依硫磷酸调控人类基因表达谱的预测及生物信息学分析

本研究对依硫磷酸调控人类基因表达谱进行生物信息学分析,预测其可能具有的新生物学作用,为深入研究依硫磷酸的药理学作用及方法提供指导。以依硫磷酸(amifostine)为关键词,在互联网开放性数据库包括GEO、Affymetrix基因芯片表达数据库、人类基因组数据库(GenBank)、基因表达数据库SAGE、GeneCard、InterPro、ProtoNet、UniProt和BLOCKS中搜索,并进一步对筛选出的基因表达数据库进行有效性检验、基因表达差异和聚类分析。结果表明,仅在GEO数据库中筛选出1个与依硫磷酸相关的基因表达谱数据库(accession:GSE3212)。有效性检验及基因表达差异分析显示,依硫磷酸处理K562细胞后,分类全基因组仅2.14%(460/192000)的基因在转录水平的表达具有显著差异(p<0.01)。基因注释分析表明,460条差异基因中有139条为已知基因,其中77条表达上调,62条表达下调;13条为依硫磷酸处理后新表达的基因,5条为依硫磷酸处理后表达完全受抑的基因。聚类分析显示,139条基因分属11大类,主要生物学功能与造血及免疫调控、凋亡和细胞周期相关。结论 :生物信息学方法可用于依硫磷酸基因表达谱分析。依硫磷酸对人类基因表达谱具有调控作用,可能对造血及免疫、凋亡和细胞周期等生物学过程具有重要影响,需进一步在实验水平加以验证。     

Increased expression of paxillin is found in human oesophageal squamous cell carcinoma: a tissue microarray study

Oesophageal cancer is one of the most common cancers worldwide. Currently, the tumour, node, metastasis (TNM) staging system is the primary method for determining its extent and prognosis, however, data suggest this system does not predict prognosis accurately. Research has, therefore, concentrated on searching for specific biomarkers. Paxillin has been shown to play an important role in controlling cell spread and migration. Its over-expression is considered to correlate with the prognosis of some types of cancers, however, the relationship between paxillin expression and clinical outcome in oesophageal cancer has not been investigated. This study determined the expression of paxillin by immunohistochemistry on the tissue microarray of 100 oesophageal squamous cell cancer patients followed up for a mean of 55 months. Paxillin was over-expressed in tumours in 27/100 cases, compared with 6/100 cases for adjacent non-tumoural cells. No correlation occurred between expression of paxillin and overall patient survival, hence paxillin is not an effective prognostic marker in these patients.     

弥漫型胃癌基因表达谱聚类分析

目的:从转录组水平识别弥漫型胃癌与正常胃黏膜间的基因表达差异,探讨胃癌分子发生、发展的机制。方法:收集22例弥漫型胃癌患者的胃癌组织及其相应远切端的正常胃黏膜。采用含14592个点的cDNA表达谱芯片建立胃癌基因表达谱。差异表达基因筛选标准为该基因在50%以上样本中肿瘤比正常组织荧光强度大2倍或以上(P<0.05)。采用系统聚类、方差分析(P<0.05)等方法进行差异表达分析,实时定量RT-PCR方法验证芯片结果。结果:胃癌组织与正常胃黏膜间的差异表达基因共357个,其中表达上调者153个;表达下调者204个。上调基因的功能主要与细胞骨架运动、基质重建和细胞黏附、细胞周期调控及信号传导相关;下调基因主要与消化功能、细胞免疫防御、代谢、凋亡抑制及电子传递相关。实时定量PCR验证结果与芯片结果一致。结论:运用cDNA芯片进行弥漫型胃癌基因表达谱分析,有助于从分子水平全方位理解弥漫型胃癌发病机制及生物学特性,也有助于发现新的分子诊断指标和基因治疗靶标。     

Prognostic value of protease-activated receptor 2 expression in oesophageal squamous cell carcinoma

