Analysis of cyclooxygenase-2 expression in human breast cancer: high throughput tissue microarray analysis

Purpose The objective of this study was to evaluate breast carcinomas for the expression of cyclooxygenase-2 (Cox-2) using a tissue microarray (TMA) and to determine its clinical and prognostic relevance.Methods We analyzed Cox-2 expression in 600 samples from 200 breast carcinomas immunohistochemically performing TMA technology and semiquantitative analysis. Results were correlated with various clinicopathological variables and follow-up data. Expression of estrogen receptor, progesterone receptor, Ki-67, and Her-2/neu-oncogene was analyzed and correlated with Cox-2 status.Results We observed a moderate or strong cytoplasmic staining for Cox-2 in 78 (40.6%) of breast carcinomas. Increased Cox-2 expression corresponded to higher pT stage (P=0.038), amplification of Her-2/neu (P=0.032), lymphovascular invasion (P=0.006), a high MIB-1 labeling index (LI) (P<0.001), and histological grading (P=0.013). We also observed an inverse relationship between strong Cox-2 expression and estrogen and progesterone receptor content of tumors (P=0.037 and P=0.010). However, we could not demonstrate a significant association between Cox-2 staining and overall survival or disease free survival time.Conclusions These results suggest that Cox-2 expression is significantly associated with less differentiated and more aggressive breast carcinomas and might therefore be a useful prognostic indicator as well as a target for therapy.     

Identification of Annexin A1 protein expression in human gastric adenocarcinoma using proteomics and tissue microarray

AIM:To study the differential expression of Annexin A1(ANXA1)protein in human gastric adenocarcinoma.This study was also designed to analyze the relationship between ANXA1 expression and the clinicopathological parameters of gastric carcinoma.METHODS:Purified gastric adenocarcinoma cells(GAC)and normal gastric epithelial cells(NGEC)were obtained from 15 patients with gastric cancer by laser capture microdissection.All of the peptide specimens were labeled as18O/16O after trypsin digestion.Differential protein expressions were quantitatively identified between GAC and NGEC by nanoliter-reverse-phase liquid chromatography-mass/mass spectrometry(nanoRPLC-MS/MS).The expressions of ANXA1 in GAC and NGEC were verified by western blot analysis.The tissue microarray containing the expressed ANXA1 in 75 pairs of gastric carcinoma and paracarcinoma specimens was detected by immunohistochemistry(IHC).The relationship between ANXA1 expression and clinicopathological parametes of gastric carcinoma was analyzed.RESULTS:A total of 78 differential proteins were identified.Western blotting revealed that ANXA1 expression was significantly upregulated in GAC(2.17/1,P<0.01).IHC results showed the correlations between ANXA1protein expression and the clinicopathological parameters,including invasive depth(T stage),lymph node metastasis(N stage),distant metastasis(M stage)and tumour-lymph node metastasis stage(P<0.01).However,the correlations between ANXA1 protein expression and the remaining clinicopathological parameters,including sex,age,histological differentiation and the size of tumour were not found(P>0.05).CONCLUSION:The upregulated ANXA1 expression may be associated with carcinogenesis,progression,invasion and metastasis of GAC.This protein could be considered as a biomarker of clinical prognostic prediction and targeted therapy of GAC.     

COX-2 expression in gastric cancer and its relationship with angiogenesis using tissue microarray

