Analysis of serum biomarkers in human knee osteoarthritis

AIM: To analyze the proteomic components of the sera from knee osteoarthritis patients and normal people, and to search proteins that might serve as serum biomarkers for osteoarthritis diagnosis, treatment or pathogenesis. METHODS: Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was applied to analyze the sera obtained from the patients with knee osteoarthritis (n=4) and normal controls (n=4). The differentially expressed proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western blotting analysis was applied to confirm the results. RESULTS: Comparative proteomic data of serum from the patients with osteoarthritis was successfully obtained. Eight differentially expressed protein spots were observed. Five up-regulated and 3 down-regulated proteins were identified. Western blot analysis confirmed that α₂-macroglobulin was increased. CONCLUSION: There are significant differences between serum proteins obtained from the patients with knee osteoarthritis and normal controls. α₂-Macroglobulin might be utilized as potential biomarkers for the diagnosis and treatment of osteoarthritis.     

The proteome research of human lens epithelial cells by two-dimensional gel electrophoresis and mass spectrometry

AIM: To establish the techniques in the proteome research of human lens epithelial cells,including the techniques of two-dimensional electrophoresis and mass spectrometry.METHODS: Total protein of cultured human lens epithelial cells was extracted with two kinds of different methods.The proteins were separated using immobilized pH gradients 2-DE and visualized by silver staining.The digitized images obtained by GS-800 scaner were then analyzed with PDQuest software in order to establish the differential expression profiles.The differential expressed protein spots were cut from the gels using proteomework spot cutter and subjected to in-gel digestion with trypsin.The digested peptide separation was conducted by a Finnigan LCQ MS coupled with a Surveyor HPLC system. RESULTS: A high resolution and reproducible 2-ED image was successfully obtained.The maps of 2-DE showed that lens proteins were in the section of pH 4-7 which the relative molecular weight was 17-72 kD.Relative molecular weight of more abundant proteins was localized at 19-50 kD,as well as the isoelectric points were found to lie between PI 5-7.Two of these proteins were identified by mass spectrometry and database queries.CONCLUSION: A stable protein maps of human lens epithelial cells is constructed.The technique will be used in human lens research to characterize physiological processes and diseases.     

Changes of expressive proteome of renal tissues in chronic intermittent hypoxic rats

AIM: To obtain the two-dimensional gel electrophoresis maps of renal tissue proteins for identifying the differentially expressed proteins in the chronic intermittent hypoxic rats. METHODS: The rat model of chronic intermittent hypoxia was established. The rats lived in the normoxia environment were used for control. The proteins in the renal tissues underwent two-dimensiona1 gel electrophoresis with immobiline pH gradient isoelectric focusing as the first and vertical SDS-PAGE as the second dimension. Analysis of 2-DE maps was performed to determine the differential expression of proteins between the two groups by the software of ImageMaster 2D Platinum V5.0, in which 4 protein spots expressed differentially were picked up for further identification by MALDI-TOF-MS. RESULTS: Matched and compared with those in control group, 112 protein spots were determined in chronic intermittent hypoxia group. By MALDI-TOF-MS, 4 protein spots with the highest differentially expressive levels were identified as ATP synthase delta subunit mitochondrial precursor, hexokinase, catechol O-methyltransferase and apurinic/apyrimidinic endonudease/redox factor-1. The functions of these identified proteins are involved in cellular energy metabolism, apoptosis, signal transduction, anti-cell injury and hormone metabolism. CONCLUSION: There are obvious differences in expressive proteomes in renal tissue between normoxic and chronic intermittent hypoxic rats. Proteomics can serve as a new approach in the study of obstructive sleep apnea-hypopnea syndrome for discovering new therapeutic targets.     

蛋白质组学研究技术及其在果树学中的应用

蛋白质组研究是当今生命科学发展的一个新的增长点,它能阐明基因组所表达的真正执行生命活动的全 部蛋白质的表达规律和生物功能。简要介绍了蛋白质组学产生的科学背景、研究方法和研究内容。蛋白质组学研究 方法主要有双向聚丙烯酰胺凝胶电泳(2D-PAGE)、质谱(Mass-spectrometric)技术、蛋白质芯片(Protein chips)技术、酵母 双杂交系统(Yeast two-hybrid system)、双向高效柱层析和生物信息学等。其应用的范围包括植物群体遗传学、在个体 水平上植物对生物和非生物环境的适应机制、植物的发育和组织器官的分化过程,以及不同亚细胞结构在生理生态 过程中的作用等诸多方面。同时展望了植物蛋白质组学研究前景以及蛋白质组学技术在果树学中的应用前景。     

Proteomics and its application in medicine of reproduction

With the development of proteomics, the application of proteomics in the research for the medicine of reproduction becomes more and more widely, especially in physiology, pathology, toxicology of reproduction and drug discovery. Significant performance has been achieved for instance, PHGPx is identified in mitochondrial capsule by proteomics approach. 53 proteins involved in the spermatogenesis of rat are identified. The toxicological effect of TCDD on placenta of rat has also been conducted. As a kind of tools of molecular biology, proteomics approach brings into full play.     

