多功能蛋白ASPP2在肿瘤发生发展中的作用

恶性肿瘤的发生发展是一个多因素、多步骤参与的复杂过程,临床上肿瘤治疗存在复发率高且易耐药等现象,寻找肿瘤易转移、易复发及耐药的干预靶点对恶性肿瘤的诊断和治疗意义重大。多功能蛋白p53促凋亡刺激蛋白2 (ASPP2)是一种单倍体不足肿瘤抑制因子,自被发现以来其在肿瘤中的作用备受关注。ASPP2在多种恶性肿瘤中的表达均明显下调,且其下调水平与肿瘤晚期及不良预后相关,表明其在肿瘤发生发展中扮演重要角色。本文主要综述ASPP2在肿瘤转移、耐药和代谢等方面的研究进展,为以ASPP2为治疗靶点的研究提供理论依据。     

乳腺癌细胞株中MKK4的表达及其对细胞转移的影响和机理研究

目的：观察乳腺癌细胞株中MKK4蛋白的表达水平,研究MKK4蛋白表达对乳腺癌细胞运动能力及EMT标志物的影响,确定MKK4在肿瘤细胞EMT转化及肿瘤转移中的作用,为肿瘤转移机制研究提供一定的基础资料,为肿瘤防治奠定一定的理论基础。方法：通过体外细胞培养技术收集系列乳腺癌细胞株的培养裂解液,利用Westernblot技术检测细胞培养裂解液中MKK4及EMT标志物的表达水平,构建M鼬（4表达水平与细胞转移能力的对应图;采用siRNA技术,干扰MKK4高表达乳腺癌细胞株MKK4的表达,Westernblot技术观察MKK4低表达后,EMT标志物的变化,同时,构建MKK4质粒,转染MKK4低表达乳腺癌细胞株,Westernblot技术观察MKK4高表达后,EMT标志物的变化。并采用MTT法、Transwell、划痕法观察MKK4高表达后细胞增殖、运动、迁移等能力的变化。结果：乳腺癌细胞株中MKK4蛋白的表达水平与乳腺癌细胞运动能力有一定的相关性,并与EMT标志物的表达具有相关性,MKK4蛋白表达越高,细胞运动能力越差。干扰或转染技术影响乳腺癌细胞株中MKK4的表达后,细胞EMT标志物的表达也相应变化,乳腺癌细胞株中MKK4高表达后,其细胞增殖明显抑制,运动迁移能力也相应下降。结论：乳腺癌细胞中MKK4蛋白的表达水平与乳腺癌细胞EMT转化及运动能力有一定的相关性,MKK4蛋白表达越高,细胞运动能力越差。     

乳腺癌细胞株中MKK4 的表达及其对细胞转移的影响和机理研究

目的:观察乳腺癌细胞株中MKK4蛋白的表达水平,研究MKK4蛋白表达对乳腺癌细胞运动能力及EMT标志物的影响,确定MKK4在肿瘤细胞EMT转化及肿瘤转移中的作用,为肿瘤转移机制研究提供一定的基础资料,为肿瘤防治奠定一定的理论基础。方法:通过体外细胞培养技术收集系列乳腺癌细胞株的培养裂解液,利用Western blot技术检测细胞培养裂解液中MKK4及EMT标志物的表达水平,构建MKK4表达水平与细胞转移能力的对应图;采用siRNA技术,干扰MKK4高表达乳腺癌细胞株MKK4的表达,Western blot技术观察MKK4低表达后,EMT标志物的变化,同时,构建MKK4质粒,转染MKK4低表达乳腺癌细胞株,Western blot技术观察MKK4高表达后,EMT标志物的变化。并采用MTT法、Transwell、划痕法观察MKK4高表达后细胞增殖、运动、迁移等能力的变化。结果:乳腺癌细胞株中MKK4蛋白的表达水平与乳腺癌细胞运动能力有一定的相关性,并与EMT标志物的表达具有相关性,MKK4蛋白表达越高,细胞运动能力越差。干扰或转染技术影响乳腺癌细胞株中MKK4的表达后,细胞EMT标志物的表达也相应变化,乳腺癌细胞株中MKK4高表达后,其细胞增殖明显抑制,运动迁移能力也相应下降。结论:乳腺癌细胞中MKK4蛋白的表达水平与乳腺癌细胞EMT转化及运动能力有一定的相关性,MKK4蛋白表达越高,细胞运动能力越差。     

