卵巢癌铂类耐药细胞株与敏感株间差异蛋白的筛选

目的:筛选卵巢癌铂类耐药细胞和敏感细胞间的差异蛋白质.方法:提取卵巢癌耐卡铂细胞SKOV3/CB和亲本细胞SKOV3的总蛋白,用二维液相色谱技术(ProtemeLabTM PF-2D)分离、筛选差异蛋白,用电喷雾离子化串联质谱(ESI-MS/MS)分析鉴定蛋白质.结果:共分离筛选出差异蛋白54个,其中SKOV3/CB表达上调的34个,SKOV3表达上调的20个.初步鉴定出5个SKOV3/CB表达上调的差异蛋白,即硫氧还原蛋白(thioredoxin,Trx)、Myosin regulatory light polypeptide 9(MYL9)、AX887247 NID、stanniocalcin homolog、human regulator ofG-protein signaling 5(RGS-5).这些蛋白涉及氧化还原、凋亡调控、甲基化及信号传导等多方面.结论:卵巢癌铂类耐药细胞和敏感细胞间存在蛋白表达差异,这些差异蛋白可能通过多种机制介导了卵巢癌对铂类的耐药.     

肺耐药相关蛋白在卵巢癌中的表达及其临床意义

背景与目的：有文献表明,肺耐药相关蛋白（lung resistant-related protein,LRP）表达水平与对顺铂、环磷酰胺的敏感程度呈负相关,LRP的过度表达可以预测卵巢癌患者的化疗敏感性及预后,并且对缓解期和生存期是一项独立的预后因素。本文结合药敏实验探讨肺耐药相关蛋白在铂类药物耐药中的作用,及其与卵巢癌预后的关系。方法：①采用三磷酸腺苷生物发光法（ATP-bioluminescence assay）对50例新鲜上皮性卵巢癌组织进行药物敏感实验,并对患者进行3个化疗疗程的随访;②采用免疫组化SP法测定其石蜡标本中LRP的表达情况。结果：①LRP非过度表达为24％,过度表达占76％;②LRP在卵巢癌中为非过度表达、百铂敏感的为9你（75％）,耐药的为3例（25％）,但无显著差异;③LRP在期别晚、组织分化差的卵巢癌组织标本中过度表达明显增多,且有统计学意义。结论：LRP的表达水平与肿瘤的分期、分化程度及化疗敏感性有一定关系,有可能成为筛选化疗药物、判断病人预后的指标。     

卵巢上皮癌组织中hMSH2的表达及其与化疗耐药的关系

目的 检测不同化疗敏感程度的卵巢上皮癌组织中hMSH2基因的表达,分析其与化疗耐药及预后的关系。方法 80例新鲜卵巢上皮癌组织均由手术中取得,采用ATP-TCA技术检测卵巢癌组织对八种药物：紫杉醇、卡铂、拓泊替康、多西他赛、吉西他滨、环磷酰胺体内代谢产物、依托泊苷及博来霉素的体外敏感性。采用Real-time PCR及Western blot法分别检测80例卵巢上皮癌组织中hMSH2 基因mRNA和蛋白水平的表达情况。结果 对紫杉醇耐药的卵巢上皮癌组中,hMSH2基因在mRNA及蛋白水平的表达均明显高于敏感组（P<0.01）,在对卡铂、拓泊替康、多西他赛耐药组中hMSH2表达水平也明显高于敏感组（P<0.05）;hMSH2基因的相对表达值与卡铂、拓泊替康、多西他赛的敏感度系数有显著相关性（P≤0.05）。早期卵巢癌患者组织的hMSH2表达水平明显低于晚期卵巢癌组织（P<0.05）。高、中分化卵巢癌组织hMSH2蛋白表达水平明显低于低分化卵巢癌组织（P=0.000）。原发卵巢癌患者组织的hMSH2表达水平明显低于复发卵巢癌患者组织（P<0.01）。结论 hMSH2基因的高表达与卵巢癌化疗耐药及预后相关,化疗可能诱导hMSH2基因的高表达;下调hMSH2基因的表达或许是逆转肿瘤耐药的新途径。     