山葡萄花序蛋白质双向电泳技术体系的建立

【目的】为开展山葡萄花序蛋白质组学研究。【方法】以山葡萄(Vitis amurensis Rupr)花序为试材,通过对蛋白质提取方法、裂解液成分、胶条pH梯度的改进,建立了适于山葡萄花序蛋白质组学研究的最佳双向电泳技术体系。【结果】采用酚提取法提取花序蛋白质,用含有硫脲和DTT的裂解液Ⅱ裂解,选用17 cm pH 4～7的IPG胶条在适宜的双向电泳条件下分离,可以获得分辨率高、背景清晰且重复性好的双向电泳图谱,可以检测出1 088个清晰的蛋白点。【结论】酚提取法适于山葡萄花序蛋白质的提取,硫脲和DTT可增强花序蛋白质的溶解性。     

Differential analysis of serum proteomics in Crohn's disease treated with infliximab

AIM: To identify the serum proteins that might serve as biomarkers for predicting mucosal healing (MH) in the patients with Crohn's disease (CD) treated with infliximab (IFX). METHODS: We collected serum samples before treatment (0 week, group A) and 14 weeks after treatment (group B) from 7 CD patients with IFX treatment who had achieved MH, as well as the serum samples from 7 CD patients who had not achieved MH (0 week, group C; 14 weeks, group D). Two-dimensional fluorescence difference gel electrophoresis was applied to analyze and compare the results of serum profiles between groups A and B, C and D, A and C, B and D. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry and bioinformatics tools were utilized to preliminarily identify and figure out the differentially expressed proteins. RESULTS: (1) In total, there were 44 differentially expressed spots, 36, 3, 10 and 31 differentially expressed spots were detected while comparing A with B, C with D, A with C and B with D, respectively. (2) Among those spots, 17, 2, 2 and 15 proteins were identified, respectively. In total, there were 19 differentially expressed proteins, including apolipoprotein E, apolipoprotein A-I, complement factor H, and so on. (3) Protein functional association networks were carried out based on STRING database. CONCLUSION: The serum protein profiles obviously change after IFX treatment in MH CD patients, and the serum protein profiles of MH patients are different from that of non-MH patients after IFX treatment. The 19 proteins we identified may serve as potential biomarkers for predicting MH in CD patients with IFX treatment.     

桃叶片总蛋白双向电泳技术的研究

采用改进的O'Farrell双向电泳系统,研究适于桃树叶片蛋白质组分析的双向电泳方法。通过对不同蛋白样品提取方法、不同pH梯度范围、不同上样量的比较,探索出一套适合桃树叶片总蛋白分析的双向电泳方法,即以TCA/丙酮沉淀法提取叶片蛋白质样品,采用两性电解质配比pH3～10︰pH5～7为1∶4的IEF胶条,按90μg蛋白上样量,并结合适宜的双向电泳参数,可获得背景清晰、重复性较好、蛋白分辨率高的双向电泳图谱。经此方法分离的桃叶片蛋白质经串联质谱分析(MS/MS)得到了较好的鉴定。     

黄瓜杂交二代抗黄瓜白粉病的蛋白质组学初步分析

通过分组分离法建立F₂代抗感池, 利用2-DE差异显示和质谱分析研究了抗病池和感病池的差异蛋白质组。抗感池经双向电泳检测到近500个蛋白点, 质谱分析鉴定出9种抗感池表达差异蛋白点,分别为抗病基因蛋白( SSP3110) , 14-3-3脑蛋白家族蛋白( SSP1411) , 粪卟啉原氧化酶Ⅲ ( SSP5513) , 碳酸酐酶( SSP5412 ) , α - 半乳糖苷酶( SSP5710 ) , 推定的蛋白( SSP6002 ) , 17.8 kDa 的热激蛋白( SSP0710) , 假想蛋白( SSP3111) , ( S) - 2 - 羟酸氧化酶( SSP4610) 。其中有3种蛋白的性质及生物学功能未知, 其余6种蛋白分别与光合作用、呼吸作用、抗病反应及信号转导等有密切关系。这些蛋白都可能是与抗性有关或与发育相关的蛋白网络中的一部分, 它们在黄瓜抗病反应中起着不同的作用。     