AIM： To explore the expression and clinicopathological significance of cyclooxygenase-2 （COX-2） and microvessel density （MVD） in gastric carcinogenesis, and to investigate their roles in the invasion and the relationship between biological behaviors and prognosis of gastric cancer.
METHODS： Using Envision immunohistochemistry, COX-2 and CD34 expressions in gastric cancer tissue array were examined. MVD was counted and the relationship between the biological behaviors and prognosis was analyzed.
RESULTS： The expression of COX-2 in gastric cancer tissue was significantly higher than that in normal mucosa （χ^2 = 12.191, P 〈 0.05）. The over-expression of COX-2 in gastric cancer was obviously related to metastasis and depth of invasion （χ^2 = 6.315, P 〈 0.05）, but not related to the histological type and Borrmann type （χ^2 = 5.391 and χ^2= 2.228, respectively）. Moreover, MVD in gastric cancer tissues was significantly higher than that in the normal mucosa （65.49 ± 20.64 vs 36.21 ± 18.47, t/F = 7.53, P 〈 0. 05）. MVD was related to the histologic type and metastasis （t/F= 3.68 and t/F = 4.214, respectively, P 〈 0. 05）, but not related to the depth of invasion and Borrmann type （t/F = 0.583 and t/F = 0.459, respectively）. MVD in COX-2-positive tissues was markedly higher compared to COX-2-negative tissues, indicating a positive correlation between COX-2 expression and MVD （t = 13.12, P 〈 0. 05）.
CONCLUSION： Tissue microarray （TMA） is a powerful tool for rapid identification of the molecular alterations in gastric cancer. COX-2 expression, via inducingangiogenesis, may play an important role in gastric carcinogenesis. It could be served as a determinant factor for clinical prognosis and curative effect.     

组织和血清癌胚抗原含量比值对大肠癌的诊断意义

应用放射免疫法测定活检组织及血清的组织癌胚抗原（CEA）含量，评价CEA含量对大肠癌的诊断价值。16例大肠癌患者同时测定病变组织（1组）、癌旁组织（2组）及其血清（4组）的CEA含量，并设31例非大肠癌患者大肠组织（3组）及其血清（5组）的CEA含量为对照。结果显示1组和其他四组比较有显著性差异（P〈0．01）。组织CEA含量／自身血清CEA含量〉8为阳性，则1组、2组、3组的阳性率分别为75．00％、0、12．90％；1组与另两组间比较均有显著性差异（P〈0．01）。另外大肠癌的血清CEA异常升高的比率为5．88％。本文结果提示对大肠癌患者测定活检组织中CEA含量明显优于血清的CEA测定。组织与血清CEA含量比值的明显增高对大肠癌的诊断呵提供佐证。     

利用组织芯片技术研究COX-2在胃癌中的表达及其与血管生成的关系

目的:研究COX-2在胃癌中的表达及其与血管生成的关系,探讨其在胃癌转移中的作用及与胃癌病理生物学行为的关系．方法:采EnVision方法检测胃癌组织芯片中 COX-2的表达,用CD34进行微血管内皮细胞染色,计算微血管密度(MVD),分析其相关性．结果:COX-2在胃癌中的表达明显高于正常胃黏膜(P=0．001)．COX-2的高表达与胃癌的转移(P=0．019)和胃壁浸润深度(0．031)呈正相关,与胃癌的组织病理分型无关(P=0．495), 与Borrmann分型无关(P=0．109)组织MVD (65．49±20．64)明显高于正常胃黏膜组织 (36．21±18．47,P=0．001)．MVD值与胃癌的组织病理分型(P=0．003)和转移有关(P=0．043), 与胃癌胃壁浸润深度(P=0．627)和Borrmann 分型(P=0．634)无明显相关性．COX-2表达阳性组的MVD指数明显高于COX-2表达阴性组 (68．59±19．8 vs 25．82±7．76．P<0．05),COX-2 表达与MVD呈正相关(P=0．001)．结论:组织芯片技术对于快速检测胃癌及其他肿瘤的分子病理学改变是一个强有力的工具．COX-2表达可能通过促进血管形成对胃癌的发生、发展起重要作用,其可作为判断预后和指导治疗的有效指标．     

