两个新的前列腺癌相关分泌蛋白的鉴定和表达

为了获得代表不同前列腺癌进展阶段的细胞系的胞外蛋白表达谱, 验证其中差异表达蛋白是否为分泌蛋白, 在细胞水平看其是否有作为前列腺癌血清标志物的潜质, 文章利用双向电泳寻找胞外蛋白中差异表达的点, 并质谱鉴定其是何种蛋白质。应用RT-PCR方法分析候选分子在8种细胞系中的表达和对雄激素刺激的应答, 构建了候选分子的真核表达载体, 瞬时转染293T细胞, 应用标签抗体Western blotting方法检测验证细胞培养基中候选分子的表达。结果表明: 筛选出两个C4-2胞外高表达的分子-- 磷酸丙糖异构酶-1(Triosephosphate isomerase 1, TPI1)和多配体聚糖结合蛋白(Syndecan binding protein, syntenin, ST1); 转录水平发现它们与前列腺癌恶性程度相关, 并且后者受雄激素的作用下调; 二者均为分泌蛋白。磷酸丙糖异构酶-1和多配体聚糖结合蛋白均有作为指示前列腺癌发展阶段的血清标志物的潜质。     

异烟肼耐药结核分枝杆菌感染人源巨噬细胞的蛋白质组学研究

利用双向电泳技术,对人源巨噬细胞U937感染异烟肼耐药结核分枝杆菌前后的全细胞蛋白表达图谱进行差异比较和分析,发现其中产生差异的有32个蛋白质斑点,利用基质辅助激光解吸/电离飞行时间质谱技术,对其中5个表达明显上调的蛋白质斑点进行分析鉴定,获得5个明确的肽质量指纹图谱,通过在数据库中进行检索分析,确定这5个蛋白质分别为热休克蛋白105_β、凋亡抑制蛋白-1、磷酸甘油酸变位酶1、组织蛋白酶B、桥粒胶蛋白3.上述发现有助于了解耐药结核分枝杆菌入侵早期导致的巨噬细胞蛋白质组表达变化,为深入研究耐药结核分枝杆菌-宿主相互作用提供了探索方向.     

EphA3受体截短突变体variant b在前列腺癌细胞中的分泌表达

目的:验证EphA3受体截短突变体variant b是否是一种分泌蛋白,并探索其内源分泌的特点,在细胞水平看其是否有作为前列腺癌血清标志物的潜质。方法:构建EphA3 variant b真核表达载体,应用标签抗体、特异性抗体和Western印迹检测前列腺癌细胞培养液中EphA3 variant b的表达,用RT-PCR方法分析EphA3 variant b在8种细胞系中的表达谱和对雄激素刺激的应答。结果:验证了EphA3 variant b是一种分泌蛋白,在雄激素受体阳性的前列腺癌细胞系中特异性表达,雄激素以剂量依赖方式诱导EphA3 variant b表达。结论:EphA3 variant b是一种分泌蛋白,其表达与雄激素受体信号通路相关,有作为前列腺癌血清标志物的潜质。     

用基因芯片筛选前列腺癌LNCaP和C4-2细胞系中差异表达的基因

目的：筛选与前列腺癌进展相关的功能基因。方法：利用Affymetrix公司的人类金基因组U133A芯片,筛选在前列腺癌细胞系LNCaP和C4-2中差异表达的基因,并用RT-PCR方法验证部分差异基因的表达。结果：芯片筛选结果表明,与LNCaP细胞系相比,C4-2细胞系中217个基因转录本表达上调,101个基因转录本表达下调;对其中45个基因进行了RT-PCR验证,证明35个基因转录本的表达结果与芯片筛选一致。结论：筛选出了在LNCaP和C4-2细胞系中显著差异表达的基因35个,为进一步研究前列腺癌进展的机制奠定了基础。     