Screening and identification of specific serum biomarkers of Parkinson disease

AIM：To screen the possible serum biomarkers of Parkinson’s disease. METHODS：The surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to screen the serum samples from 44 cases of Parkinson’s disease and 60 control subjects. The differentially expressed protein peaks were selected and isolated with high-performance liquid chromatography (HPLC), and processed with enzyme before analysis by liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS). The data mining was performed by Xcalibur program component BioWorks 3.2. RESULTS：Three differentially expressed protein peaks were selected as potential serum biomarkers from the patients of Parkinson’s disease: the protein at 8 937 m/z peak showed significant increase (27.47±16.58 in Parkinson’s disease group, and only 5.01±3.47 in control group), and the proteins at 6 636 and 8 697 m/z peaks showed significant decreases (5.43±2.66 and 20.22±9.57, respectively, in Parkinson’s disease group, and 18.85±7.56 and 51.13±26.22, respectively, in control group). The proteins at 6 636, 8 697 and 8 937 m/z peaks were identified as apolipoprotein C-I, apolipoprotein C-III and complement 3a,respectively. Combined use of these 3 biomarkers effectively distinguished the subjects between Parkinsons disease group and control group. The detection rate of the patients with Parkinsons disease was 90.0% (27/30), and the detection rate of the healthy sibkects was 92.5% (37/40). CONCLUSION：The apolipoprotein C-I, apolipoprotein C-III and complement component 3a identified as potential markers of Parkinson’s disease have diagnostic value in clinical application.     

橡胶树死皮病黄色体差异表达蛋白的初步分析

橡胶树"死皮病"给橡胶种植业带来严重的危害.为了更好地了解和阐明死皮病发生、发展的分子机制,研究应用双向凝胶电泳技术(two-dimensional gel electrophshiya/oresis,2-DE)比较橡胶树死皮株与健康株胶乳黄色体蛋白质组表达的差异.采用TCA/丙酮沉淀法提取橡胶树死皮株与健康株黄色体蛋白质并采用固相pH梯度(immobjlized pH graalient,IPG)双向凝胶电泳分离两种材料蛋白质,凝胶经银染显色后,用PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质.成功获得橡胶树死皮株与健康株胶乳黄色体的双向凝胶电泳图谱.鉴定出13个蛋白差异点,其中10个上调表达,3个下调表达.并应用质谱技术鉴定了其中部分表达差异的蛋白质点,对渗调蛋白进行功能分析,认为渗调蛋白在死皮株中表现下调的情形可能与死皮病的发生有一定的关系.     

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.     

新红星苹果花芽蛋白质提取及双向电泳的改良方法

以新红星苹果花芽为材料,探索适合蛋白质的提取方法及其双向电泳的条件。采用物理和化学2种方法充分裂解植物组织细胞,并用低温操作加入PVP等去除植物大量的酚类物质,最后离心去除不溶性杂质的苹果蛋白质组双向电泳(2-DE)样品制备方法,第1向为ISO-DALT等电聚焦,第2向为垂直平板SDS-PAGE。通过对样品制备、双向电泳参数的选择及染色等方面进行优化,电泳图谱可分辨出蛋白质斑点数在519个以上,蛋白质相对分子质量约为14.0～97.0ku,主要分布为14.0～67.0ku;等电点约为3.5～9.0,主要分布在4.0～7.0。     

辣椒CMS型雄性不育系与保持系花期叶片蛋白质组分析

采用双向凝胶电泳和计算机辅助分析方法,比较辣椒细胞质雄性不育系W9A与保持系W9B花期叶片蛋白质组,发现不育系与保持系花期叶片存在蛋白质(多肽)的差异,可能与细胞质雄性不育的形成有关。     

白粉病菌胁迫下黄瓜R17抗病品系叶片蛋白质组分析

 以黄瓜抗白粉病品系R17为试材,在接种白粉病菌24、48和72 h后提取叶片蛋白质,采用双向电泳技术研究白粉病菌胁迫条件下黄瓜叶片蛋白质组的变化。结果表明：黄瓜幼苗在白粉病菌胁迫下,24、48和72 h的叶片双向电泳图谱与未接种对照组均存在一定差异。通过质谱分析和数据库搜索,共鉴定出7种表达差异蛋白点,它们分别为磷脂酰肌醇-4-磷酸5-激酶、假拟蛋白g5bf、PSⅡ聚光复合体叶绿素a/b结合蛋白、铁氧还蛋白NADP氧化还原酶、XS结构域所含蛋白、肌球蛋白XI和苹果酸脱氢酶。这些蛋白都可能是与应答白粉病菌胁迫相关的蛋白质网络中的一部分,它们在黄瓜抗病反应中起不同的作用。     