Protease-activated receptor-2 (PAR-2) is essential for the initiation and development of tumours, suggesting that the detection of PAR-2 expression might serve as a clinical marker in the prediction or diagnosis of clinical outcomes of malignant neoplasia. Using immunohisto chemical methods, this study investigated whether the detection of PAR-2 protein had clinical implications for patients with oesophageal squamous cell carcinoma (OSCC). PAR-2 protein was present at a high level in primary OSCC sites but at a low level in normal oesophageal tissue. The level of PAR-2 protein in tumours was significantly correlated with the clinical stage and histological grade of disease. Patients with tumours highly positive for PAR-2 protein had a significantly worse prognosis than those with lower PAR-2 levels. Thus, the over-expression of PAR-2 is a characteristic feature of OSCC and suggests that the immuno histochemical detection of raised levels of PAR-2 may be a potentially useful prognostic indicator.     

Differential gene expression identifies subgroups of renal cell carcinoma

Clear cell carcinoma of the kidney, the most common subtype of renal cell cancer, displays different biological behavior in different patients. This heterogeneity cannot be recognized by light microscopy. In this study, gene expression in 16 clear cell renal cell carcinoma samples and 17 non-malignant tissue types comprising 539 samples was determined using oligonucleotide microarrays representing approximately 40,000 known genes and ESTs. Differences in gene expression were quantified as the fold change in gene expression between the various sets of samples. A set of genes was identified that was overexpressed in the renal cell carcinoma samples compared with the normal kidney samples. Principle component analysis of the set of renal cell carcinomas using this set of genes overexpressed in renal cell cancer revealed the existence of 2 major subgroups among the renal carcinomas. A series of principle component analyses of the set of renal cell carcinomas using different gene sets composed of genes involved in different metabolic pathways also revealed the same 2 major subgroups of the renal cell cancers. Eisen clustering using the same genes also revealed the same 2 major renal cell cancer subsets. Review of the pathology suggested that these 2 subgroups differed in pathologic grade. Genes differentially expressed between the 2 renal cell cancer subsets were identified. Examination of gene expression in each renal cell cancer subset and the pool of renal cell carcinoma samples compared with that in 17 different normal tissues revealed genes specifically overexpressed in renal cell cancer compared with these normal tissues. The authors conclude that gene expression patterns may be useful in helping to further classify subtypes of renal cell carcinoma that may have clinical significance. In addition, the genes identified as overexpressed in each set of clear cell renal cell carcinomas compared with normal tissues may represent useful targets for therapy.     

早期食管鳞癌患者中微小RNA的表达差异及其临床意义研究

目的探讨早期食管鳞癌患者中微小RNA的表达差异及其临床意义。方法选取2013年4月至2016年4月收治的65例早期食管鳞癌患者作为食管鳞癌组,以及同期65例健康人作为对照组,分析微小RNA对早期食管鳞癌的诊断意义,以及微小RNA表达与化疗敏感性的关系。结果 microRNA-21、microRNA-205在食管鳞癌组中的表达水平明显高于对照组(P0.05)。microRNA-21、microRNA-205对食管鳞癌的诊断灵敏度分别为89.23%、84.62%,特异度分别为96.92%、95.38%。癌组织microRNA-21、microRNA-205高表达的患者化疗耐受性明显高于低表达患者(P0.05)。结论微小RNA可协助临床早期诊断食管鳞癌,并且可根据微小RNA的表达量,评估患者化疗的敏感性,以指导临床医师优化治疗策略。     

Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis

ObjectiveWe seek to identify the differentially expressed miRNAs in the clear cell subtype (ccRCC) of kidney cancer.Design and methodsWe performed a miRNA microarray analysis to compare the miRNA expression levels between ccRCC tissues and their normal counterpart. The top dysregulated miRNAs were validated by quantitative RT-PCR analysis. Bioinformatics analysis was also performed.ResultsA total of 33 dysregulated miRNAs were identified in ccRCC, including 21 upregulated miRNAs and many of these miRNAs have been reported to be dysregulated in other malignancies and have a potential role in cancer pathogenesis. The miRNAs showed a significant correlation with reported chromosomal aberration sites. We also utilized target prediction algorithms to identify gene targets. Preliminary analyses showed these targets can be directly involved in RCC pathogenesis.ConclusionWe identified miRNAs that are dysregulated in ccRCC and bioinformatics analysis suggests that these miRNAs may be involved in cancer pathogenesis and have the potential to be biomarkers.     