利用组织芯片技术研究P73和mP53基因编码蛋白在胃癌中的表达及意义

目的:构建胃癌及其癌前病变组织芯片,采用免疫组化方法观察研究P73和mP53基因编码蛋白在胃癌中的表达并探讨其临床病理学意义.方法:收集2003-2004年辽宁省肿瘤医院和中国医大附属一院104例胃癌及癌前病变组织标本构建两个组织芯片蜡块,组织样品直径为1 mm.采用SABC免疫组化方法检测胃癌组织中P73和mP53蛋白的表达,观察分析其与胃癌病理生物学行为的关系.结果:P73基因编码蛋白在胃癌、肠上皮化生、不典型增生病变组织中的阳性表达率显著高于远癌正常胃黏膜(90.1%,44.0%,80.0%vs 17.9%,P＜0.01).Borrman Ⅲ/Ⅳ型胃癌P73蛋白阳性表达率(92.9%/100%)显著高于BorrmanⅡ型胃癌(57.1%)(P＜0.05).伴转移胃癌组P73蛋白阳性表达率(淋巴结转移组94.4%,肝转移组100%,卵巢转移100%)显著高于无转移组(76.2%)(P＜0.05).胃癌组织中P73蛋白表达与mP53蛋白表达密切相关(χ2=9.6736,P＜0.01).结论:P73蛋白表达与胃癌恶性病理生物学行为密切相关,其虽与抑癌基因P53同源,但与mP53蛋白表达呈正相关,提示其可能作为P53的一种模拟突变体在胃癌发生、发展中起作用.     

基因芯片在肺癌发生相关基因筛查中的应用

目的 研究人肺癌发生相关基因表达谱,探讨肺癌发生的分子机制。方法 选取1280个与肿瘤相关的基因克隆。制备成肿瘤相关cDNA芯片。提取人肺癌组织以及相应正常组织RNA,反转录后标记为cDNA探针,与cDNA芯片杂交,经扫描及Quantarray3．0软件分析后比较两种组织中的差异表达基因。结果 共筛查出差异表达基因52条。其中表达上调基因35条,下调基因17条;按照基因功能可分为运输载体,代谢相关基因、细胞信号转导分子、细胞骨架、转录调控因子以及未知功能基因。结论 基因芯片可用于肺癌相关基因表达谱的筛查,为明确肺癌发生机制提供重要参考。     

Substantially reduced expression of PIAS1 is associated with colon cancer development

Purpose  Protein inhibitors of activated STATs (PIAS) regulate the interferon-gamma (IFN-γ) signaling pathway, which has numerous effects on tumor development and tumor cell biology. PIAS’s also regulate STAT family members not directly involved in IFN-γ signaling. This project was designed to assess PIAS1 expression in colon cancer. Methods  To determine whether PIAS1, one of the PIAS family members, or IFN-γ signaling pathway components could be used to stratify colon tumors, we stained tissue microarrays for PIAS1, interferon regulatory factor-1 (IRF-1) and STAT1α. Results  PIAS1 staining of the colon cancer tissue microarrays indicated a strong correlation of normal colon cells, and adenomas, with high expression of both PIAS1 and IRF-1. Conclusion  The PIAS1 results in particular may represent a basis for new approaches for efficiently distinguishing adenomas from colon cancer.     

Correlation of bFGF expression in renal cell cancer with clinical and histopathological features by tissue microarray analysis and measurement of serum levels

The prognostic value of bFGF for surgically treated renal cell cancer (RCC) patients was evaluated by immunohistochemistry (IHC) and the tissue microarray technique (TMA). Additionally, preoperative serum bFGF levels were correlated to tumour stage and the presence of metastases at initial diagnosis. Serum levels of bFGF were measured by ELISA in 39 healthy volunteers, in 37 patients with benign urologic diseases and in 74 RCC patients, 26 of whom revealed lymph node or distant metastases. bFGF expression as detected by IHC was investigated in 777 tissue cores from 259 different RCC patients [median follow-up: 138 (36–240) months]. Eighty eight patients died from tumour progression. For each patient, the TMA slides contained a tissue core from the primary tumour, its invasion front and the normal renal parenchyma. bFGF serum levels were higher in RCC patients vs healthy volunteers (P<0.01) and vs patients with benign urologic diseases (P<0.01). Metastasized patients revealed higher bFGF serum levels than organ-confined specimens (P<0.01). As detected by IHC only increased bFGF expression in the invasion front tissue correlated with the patients’ long-term survival (log rank test) (P=0.03). In multivariate analysis regional LN metastases (P<0.01), the histological grading (P<0.01), and an increased bFGF expression in the invasion front (P=0.04) independently predicted the patients’ clinical prognosis. Not the expression of bFGF in the primary tumour but in its invasion front reflects the aggressiveness of RCC, hereby indicating a different biological potential within both areas. The value of bFGF serum levels as indicators of systemic tumour dissemination remains to be determined.     