ROCK蛋白与肿瘤

ROCK蛋白作为Rho亚家族下游最重要的效应分子之一,主要通过调节肌动蛋白在调控细胞的形态、极性、细胞骨架重构和细胞迁移等多个方面发挥生理功能。研究发现ROCK蛋白在肿瘤的发生发展中起着重要作用,主要参与调控肿瘤细胞的生存与凋亡,以及恶性肿瘤的侵袭与转移。文章论述了ROCK蛋白与肿瘤关系的研究进展,为寻找新的抗癌药物治疗靶点提供依据。     

上皮间质转化在肿瘤转移中的研究进展

转移侵袭是恶性肿瘤的基本特征和重要标志,也是导致患者死亡的最主要原因。研究表明,超过80%的恶性肿瘤患者最终死于肿瘤转移。本文综述了DNA甲基化、DNA羟甲基化、组蛋白修饰等表观遗传机制在上皮间质转化中的作用机制,以及肿瘤转移相关信号通路在上皮间质转化中的研究进展。其机制的阐明有可能为肿瘤治疗提供新的方向。     

淋巴道转移相关标志物与癌

肿瘤早期的淋巴道转移是恶性肿瘤患者高死亡率的主要原因,其机制复杂,一直是肿瘤研究的难点. 肿瘤淋巴道转移特异性蛋白标记物的发现有助于揭示恶性肿瘤早期发病机制、早期临床诊断和治疗. 本文综述了近年来肝癌、胃癌、食管癌、结肠癌、乳腺癌、肺癌、鼻咽癌和前列腺癌淋巴道转移研究所取得的蛋白质组学成果,并对发现的与以上几种肿瘤淋巴道转移相关的蛋白标记物及其在肿瘤的发生、浸润、转移及病人预后中作用及机制进行了探讨.     

采用蛋白质组学技术筛选大肠癌转移相关蛋白

采用对同一亲本来源、不同转移潜能细胞株SW480和SW620的蛋白质表达谱进行双向凝胶电泳和质谱技术分析,并在蛋白质和mRNA水平进行验证,成功鉴定了10个大肠癌转移相关蛋白,其中SW620细胞株表达上调的蛋白质有磷酸甘油酸变位酶1,磷脂酰乙醇胺结合蛋白和高迁移率族蛋白B-1,而热休克蛋白27,膜联蛋白Ⅰ,甲硫腺苷磷酸化酶,切丝蛋白1和表皮型脂肪酸结合蛋白在SW620中表达下调.大多数差异蛋白质功能涉及肿瘤细胞生长、运动、粘附、凋亡等过程,研究结果为阐明大肠癌转移机制及寻找预测大肠癌转移的潜在标志物提供了理论依据.     

癌蛋白YAP1的研究进展

Yes相关蛋白1(Yes-associated protein 1,YAP1)是Hippo信号通路(Hippo pathway)中的一个分子.早期研究人员发现,在Hippo信号通路正常的情况下,YAP1处于非激活状态;当Hippo信号通路中的某些分子出现突变时,YAP1处于超激活状态.此时,超激活状态下的YAP1可以促进细胞增殖、转移、生存(survival)以及维持干细胞活性.由于YAP1的超激活可以促进肿瘤的发生与发展,因此,YAP1被定义为一个癌蛋白.近期,研究者发现,YAP1的突变体与小细胞肺癌病人的存活率有一定关系,YAP1与链蛋白(catenin)、Kras相互作用,调节肿瘤细胞的转移侵袭能力,此外,部分micro RNA也与YAP1有相互作用.基于YAP1的功能,可以制定一些抗癌策略,寻找一些抗癌靶点.本文对当前YAP1的研究进行综述,为肿瘤治疗的基础及临床研究提供一些依据.     