Survivin在宫颈鳞癌中的表达及临床意义

目的 探讨宫颈鳞癌组织中survivin基因表达的特征及临床意义。方法 采用免疫组织化学方法（PV法）检测54例宫颈鳞癌，16例正常官颈组织中Survivin基因的表达，并分析其临床病理特征。结果 Survivin在正常宫颈组织中不表达，在官颈鳞癌组织中阳性表达率与年龄、临床分期及淋巴结转移有关。结论 Survivin基因在官颈鳞癌组织中的稳定表达。预示肿瘤有较高的侵袭性和不良预后，可能成为有效的预测官颈鳞癌侵袭性的监测指标及判断预后的指标，对指导临床治疗具有重要意义。     

基质金属蛋白酶2的表达在中晚期食管癌放射治疗中的临床意义

目的 探讨食管癌中基质金属蛋白酶2(MMP-2)表达与临床病理因素间的关系及其对单纯放疗预后的影响.方法 应用免疫组化法检测59例食管癌和41例食管癌旁组织中MMP-2表达的情况,分析该蛋白表达与临床病理因素间的关系及其对食管癌单纯放疗预后的影响.结果 59例食管癌组织中MMP-2阳性表达率为59.32%,41例癌旁组织中阳性表达率为31.71%,两者间差异有统计学意义(P=0.007);胸部CT扫描显示病变最大浸润深度、气管和(或)支气管受侵、主动脉受侵及椎前三角受侵等与MMP-2表达均有关系.MMP-2表达对食管癌单纯放疗的预后有显著影响.结论 MMP-2高表迭与食管癌进展有关,很可能促进肿瘤侵袭转移,可作为食管癌放疗预后的一个评价指标.     

Differential protease expression by cutaneous squamous and basal cell carcinomas.

To assess the postulated role of plasminogen activation in tumor invasion, we have investigated the cellular sites of synthesis for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their inhibitors (PAI-1 and PAI-2) in two human cutaneous neoplasia that differ in their metastatic potential. The combined use of zymography on tissue sections and in situ hybridization demonstrates that uPA is produced by malignant cells of squamous cell carcinomas (SCC) but not by basal cell carcinomas (BCC), whereas tPA is detected exclusively in nonmalignant dermal tissue. In addition, we show that SCC neoplastic cells simultaneously produce variable amounts of PAI-1, and that PAI-1 production correlates inversely with uPA enzymatic activity. These observations establish that invasive human malignant cells in vivo can activate plasminogen through uPA production during the early phases of tumor growth; they also demonstrate that the proteolytic activity of tumor cells can be modulated by the concomitant production of PAI-1. Because SCC have a higher invasive and metastatic potential than BCC, our findings lend further support to the involvement of plasminogen activation in malignant behavior.     

食管鳞癌中分泌性富含半胱氨酸的酸性蛋白基因差异表达分析

目的探讨分泌性富含半胱氨酸的酸性蛋白(SPARC)在食管鳞癌(ESCC)增殖、浸润、转移中的作用和意义。方法用半定量逆转录聚合酶链反应(RTPCR)分析SPARC在24份ESCC组织和其相邻的正常黏膜组织的差异表达量及基因表达变化趋势。用酶联免疫吸附试验(ELISA)检测40名正常对照者和32例ESCC患者手术前后血浆中SPARC水平变化。结果经RTPCR分析SPARC在24份配对标本中的表达,其中19份ESCC组织中的表达明显高于其配对的正常黏膜组织,5份差异表达不明显;而在不同分化程度组织中的表达差异无统计学意义(P=0.663),淋巴结转移组与未转移组的差异有统计学意义(P<0.05)。正常人血浆SPARC水平[(644±124)ng/ml]与ESCC术前患者[(613±119)ng/ml]的差异无统计学意义(P=0.829);ESCC患者术后1周血浆SPARC水平[(245±45)ng/ml]明显低于术前(P<0.01),而19例ESCC术前患者血浆SPARC水平与术后3个月[(661±182)ng/ml]的差异亦无统计学意义(P=0.935);不同分化程度的ESCC患者血浆SPARC水平差异无统计学意义(P>0.05)。结论组织中SPARC水平与ESCC的发生与发展密切相关,有淋巴结转移时SPARC表达升高,血浆SPARC水平对ESCC诊治无指导意义。     