微阵列技术在结肠癌研究中的应用

结肠癌细胞在长期的演进过程中受多种基因的调控,微阵列技术以其自动微型化、高速化、高通量、高度并行性及敏感性的优势,逐渐成为基因水平研究结肠癌的重要工具。它可以随意获取结肠癌生长各期与结肠癌相关的基因表达模式,确定结肠癌发生发展中的基因突变位点;根据基因型将结肠癌分类,寻找新的药物作用靶点以及进行药物筛选,在研究结肠癌发生的分子水平机制及临床诊断治疗中都具有非常重要的应用价值。本文介绍了微阵列技术的应用原理和意义,对这项技术在结肠癌研究中的应用进展情况,及现存问题和应用前景进行了评价。     

PUMA蛋白在结肠癌组织中的表达及其临床意义

目的检测PUMA蛋白在结肠癌组织和癌旁正常组织中的表达水平,从而探讨其在结肠癌发生、发展中的作用及其临床意义。方法针对40例结肠癌组织标本及30例癌旁正常组织标本,通过免疫组化方法对其PUMA蛋白的表达进行测定。结果 PUMA蛋白在结肠癌组织中的表达阳性率为22.5%（9/40）,在癌旁正常结肠组织中表达阳性率为66.7%（20/30）,PUMA蛋白的表达与结肠癌临床病理分期、肿瘤病理分化程度、是否伴有淋巴结转移有关。结论 PUMA蛋白在结肠癌的发生、发展中起重要作用,可能成为结肠癌的分子标记物或结肠癌治疗的新分子靶点。     

应用基因芯片研究康莱特对人胰腺癌Patu-8988细胞的凋亡诱导作用

目的 探讨康莱特注射液诱导人胰腺癌细胞(Patu-8988)凋亡过程中相关基因表达的变化。方法 通过流式细胞仪Annexin V／PI双染法研究康莱特诱导Patu-8988细胞凋亡的过程，应用基因芯片技术分析加药前后凋亡相关基因的表达差异，并以Western印迹对部分基因蛋白产物表达进行验证。结果康莱特对Patu-8988细胞的凋亡诱导作用呈时间依赖性。康莱特作用24h后，在96条有关凋亡的目的基因中共有17条基因表达发生大于3倍的显著变化，其中表达上调基因12条，下调基因5条。Western印迹表明P53、Bcl-2、Bax、Caspase-3等蛋白表达改变与基因芯片结果一致，同时Caspase-3底物多聚ADP核糖多聚酶被降解。结论 康莱特可使Patu-8988细胞中多个凋亡相关基因的表达发生改变，进一步揭示了其诱导胰腺癌细胞凋亡的作用机制。     

应用芯片技术筛查人结肠癌羟基喜树碱多药耐药相关微RNA的研究

目的 探讨新一类调控分子微RNA(miRNA)在人结肠癌羟基喜树碱多药耐药中的作用,为逆转肿瘤化学治疗的多药耐药性提供新的靶点.方法 运用miRCURYTM LNA Array V8.1版芯片检测人结肠癌细胞SW1116和人结肠癌羟基喜树碱多药耐药细胞SW1116/HCPT的miRNA表达谱,筛选出具有表达差异的miRNA(2倍以上).通过PiTcar,Targetscan,MIRanda等算法在线搜寻差异表达miRNA的靶基因.利用茎环特异miRNA引物反转录特异的miRNA,并采用荧光定量PCR法对个别差异表达的miRNA进行验证.结果 用于芯片分析的总RNA的吸光度比值分别为1.93(SW1116)和1.94(SW1116/HCPT).琼脂糖变性凝胶电泳进一步验证总RNA质量符合芯片要求.通过芯片检测后发现SW1116/HCPT相比SW1116表达下调的miRNA共有28条,表达上调的有36条.根据靶基因预测结果,我们选取其中2条表达下调hsa-miR-452,hsa-miR-373*和1条表达上调hsa-miR-506进行荧光定量PCR验证,hsa-miR-452和hsa-miR-506的结果与芯片相符合,但hsa-miR-373*表达量不足以由荧光定量PCR检测出.结论 miRNA表达量的改变可能在人结肠癌羟基喜树碱多药耐药中起有关键作用.     