利用基因芯片技术筛选人卵巢癌耐药差异表达基因

摘 要：［目的］ 比较卵巢上皮癌铂类耐药和非铂类耐药细胞间的基因表达差异,并进行相关基因的筛选和鉴定。［方法］ 采用人类全基因组表达谱芯片对卵巢癌铂类耐药细胞SKOV3/CB和亲本细胞SKOV3进行差异基因的筛选和分析;用半定量逆转录聚合酶联反应(RT-PCR)对部分芯片结果进行核对验证。［结果］ 共分离筛选出差异基因3506个,SKOV3/CB比SKOV3上调的差异基因共有1712个,其中上调10倍以上的共有163个,下调的差异基因共有1794个,其中下调10倍以上的共有70个。SKOV3/CB比SKOV3上调10倍以上的3个差异基因,即ZNF198、ERCC5和BIRC3经半定量RT-PCR验证,表明上述3个基因在耐药细胞株中的表达的确高于敏感细胞株。［结论］ 卵巢癌的多药耐药是一个多种基因参与的复杂过程,ZNF198、ERCC5和BIRC3参与卵巢癌铂类耐药。     

基于iTRAQ蛋白质组学技术对前列腺癌血清标志物的筛查

目的:应用同位素标记相对和绝对定量（iTRAQ）蛋白质组学技术筛选前列腺癌血清中的差异表达蛋白质,提供新的候选标志物。方法:收集4组患者的外周血清标本:良性前列腺增生（BPH）（n=10）、高级别前列腺上皮内瘤变（HGPIN）（n=10）、局限性前列腺癌（localized PCa）（n=10）以及伴有远处转移的前列腺癌（metastatic PCa）（n=10）。每组10例患者的血清样本进行等体积混合后,应用iTRAQ技术联合液相串联质谱分析（LC-ESI-MS/MS）对蛋白质进行鉴定和相对定量。结果:在患者外周血清中共鉴定到蛋白质825个。相对于良性前列腺增生,在前列腺癌组中表达差异在1.2倍以上的蛋白质13个,其中9个表达上调,4个表达下调。结论:基于iTRAQ技术的蛋白质组学方法有助于鉴定出前列腺癌相关的差异蛋白质,为进一步探索前列腺癌肿瘤标志物提供了新的思路和线索。     

应用iTRAQ技术筛选预测食管鳞癌放化疗疗效优势蛋白的研究

目的:利用同位素标记相对和绝对定量（iTRAQ）技术筛选预测食管鳞癌放化疗疗效的血清差异蛋白，以指导临床个体化治疗。方法:纳入52例诊断明确的局部晚期和晚期食管鳞癌患者，行规范放化疗，判断疗效及长期随访，同时收集患者治疗前、后血清。应用iTRAQ技术筛选放化疗疗效相关的血清差异蛋白。应用Uniprot、KEGG等数据库对差异蛋白进行生物学分析，筛选出与肿瘤相关、具有核心作用且介导肿瘤相关通路的优势蛋白。结果:疗效评价结果:放化疗敏感组（CR+PR）食管癌患者占59.62%（31/52例）；放化疗抗拒组（SD+PD）占40.38%（21/52例）。放化疗敏感组对比放化疗抗拒组:共筛选出17个有显著差异的差异蛋白，其中上调蛋白为脂联素（ADIPOQ）、IgL C7、血清转铁蛋白（TF）、中性粒细胞防御素（DEFA1）共4个，下调蛋白为聚合物免疫球蛋白受体（PIGR）、单核细胞分化抗原14（CD14）等13个。KEGG通路分析显示，TF、CD14可能分别参与HIF-1、MAPK信号通路的调节。结论:食管鳞癌患者外周血中能够筛选出预测放化疗疗效的潜在优势蛋白，并参与一些关键通路；TF、ADIPOQ、PIGR、CD14对于食管鳞癌放化疗疗效具有潜在的预测价值。     