Ezrin蛋白信号转导通路及其对肿瘤侵袭转移的最新进展

肿瘤转移是一个多阶段、多途径、涉及多基因及其信号通路变化的一系列复杂过程。了解肿瘤转移相关基因的信号传导通路以及对肿瘤转移的作用机制,为寻找抑制肿瘤转移的关键靶点具有重要的意义。Ezrin高表达与肿瘤转移密切相关,它可通过改变肿瘤细胞极性及细胞运动、调节肿瘤细胞间黏附及细胞与细胞外基质黏附、参与肿瘤细胞内信号转导而影响恶性肿瘤转移。Ezrin过度表达可以破坏正常细胞内信号传递网络的平衡,其中主要涉及的为细胞信号转导相关分子(Rho)及受体酪氨酸蛋白激酶等信号传导途径。Ezrin借助于细胞内错综复杂的信号转导网络调控细胞的形态构成、黏附、吞噬、运动、血管形成等一系列的生物学过程,最终实现肿瘤细胞的侵袭和转移。本文就Ezrin蛋白的信号转导通路及其对肿瘤转移作用的研究进展做一综述。     

SELDI-TOF-MS技术在临床肿瘤蛋白质组学研究中的应用

恶性肿瘤严重地威胁着人类的健康和生命。多年来,人类一直在为治疗肿瘤而奋斗。众所周知,恶性肿瘤的早期发现、早期诊断、早期治疗是提高大多数恶性肿瘤患者治疗效果的关键。同时,肿瘤的早期发现也能减少肿瘤转移和播散的机会。癌症发现、确诊得越早,治疗成功的可能性就越大,患者的生存率也就会越高。目前,SELDI-TOF-MS技术在临床肿瘤蛋白质组学研究中已经得到了普遍应用,研究的范围几乎覆盖所有的常见肿瘤,为临床肿瘤的蛋白质组学研究奠定了良好的基础。以下概要介绍SELDI—TOF-MS技术在部分肿瘤中的研究应用。     

SELDI-TOF-MS筛选慢性阻塞性肺疾病血清标志蛋白的初步研究

目的:利用表面增强激光解吸电离飞行时间质谱技术(SELDI-TOF-MS)筛选慢性阻塞性肺疾病(COPD)血清特异标志物。方法:应用SELDI-TOF-MS技术检测30例COPD稳定期患者和30例健康对照者血清蛋白指纹图谱,采用Biomarker pattern软件进行分析,建立COPD的诊断模型。结果:COPD患者血清蛋白图谱与对照组相比,在相对分子质量2000-15 000范围内共检测到75个蛋白峰,发现19个有统计学差异的蛋白峰(P0.05)。通过对COPD组与对照组间的数据作进一步分析,经BPS软件分析,建立质荷比(M/Z)3 167、4 645的差异蛋白组成的诊断模型,其诊断敏感度为96.67%,特异度为96.67%。结论:SELDI-TOF-MS技术是一种快速、简单易行、用量少和高通量的分析方法。能直接筛选出COPD血清中特异表达标志物,用特异表达标志物建立的诊断模型能有效区分COPD患者与健康对照者,有望成为COPD诊断的辅助指标。     

Proteomic analysis of lymph

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.     

Differential expression of proteomics models of colorectal cancer, colorectal benign disease and healthy controls

Background

Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. The hypersensitive analytical technique of proteomics can detect molecular changes before the tumor is palpable. The surface-enhanced laser desorption/ionization-time of flight-mass spectra (SELDI-TOF-MS) is a newly-developed technique of evaluating protein separation in recent years. The protein chips have established the expression of tumor protein in the serum specimens and become the newly discovered markers for tumor diagnosis. The objective of this study was to find new markers of the diagnosis among groups of CRC, colorectal benign diseases (CBD) and healthy controls. The assay of SELDI-TOF-MS with analytical technique of protein-chip bioinformatics was used to detect the expression of protein mass peaks in the sera of patients or controls. One hundred serum samples, including 52 cases of colorectal cancer, 27 cases of colorectal benign disease, and 21 cases of healthy controls, were examined by SELDI-TOF-MS with WCX2 protein-chips.

Results

The diagnostic models (I, II and III) were setup by analyzed the data and sieved markers using Ciphergen - Protein-Chip-Software 5.1. These models were combined with 3 protein mass peaks to discriminate CRC, CBD, and healthy controls. The accuracy, the sensitivity and the particularity of cross verification of these models are all highly over 80%.

