肺癌与肺良性疾病血清蛋白质双向电泳差异分析

目的:分析肺癌与肺良性疾病差异表达的血清蛋白质.方法:以肺癌和肺良性疾病患者血清标本为研究对象,利用双向电泳(2-DE)分离2组血清蛋白质,银染显色,Umax扫描仪获取图像,Melanie4软件分析图像,寻找肺癌相关差异蛋白质.结果:建立了较稳定的肺癌和肺良性疾病血清蛋白质2-DE图谱,平均蛋白质点数约400个.软件分析显示2组共有6个差异蛋白质点,肺癌组量上调的差异点5个,量下调的差异点1个.结论:差异蛋白质的分离为进一步的肺癌相关血清蛋白质的鉴定和其他肿瘤血清蛋白质组学研究奠定了良好的工作基础.     

结肠癌肝转移患者血清双向电泳图谱的建立及优化

目的建立并优化结肠癌肝转移患者血清蛋白质组双向电泳分离技术。方法对2-DE的各种条件进行调整、优化,通过双向电泳图谱比较各种方法对血清蛋白的分离效果。结果采用pH4~7胶条,含尿素9M、硫脲2M的上样缓冲液,12.5%的SDS-PAGE胶提高了血清蛋白的分离效果。结论血液中高丰度蛋白质的去除能提高了2-DE图谱的斑点数和分辨率。成功建立了血清蛋白质的纯化技术,为寻找疾病的血清学标志物的研究奠定基础。     

先天性甲状腺功能减退症大脑蛋白质双向电泳分析

目的:比较新生正常大鼠和先天性甲状腺功能减退症(CH)大鼠大脑蛋白质表达的差异,为探讨CH致脑发育障碍发病机制提供线索.方法:制作CH仔鼠动物模型,于出生时称体质量后处死仔鼠,取大脑皮质,应用双向电泳(2-DE)技术分析正常仔鼠与CH仔鼠大脑蛋内质表达的差异情况.放射免疫分析法检测2组大鼠血清游离三碘甲腺原氨酸(FT3)、游离甲状腺素(FT4),水平.结果:CH组仔鼠体质量、FT3、FT4水平均低于正常组仔鼠.建立了稳定的正常仔鼠与CH仔鼠大脑皮质2-DE图谱,发现2组共有8个蛋白质点存在差异.与正常仔鼠相比,CH仔鼠大脑有3个蛋白质点量上调,2个蛋白质点量下调,有3个蛋白质点仅出现于CH仔鼠组.结论:CH仔鼠大脑差异蛋白表达可能与CH致脑发育障碍的发生、发展密切相关.     

蜂蜇伤战士血清相关蛋白分析

目的分析蜂蜇伤战士与正常人血清中蛋白质图谱之间表达差异。方法分别收集来源于解放军181医院于2008年10月—2010年2月收集的11例蜂蜇伤国防战士标本和8例健康人的血清标本进行双向凝胶电泳（2-DE）检测。通过基质辅助激光解析飞行时间质谱（MALDI-TOF-MS）分析鉴定蜂蜇伤患者的血清蛋白质中存在显著差异的蛋白质点。结果在蜂蜇伤战士的血清中存在5个新增蛋白质点,3个蛋白点上调,7个蛋白点下调,其中包括KRT10角蛋白Ⅰ型、KRT1角蛋白Ⅱ型、触珠蛋白相关蛋白、触珠蛋白HP、HPR蛋白、α2-巨球蛋白、β血影蛋白brain1。结论采用固相pH梯度2-DE分离鉴定血清蛋质组能够获得很好的实验重复性。蜂蜇伤患者与正常人血清存在差异表达蛋白,为进一步研究蜂蜇伤相关生物标记提供理论基础及指导。     