高效絮凝菌A9在不同碳源培养条件下差异表达蛋白鉴定及分析

研究了高效絮凝菌A9在普通培养基、葡萄糖培养基和甘露糖培养基的培养条件下蛋白质组表达的差异。从这3种不同培养基培养的菌体中提取蛋白,进行蛋白质双向电泳,胶图分析后选取差异蛋白进行质谱鉴定。胶图分析结果表明:絮凝菌A9在不同碳源培养条件下蛋白质表达有较大差异。通过质谱分析,共有54个蛋白得到成功鉴定,包括二氢硫辛酸脱氢酶、磷酸丙糖异构酶、果糖-1,6-二磷酸酶、甘油醛-3-磷酸脱氢酶、磷酸甘油酸激酶、顺乌头酸酶、磷酸酰基转移酶、肽基脯氨酸顺反异构酶、延伸因子G等。这些差异蛋白的功能主要与能量代谢、糖类合成和代谢、脂类运输和代谢、蛋白翻译后修饰以及核糖体结构和生成有关。本文从蛋白水平阐明了絮凝菌A9在不同碳源培养条件下部分蛋白表达差异,为优化菌株絮凝剂培养条件提供了依据。     

大鼠侧脑室注射瘦素对卵巢摘除给予雌激素子宫腔液2-DE图谱分析

瘦素(leptin)通过动物下丘脑-垂体轴对其生殖活动予以调节,但瘦素通过下丘脑对子宫调节作用仍不清楚.本研究在大鼠侧脑室微量注射瘦素6 h后,采用双向凝胶电泳(2-DE)结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术分离、鉴定了卵巢摘除给予雌激素的子宫腔液蛋白质表达谱.我们获得了分辨率较高、重复性较好的2-DE图谱|质谱分析结合SwissProt蛋白数据库检索,共鉴定了24个差异表达的蛋白质与生理盐水注射（对照）比较,侧脑室微量注射瘦素引起22个蛋白质分子显著上调,2个显著下调.重要的是,在上调的蛋白质分子中包括转铁蛋白、基质金属蛋白酶7、膜联蛋白A1、补体B因子、磷酸甘油酸酯变位酶1、烯醇酶、冠蛋白-1A等15个蛋白质分子,下调的2个蛋白质分子是补体C3、溶解物载体家族4成员3.这些蛋白质分子涉及细胞分化、细胞迁移、细胞凋亡,以及炎症反应等功能.实验结果提示,瘦素可能通过下丘脑-垂体轴参与免疫调节、雌性生殖及炎症反应等,从而对子宫发挥调节作用.本研究将为加深对瘦素调节子宫作用的认识,并为深入研究女性生殖系统疾病提供新的启示.     

正常与脑缺血大鼠的脑皮质蛋白质差异分析鉴定

Wistar大鼠随机分为正常组和模型组,采用改进的线栓法制备模型,在规定的时间点快速断头取脑,分离脑皮质组织,提取蛋白质后双向电泳展示,以ImageMaster 2D Elite v301软件对2_DE图谱进行差异表达分析,目标蛋白点用基质辅助激光解析电离质谱测定肽质量指纹图进行鉴定。线粒体应激70蛋白前体、血小板活化因子乙酰基水解酶Ibβ亚单位、ADP核糖基化因子蛋白3、电压依赖性阴离子选择通道蛋白1、泛素C末端水解酶同工酶L1、突触结合蛋白等11个蛋白在模型6h组表达上调,谷胱甘肽S-转移酶omega 1、 谷胱甘肽S-转移酶P、Cu-Zn超氧化物歧化酶、 ATP合酶D链、G蛋白β亚单位1、微管蛋白β链15、苹果酸脱氢酶等15个蛋白在模型6h组表达上调。胆绿素还原酶B、细胞因子A4前体为模型组新出现点,腺苷酸激酶同工酶1在模型组消失,Thiore doxin peroxidase 1在模型组分为2个点。以双向电泳技术得到分辨率较好的电泳图谱,并初步鉴定脑缺血后差异表达蛋白,为深入研究缺血性脑损伤病理机制奠定了基础。     