铂类耐药相关基因在卵巢癌顺铂耐药形成过程中表达量的动态变化

目的了解卵巢癌患者在铂类治疗过程中产生获得性耐药相关基因表达动态变化的情况,为临床预后的预测及个体化治疗奠定理论基础。方法采用Benjamini—Hochberg（BH）法对源于TCGA数据库（The Cancer Genome Atlas）的256例敏感和耐药患者的卵巢癌全基因表达谱数据进行差异表达基因筛选。采用DAVID软件中的KEGG模块对筛选出的差异基因进行通路富集,筛选出P〈0．05的通路,对筛选出的通路所包含的基因采用成组t检验找出差异显著的基因（P〈0．05）。对筛选出的基因采用COREMINE工具进行文本挖掘,找出与多药耐药及肿瘤耐药存在线性相关的基因,将其与患者预后进行分析,确定存在差异显著的基因。采用实时荧光定量PCR法检测所筛选出的基因在裸鼠诱导耐药模型不同给药阶段的表达变化情况。结果BH法共筛选出306个差异表达基因,其中上调基因110个,下调基因196个。DAVID软件的KEGG模块分别筛选出5条上调及4条下调通路,成组t检验筛选出有差异的基因共37个,其中上调基因18个,下调基因19个。COREMINE工具检索出与多药耐药及肿瘤耐药存在线性相关的上调基因为ITPA、IMPDH2、RPS7、PDE5A和PDE4D,下调基因为GNAS、CVFR、BUB3、PRKDC、RBL2、SMC1A、CALR、CCNE2及CHEK1。生存分析结果提示CALR及PRKDC基因的表达与患者生存时间有关,CALR和PRKDC基因高表达组的患者生存时间长于低表达组。qRT-PCR检测发现,CALR和PRKDC基因随顺铂注射次数增多,在SKOV3-GFP及SKOV3／DDPii肿瘤组织中的表达量逐渐降低。结论CALR与PRKDC基因可能与卵巢癌铂类耐药及患者的预后密切相关,且随着耐药的产生,CALR和PRKDC基因的表达量逐渐降低。     

A2M介导Fas信号传导途径提高卵巢癌对铂类化疗的敏感性研究

目的 探讨A2M基因的表达与卵巢癌患者耐药及预后的关系及其对Fas信号传导通路的影响。方法 利用癌症基因图集（The Cancer  Genome Atlas,TCGA）数据库中铂类敏感和耐药的卵巢癌患者的基因表达谱,分析A2M基因在铂类敏感和耐药患者中的表达差异。利用已构建的带有绿色荧光标记的卵巢癌SKOV3敏感细胞（SKOV3-GFP）和顺铂耐药细胞（SKOV3-GFP/DDPⅡ）进行裸鼠皮下种植,成瘤后分次予以顺铂体内干预,以实时荧光定量PCR（real-time quantitative PCR,qRT-PCR）技术检测不同次数顺铂干预后的肿瘤组织中A2M mRNA与Fas信号传导通路上重要节点基因的表达水平。用双变量线性回归分析A2M mRNA与所检测基因的表达量的相关性。结果 相对于铂类敏感病例,A2M mRNA在铂类耐药患者中的表达下降（P<0.05）,A2M mRNA的表达与耐药相关（P=0.02）,与卵巢癌患者总生存期、化疗间隔生存期和无疾病进展生存期高度相关（P均<0.001）,其表达量越高,患者生存期越长。A2M mRNA以及细胞凋亡相关的Fas信号传导通路上重要节点基因Fas、FADD、caspase10、caspase9 和caspase3随顺铂注射次数的增加,在耐药的移植瘤组织中相对低表达（P<0.001）;而其下游与DNA修复相关的基因PARP1随顺铂注射次数的增加,在耐药的移植瘤组织中相对高表达（P<0.05）。A2M mRNA的表达与Fas信号传导通路上节点基因的表达存在线性相关（P<0.05）。结论 A2M可能是卵巢癌顺铂耐药的潜在信号分子,它可能维持肿瘤细胞对铂类药物的敏感性。其在耐药细胞中的低表达可能通过某种机制抑制下游的Fas信号传导通路,使其传导失调,最终引起PARP1的表达上调,促进DNA修复,抑制细胞凋亡,诱发卵巢癌细胞对顺铂的耐药。     