Cytokeratin expression in subungual squamous cell carcinoma

Cytokeratin expression in subungual squamous cell carcinoma was investigated in order to evaluate the origin and state of differentiation of the tumour. The tumour nests contained cytokeratin 14, 16 and 17, which were also expressed in the nail bed. Therefore, cytokeratin expression in subungual squamous cell carcinoma may reflect its indolent clinical prognosis.     

RUNX3蛋白在食管鳞状细胞癌中的表达

目的：探讨 RUNX3蛋白在食管正常黏膜组织以及鳞癌组织中的表达情况及意义。方法采用免疫组织化学 SP 法对 RUNX3蛋白在20例食管正常黏膜以及48例鳞状细胞癌组织中的表达进行检测。结果食管鳞状细胞癌中 RUNX3蛋白的阳性率为37.5%(18/48),明显低于正常黏膜组织中的阳性率80.0%(16/20)(P <0.05)。RUNX3蛋白表达与食管鳞状细胞癌患者的年龄、性别、肿瘤大小、病理学分级以及临床分期均无明显相关性(P >0.05),然而在淋巴结转移组中 RUNX3的阳性率为15.4%(4/26),明显低于在无淋巴结转移组中 RUNX3的阳性率63.6%(14/22)(P <0.05)。结论 RUNX3蛋白在食管鳞状细胞癌组织中的表达明显低于其在正常黏膜组织中的表达,提示 RUNX3可能与肿瘤的发生发展有关。     

Evaluation of quality-control criteria for microarray gene expression analysis

BACKGROUND: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. METHODS: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A(260)/A(280) ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. RESULTS: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). CONCLUSIONS: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.     

食管鳞癌组织和细胞系中富含半胱氨酸的酸性分泌蛋白表达分析

目的研究富含半胱氨酸的酸性蛋白（SPARC）在食管鳞癌的增殖、浸润和转移中的作用及临床意义.方法用免疫印记法（WB）分析食管鳞癌组织和其相邻的正常黏膜组织及食管鳞癌不同细胞系,研究SPARC蛋白表达的趋势.结果 WB分析SPARC在30份配对组织标本中的表达, 其中22份食管鳞癌组织中的表达明显高于其相应的正常黏膜组织, 8份中差异表达不明显 [R=73.3%（有差异例数 /总例数）];食管鳞癌组织中的表达与其相应正常黏膜组织分化程度方面的差异无统计学意义（P〉0.05）,而与淋巴结转移组的差异有统计学意义（P〈0.05）.食管鳞癌不同细胞系中SPARC表达与组织分化程度无关.结论食管鳞癌组织中的SPARC表达与淋巴结转移明显相关,而其在食管鳞癌不同细胞系中的表达与组织分化程度无关.     

鳞状上皮恶性肿瘤组织中HPV16 E5基因的克隆和序列分析

目的克隆高危型人乳头状瘤病毒(HPV)E5基因,并对HPV16E5基因进行序列分析。方法收集10例安徽省立医院妇产科宫颈癌患者手术中癌变部位病理标本,提取总DNA,设计两对嵌套特异性引物,对模板进行PCR扩增,纯化的目的片段与pMD18-T载体连接后转化大肠杆菌TG1,筛选阳性克隆并测序。结果获得两个样本的E5基因HPV-E5-6和HPV-E5-7,全长均为252 bp,两者测序结果完全一致。构建HPV-E5-6/7与HPV16其它E5基因的系统关系树,显示出他们与HPV16E5(AF120701)亲缘关系最近。结论HPV16E5序列保守性强,特异性引物可有效检测HPV16E5基因,E5基因的获得为进一步研究它的功能建立了良好基础。