吗啡成瘾和正常大鼠的前额叶皮质表达蛋白质组变化的初步研究

目的建立和比较吗啡成瘾与正常大鼠前额叶皮质(PFC)蛋白质双向电泳图谱,寻找和鉴定吗啡成瘾大鼠PFC中的差异蛋白表达谱。方法以固相pH梯度等电聚焦为第一向和垂直SDS-PAGE为第二向,分别对正常对照大鼠和吗啡成瘾大鼠的PFC蛋白质样品进行二维分离,2-DE图谱经ImageMaster 2D Platinumv5.0软件分析,选取4个差异蛋白点用基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)进行鉴定。结果通过对2-DE图谱蛋白斑点的匹配及对比分析,与吗啡成瘾相关的差异表达蛋白斑点为87个;经质谱鉴定出2个有意义的差异表达的蛋白斑点:Snap25亚型β-Snap25突触相关蛋白25、β-肌动蛋白。结论吗啡成瘾组与正常对照组大鼠PFC蛋白表达存在差异;初步鉴定了大鼠前额叶皮质中与吗啡成瘾相关的差异蛋白,其表达的变化可能通过多种途经影响PFC神经元功能,为我们研究阿片类物质依赖作用机制提出了新的思路和方向。     

双向电泳分析叶酸对神经干细胞蛋白表达的影响

目的:探讨叶酸缺乏对胚胎大鼠神经干细胞(NSCs)蛋白质表达的影响。方法:采用无血清体外细胞培养方法,培养胚胎大鼠NSCs并进行鉴定,采用叶酸缺乏培养基培养NSCs,根据叶酸添加终浓度将细胞分为对照(Con?trol)组、叶酸缺乏(Folate-D)组、叶酸补充(Folate-L)组,各组叶酸终浓度分别为4、0.6、8mg/L。应用双向电泳(2-DE)技术分析各组蛋白质表达的差异。结果:建立了稳定可重复的NSCs2-DE图谱,与Control组相比,Folate-D组2个蛋白点表达量降低,4个蛋白点表达量升高;Folate-L组2个蛋白点表达量降低,1个蛋白点表达量升高。结论:叶酸缺乏或补充叶酸影响NSCs蛋白表达,差异蛋白分离为进一步研究叶酸对NSCs增殖分化调控机制奠定了基础。     

心肾综合征阳虚水泛证与气虚血瘀证患者血清蛋白质组学研究

目的 建立心肾综合征(CRS)阳虚水泛证、CRS气虚血瘀证及健康对照的血清蛋白质组表达图谱,初步鉴定其差异表达的蛋白质.方法 选择CRS患者35例,其中阳虚水泛证组20例;气虚血瘀证组15例.另选健康者12例作为对照.各组血清去除白蛋白,测定血清蛋白浓度.以双向凝胶电泳分离各组血清蛋白质,考马斯亮蓝染色,扫描凝胶成像后,用PDQuest软件对各组血清蛋白质组表达图谱进行差异分析,选定差异点进行基质辅助激光解吸电离飞行时间质谱(Matrix-assisteD Laser DesorptioN/ioNizatioN-Time of Flight-Mass SpeCtrometry,MALDI-TOF-MS)分析,MasCot软件搜索MSDB和SWISS-PROT数据库鉴定蛋白质.结果 初步建立CRS阳虚水泛证、CRS气虚血瘀证及健康对照组3组血清蛋白质表达图谱,分别获得蛋白质点95、87 和79 个;13 个蛋白质点在3 组血清中呈现差异表达;初步鉴定其中的11 个蛋白质分别为:生长分化因子15 前体、N 末端B 型钠尿肽、高敏C 反应蛋白、半乳凝素3、肾损伤分子-1、白细胞介素-18、促肾上腺髓质素、髓过氧化物酶、胱抑素C、脂联素、骨保护素.结论 得到鉴定的11 个蛋白质可能与心肾综合征阳虚水泛证、CRS 气虚血瘀证发生发展密切相关,但其具体机制有待进一步研 究.     

