,共鉴定出了9个差异蛋白.分别为M2型丙酮酸激酶、ATP合成酶α亚单位、热休克蛋白60、Toll样受体9、含黄素单加氧酶、钙网硬蛋白前体、锰超氧化物岐化酶、nm23-H1、G-蛋白偶连受体激酶5;其中4个蛋白在高转移细胞株MHCC97-H中表达升高,5个蛋白在低转移细胞株MHCC97-L表达升高.结论:这些差异蛋白可能在肝癌的转移中起关键作用.     

IL-1β致乙酸性胃溃疡复发大鼠的比较蛋白质组学分析

目的:筛选胃溃疡复发相关的蛋白质,进一步揭示胃溃疡复发的分子机制.方法:以乙酸制备胃溃疡模型,并用IL-1β诱导复发.采用二维凝胶电泳(2-DE)技术分离胃溃疡复发大鼠胃溃疡处组织及正常大鼠相应处胃组织的总蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)得到相应的肽质量指纹图(PMF),搜索数据库鉴定蛋白质.结果:获得分辨率较高、重复性较好的胃溃疡复发大鼠和正常大鼠胃组织的2-DE图谱,质谱分析共鉴定了12个差异蛋白质点,其中7个表达上调,含热休克蛋白27(HSP27)、葡萄糖调节蛋白(GRP78)、L-乳酸脱氢酶轻链、磷酸甘油醛脱氢酶、血红蛋白β链、过氧化物酶2、过氧化物酶1;5个表达下调,含膜联蛋白A2、热休克蛋白60、氯离子通道蛋白1、黏着斑蛋白、凝溶胶蛋白.随即采用免疫组化法检测差异蛋白质HSP27和GRP78在两类组织中的表达,结果显示胃溃疡复发组HSP27和GRP78阳性细胞百分率均显著高于正常对照组(HSP27:27.90%±5.34% vs 22.10%3.67%,P＜0.05;GRP78:43.00%±4.52% vs 26.30%±3.95%,P＜0.01).结论:差异蛋白如HSP27和GRP78等可能以不同的方式参与了胃溃疡的复发过程,为揭示胃溃疡复发的机制提供了线索.     

IL-1beta致乙酸性胃溃疡复发大鼠的比较蛋白质组学分析

目的筛选胃溃疡复发相关的蛋白质,进一步揭示胃溃疡复发的分子机制.方法以乙酸制备胃溃疡模型,并用IL-1β诱导复发.采用二维凝胶电泳(2-DE)技术分离胃溃疡复发大鼠胃溃疡处组织及正常大鼠相应处胃组织的总蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)得到相应的肽质量指纹图(PMF),搜索数据库鉴定蛋白质.结果获得分辨率较高、重复性较好的胃溃疡复发大鼠和正常大鼠胃组织的2-DE图谱,质谱分析共鉴定了12个差异蛋白质点,其中7个表达上调,含热休克蛋白27(HSP27)、葡萄糖调节蛋白(GRP78)、L-乳酸脱氢酶轻链、磷酸甘油醛脱氢酶、血红蛋白β链、过氧化物酶2、过氧化物酶1;5个表达下调,含膜联蛋白A2、热休克蛋白60、氯离子通道蛋白1、黏着斑蛋白、凝溶胶蛋白.随即采用免疫组化法检测差异蛋白质HSP27和GRP78在两类组织中的表达,结果显示胃溃疡复发组HSP27和GRP78阳性细胞百分率均显著高于正常对照组(HSP2727.90%±5.34% vs 22.10%3.67%,P＜0.05;GRP7843.00%±4.52% vs 26.30%±3.95%,P＜0.01).结论差异蛋白如HSP27和GRP78等可能以不同的方式参与了胃溃疡的复发过程,为揭示胃溃疡复发的机制提供了线索.     

人小脑与额叶的比较蛋白质组学分析

目的对小脑及额叶进行比较蛋白质组学研究以探讨其对变性损伤敏感性不同的原因。方法死亡24h内新鲜取材尸检小脑及额叶组织,经提取蛋白质,以固相pH梯度等电聚焦为第一向,SDS PAGE垂直电泳为第二向进行双向电泳(2DE),图像分析软件分析电泳图谱,MALDI TOF质谱或MALDI TOF/TOF串联质谱鉴定蛋白质。结果发现3个蛋白质点在小脑表达量增加,7个蛋白质点在额叶表达量增加。以质谱鉴定了3个小脑中表达量增加的蛋白质点分别为抗氧化蛋白2、肌酸激酶前体(线粒体)和果糖二磷酸醛缩酶C,2个在额叶增加的蛋白质点为胶质纤维酸性蛋白、丙酮酸激酶。30个表达量在额叶及小脑没有显著变化的蛋白质点通过MALDI TOFMS/TOFTOF MSMS和蛋白质数据库检索得到鉴定。结论本研究结果进一步丰富了人脑蛋白质组数据库。额叶与小脑的差异表达蛋白质为深入理解脑老化及变性痴呆分子机制提供了有益的线索。     