应用基因芯片技术筛选胰腺癌相关基因

目的应用基因芯片技术筛选胰腺癌相关基因。方法将14000种人类基因PCR产物按微矩阵排列点样于化学涂层的载玻片上，制成基因芯片。按一步法抽提4例胰腺癌和癌旁正常胰腺组织的总RNA，将等量的RNA分别逆转录合成荧光分子掺人的cDNA一链作探针，混合后杂交上述基因芯片。经严格洗片后用ScanArray 4000扫描仪扫描芯片荧光信号图像，每点上两种荧光信号的强度分别代表Cy5-dCTP和Cy3-dCTP的量，获得的荧光信号图像用计算机分析。结果按差异显著性标准，从14000个基因中筛选出在胰腺癌组织中共同差异表达基因189条，其中已知基因101条，新基因88条。在筛选出的已知基因中，有50条表达上调，51条表达下调。结论基因芯片技术的肿瘤基因表达谱分析能够高通量筛选胰腺癌相关基因。并高效对基因功能进行研究。胰腺癌基因表达谱的分析有助于认识肿瘤发病机制。     

结肠癌中骨桥蛋白与环氧化酶-2的表达及临床意义

目的研究骨桥蛋白(OPN)与环氧化酶-2(COX-2)在结肠癌组织中的表达水平,并探讨两者与结肠癌转移浸润的关系。方法应用免疫组化法检测60例结肠癌组织和16例癌旁正常组织中OPN与COX-2的表达,并用SPSS 16.0统计软件分析其表达水平与临床病理特征的关系及二者的表达相关性。结果 OPN在结肠癌组织中表达的阳性率(70.0%)高于癌旁正常结肠组织(18.8%),差异具有统计学意义(P<0.01);COX-2在结肠癌组织中表达的阳性率(75.0%)高于癌旁正常结肠组织(37.5%),差异具有统计学意义(P<0.01)。OPN和COX-2蛋白的表达与患者的性别、年龄、肿瘤大小无关,与肿瘤分期、淋巴结转移有关(P<0.05),OPN与COX-2蛋白在结肠癌组织中表达呈正相关。结论 OPN和COX-2在结肠癌的发生发展中起重要作用,联合检测可作为判断结肠癌恶性程度和预后的指标。     

组织芯片检测硒结合蛋白1在直肠癌中的表达及临床意义

目的探讨硒结合蛋白(selenium binding protein 1,SBP1)在直肠癌中的表达及其临床意义。方法采用组织芯片技术和免疫组化SP法检测65例直肠腺癌组织、5例正常直肠黏膜组织中SBP1的表达,并分析其与直肠癌临床病理参数的关系。结果 SBP1在直肠腺癌中的阳性表达率为63.08%,而在5例正常直肠黏膜组织中的4例表达为阳性。SBP1的表达与直肠癌的浸润深度和淋巴结转移相关(P0.05),与性别、年龄、分化程度和TNM分期无关(P0.05)。结论 SBP1在直肠癌组织中的低表达与浸润深度和淋巴结转移有关,可能作为一种新的判断转移和预后标志物。     