Ⅰ期非小细胞肺癌相关血清差异表达蛋白的研究

目的:寻找Ⅰ期非小细胞肺癌(NSCLC)与健康人血清的差异表达蛋白.方法:收集天津市胸科医院经手术、病理证实的6例Ⅰ期NSCLC患者血清,等量混合后设为肺癌组;同期15例健康体检血清,等量混合后设为健康对照组.利用双向凝胶电泳(2-DE)和基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)分离、筛选和鉴定2组间的差异表达蛋白.结果:成功获得了2组的双向凝胶电泳图谱,PDquest软件分析显示,2组间差异大于2倍的蛋白质点共有128个,质谱鉴定后确定了10种蛋白质,其中7种表达量上调,分别为α1-酸性糖蛋白、C1酯酶抑制物、血管紧张素原、转铁蛋白、转甲状腺素蛋白、间-α-胰蛋白酶抑制剂重链H2及无花果酶-3;3种表达量下调,分别为纤连蛋白、补体C4-A及补体C3.结论:应用2-DE及MALDI-TOF-MS在Ⅰ期NSCLC血清中鉴定出4种与肿瘤关系密切的差异表达蛋白,可作为开发NSCLC早期诊断标志物的候选蛋白.     

脑脊液S-100B与儿童病毒性脑炎脑脊液蛋白组学关系的研究

目的分析病毒性脑炎(VE)患儿和对照组儿童脑脊液(CSF)蛋白质组的表达差异,筛选VE特异性蛋白,探讨其与VE的关系,为VE的早期诊断提供线索。方法以三氯醋酸/丙酮沉淀法提取VE患儿和对照组儿童CSF总蛋白,固相pH梯度二维聚丙烯酰胺凝胶电泳(2-DE)技术分离蛋白质,应用双向电泳凝胶分析软件(PDQuest 7.3.1)对2-DE凝胶进行量化比较分析,识别差异表达明显的蛋白质点,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)得到肽质量指纹图谱(PMF),在Mascot数据库中进行蛋白质匹配,鉴定部分差异蛋白质。结果VE患儿和对照组CSF蛋白2-DE凝胶图谱上分别平均显示442和401个点,有23个点蛋白质表达存在2倍以上的差异,选取5个表达明显上调的蛋白质点进行质谱鉴定,成功鉴定出钙结合蛋白S100B。结论钙结合蛋白S100B在VE患儿和无中枢神经系统病变的对照组儿童CSF的蛋白表达上存在明显差异,可能是VE密切相关的疾病特异性蛋白,并有可能成为VE诊断的分子标志物。     

应用SELDI-TOF-MS技术分析肝癌患者血清蛋白质谱的手术前后变化

目的研究肝癌患者血清蛋白质谱的变化,从而筛选特异性生物标志物,建立肝癌诊断模型。方法选用IMAC30蛋白质芯片和SELDI-TOF-MS蛋白质芯片技术,对47例原发性肝癌患者手术前后和81名正常人的血清蛋白质谱进行分析。获得的蛋白质谱采用Ciphergen公司的Biomarker wizard和Biomarker Patterns软件分析,建立区分肝癌患者与正常人血清蛋白质谱诊断模型。结果通过对肝癌患者术前血清与正常人血清蛋白质谱分析,发现共有28个蛋白质表达量差异显著(P<0.05)。并获得分子量为9492.93Da的蛋白质组成的模板,可将肝癌与正常人正确分组,其敏感性为97.9%(46/47),特异性为97.5%(79/81)。术后血清蛋白质谱中,原高表达的蛋白质明显下调。结论血清中可以筛选到诊断肝癌的特异性生物标志物并可用于预后的判断。SELDI-TOF-MS蛋白质芯片技术为建立蛋白质模板用以早期诊断肝癌提供了可靠的技术平台。     

甲基苯丙胺致PC12细胞的差异蛋白表达

目的探讨甲基苯丙胺(METH)作用于PC12细胞后的蛋白质表达变化。方法 METH2.5 mmol·L-1作用PC12细胞24 h后,提取细胞总蛋白质,丙酮沉淀法纯化蛋白,Braford法对蛋白质进行定量,并对蛋白质进行双向凝胶电泳,Image scannerⅢ透射扫描仪获取凝胶电泳图谱。应用Image Master 7.0软件对获得的双向凝胶电泳图谱进行差异性蛋白质点分析,并对相应差异蛋白点用高端基质辅助激光解析-飞行时间(MALDI-TOF)串联质谱仪进行差异蛋白质鉴定。结果应用双向凝胶电泳结合质谱分析技术,METH作用PC12细胞24 h后,共鉴定出18个差异表达蛋白质点,其中8个差异蛋白点在METH作用后表达增强,10个蛋白点表达减弱。这些蛋白质主要包括细胞骨架相关蛋白、分子伴侣、氧化应激和凋亡相关的蛋白以及与能量代谢相关的酶类。结论 METH诱导PC12细胞18个蛋白差异表达。     