Differential proteomic analysis of a highly metastatic variant of human breast cancer cells using two-dimensional differential gel electrophoresis

Distant metastasis represents the major lethal cause of breast cancer. To understand the molecular mechanisms of breast cancer metastasis and identify markers with metastatic potential, we established a highly metastatic variant of parental MDA-MB-231 cells (MDA-MB-231HM). Using two-dimensional electrophoresis (2-DE), we performed a proteomic comparison of the two kinds of cells. As much as 51 protein spots were differentially expressed between the selected variant and its parental counterpart in at least 3 experiments. Ten unique proteins were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and database searching software. Among them, nine proteins were up-regulated in MDA-MB-231HM cells, including Macrophage-capping protein (CapG), Galectin-1, Chloride intracellular channel protein 1, Endoplasmic reticulum protein ERp29 precursor, Stathmin-1 (STMN1), Isoform 1 of uridine–cytidine kinase 2(UCK2), Rho GDP-dissociation inhibitor 2 (ARHGDIB), isocitrate dehydrogenase [NADP] cytoplasmic (IDH1), and N-myc downstream regulated gene 1 (NDRG1) protein. Only transgelin-2 was down-regulated. Differential expression was confirmed for three proteins including CapG, STMN1, and transgelin-2 by Western blotting analysis. Transgelin-2 was chosen for further verification by immunohistochemistry. The results suggested that 2-DE would be an efficient way to screen the proteins responsible for specific biological function. Furthermore, the findings imply that different proteins may be involved in the metastatic process in breast carcinomas.     

APP转基因小鼠与正常小鼠脑蛋白质组双向电泳图谱的比较

目的探讨APP转基因小鼠与正常C57小鼠脑蛋白质组双向电泳(2-DE)图谱差异,从蛋白质水平初步探索老年性痴呆发病机制。方法APP转基因小鼠与正常C57小鼠脑组织经蛋白提取后,分别以固相pH梯度等电聚焦为第一向,SDS-PAGE垂直电泳为第二向进行2-DE。图像分析软件ImageMaster 2D Elite分析电泳图谱。结果APP转基因小鼠与正常小鼠脑组织2-DE图谱分别检测出976和947个蛋白点。对两张电泳图进行匹配后,发现有16个蛋白点仅在APP转基因小鼠脑蛋白2-DE图谱检测到表达,而有7个蛋白点只在正常小鼠检测到。部分蛋白在2组小鼠脑组织中含量发生了明显变化。结论差异点的发现初步建立了差异表达蛋白质组学的技术方法;为研究阿尔茨海默病机制及研发新药提供了有益的线索。     

Proteomic analysis of functional dyspepsia in stressed rats treated with traditional Chinese medicine "Wei Kangning"

Background and Aim: Chinese traditional medical science is generally used as a therapeutic method against functional dyspepsia (FD) in China. Although great effort is made to understand the pharmaceutical mechanisms of Chinese traditional medicine, such as typical traditional Chinese medicine, Wei Kangning, there are still many mysteries to be uncovered. Methods: The model of FD was established by stimulating rats via tail damping and the rats were treated with traditional Chinese medicine, Wei Kangning. The proteins of the rat gastrointestinal tissues were extracted and run by 2‐DE, then the differential proteins were identified using matrix‐assisted laser desorption ionisation time‐of‐flight mass spectrometry and validated with Western blotting or fluorescent quantitation polymerase chain reaction. Results: A total of 228 unique proteins in FD model rats were detected with significant changes in their expression levels corresponding with traditional Chinese medicine, Wei Kangning, administration. Twenty‐eight of these proteins were identified, which are involved in many biological functions, such as organism antioxidant enzymes, energy metabolism, glutathione S‐transferase, pi2, superoxide dismutase 2 and alpha‐enolase and so on. Conclusions: These proteomic results presented therefore provide additional support to the hypothesis that glutathione S‐transferase, pi2, superoxide dismutase 2, α‐enolase and voltage‐dependent anion channel are the targets of FD treated with traditional Chinese medicine, Wei Kangning.     

