神经胶质瘤蛋白质双向电泳技术的建立

目的建立神经胶质瘤双向电泳技术。方法利用不同体积的裂解液提取大鼠脑组织中的蛋白质，并通过不同的蛋白质上样量（1、2、3mg），进行双向电泳，考马斯亮蓝染色，图谱分析。结果在等电点3-10，相对分子质量6．5-200KDa范围内分离得到蛋白质斑点为，1mg蛋白质上样量为574个蛋白质斑点，2mg为798个，3mg为803个斑点。2mg蛋白质上样量的双向电泳图谱更清晰，分离更好。结论成功建立了神经胶质瘤蛋白质的双向电泳技术。     

HSP70、Galectin7在放疗敏感性不同宫颈癌组织中表达的研究

目的:观察HSP70和Galectin7在对放疗敏感性不同宫颈癌组织中的表达及临床病理学意义。方法:收集治疗前早期宫颈癌组织标本,病理诊断均为中分化鳞状细胞癌,置于-80℃超低温冰箱保存。在放疗后,根据WHO实体瘤放疗疗效判断标准,从中选择对放疗敏感的5例(高敏感组)和对放疗不敏感的5例(低敏感组),提取其组织总蛋白,进行双向凝胶电泳和质谱分析鉴定差异表达蛋白,分别应用Western Blot和免疫组织化学方法检测组织中蛋白的表达。结果:建立了分辨率高的宫颈癌放疗高敏感组和低敏感组的双向凝胶电泳图谱,差异表达蛋白经鉴定得到高敏感组高表达的Galectin7蛋白以及低表达的HSP70蛋白。Western Blot检测结果显示Galectin7在高敏感组的表达强于低敏感组,HSP70在低敏感组的表达强于高敏感组。免疫组化结果显示,HSP70在高敏感组的阳性表达率为21.7%(13/60),低敏感组的阳性表达率为85.7%(30/35),差异有统计学意义(χ2=36.5997,P<0.0001);Galectin7在高敏感组的阳性表达率为91.7%(55/60),在低敏感组中的阳性表达率为40%(14/35),差异有统计学意义(χ2=29.6853,P<0.0001),并且Galectin7在高敏感组的表达强度高于低敏感组,HSP70在低敏感组的表达强度高于高敏感组,差异均有统计学意义。结论:HSP70和Galectin7的表达与宫颈癌放疗敏感性有关。     

Proteome analysis of the thalamus and cerebrospinal fluid reveals glycolysis dysfunction and potential biomarkers candidates for schizophrenia

Schizophrenia (SCZ) is the result of DNA alterations and environmental factors, which together lead to differential protein expression and ultimately to the development of the illness. The diagnosis is based on clinical symptoms, and the molecular background of SCZ is not completely understood. The thalamus, whose dysfunction has been associated with SCZ based in diverse lines of evidences, plays for instance a pivotal role in the central nervous system as a relay center by re-distributing auditory and visual stimuli from diverse brain regions to the cerebral cortex. We analyzed the proteome of postmortem mediodorsal thalamus (MDT) samples from 11 SCZ patients and 8 non-SCZ individuals by using quantitative shotgun-mass spectrometry and two-dimensional gel electrophoresis. Our analyses identified 551 proteins, 50 of which showed significant differential expression. The main pathways affected by the differentially expressed proteins include energy metabolism, oligodendrocyte metabolism, and cytoskeleton assembly. The potential protein biomarkers candidates myelin basic protein and myelin oligodendrocyte protein were validated by Western blot in the MDT samples and also in cerebrospinal fluid from a separate set of samples of 17 first-episode SCZ patients and 10 healthy controls. The differential expression of μ-crystallin, protein kinase C-gamma, and glial fibrillary acidic protein were confirmed in MDT. Because we found several glycolysis enzymes to be differentially expressed, we measured the levels of pyruvate and NADPH and found them to be altered in MDT. The protein changes described here corroborate the importance of myelin/oligodendrocyte and energy metabolism in SCZ and highlight new potential biomarkers candidates that may contribute to the understanding of the pathogenesis of this complex disease.     