应用MALDI-TOF-MS筛选紫杉醇耐药乳腺癌组织蛋白标志物

目的:应用基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）系统分析紫杉醇耐药MMTV-PyMT乳腺癌小鼠组织蛋白差异谱,筛选具有潜在诊断意义的组织蛋白标志物。方法:取8只雌性MMTV-PyMT转基因小鼠,10周龄时给予紫杉醇（10 mg/kg）治疗,每周给药2次,给药4周后处死。测量肿瘤体积,分别取肿瘤体积较小和较大的4只小鼠作为紫杉醇药物敏感组和非敏感组（耐药组）。采用免疫组织化学法（IHC）检测两组肿瘤组织增殖相关因子表达情况,细胞凋亡检测法（TUNEL）检测两组肿瘤中细胞凋亡情况,苏木精-伊红（HE）染色法观察两组肿瘤组织细胞损伤程度,蛋白质印迹法（Western blot）检测两组肿瘤组织凋亡相关蛋白的表达。采用MALDI-TOF-MS系统对两组组织中的蛋白或多肽表达谱进行检测,运用ClinProTools分析软件进行分析,筛选出12个差异分子峰,建立诊断预测模型。结果:相对于紫杉醇敏感乳腺癌组织,紫杉醇耐药组乳腺癌组织细胞增殖数目明显增加,细胞凋亡明显减少。耐药组凋亡蛋白BAX和Cleaved Caspase-3表达明显减少,肿瘤细胞无明显损伤。紫杉醇敏感与耐药乳腺癌组织,共筛选出12个差异明显的分子峰值,其中耐药组有9个上调,有3个下调,筛选出5个差异最大的分子峰建立紫杉醇耐药预测模型。结论:应用MALDI-TOF-MS系统可检测乳腺癌紫杉醇敏感组与耐药组肿瘤组织蛋白差异,筛选的差异组织蛋白可能成为诊断紫杉醇耐药乳腺癌的分子标志物。     

卡铂耐药蛋白S100A4在上皮性卵巢癌中的表达及与化疗耐药的关系

目的:检测S100A4蛋白在上皮性卵巢癌中的表达及其与化疗耐药的关系。方法:选择81例卵巢上皮性癌组织,分为耐药组40例,敏感组41例,应用免疫组织化学法检测S100A4在卵巢上皮性癌组织中的表达情况;MTT法测定卡铂对细胞的半数抑制浓度（IC50）,采用Western Blot检测卡铂化疗敏感的卵巢癌细胞株SKOV3及卡铂化疗耐药的卵巢癌细胞株SKOV3/CB中S100A4的表达情况。结果:S100A4分别在卵巢癌组织和卵巢癌细胞耐药组中的表达高于敏感组,与卵巢癌的化疗耐药有关（P<0.05）。S100A4在胞浆/胞核或胞核表达耐药组中高于敏感组,有统计学意义。卡铂对SKOV3组和SKOV3/CB组的IC50分别为28.83μmol/L和69.11μmol/L,因此SKOV3/CB对卡铂的耐药指数(RI)为2.4。结论:S100A4的表达与上皮性卵巢癌的化疗耐药密切相关,为卵巢癌患者个体化治疗提供依据。     

上皮细胞黏附分子表达与乳腺癌淋巴结转移的相关性

目的 探讨上皮细胞黏附分子(Ep-CAM)在乳腺癌中的表达及意义.方法 收集经病理学确诊的乳腺癌新鲜组织标本23例及配对的淋巴结转移灶.应用同位素标记的相对和绝对定量(iTRAQ)蛋白质组学技术筛选及鉴定乳腺癌原发灶和淋巴结转移灶的差异表达蛋白,在4例新鲜乳腺癌原发灶组织和配对淋巴结转移灶中进行Western blotting检测Ep-CAM的表达.采用免疫组织化学法检测252例乳腺病变标本中Ep-CAM的表达.结果 定量蛋白质组学检测结果显示乳腺癌原发灶和淋巴结转移灶存在差异表达蛋白,其中Ep-CAM在转移灶的表达高于原发灶,Western blotting显示Ep-CAM在转移灶的表达(1.46±0.22)高于原发灶(1.16±0.09),变化趋势与蛋白质组学结果一致.免疫组织化学检测结果显示,Ep-CAM在淋巴结转移灶中的阳性表达率(93.16％,109/117)显著高于无淋巴结转移的乳腺癌原发灶(72.73％,64/88),差异有统计学意义(x2=15.921,P=0.000);有淋巴结转移的乳腺癌原发灶Ep-CAM阳性表达率为72.65％(85/117),较配对淋巴结转移灶阳性表达率低,差异有统计学意义(P=0.001).结论 Ep-CAM在乳腺癌原发灶和淋巴结转移灶呈差异表达,该蛋白可能与乳腺癌淋巴结转移有关.     