羊种布鲁氏菌疫苗株M5和强毒株16M的比较蛋白质组学研究

目的阐明羊种布鲁氏菌疫苗株M5致弱的分子基础,更加深入地理解布鲁氏菌的毒力机制。方法利用双向电泳技术对相同条件下培养的羊种布鲁氏菌疫苗株M5和强毒株16M总蛋白进行分离,两株间差异蛋白点利用基质辅助激光解吸/电离串联飞行时间质谱技术进行鉴定。每个蛋白质点的肽指纹图谱均使用Mascot在NCBInr蛋白质数据库中进行检索。结果共成功鉴定了13个差异蛋白,代表了8种不同的开放阅读框(ORFs),功能涉及能量代谢,应激,物质运输等。结论上述发现为羊种布鲁氏菌疫苗株M5致弱分子基础的阐明提供了新依据。     

Identification of proteins differentially expressed by melatonin treatment in cerebral ischemic injury – a proteomics approach

Abstract:  We previously reported that melatonin protects neuronal cells against ischemic brain damage. In this study, we identified proteins that were differentially expressed by melatonin treatment during ischemic brain injury. Rats were subjected to cerebral ischemia by middle cerebral artery occlusion (MCAO). Adult male rats were treated with melatonin (5 mg/kg) or vehicle prior to MCAO and brains were collected at 24 hr after MCAO. Proteins derived from the cerebral cortex were analyzed using two-dimensional gel electrophoresis. Protein spots with a greater than 2.5-fold change in intensity were identified by mass spectrometry. Among these proteins, γ-enolase, stathmin, thioredoxin, peroxiredoxin-6, hippocalcin, protein phosphatase 2A, adenosylhomocysteinase, ubiquitin carboxy-terminal hydrolase L1, and NAD-specific isocitrate dehydrogenase subunit α were significantly decreased in the vehicle-treated group in comparison to the melatonin-treated group. The identified proteins consist of cell differentiation and stabilization proteins, as well as an antioxidant enzyme. In contrast, dehydroprimidinase-related protein 2 (DRP-2), a target of protein oxidation in neurodegeneration, was significantly increased in vehicle-treated animals, while melatonin prevented the injury-induced increase of DRP-2. Thus, the results of this study suggest that melatonin prevents cell death resulting from ischemic brain injury and that its neuroprotective effects are mediated by both the up- and down-regulation of various proteins.     

DJ-1蛋白在晚发型阿尔茨海默病蛋白组学的表达

目的 从蛋白质水平初步探讨晚发型阿尔茨海默病发病机制,寻找并鉴定与之有关的生物标志物.方法 选择经病理证实的8例晚发型阿尔茨海默病患者及5例正常老年人颞叶脑皮质为研究对象.分别进行双向凝胶电泳后采用基质辅助激光解析-电离-飞行时间质谱及电喷雾电离串联质谱法进行质谱分析,搜索数据库鉴定蛋白质.结果 分别获得两组脑组织双向凝胶电泳蛋白质组表达图谱,DJ-1蛋白在疾病组明显上调.结论 DJ-1蛋白可能成为具有临床诊断意义的晚发型阿尔茨海默病潜在标志物,从而帮助开发靶向目的 蛋白质的新型抗神经退化药物.     

Identification of GYRKPPFNGSIFamide (crustacean-SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis

A new concept relating to the purification protocol for biological proteins and peptides has been designed as "topological mass spectrometry analysis," in combination with MALDI-TOF MS using slices of tissues, chromatographic purification from the extract of tissues, molecular cloning for the determination of the precursor structure, and capillary LC-MS/MS analysis for elucidation of its posttranslational modifications. In an actual application, we identified an alpha-amidated neuropeptide from the red swamp crayfish (Procambarus clarkii) brain. Initially, an MS number of around m/z 1382 was found by the direct MALDI-TOF MS analysis with slices of the accessory lobe of the brain. After two steps of reversed-phase HPLC separation with brain extract, the structure of a 1381 Da peptide was sequenced to the GYRKPPFNGSIFamide (named crustacean-SIFamide). Subsequently, the cDNA has been characterized and encodes a 76 amino acid precursor protein that contains a signal sequence, one copy of GYRKPPFNGSIFG and one additional peptide. The RT-PCR analysis implied that the mRNA of the neuropeptide was expressed throughout the nervous system of the crayfish. Furthermore, immunostaining demonstrated that the neuropeptide is distributed in the olfactory lobe, accessory lobe, olfactory globular tract, and olfactory lobe cells. In addition, database searches revealed that there are homologous sequences of the AYRKPPFNGSIFamide in the genome library of fruitfly Drosophila melanogaster and AYRKPPFNGSLFamide isolated from the grey fleshfly Neobellieria bullata, and GYRKPPFNGSIFamide isolated from the giant tiger prawn Penaeus monodon. These results suggested that the neuropeptide family might be widely distributed in arthropods and plays a significant role in the nervous system.