A proteomic investigation of Streptococcus agalactiae grown under conditions associated with neonatal exposure reveals the upregulation of the putative virulence factor C protein β antigen

Streptococcus agalactiae is a major neonatal pathogen that is able to adapt to a variety of host environments, including both rectal and vaginal maternal carriage, growth in amniotic fluid and at various neonatal body sites. As such it is important to elucidate the patterns of protein expression that are associated with S. agalactiae growth under these different in vivo conditions. To this end, we have grown S. agalactiae strain A909 under in vitro conditions reflecting those associated with maternal vaginal carriage (low pH, low oxygen, nutrient stress) and those associated with exposure to body fluids during invasive disease (neutral pH, aeration, nutrient sufficient). The protein profiles of bacterial cells grown under each of these conditions were compared using a proteome approach. A total of 76 proteins were reproducibly identified 16 of which were shown to be differentially expressed. The putative virulence factor C protein β and several proteins linked to resistance to oxidative stress were found to be upregulated under the conditions hypothesised to reflect those associated with foetal exposure to S. agalactiae. Thus, these data add to the currently limited understanding of the molecular basis of S. agalactiae GBS adaptation to different environmental conditions.     

转化生长因子β1诱导肾小管上皮细胞转分化的蛋白质组学研究

目的 研究转化生长因子（TGF）β1诱导肾小管上皮细胞（NRK52E）转分化（EMT）过程中相关靶蛋白的表达变化。 方法 应用比较蛋白质组学方法。用双向凝胶电泳（2-DE）对TGF-β1刺激组和对照组 NRK52E细胞总蛋白进行分离。两组间差异蛋白点用质谱和数据库搜索鉴定，并用RT-PCR和Western 印迹在蛋白质和mRNA水平上进一步检测验证。 结果 鉴定出22个差异蛋白，包括与细胞骨架相关蛋白、参与物质转化和能量代谢的酶类、与蛋白翻译后修饰相关蛋白、细胞因子及其他蛋白等。应用RT-PCR验证了转胶蛋白（transgelin）、ATP合成酶α亚型、苹果酸脱氢酶、丝氨酸（半胱氨酸）蛋白酶抑制因子、泛素结合酶9（Ubc9）和表皮生长因子8在mRNA水平的表达差异，与2-DE结果一致。用Western 印迹验证了transgelin、ATP 合成酶α亚型两种蛋白的表达差异，亦与2-DE结果一致。 结论 TGF-β1刺激NRK52E细胞EMT发生过程中可诱导包括与细胞骨架相关蛋白、与翻译后修饰相关蛋白、代谢酶类及细胞因子等多种蛋白表达变化。本研究结果有助于深入理解EMT的具体分子机制及阐明肾脏疾病慢性进展的机制。     

Proteomic analysis of dorsolateral prefrontal cortex indicates the involvement of cytoskeleton, oligodendrocyte, energy metabolism and new potential markers in schizophrenia

Schizophrenia is likely to be a consequence of serial alterations in a number of genes that, together with environmental factors, will lead to the establishment of the illness. The dorsolateral prefrontal cortex (Brodmann’s Area 46) is implicated in schizophrenia and executes high functions such as working memory, differentiation of conflicting thoughts, determination of right and wrong concepts, correct social behavior and personality expression. We performed a comparative proteome analysis using two-dimensional gel electrophoresis of pools from 9 schizophrenia and 7 healthy control patients’ dorsolateral prefrontal cortex aiming to identify, by mass spectrometry, alterations in protein expression that could be related to the disease. In schizophrenia-derived samples, our analysis revealed 10 downregulated and 14 upregulated proteins. These included alterations previously implicated in schizophrenia, such as oligodendrocyte-related proteins (myelin basic protein and transferrin), as well as malate dehydrogenase, aconitase, ATP synthase subunits and cytoskeleton-related proteins. Also, six new putative disease markers were identified, including energy metabolism, cytoskeleton and cell signaling proteins. Our data not only reinforces the involvement of proteins previously implicated in schizophrenia, but also suggests new markers, providing further information to foster the comprehension of this important disease.