蛋白质组学及其研究进展

综述蛋白质组学的定义、研究策略、研究内容、主要研究技术及其在基础研究、农业、疾病研究、药物开发等方面的应用。     

基于组学的作物杂种优势研究进展

杂种优势,又称杂种活力,是指来源于物种内不同品种或者不同物种间杂交后代在生物产量、生长势以及育性等方面均优于亲本的现象。目前对于杂种优势形成的遗传机制一直停留在假说层面。分子标记技术的飞速发展为杂种优势的遗传机制研究提供了新的途径,在不同作物中,已证实显性、超显性和上位性效应对杂种优势的形成均有贡献。随着基因组、转录组、蛋白组等多组学技术及表观遗传学技术的兴起,为在全基因组水平研究杂种优势提供了机会,更有利于从不同角度对杂种优势形成的遗传基础进行全面解析。本研究从杂种优势位点定位,杂交种相对于亲本的转录组水平,蛋白质水平及表观调控改变等方面进行了概述,为全面深入理解杂种优势及更好地研究、利用杂种优势提供帮助。     

家蚕品种金秋及其杂交亲本的蛋白质表达谱分析

 【目的】探讨家蚕杂交育成品种及其亲本蛋白水平的差异,从蛋白水平分析育成品种的基因结构,为阐明杂交育种成败的分子机理创造条件。【方法】采用蛋白质组研究技术,分析杂交亲本及其育成品种的丝腺、血淋巴和中肠的蛋白质表达谱。【结果】3种组织器官中育成品种金秋与两个亲本的匹配蛋白斑点比例只达到70%左右,剩余30%左右是各个品种的特异蛋白斑点;在匹配蛋白斑点中,还有9%～24%的蛋白表现上调和下调。这些特异蛋白可能是两个亲本的基因在杂交育成品种中产生了互作而形成的新蛋白,也可能是对相同的蛋白进行了不同的修饰而获得的修饰蛋白。【结论】新品种的育成,除了汇集亲本的优良基因外,还有赖于基因互作产生的新功能蛋白的作用。     

Study of annexin A2 expression in gastric cancer by proteomics and tissue microarray

AIM: To investigate the differential expression of annexin A2 (ANXA2) in gastric carcinoma and to analyze the relationship between ANXA2 expression and clinicopathological parameters of gastric carcinoma. METHODS: Pure gastric adenocarcinoma cells (GAC) and normal gastric epithelial cells (NGEC) in 15 patients with gastric cancer were acquired by laser capture microdissection (LCM). All peptide specimens after trypsin digestion were labeled with ¹⁸O/¹⁶O. Quantitatively identification of differential expression of the proteins betweem GAC and NGEC was performed by Nano-RPLC-MS/MS. The expression of ANXA2 in the 2 kinds of tissues was detected by Western blotting. Tissue microarray containing 75 pairs of gastric carcinoma and para-carcinoma tissues was used and the expression of ANXA2 in these specimens was detected by the method of immunohistochemistry (IHC). The relationship between ANXA2 expression and clinicopathological parameters of the pateints with gastric carcinoma was analyzed. RESULTS: A total of 78 differential proteins were identified and ANXA2 was up-expressed in GAC (2.32∶ 1), which was confirmed by Western blotting (P<0.01). The results of IHC showed that the correlations between the expression level of ANXA2 protein and invasive depth (T stage), lymph node metastasis (N stage), histological differentiation, TNM stage and the size of tumor were observed (P<0.01), but the correlations between the ANXA2 expression and sex, age and distant metastasis (M stage) were not found (P>0.05). CONCLUSION: The up-expressed ANXA2 may play an important role in the biological behavior of gastric cancer.     

cPKCγ-HSP60 signal pathway participates in hypoxic preconditioning in mice

AIM: To identify the proteins interacted with conventional protein kinase Cγ (cPKCγ) which are involved in hypoxic preconditioning (HPC), and to investigate the role of cPKCγ-interacted heat-shock protein 60 (HSP60) in the development of HPC and ischemia (I).METHODS: Healthy male BALB/c mice were randomly divided into normoxia(Norm) and HPC groups. Based on whether middle cerebral artery occlusion (MCAO) was performed, the mice were divided into Norm+sham group, Norm+I group, HPC+sham group and HPC+I group ( all n=6). Immunoprecipitation, two-dimensional electrophoresis and mass spectrometry were applied to identify the proteins interacted with cPKCγ. The changes of HSP60 expression in the brain of the mice after HPC and MCAO were analyzed by Western blotting.RESULTS: The interaction of cPKCγ and HSP60 was confirmed by co-immunoprecipitation result. Compared with Norm groups, the expression level of cPKCγ-interacted HSP60 obviously increased in particulate fraction of cerebral cortex in HPC mice. In the MCAO ischemic animals, the level of HSP60 expression was significantly higher in ischemic core and penumbra in Norm+I group and HPC+I group than that in Norm+sham group. HSP60 expression in ischemic core was lower in HPC+I group than that in Norm+I group.CONCLUSION: cPKCγ-HSP60 signal pathway might be involved in the development of cerebral hypoxic preconditioning in MCAO mice.     