双向电泳技术筛选乙肝患者血清中标志蛋白

利用双向电泳技术进行筛选乙肝患者血清中的标志蛋白,通过对比乙肝患者和健康人血清中蛋白质的双向电泳图谱,得到了11个显著的差异点,其中在乙肝患者血清中表达量上调有6个点,表达量下调有2个点;乙肝患者血清中新出现2个点,消失1个点。本研究为乙肝的诊断和治疗提供新的途径。     

重组hFGF-10腺病毒对HaCat细胞影响的蛋白质组学研究

研究重组腺病毒导入人成纤维细胞生长因子10 (hFGF-10) 对HaCat细胞蛋白质组的影响, 由鉴定的差异蛋白质推测hFGF-10对HaCat细胞作用的可能机制。采用双向凝胶电泳结合串联飞行时间质谱技术, 对空载体腺病毒Ad感染细胞和重组腺病毒rAd-hFGF-10感染细胞的总蛋白图谱上的差异蛋白点进行鉴定, 并通过半定量RT-PCR和Western blotting在转录和翻译水平对差异蛋白进行确证。结果显示, 获得了蛋白质分离效果较好的双向凝胶电泳图谱, 鉴定了4种与细胞凋亡、细胞骨架调控、蛋白质降解等相关的差异蛋白质, 并在转录和翻译水平确证了差异蛋白质VDAC2在导入目的基因hFGF-10的HaCat细胞中表达上调, 提示VDAC2可能在hFGF-10的生物学功能中发挥作用。     

Specific identification of three low molecular weight membrane-associated antigens of Helicobacter pylori

BACKGROUND: A large number of Helicobacter pylori proteins are antigenic, but antibodies to these proteins persist in spite of the eradication of the infection. METHODS AND RESULTS: The analysis of sera from H. pylori-infected and non-infected patients, before and 3 and 5 months after eradication, showed that the antibody response against unknown H. pylori antigens at 32, 30, 22 and 14 kDa in sodium dodecylsulphate polyacrylamide gel electrophoresis decreased by > or = 60% at 3 months and > or = 70% at 5 months after treatment. Two-dimensional gel electrophoresis and mass spectrometry allowed the identification of eight proteins at these positions: neuraminyl-lactose-binding haemagglutinin precursor, 3-oxoadipate CoA-transferase subunit A, elongation factor P, peptidoglycan-associated lipoprotein precursor, hypothetical protein HP0596, adhesin-thiol peroxidase, 50S ribosomal protein L7/L12 and subunit b' of the F(0) ATP synthase. Three of these eight, expressed as recombinant proteins (32 kDa neuraminyl-lactose-binding haemagglutinin precursor, 30 kDa peptidoglycan-associated lipoprotein precursor and 22 kDa hypothetical protein HP0596), reacted specifically with sera from infected patients, while the 14 kDa 50S ribosomal protein L7/L12 cross-reacted with one out of five sera from H. pylori-negative patients. The other recombinant proteins did not show significant immunoreactivity. CONCLUSIONS: Four low molecular weight antigens were identified by these methods, three of which were specific. Immunoreaction with these three proteins (neuraminyl-lactose-binding haemagglutinin precursor, peptidoglycan-associated lipoprotein precursor and hypothetical protein HP0596) could provide a serological assessment not only of H. pylori infection, but also of eradication.     