基于二氧化硅、百草枯、博莱霉素建立3种大鼠肺纤维模型的差异蛋白质组学分析

目的 以二氧化硅、百草枯、博莱霉素诱导的大鼠肺纤维化(PF)模型为研究对象,以期在3种大鼠PF模型中探究存在差异表达的参与PF发生、发展相关的关键蛋白.方法 应用同位素标记相对与绝对定量技术(iTRAQ)结合液相色谱-串联质谱(LC-MS/MS)分析3种大鼠PF模型肺组织蛋白表达谱;采用Proteome Discove...     

基于iTRAQ标记结合2D nano HPLC-ESI-OrbiTrap MS/MS技术筛选卵巢癌血清标记物

  目的  利用相对和绝对同位素标记（iTRAQ）和纳升级两维高效液相色谱-电喷雾-OrbiTrap质谱（2D nano HPLC-ESI-OrbiTrap MS/MS）蛋白组学方法探寻卵巢癌血清标记物,以提高早期卵巢癌诊断率。  方法  收集卵巢癌患者血清20例（卵巢癌组）,CA125异常的卵巢良性囊肿16例（良性囊肿组）,健康者20例（正常对照组）,每组取10例组内等量混合后去除高丰度蛋白,iTRAQ试剂标记,进行强离子交换柱分离多肽、nanoLC分离并在线连接电喷雾串联OrbiTrap质谱分析,筛选出重要的差异蛋白。随后采用Western blot方法,将筛选出的重要差异蛋白进行56例临床血清样本逐例验证。  结果  共鉴定出差异蛋白326个。其中重点筛选出了8个重要的差异蛋白:蛋白PARD3、SRP1-alpha、LAMA4、LRP16、IgSF2、NRAMP1、NF-E2-related factor 1和APOA4;验证了3个重要差异蛋白:PARD3、NRAMP1、APOA4。  结论  应用iTRAQ标记的定量蛋白质组学技术筛选出了8个重要的差异蛋白,可能是潜在的肿瘤标记物。其中APOA4蛋白有可能成为区分恶性与良性卵巢疾病的生物标志物。      

Whole genome expression profiling of advance stage papillary serous ovarian cancer reveals activated pathways

Ovarian cancer is the most lethal type of gynecologic cancer in the Western world. The high case fatality rate is due in part because most ovarian cancer patients present with advanced stage disease which is essentially incurable. In order to obtain a whole genome assessment of aberrant gene expression in advanced ovarian cancer, we used oligonucleotide microarrays comprising over 40,000 features to profile 37 advanced stage papillary serous primary carcinomas. We identified 1191 genes that were significantly (P < 0.001) differentially regulated between the ovarian cancer specimens and normal ovarian surface epithelium. The microarray data were validated using real time RT-PCR on 14 randomly selected differentially regulated genes. The list of differentially expressed genes includes ones that are involved in cell growth, differentiation, adhesion, apoptosis and migration. In addition, numerous genes whose function remains to be elucidated were also identified. The microarray data were imported into PathwayAssist software to identify signaling pathways involved in ovarian cancer tumorigenesis. Based on our expression results, a signaling pathway associated with tumor cell migration, spread and invasion was identified as being activated in advanced ovarian cancer. The data generated in this study represent a comprehensive list of genes aberrantly expressed in serous papillary ovarian adenocarcinoma and may be useful for the identification of potentially new and novel markers and therapeutic targets for ovarian cancer.     