Conclusions

The SELDI-TOF-MS is a useful tool to help diagnose colorectal cancer, especially during the early stage. However, identification of the significantly differentiated proteins needs further study.     

Identification of novel and downregulated biomarkers for alcoholism by surface enhanced laser desorption/ionization-mass spectrometry

Since personal and verbal reporting of alcohol use is not necessarily accurate, objective markers to assess alcohol consumption are required. The currently available markers, however, are limited in sensitivity and specificity for screening of excessive alcohol drinkers. Therefore, searches for novel markers are warranted. Recently, surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) has been successfully used to detect disease-associated proteins in complex biological specimens. We used the ProteinChip SELDI technology to generate comparative protein profiles of the consecutive serum samples obtained during abstinence from a total of 16 chronic alcoholic patients hospitalized for a rehabilitation program. We recognized two peaks (5.9 and 7.8 kDa), both of which had been downregulated on admission, the expression level of which significantly increased after a one-week abstinence. These changes were also seen in nonresponders of gamma-glutamyltransferase. These two proteins were partially purified and subjected to amino acid sequencing. The 5.9 kDa protein was identified as a fragment of fibrinogen alphaE chain and the 7.8 kDa was a fragment of apoprotein A-II. These novel protein fragments may be promising biomarkers for excessive alcohol drinking.     

评估SELDI-TOF-MS技术检测肾移植术后病人尿液的能力

目的:研究表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术检测肾移植术后病人尿液中蛋白质的能力。方法:根据尿液标本中蛋白质浓度进行稀释处理,采用SELDI-TOF-MS技术,用3种不同芯片(NP20、CM10、IMAC30)与蛋白质浓度的不同组合分别检测肾移植术后病人的尿液。结果:采用NP20/蛋白质未稀释、CM10/蛋白质浓度约0.1g/L、IMAC30/蛋白质浓度约2g/L时,质谱图中蛋白质峰的个数和丰度达到最佳;单一芯片中NP20捕获质荷比为5000～20000的蛋白质能力最佳,而CM10和IMAC30在质荷比为2000～5000时有较大的捕获能力;3种芯片中CM10具有最大的捕获蛋白质的能力。结论:3种芯片检测尿液中蛋白质的能力不同;为了更好地发现疾病特异性标志物,最好多种芯片同时检测,且在各种芯片得到最佳质谱图时的浓度进行检测。     

Identification of serum proteins involved in pancreatic cancer cachexia

AimsTreatment of cachexia requires pharmacological intervention which, in turn, requires knowledge of the mediators and processes. Cachexia markers that are specifically expressed in pancreatic cancer and secreted into the blood circulation have yet to be identified. The aim of our study was to investigate the serum protein profiles and protein alterations associated with cachexia and to identify potential disease protein biomarkers indicative for this syndrome.Main methodsSerum samples from cachectic and non-cachectic patients undergoing pancreatic cancer (PaCa) surgery and controls were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). The identity of detected discriminatory markers was determined by a combination of protein fractionation, chromatographic purification steps, gel electrophoresis, and mass spectrometry.Key findingsUsing Cu-IMAC array and CM-10 array based SELDI-TOF-MS. we identified eleven up- and four down-regulated proteins associated with cachexia. CiphergenExpress analysis revealed four disease-associated protein features (38559 Da, 9138 Da, 8925 Da and 3358 Da) that were elevated by a factor of 2.3, 1.7, 1.4 and 1.4, respectively. Zinc-α2-glycoprotein (ZAG), apolipoproteins apo C-II and apo C-III and glucagon-like peptide-1 (GLP-1) were identified as markers for PaCa-associated cachexia syndrome. ZAG levels were additionally evaluated in serum and tissue samples by ELISA and immunohistochemistry and the obtained data confirmed the SELDI-TOF-MS results.SignificanceThe identified proteins could be routinely and reliably measured in the serum of patients and provide an elegant non-invasive approach for early diagnosis of cachectic pancreatic cancer patients. Controlling ZAG and GLP-1 activity could be beneficial in the management of cancers and cachexia-induced conditions.     