前列腺癌细胞中雄激素调控核糖体蛋白和氨酰-tRNA合成酶蛋白棕榈酰化修饰

作者对雄激素R1881或DMSO处理的LNCaP细胞进行炔基棕榈酸代谢标记,之后利用点击化学反应形成的共价键富集棕榈酰化修饰蛋白,并对富集蛋白进行质谱定量分析,从而筛选、鉴定棕榈酰化修饰水平受雄激素诱导的蛋白。结果发现,雄激素在LNCaP细胞内促进核糖体蛋白RPL12、RPS4X和谷氨酰脯氨酰-tRNA合成酶(EPRS)棕榈酰化修饰,在细胞上清中促进甘氨酰-tRNA合成酶(GARS)棕榈酰化修饰。对RPL12、RPS4X和EPRS棕榈酰化调控机制的研究将为前列腺癌的治疗提供新的指导思路;雄激素诱导下细胞内RPL12、RPS4X和EPRS棕榈酰化修饰水平的升高可以作为相关的肿瘤标志物,细胞上清中GARS棕榈酰化修饰水平的升高对于前列腺癌的早期筛查具有重要的参考价值。     

MSX1通过与GRP78相互作用促进前列腺癌细胞PC-3增殖

同源异型框蛋白在发育中具有非常重要的作用,而同源异型框蛋白的异常表达和很多人类疾病相关,其中就包括前列腺癌的发生发展。我们通过分析TCGA数据库中前列腺癌病人样本MSX1的表达量发现,在肿瘤样本中MSX1表达量显著高于非肿瘤样本。因此我们检测正常永生化前列腺基底细胞系WPMY-1、雄激素依赖的前列腺癌细胞系LNCa P、雄激素非依赖的前列腺癌细胞系PC-3中MSX1的表达量,发现在雄激素依赖的前列腺癌细胞系LNCa P中MSX1表达量最高。为了探究MSX1在前列腺癌细胞中的作用,我们选取MSX1表达量较低且不受雄激素调控的PC-3细胞作为研究对象。研究表明过表达MSX1可以显著促进PC-3细胞的增殖。我们通过免疫沉淀技术确定了MSX1与GRP78蛋白相互作用,而GRP78蛋白可以调控AKT、ERK1/2等细胞周期相关蛋白从而调控细胞周期等细胞进程。因此推测MSX1通过和GRP78相互作用从而促进前列腺癌细胞增殖。我们的研究为阐明MSX1在前列腺癌发生发展中作用机理提供了一定的理论参考依据。     

敲低前列腺癌相关基因PC-1的表达抑制前列腺癌细胞C4-2雄激素非依赖性生长

目的:研究敲低癌基因D52家族成员PC-1的表达对前列腺癌细胞雄激素非依赖性生长的影响。方法:利用RNA干扰技术构建PC-1稳定低表达的C4-2细胞株;利用四唑盐(MTT)比色实验检测敲低PC-1基因表达对C4-2细胞雄激素非依赖生长的影响。结果:敲低PC-1表达抑制前列腺癌C4-2细胞的生长,并降低了C4-2细胞雄激素非依赖性生长的能力。结论:PC-1基因参与了前列腺癌向雄激素非依赖阶段发展和维持的过程,为进一步研究PC-1在促进前列腺癌细胞发生发展过程中的作用奠定了基础。     

Proteomic analysis of cisplatin resistance in human ovarian cancer using 2-DE method

Platinum-based chemotherapy, such as cisplatin, is the primary treatment for human ovarian cancer. However, overcoming drug resistance has become an important issue in cancer chemotherapy. In this study, we performed 2-DE and ESI-Q-TOF MS/MS analysis to identify differential proteins expression between cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780-CP) ovarian cancer cell lines. Of the 14 spots identified as differentially expressed (±over twofold, P < 0.05) between the two cell lines, ten spots (corresponding to ten unique proteins) were positively identified by ESI-Q-TOF MS/MS analysis. These proteins include capsid glycoprotein, fructose-bisphosphate aldolase C, heterogeneous nuclear ribonucleoproteins A2/B1, putative RNA-binding protein 3, Ran-specific GTPase-activating protein, ubiquitin carboxyl-terminal hydrolase isozyme L1, stathmin, ATPSH protein, chromobox protein homolog3 and phosphoglycerate kinase 1. The proteins identified in this study would be useful in revealing the mechanisms underlying cisplatin resistance and also provide some clues for further research.     