Proteomic Analysis of Serum of Women with Elevated Ca-125 to Differentiate Malignant from Benign Ovarian Tumors

Clinically, elevated cancer antigen 125 (CA-125) in blood predicts tumor burden in a woman’s body, especiallyin the ovary, but cannot differentiate between malignant or benign. We here used intensive modern proteomicapproaches to identify predictive proteins in the serum of women with elevated CA-125 to differentiate malignantfrom benign ovarian tumors. We identified differentially expressed proteins in serum samples of ovarian cancer(OC) patients, benign ovarian tumor (BT) patients, and healthy control women using mass spectrometry-basedquantitative proteomics. Both the OC and BT patients had elevated CA-125. Quantitation was achieved usingisobaric tags for relative and absolute quantitation. We obtained 124 quantified differential serum proteins in OCcompared with BT. Two proteins, apolipoprotein A-4 (APOA4) and natural resistance-associated macrophage1, were verified using Western blotting. Proteome profiling applied to OC cases identified several differentialserum proteins in the serum of women with elevated CA-125. A novel protein, APOA4, has the potential to bea marker for malignant tumor differentiation in the serum of women with elevated CA-125.     

Comparative proteome analysis of human adenocarcinoma

This study was designed to use comparative proteomics technology to find the differentially expressed proteins between human lung adenocarcinoma and paired normal tumor-adjacent lung tissues. The total proteins of 20 human lung adenocarcinoma tissues and paired normal tumor-adjacent lung tissues were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE) and Coomassie Blue staining. The differentially expressed proteins were analyzed with image analysis software and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and quadrupole time of flight mass spectrometry (Q-TOF-MS/MS). (1) Well-resolved, highly reproducible 2-DE patterns of human lung adenocarcinoma and paired normal tumor adjacent lung tissues were obtained. (2) PDQUEST 2D image analysis software was used to analysis 20 cases of lung adenocarcinoma and paired normal lung tissues, 1006 ± 54 spots were matched among tumor tissues and normal lung tissues. Twenty-eight differentially expressed spots were screened. (3) Twenty non-redundant differentially expressed proteins were identified by mass spectrometry, thirteen proteins were up-regulated, and seven proteins were down-regulated, some proteins were involved in the regulation of cell signal transduction or metabolized enzyme of cell. (4) To validate the results screened by proteome research, immunohistochemistry was used to validate several lung adenocarcinoma differentially expressed proteins including 14-3-3 sigma, annexin 1, and manganese superoxide dismutase. The results showed that these three proteins were really differentially expressed between lung adenocarcinoma and paired normal lung tissues. These results will provide scientific foundation and new clue for the research of human lung adenocarcinoma.     

Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancer

Individual classes of human endogenous retrovirus (HERV) genes and proteins are expressed in cancer, but expression of more than one type of HERV is rare. We report here the expression of multiple HERV genes and proteins in ovarian cell lines and tissues. Expression of HERV-K env mRNA was greater in ovarian epithelial tumors than in normal ovarian tissues (N = 254). The expression of this protein on the surface and in the cytoplasm of ovarian cancer cells was confirmed using anti-HERV-K specific antibody by flow cytometric analysis. The frequency of expression of HERV-K env protein in multitissue microarrays (N = 641) was determined by immunohistochemistry and a significant correlation with tumor histotype was found. A significantly increased expression of HERV-K was observed in tumors with low malignant potential and low grade, relative to expression in normal ovarian tissues. The increase in expression of HERV-K env protein took place in a stepwise fashion in serous papillary adenocarcinoma. Interestingly, we found that other classes of HERV env mRNAs, including ERV3 and HERV-E, are expressed in the same ovarian cancer tissues that expressed HERV-K. Furthermore, anti-HERV antibodies including anti-ERV3 (30%), anti-HERV-E (40%) and anti-HERV-K (55%) were detected in patients with ovarian cancer, but not in normal female controls. HERV env proteins are frequently transcribed and translated in ovarian epithelial tumors, and multiple HERV families are detectable in ovarian cancer. HERV env proteins, and especially those expressed on the cell surface, may serve as novel tumor targets for detection, diagnosis and immunotherapy of ovarian cancer.     

WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma

BACKGROUND: Ovarian clear cell adenocarcinoma is associated with one of the poorest prognoses among human epithelial ovarian cancers. The authors hypothesized that Wilms tumor suppressor 1 gene (WT1) sense and antisense (WT1-AS) expression and their promoter methylation status could characterize ovarian clear cell adenocarcinoma from ovarian serous adenocarcinoma. METHODS: To test this hypothesis, ovarian cancer cell lines and 42 cancer tissues (17 clear cell and 25 serous adenocarcinoma) were analyzed for expression and methylation of WT1 and WT1-AS genes. RESULTS: These experiments demonstrated that all serous adenocarcinoma tissues expressed both WT1 and WT1-AS genes, although expression of these genes was lacking in clear cell adenocarcinoma. The WT1 and WT1-AS promoter were significantly methylated in clear cell adenocarcinoma (88.2% and 88.2%, respectively) compared with serous adenocarcinoma (24.0% and 20.0%, respectively). Significant correlation between methylation and mRNA expression status was observed for each gene. Also in agreement with these data, WT1 and WT1-AS negative ovarian cancer cell lines reexpressed these genes after treatment with the demethylating agent, 5-aza-2'-deoxycytidine. CONCLUSIONS: The current study shows that CpG hypermethylation is an important mechanism of WT1 and WT1-AS gene inactivation in ovarian clear cell adenocarcinoma. This is the first report that has demonstrated differential expression and methylation of WT1-AS in ovarian clear cell and serous adenocarcinomas. This study presents new molecular characterizations between these two types of adenocarcinoma and may provide insight as to why clear cell adenocarcinoma has a poorer prognosis than serous adenocarcinoma of the ovary.     

应用SELDI-TOF-MS技术筛选卵巢癌淋巴结定向高转移特性细胞株差异表达蛋白

背景与目的:人卵巢浆液性乳头状腺癌细胞株SKOV3及其在裸鼠体内建立的淋巴结定向高转移亚克降第二代SKOV3-pm2、第三代SKOV3-pm3细胞株,为卵巢癌转移分子机制的研究提供了细胞模型.本研究应用飞行时间质谱技术结合蛋白芯片分析筛选卵巢癌淋巴结定向高转移与非定向转移细胞株之间的蛋白质表达差异.方法:采用动物实验测定SKOV3、SKOV3-pm2和SKOV3-pm3三种细胞株的淋巴结转移率.应用表面增强激光解吸离子化-飞行时间质谱(surface-enhanced laser desorption and ionization-time of flight-mass spectrometry,SELDI-TOF-MS)技术检测各细胞株提取的胞浆总蛋白和分泌蛋白,每种细胞分别采用CM-10型弱阳离子交换芯片和IMAC-3型固定金属亲和芯片检测.应用Ciphergen Protein Software 3.2.0软件和Biomarker Wizard软件对采集3组细胞的蛋白峰进行比较.峰强度相差0.5倍以上者定义为差异蛋白.结果:动物实验显示.细胞株SKOV3及高转移亚克隆SKOV3-pm2、SKOV3-pm3的淋巴结转移率分别为20%、90%和100%,前者与后二者之间的差异有统计学意义(P<0.05).获得CM-10和IMAC3两种蛋白芯片上SKOV3、SKOV3-pm2、SKOV3-pm3细胞株蛋白质谱图谱,对比发现:质荷比(m/z)为6971、7475、9089、9453、10 103、11 655的胞浆蛋白及质荷比(m/z)为4746的分泌蛋白在上述三种细胞株中存在不同程度差异表达.结论:应用SELDI-TOF-MS技术结合固定金属亲和芯片和弱阳离子交换芯片,可有效筛选卵巢癌淋巴结定向高转移特性细胞株中的特异性表达蛋白;寻找到的差异蛋白与淋巴结定向高转移潜能密切相关.     

iTRAQ技术在消化道肿瘤研究中的应用进展

近年来,同位素标记相对和绝对定量(Isobaric tags for relative and absolute quantification,iTRAQ)技术已成为蛋白质组学定量研究中一种强有力的工具,尤其在肿瘤相关领域备受关注,其突出优势为测定蛋白范围广泛、分析结果可靠、精确度高、重复性好等。大量文献也表明,iTRAQ技术在消化道肿瘤,如食管癌、胃癌、肝癌等肿瘤标志物筛查研究、肿瘤发生发展机制研究以及肿瘤治疗预后探索等方面得到广泛应用。在大规模定量生物学时代,iTRAQ技术在分子水平上深入了解消化道肿瘤的相关机制研究中越来越不可或缺。因此本文对iTRAQ技术在消化道肿瘤中的研究进展进行简要综述。