Identification of specific serum biomarkers in gastric adenocarcinoma patients

AIM: To screen the possible serum biomarkers for the diagnosis of gastric adenocarcinoma. METHODS: The surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to screen the serum samples from 109 cases of gastric adenocarcinoma and 106 control subjects (60 healthy subjects, 30 patients with chronic superficial gastritis and 16 cases of chronic atrophic gastritis). The differentially-expressed protein peaks were selected and isolated with high-performance liquid chromatography (HPLC), and processed with enzyme before liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS) analysis. The data mining was performed with software Xcalibur program component Bioworks 3.2. RESULTS: Three differentially-expressed protein peaks were selected as potential serum biomarkers of gastric adenocarcinoma patients.The m/z peak at 5 906.5 showed the increase (8.53±4.33 in cancer group, and 0.88±0.31 in control group). The m/z peaks at 6 635.7 and 8 716.3 showed the decrease (6.54±2.44 and 0.93 ± 0.29, respectively, in cancer group and 17.56±4.43 and 2.16±0.98, respectively, in control group, P<0.01). The 3 m/z peaks were identified as fibrinogen α-chain, apolipoprotein A-II and apolipoprotein CI,respectively. The combined use of the 3 biomarkers distinguished the samples in the cancer patients out of the controls at a sensitivity of 93.85% (61/65) and a specificity of 94.34% (50/53). CONCLUSION: The fibrinogen α-chain, apolipoprotein A-II and apolipoprotein CI identified as potential markers for gastric adenocarcinoma show diagnostic values in clinical application.     

桃果实蛋白质双向电泳影响因素的研究

以‘大久保’桃（^{Prunus persica} L.'Okubao'）果实为试材,通过比较不同上样量、不同上样缓冲液、不同平衡缓冲液、不同染色方式等条件下的双向电泳图谱,探讨以上各因素对桃果实双向电泳图谱效果的影响。结果表明考马斯亮蓝染色后的电泳图谱虽然蛋白质点的数量少于银染色后的图谱,但背景较为清晰,适用于蛋白质组分析。采用固相pH梯度17 cm胶条,100 μg蛋白质上样量,样品干粉溶于375 μL含有硫脲、磺基三甲基胺乙内脂3~10的上样缓冲液中,等电聚焦后用含有磷酸三丁酯的平衡缓冲液还原,进行SDS-PAGE,银染色得出的双向电泳图有蛋白点1 209个,纹理较少,适用于桃果实蛋白质组学分析且兼容于下游技术的蛋白质质谱鉴定。     

枇杷叶片和果实总蛋白质提取及双向电泳的优化方法

以枇杷叶片和果实为材料,探索适于枇杷总蛋白质分析的双向电泳方法.比较2种不同蛋白质提取方法对双向电泳结果的影响,并对双向电泳蛋白质上样量、等电聚焦条件等进行了优化,探索出一种适于枇杷叶片和果实总蛋白质分析的高分辨率、高重复性双向电泳方法.采用酚抽法提取枇杷叶片和果实蛋白质,以24 cm、pH 4~7的IPG胶条,按15...     

油桃裂果研究进展

从品种特性、果实生长发育特点、果实细胞组织结构等内部因素和气象因子、土壤条件、栽培措施等外部因素方面综述了油桃裂果的影响因素,寻找引起油桃裂果的主要原因,并提出防止油桃裂果相应的技术措施。首先从园地选择、品种选择方面尽量做到适地适栽,因地制宜,另外果园增施有机肥,提高土壤透水性和保水性,适时灌水,及时排水,使水分保持均衡状态,合理修剪,加强病虫害防治,严格疏花疏果,实行套袋栽培等一系列科学化管理均是防止油桃裂果和果锈的有效技术措施。     

农艺和生物节水技术在果树上的研究进展

本文综述了农艺节水技术和生物节水技术在果树中的研究进展,提出高水分利用效率树种、品种的选择,节水生化制剂的应用,水肥耦合高效利用技术以及调亏灌溉和分根区交替灌溉等节水灌溉制度。并指出了农业节水技术的发展方向。