利用组织芯片技术研究PTEN和VEGF在胃癌中的表达及意义

目的研究PTEN和血管内皮生长因子（vascular endothelial growth factor，VEGF）在胃癌中的表达及临床意义。方法应用组织微阵列仪制作97孔胃癌组织芯片（tissue microarray）。用免疫组织化学S—P法检测PTEN、VEGF在72例胃癌和25例正常胃黏膜中的表达。结果胃癌组织中PTEN蛋白阳性表达率显著低于正常胃黏膜（45．8％ VS 100％，P〈0．01）；VEGF的阳性表达率显著高于正常胃黏膜（75％VSl2％，P〈0．01），PTEN在胃癌中的表达与VEGF呈负相关（P〈0．01）。PTEN、VEGF的表达在中高分化腺癌分别为68．8％、62．5％（P〉0．05），在低分化及未分化腺癌分别为27．5％、85．0％（P〈0．05）；伴淋巴结转移者分别为31．6％、86．9％（P〈0．05），无淋巴结转移者分别为61．8％、61。8％（P〉0．05）；临床病理分期Ⅰ＋Ⅱ期分别为57．1％、61．9％（P〉0．05），Ⅲ＋Ⅳ期分别为30．0％、93．3％（P〈0．05）；与性别、年龄、肿瘤大小和组织分型无显著差异（P〉0．05）。结论PTEN失活或蛋白表达降低、VEGF的高表达与胃癌临床病理特征和生物学行为有密切关系。PTEN在低分化或未分化以及伴淋巴结转移和临床Ⅲ＋Ⅳ期胃癌中的表达与VEGF呈负相关。联合检测PTEN、VEGF对胃癌的恶性程度及预后判断具有一定的临床参考意义。应用组织芯片大规模高效检测临床组织样本是可行的，具有快速、准确、方便经济的特点。     

hFRNK基因对胃泌素诱导的人结肠癌细胞侵袭力的影响

目的 观察腺病毒介导hFRNK基因对胃泌素所诱导的人结肠癌Colo320WT细胞侵袭力的影响.方法 试验分为胃泌素组、hFRNK组和对照组,胃泌素组用100 μmol/L胃泌素诱导结肠癌Col0320WT细胞12 h;hFRNK组,首先用脂质体瞬时转染腺病毒受体pCR3.1-CAR于Col0320WT细胞48 h,然后用100 μmol/L胃泌素干预结肠癌Colo320WT细胞12 h,再用重组腺病毒(pAdhFRNK)感染细胞;对照组为未经处理的Colo320WT细胞.用免疫印迹检测hFRNK基因黏着斑激酶(FAK)397位酪氨酸(FAKTyr397)的磷酸化表达,激光共聚焦显微镜观察FAKTyr397在细胞板状伪足的表达情况,免疫共沉淀检测hFRNK基因对四联信号复合物FAK-Src-Doek180一p130Cas形成的影响,Pull-down法检测hFRNK对Rac蛋白活性的影响.结果 胃泌素诱导后,磷酸化FAKTyrr397明显增强;与胃泌素组相比,hFRNK组中FAKTyr397表达下降,FAKTyr397定位到细胞板状伪足的量明显减少,FAK、Src、Dockl80和p130Cas 四联信号复合物没有形成,Rac的活性降低.结论 hFRNK基因可阻断胃泌素引起的FAK的磷酸化,阻断FAKTyr397摹积到细胞的板状伪足,阻止四联信号复合物FAK-Src-Dock180-p130Cas的形成以及Rac的活化,为hFRNK基因防治肿瘤的侵袭和转移提供理论依据.     

白介素-8、白介素-15在结肠癌患者结肠黏膜的表达及意义

目的:研究结肠癌患者结肠黏膜中白介素-8(IL-8)、白介素-15(IL-15)的表达并探讨其临床应用价值．方法:采用免疫组化法检测结肠癌患者结肠黏膜中IL-8,IL-15的表达情况．结果:IL-8染色阳性44例,占66．7％．IL-15染色阳性40例,占60．6％．IL-8和IL-15表达水平与结肠癌临床分期(IL-8:r=0．437,P=0．006; IL-15:r=0．317,P=0．014)、浸润深度(IL-8: r=0．332,P=0．003;IL-15:r=0．312,P= 0．015)、淋巴结转移(IL-8:r=0．316,P=0．042; IL-15:r=0．236,P=0．017)、病理分级(IL-8:r =0．826,P=0．0001;IL-15:r=0．368,P=0．001)均呈显著正相关．结论:检测IL-8,IL-15的表达可做为判断结肠癌恶性程度有价值的指标．