巴西橡胶芽接树砧穗互作的蛋白质组初步研究

运用蛋白质组学研究技术,寻找砧木和接穗互作相关蛋白。以巴西橡胶树3种芽接组合植株的砧木与接穗结合部附近处的胶乳为试验材料,经双向凝胶电泳(2-DE)分离获得胶乳中存在的差异表达蛋白,采用质谱技术鉴定出32个差异表达蛋白,这些蛋白主要参与转录、翻译、光合作用、细胞分裂、蛋白质合成和胶乳合成等生物学过程。     

Proteomic analysis of mammary tissues from healthy cows and clinical mastitic cows for identification of disease-related proteins

To investigate the disease-related proteins and understand molecular mechanism of mastitis at the protein level, this project presents the protein changes in the mammary gland between healthy cows and clinical mastitic cows using two-dimensional gel electrophoresis (2-DE), after stained with colloidal Coomassie Bright Blue, six spots of differentially expressed protein were detected by PDQuest software and subjected to ion trap mass spectrometer equipped with a HPLC system, and five proteins were identified. Hemoglobin beta, kappa-casein and tryptophanyl-tRNA-synthetase (TrpRS) in healthy dairy cows, while hemoglobin beta, cytochrome C oxidase and annexin V in clinical mastitic cows were identified, they were involved in binding, transport and catalytic activity. The results may provide valuable information for the investigating of the host mammary immune system response to defense against pathogens at the protein level and potential protein targets for treatment.     

小峰熊蜂工蜂蛹期发育蛋白质组分析

本文采用双向凝胶电泳法对小峰熊蜂工蜂蛹期的3个发育期进行蛋白质组研究,结果表明小峰熊蜂工蜂蛹期的白眼期（A期）、褐眼期（B期）和黑眼期（C期）分别检测到77、81和59个蛋白点,特有蛋白分别为7个、6个和1个,共有蛋白为48个,A期到B期显著上调的蛋白有9个,显著下调的蛋白有3个,B期到C期显著上调的蛋白有8个,显著下调的蛋白有3个,A期到C期显著上调的蛋白有15个,显著下调的蛋白有2个。研究还发现有17个蛋白是在A期和B期表达而在C期关闭;有2个蛋白是在A期表达,B期关闭,在C期又表达。本研究初步揭示了小峰熊蜂工蜂从蛹期发育到成蜂过程中,不仅需要一些保守蛋白来调控,而目．还需要一此特异蛋白。     

Alteration and significance of protein expression profile in gastric cancer with preoperative conformal radiation therapy

AIM: To investigate the alteration and significance of the protein expression profile in gastric cancer with preoperative conformal radiation therapy. METHODS: The pathologically-diagnosed gastric cancer patients were randomly chosen to perform conformal radiation therapy before operation. The patients without conformal radiation treatment served as controls. The tumor tissues were collected after operation from the patients. The alteration of the protein expression profile was detected by 2-dimensional electrophoresis and MALDI-TOF-MS. The expression of vascular endothelial growth factor (VEGF), c-erbB-2 and epidermal growth factor receptor (EGFR) was detected by RT-PCR and Western blotting. RESULTS: Compared with the patients without conformal radiation therapy, the protein expression profile of tumor tissues from gastric cancer patients with conformal radiation therapy was obviously altered. The expression of VEGF, c-erbB-2 and EGFR was differentially reduced (P<0.01). CONCLUSION: The preoperative accurate conformal radiation therapy reduces the expression of gastric cancer-related epithelial proteins, indicating new therapeutic targets for gastric cancer.     

适用于小麦叶片蛋白质组分析的样品制备方法

 【目的】建立适用于小麦叶片蛋白质组分析的样品制备方法。【方法】以Taichung 29小麦苗期叶片为材料，分别采用改进的酚提取-甲醇/醋酸铵沉淀法及尿素/硫脲提取法提取总蛋白，进行双向电泳分离和胶体考染，并选取代表性的蛋白点进行MALDI-TOF质谱分析及数据库搜索。【结果】上述的两种方法可检测到较多的蛋白点数，分别为1 016±203和1 014±281，并能成功地完成代表性蛋白点的鉴定。【结论】作者建议，进行小麦叶片蛋白质组分析时，可采用本实验提供的两种样品制备方法。