以往研究发现。在TPA诱导永生化食管上皮细胞癌变中中性粒细胞明胶酶相关载脂蛋白（neutrophilgelatnase-associate lipocaIin，NGAK）基因过表达，但过表达机制不明。最近研究提示，食管癌细胞，NGAL启动子及其邻近区域可能存在着TPA反应元件。为了对NGAL的这-TPA反应元件进行更准确定位，采用PcR法结合嵌套缺失实验从食管癌细胞中克隆NGAL5’侧翼区-152～+84、-140～+84、-78～+84、-59～+84、-50～+84、-41～+84、-37～+84、-29～+84和-10～+84等片段，并定向插入pGLB、pGLP或pGLE等萤火虫荧光素酶报告基因表达载体中，构建了pGLB-152、pGLP-152、pGLE-152、paLB-140、pGLB-78、pGLB-59、pGLB-50、pGLB-41、pGLB-37、pGLB-29和pGLB-10等系列报告基因表达载体。将上述报告基因表达载体分别同pRL一_rK共转染食管癌细胞Ecl09，并用TpA刺激，检测TPA刺激转染ECl09的相对荧光素酶活力，综合判定NGAL-152～+84区不同长度片段的TPA反应性，对NGAL启动f区的TPA反应兀件给进一步分段定位。结果表明，NGAL启动子区的TPA反应元件位于-152～-60区段，而且应答TPA刺激的反应能力很强。生物信息学分析结果显示，NGAL工启动子区所存在的TPA反应元件很可能是一种新结构类型研究说明，NGAL在DNA序列上有应答TPA刺激的结构基础，将有助于深入到分子水平揭示TPA诱导永牛化食管上皮细胞癌变中NGAL三过表达机制，也有助于进一步认识TPA信号细胞内传递途径网络在肿瘤发生发展中的作用。

Fascin, an actin-bundling protein, induces membrane protrusions and increases cell motility in various transformed cells. The overexpressionof fascin in esophageal squamous cell carcinoma (ESCC) has been described only recently, but the roles and mechanism stillremained unclear. Here, by using RNA interference (RNAi), we have stably silenced the expression of the fascin in EC109 cells, an ESCCcell line. Down-regulation of fascin resulted in a suppression of cell proliferation and as well as a decrease in cell invasiveness. Furthermore,we revealed that fascin might have functions in regulating tumor growth in vivo. The effect of fascin on cell invasiveness correlatedwith the activation of matrix metalloproteases such as MMP-2 and MMP-9. We examined that fascin down-expression also led to adecrease of c-erbB-2 and b-catenin at the protein level. These results suggested that fascin might play crucial roles in regulating neoplasmprogression of ESCC.

为了研究反义封闭NGAI。基因表达对SHEEC食管癌细胞微丝骨架以及肿瘤细胞生物学行为的影响。以不同长度NGAL基因片段反义表达载体和硫代修饰反义寡核苷酸单链片段转染SHEEC食管癌细胞，通过C418筛选，建立一系列旨在封lⅥSHEEC食管癌细胞NGAL基因表达的亚细胞克隆在细胞内F一肌动蛋白（F-aetin）及DNA荧光双标记基础上，通过流式细胞术、激光共聚焦显微镜扫描术等技术手段检测封闭反义NGAI。基因表达后，SHEEC食管癌细胞中F-actin和DNA含量、F-actin形态结构以及肿瘤细胞生物学行为的变化特征结果显示。反义封l羽NGAL基冈表达后，SHEEC食管癌细胞F-aetin的古量明显降低，与永生化食管卜皮细胞SHEE相近，但细胞分裂增殖指数未见明显变化表明反义封}jj NGAL基因表达对SFIEEC食管癌细胞的微丝骨架有明显影响。而对SHEEC食管癌细胞的分裂增殖影响不明显激光共聚焦显微镜扫描观测显示。反义封闭NGAL基因表达可使SHEEC食管癌细胞F-actin分布均匀，F-aetin小体减少，细胞间连接重新建立。结构较紧密。主要形态结构特征与SHEE细胞趋于致提示反义封闭NGAL基因表达可对SHEEC食管癌细胞的微丝骨架F-actin产生明显影响，推测癌细胞的微丝骨架F-actin可能是NGAL基因在SHEEC食管癌细胞中发挥功能的种作用环节。