SELDI-TOF-MS of saliva: methodology and pre-treatment effects

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.     

Differential detection of S100A8 in transitional cell carcinoma of the bladder by pair wise tissue proteomic and immunohistochemical analysis

The search for novel molecular markers of tumor invasion is vital if strategies are to become more effective in the diagnostic and prognostic management of transitional cell carcinoma of the bladder. Up to 50% of tumors detected at stage 1 (pT1) progress to a higher grade even after endoscopic surgical resection, and there are currently no protein markers of this aggressive, invasive phenotype. We have combined SELDI-TOF-MS, ClinProt magnetic bead enrichment, Nano-LC-ESI-ion trap tandem mass spectrometry and immunohistochemical analysis to the study of 12 invasive bladder cancer tissue biopsies paired with normal bladder tissue samples obtained from the same patients for the definition and identification of proteins up-regulated in the tumors. We report the inflammation-associated calcium binding protein S100A8 (MRP-8, calgranulin A) to be highly expressed in tumor cells in contrast to normal urothelium in 50% of the samples, as well as two unidentified protein markers at 5.75 and 6.89 kDa that were differentially detected in 9/12 and 10/12 tumor samples, respectively. These new markers, when fully characterized, may contribute to new target proteins for the prediction of aggressive, invasive bladder tumors.     

Protein expression analysis ofChlamydia pneumoniae persistence by combined surface-enhanced laser desorption ionization time-of-flight mass spectrometry and two-dimensional polyacrylamide gel electrophoresis

The aim of this study was to examine the protein expression profiles of persistentChlamydia pneumoniae by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Although 2D PAGE is still the method of choice for separating and detecting components of complex protein mixtures, it has several distinct disadvantages; i.e., being labor-intensive and having a bias toward proteins within the dynamic range of the gel condition. Hence, SELDI-TOF-MS technology was used to complement 2D PAGE.C. pneumoniae-infected HEp2 cells were treated with or without IFN-γ, and protein expression profiles were determined at 48 h postinfection (hpi). Unfractionated monolayers were also used for protein profiling by SELDI-TOF, using two different chip surface types: weak cation exchanger and hydrophobic surface. Under IFN-γ-induced persistence,C. pneumoniae expresses an altered protein expression profile. Twenty chlamydial proteins showed differential regulatory patterns by SELDI-TOF-MS, two of which, HSP-70 cofactor, and a hypothetical protein, were identified by 2D PAGE and mass spectrometry. Two additional proteins, phosphatidylserine decarboxylase and 30S ribosomal protein S17, were exclusively identified by SELDI TOF-MS analysis, as these were not present in sufficient quantity for detection by 2D PAGE. We propose that a combination of 2D-PAGE and SELDI-TOF-MS may complement the disadvantages of each technique alone and may provide a rapid and precise screening technique.     

Evaluation of the combination of bead technology with SELDI-TOF-MS and 2-D DIGE for detection of plasma proteins

Today biomarker discovery is one of the most active aspects of proteomic investigations. However, the wide dynamic range of plasma proteins makes the analysis very challenging because high abundance proteins tend to mask those of lower abundance. Using a large bead-based library of combinatorial peptide ligands (Equalizer beads or ProteoMiner), the dynamic range of the protein concentration is compressed, the high abundance proteins present in the sample are reduced and the low abundance proteins are enriched, while retaining representatives of all proteins within the sample. In the present study, the combination of beads with surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and two-dimensional differential gel electrophoresis (2-D DIGE) technology were evaluated considering efficiency, reproducibility, sensitivity, and compatibility. The bead technology is easily compatible with both SELDI-TOF-MS and 2-D DIGE and the samples can be analyzed directly without any processing of the sample. The use of the beads prior SELDI-TOF-MS and 2-D DIGE enabled detection of many new protein spots/peaks and increased resolution and improved intensity of low abundance proteins in a reproducible fashion compared with the depletion technique. Several proteins have been identified by the combination of beads, 2-D DIGE and MS for example different kinds of complement factors and cytoskeletal proteins. Our data suggest that integration of the bead technology with our current proteomic technologies will enhance the possibility to deliver new peptide/protein biomarker candidates in our projects.