Cathepsin D is secreted from M-BE cells: its potential role as a biomarker of lung cancer

The early diagnosis of lung cancer is an effective approach to reduce the mortality caused by malignancy. To explore serum biomarkers of lung cancer at early stage, M-BE, a SV40T-transformed human bronchial epithelial cell line with the phenotypic features of early tumorigenesis at high passage, was cultured in the conditioned media to collect its secretory proteins. The proteins secreted from different passage M-BE cells were extracted and then separated by two-dimensional electrophoresis (2-DE). MALDI-TOF/TOF mass spectrometry was adopted to identify the passage-dependent 2-DE spots. Totally, 47 proteins were identified, including 23 that were up-regulated and 24 that were down-regulated. Of these proteins, cathepsin D was a typical secretory protein that exhibited the increased abundance either in culture media or in cells during passaging. Furthermore, the proteomic conclusions were validated in the clinical samples of lung cancer patients. When sandwich ELISA was used, the concentrations of cathepsin D in plasma showed significant differences between lung squamous cell carcinomas (SCC, 104 cases) and normal donors (36 cases, p 相似文献   

Proteomic investigation of metabolic shift in mammalian cell culture.

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down-regulated, and the others were up-regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down-regulated, and the precursor of the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which was up-regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, which is involved in protein turnover and degradation, was also up-regulated in the metabolically altered cells. The remaining down-regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up-regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up-regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.     

采用蛋白质组学技术筛选鼻咽癌细胞的甲基化沉默基因

为筛选鼻咽癌的甲基化沉默基因,采用二维凝胶电泳(2-DE)技术分离甲基转移酶抑制剂5-杂氮-2'-脱氧胞苷(5-aza-2-dC)处理与未处理鼻咽癌细胞5-8F的蛋白质,PDquest图像分析软件识别差异蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白质.然后采用Western blotting和RT-PCR检测差异蛋白质nm23-H1在药物处理与未处理5.8F细胞中的表达水平,采用甲基化特异性PCR(MS-PCR)检测nm23-H1基因在药物处理与未处理5-8F细胞中的甲基化水平.建立了5-aza-2-dC处理与未处理5.8F细胞蛋白质的2-DE图谱,识别了49个差异表达的蛋白质点,鉴定了33个差异表达的蛋白质,其中包括rim23.H1在内的15个蛋白质在5-aza-2-dC处理后的5-8F细胞中表达上调,而18个蛋白质表达下调.Western blotting和RT-PCR结果显示,nm23-H1在5-aza-2-dC处理5-8F细胞后表达上调,MS-PCR结果显示,在5-aza-2-dC处理5-8F细胞后nm23-H1基因甲基化水平下降,结果证实,nm23-H1基因是5-8F细胞中的甲基化沉默基因.15个5-aza.2-dC处理后表达上调的基因可能是5-8F细胞中的甲基化沉默基因,为筛选鼻咽癌甲基化失活基因提供了科学依据.     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.     

Long term anoxia in rainbow trout investigated by 2-DE and MS/MS

Twenty-four hours of N(2) induced anoxia induced global perturbations on protein expression in rainbow trout hypodermal fibroblasts cell line. Anoxia was obtained by depleting the medium of O(2) by flushing with N(2), and protein changes were studied by 2-DE coupled with MS providing quantitative measurements of a large number of proteins in one single study. The anoxic insult changed the level of 33 protein spots: 22 of these were up-regulated compared to the control situation and 11 were down-regulated. Using MS/MS sequencing 19 of the 33 protein spots that changed were identified, corresponding to a success rate of more than 50%. The identified proteins included two proteins involved in energy metabolism namely phosphoglycerate mutase and isocitrate dehydrogenase. In addition we observed the up-regulation of a cluster of proteins that contribute to cytoskeleton function. These are calpain, EB1, and Rho GDP dissociation inhibitor (GDI). The up-regulation of Rho GDI was shown to develop in a time dependent manner with no significant increase for up to 8 h of anoxia. In conclusion, this study provides a thorough investigation of the effect of anoxia in a cell line from rainbow trout.     