AIM: To separate and identify differentially expressed nuclearmatrix proteins (NMPs) between the immortalized humanesophageal epithelial cell line (SHEE) and the malignantlytransformed esophageal carcinoma cell line (SHEEC), andto provide new ways for finding specific markers and thepathogenesis of esophageal carcinoma.METHODS: SHEE and SHEEC cell lines were used to extractNMPs. The quality of NMPs was monitored by Western blotanalysis including DNA topoisomerase II, proliferation cellnuclear antigen (PCNA) and histone. NMPs of SHEE andSHEEC were analyzed by two-dimensional electrophoresis(2-DE), silver staining and PDQuest6.2 image analysissoftware. Three spots in which the differentially expressedNMPs were more obvious, were selected and analyzed withmatrix-assisted laser desorption/ionization time of flying massspectrometry (MALDI- TOF-MS) and database search.RESULTS: Western blot analysis revealed that DNAtopoisomerase IIand PCNA were detected, and the majorityof histones were deleted in NMPs of SHEE and SHEEC. After2-DE image analysis by PDQuest6.2 software, the 2-DE mapswere detected with an average of 106±7.1 spots in SHEE and132±5.0 spots in SHEEC. Most of them were matched oneanother (r=0.72), only 16 protein spots were found differingin intensity. Three NMPs including cytoskeletal tropomyosin,FK506-binding protein 6, similar to retinoblastoma bindingprotein 8 were preliminarily identified by MALDI- TOF-MS.CONCLUSION: These differentially expressed NMPs mayplay an important role during malignant transformation fromSHEE to SHEEC. Their separation and identification willcontribute to searching for specific markers and probing intothe pathogenesis of esophageal carcinoma.

AIM: To investigate the correlation between ezrin expressionand invasive phenotype formation in malignantly transformedesophageal epithelial cells.METHODS: The experimental cell line employed in thepresent study was originated form the progressive inductionof a human embryonic esophageal epithelial cell line (SHEE)by the E6E7 genes of human papillomavirus (HPV) type 18.The cells at the 35th passage after induction called SHEEIMMwere in a state of immortalized phase and used as the control,while that of the 85th passage denominated as SHEEMTrepresented the status of cells that were malignantlytransformed. The expression changes of ezrin and its mRNAin both cell passages were respectively analyzed by RT-PCRand Western blot. Invasive phenotype was assessed in vivoby inoculating these cells into the severe combinedimmunodeficient (SCID) mice via subcutaneous andintraperitoneal injection, and in vitro by inoculating them onthe surface of the amnion membranes, which then wasdetermined by light microscopy and scanning electronmicroscopy.RESULTS: Upregulated expression of ezrin protein and itsmRNA was observed in SHEEMT compared with that inSHEEIMM cells. The SHEEMT cells inoculated in SCID micewere observed forming tumor masses in both visceral organsand soft tissues in a period of 40 days with a specialpropensity to invading mesentery and pancreas, but did notexhibit hepatic metastases. Pathologically, these tumor cellsharboring larger nucleus, nucleolus and less cytoplasm couldinfiltrate and destroy adjacent tissues. In the in vitro study,the inoculated SHEEMT cells could grow in cluster on theamniotic epithelial surface and intrude into the amnioticstroma. In contrast, unrestricted growth and invasivenesswere not found in SHEEIMM cells in both in vivo and in vitroexperiment.CONCLUSION: The upregulated ezrin expression is one ofthe important factors that are possibly associated with theinvasive phenotype formation in malignantly transformedesophageal epithelial cells.