Differential expression of peroxiredoxin 6 in fetal rat testis following in utero exposure to di(n-butyl) phthalate

OBJECTIVES: To isolate and identify differentially expressed proteins in fetal rat testis following in utero exposure to di(nbutyl) phthalate (DBP). METHODS: We used the technique of proteomic analysis to compare the testis protein patterns obtained by two-dimensional gel electrophoresis from fetal rats of gestation day 19. RESULTS: We found significant differences in protein spot intensities. Subsequently several of these variant protein spots were identified by mass spectrometry. Peroxiredoxin 6 (Prdx6) was one of them. Prdx6, which expressed a higher level in DBP-treated fetal rat testes compared with normal ones, is a member of the peroxiredoxins family (Prdxs). Recently, Prdx6 had been shown to be important in protecting cells from oxidative injury. Further, immunohistochemical analyses of fetal rat testes sections were made to determine the cellular distribution of this protein, consequently a strong Prdx6 staining was found out primarily in Leydig cells. CONCLUSIONS: The present study had found several differentially regulated proteins and demonstrated the differential expression of Prdx6 in fetal rat testis following in utero exposure to DBP, when compared with controls. Combining the cellular location of Prdx6 and its function in other tissues, the results of this study could provide us with a possibility to interfere the reproductive toxicity of DBP for its specific antioxidant properties in testis tissues.     

Comparative and quantitative proteomic analysis of normal and degenerated human annulus fibrosus cells

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Identification of differentially-expressed proteins between early submucosal non-invasive and invasive colorectal cancer using 2D-DIGE and mass spectrometry

Early detection and diagnosis of colorectal cancer (CRC) are closely related to a better therapeutic outcome, and the five-year survival rate of early CRC is over 90 percent. Though endoscopic minimally invasive treatment has become a quick and effective therapy for early CRC, endoscopic biopsies are usually not deep enough to obtain tissues from the submucosal layer and it is difficult to determine whether early CRC has infiltrated into the submucosa. Therefore, in the present study, we constructed tumor models of early submucosal non-invasive CRC (SNICRC) and submucosal invasive CRC (SICRC) in Fischer-344 rats induced by N-methyl-N-nitrosourea (MNU). The differentially-expressed proteins were analyzed and identified in SNICRC, SICRC and normal control (NC) tissues using highly sensitive two dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). Proteomic data revealed 132 protein spots between SNICRC and SICRC, 162 protein spots between SICRC and NC and 154 protein spots between SNICRC and NC which were found differentially expressed. These differential spots were picked, in-gel digested and peptide mass fingerprints were obtained by MALDITOF-MS/MS. Finally, five differentially-expressed proteins in SNICRC, SICRC and NC were identified, and increases in Transgelin, peptidylprolyl isomerase A (PPIA) and tropomyosin alpha isoform d were observed, while decreases in carbonic anhydrase 2 (CAII) and an unnamed protein were detected in SICRC compared with SNICRC and NC. Furthermore, Fluorescence-based quantitative polymerase chain reaction (FQ-PCR), Western blotting and immunohistochemistry assays also revealed significant upregulation of Transgelin expression and down-regulation of CAII expression in SICRC tissues. In conclusion, 2D-DIGE is confirmed to be an efficient strategy that enables us to identify differentially expressed proteins between early SNICRC and SICRC. The potential biomarkers such as Transgelin and CAII may be used for the detection of early SICRC.     

非毒性结节性甲状腺肿蛋白质表达差异的初步研究

目的：比较非毒性结节性甲状腺（NTNG）组织和正常甲状腺组织整体蛋白质表达,以研究非毒性结节性甲状腺组织蛋白质表达差异。方法提取NTNG甲状腺组织和正常甲状腺组织总蛋白,利用双向凝胶电泳技术分离各组总蛋白;经MALDI- TOF / TOF质谱分析,搜索数据库,寻找匹配的相关蛋白质,鉴定蛋白质,获得差异蛋白质的信息。结果经过初步筛选和质谱分析共获得9个具有统计学意义的表达差异蛋白点,其中4个在非毒性结节性甲状腺高表达,5个低表达。结论 NTNG与正常腺体组织间存在多种差异表达的蛋白质,可能通过细胞外基质、细胞因子、受体信号转细胞信号传递等参与NTNG的发生和发展。