Proteome analysis of human androgen-independent prostate cancer cell lines: variable metastatic potentials correlated with vimentin expression

To better understand the molecular mechanisms of prostate cancer (PCA) dissemination and to develop new anti-metastasis therapies, key regulatory molecules involved in PCA metastasis were identified in two human androgen-independent PCA cell lines, highly metastatic 1E8-H and lowly metastatic 2B4-L cells. Through 2-DE and MS analyses, 12 proteins with different expression levels in the two cell lines were identified. The following proteins were found to be significantly up-regulated in 1E8-H cells compared with 2B4-L cells: gp96 precursor, calreticulin precursor, vimentin (VIM), Hsp90alpha, peroxiredoxin 2, HNRPH1, ezrin, T-complex protein 1, alpha subunit, and hypothetical protein mln2339. In contrast, heart L-lactate dehydrogenase H chain, annexin I, and protein disulfide isomerase were notably down-regulated in 1E8-H cells compared with 2B4-L cells. To our knowledge, this study is the first to demonstrate that up-regulation of VIM expression positively correlates with the invasion and metastasis of androgen-independent PCA.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

运用蛋白质组学技术研究与氨甲蝶呤耐药性相关的蛋白质

氨甲蝶呤（MTX）是一种重要的常用化疗药物,然而由于肿瘤细胞对其耐药性的增强而经常导致其疗效大大降低。为了寻找并鉴定与MTX耐药性相关的蛋白质从而为进一步闸明MTX的耐药机制提供线索,培养来源于小鼠NIH3T3的小鼠胚胎成纤维细胞系3T3R500与其耐300μmol／L MTX的细胞系MTX300,提取上述两种细胞系的总蛋白质,双向凝胶电泳分离蛋白质组成分,扫描并通过软件分析考马斯亮蓝染色的2-DE凝胶,选取表达差异最显著的点,胶内酶切后MALDI-TOF-MS进行肽指纹图谱（PMF）鉴定。图像分析显示,实验组和对照组的蛋白质组图谱之间,一些蛋白质点的表达有明显的变化。通过MALDI-TOF-MS和数据库查询,成功鉴定了耐药后表达变化最显著的蛋白质点为二氢叶酸还原酶（DHFR）,并通过Western blot验证了该结果,提示DHFR在MTX耐药机制中发挥重要作用。     

Profilin 1 obtained by proteomic analysis in all-trans retinoic acid-treated hepatocarcinoma cell lines is involved in inhibition of cell proliferation and migration

Previous studies have shown that all-trans retinoic acid (ATRA) suppresses growth of hepatocarcinoma cell in vitro. To understand the underlying mechanisms, we investigated the protein expression profiles by 2-DE in hepatocarcinoma cell line SMMC-7721 treated with ATRA. Our results reveal that six proteins were differently expressed in response to ATRA. Using MS and database searching, they were identified as profilin 1, phosphoglycerate kinase 1, RuvB-like 1, alpha-enolase, pyridoxal kinase and F-actin capping protein. We selected the up-regulated protein, profilin 1 (PFN1), for further studies. The PFN1 expression was increased in response to ATRA in a dose- and time-dependent manner. The PFN1 expression was reduced dramatically in four hepatoma cell lines compared to L02 cell line of non-tumor origin. The PFN1 expression was also examined in 4 cases of primary hepatocarcinoma tissues by Western blot and 30 cases by tissues microarray. It was found that the protein level of PFN1 was lower in hepatocarcinoma tissues compared to that in the adjacent tissues. Similar to ATRA, overexpression of PFN1 led to inhibition of cell proliferation and migration. Furthermore, RNAi-based PFN1 knockdown could rescue the inhibitory effect of ATRA on cell proliferation and migration. In conclusion, ATRA inhibited cell proliferation and migration through up-regulation of PFN1.