以往在研究由促癌物12-o-十四烷酰佛波醇-13-乙酯（12-o-tetradecanoylphorbol-13-acetate, TPA）诱导的人永生化食管上皮细胞恶性变中基因的差异表达情况时，曾获得中性粒细胞明胶酶相关lipocalin（netutrophil gelatinase-associated lipocalin, NGAL）等新的食管癌癌变相关基因为深入研究这些癌变相关基因在食管癌中以蛋白质—蛋白质或蛋白质—DNA相互作用为基础的功能网络调控关系，建立了一个食管癌cDNA酵母杂交文库采用Trizol试剂从一种新的人食管癌细胞（SHEEC，由人永生化食管上皮细胞转化而来）中提取细胞总RNA，采用Oligotex mRNA Kit从细胞总RNA中制备PolyA+mRNA，采用superscriptT”choice System For cDNA Synthesis Kit合成cDNA，以PolyA+mRNA为探针，化学发光法监测cDNA合成的质量，以pGADT7为载体构建了SHEEC细胞的cDNA酵母杂交文库文库的滴度为l.19×l09cfu/ml，重组片段大小主要集中在0.5～6.0kb，重组率为50%，在此基础上，应用酵母单杂交技术手段对该文库中的NF-kB出元件结合因子进行了筛选，在SD/-his/-leu/[+15mmol/L3-AT]缺陷培养基平板上共获得了约360个单克隆按照平板上酵母单克隆直径的大小，选择直径大于2 rr蚰者91个，提取质粒进行酶切鉴定和一对一酵母单杂交验证实验，结果获得了30个阳性重组子，并随机取其中的9个阳性重组子进行测序和GenBank/BLAST同源分析，发现某些基因编码的蛋白质产物在关键氨基酸残基位点上与D65和p50的NFkB元件结合域公共序列具有高度一致性这些实验结果表明，所构建的人食管癌cDNA酵母杂交文库是成功的

AIM: To search for the biomarker of cellular immortalization,the telomere length, telomerase activity and its subunitsin cultured epithelial cells of human fetal esophagus in theprocess of immortalization.METHODS: The transgenic cell line of human fetalesophageal epithelium (SHEE) was established with E6E7genes of human papillomavirus (HPV) type 18 in ourlaboratory. Morphological phenotype of cultured SHEEcells from the 6th to 30th passages, was examined byphase contrast microscopy, the telomere length wasassayed by Southern blot method, and the activity oftelomerase was analyzed by telomeric repeatamplification protocol (TRAP). Expressions of subunitsof telomerase, hTR and hTERT, were assessed by RTPCR.DNA content in cell cycle was detected by flowcytometry. The cell apoptosis was examined by electronmicroscopy (EM) and TUNEL label.RESULTS: SHEE cells from the 6th to 10th passages showedcellular proliferation with a good differentiation. Fromthe 12th to the 16th passages, many senescent andapoptotic cells appeared, and the telomere lengthsharply shortened from 23kb to 17kb withoutexpression of hTERT and telomerase activity. At the20th passage, SHEE cells overcame the senescence andapoptosis and restored their proliferative activity withexpression of telomerase and hTERT at low levels, butthe telomere length shortened continuously to thelowest of 3kb. After the 30th passage cells proliferationwas restored by increment of cells at S and G2M phasein the cell cycle and telomerase activity expressed athigh levels and with maintenance of telomere length.CONCLUSION: At the early stage of SHEE cells, telomeresare shortened without expression of telomerase and hTERTcausing cellular senescence and cell death. From the 20thto the 30th passages, the activation of telomerase andmaintenance of telomere length show a progressive processfor immortalization of esophageal epithelial cells. Theexpression of telomerase may constitute a biomarker fordetection of immortalization of cells.

AIM: To identify the differentially expressed proteinsbetween the human immortalized esophageal epithelial cellline (SHEE) and the malignant transformed esophagealcarcinoma cell line (SHEEC), and to explore new ways forstudying esophageal carcinoma associated genes.METHODS: SHEE and SHEEC cell lines were used toseparate differentially expressed proteins by two-dimensionalelectrophoresis. The silver-stained 2-D gels was scannedwith EDAS290 digital camera system and analyzed with thePDQuest 6.2 Software. Six spots in which the differentiallyexpressed protein was more obvious were selected andanalyzed with matrix-assisted laser desorption/ionization timeof flying mass spectrometry (MALDI-TOF-MS).RESULTS: There were 107±4.58 and 115±9.91 proteinspots observed in SHEE and SHEEC respectively, and themajority of these spots between the two cell lines matchedeach other (r=0.772), only a few were expresseddifferentially. After analyzed by MALDI-TOF-MS and databasesearch for the six differentially expressed proteins, One newprotein as well as other five sequence-known proteinsincluding RNPEP-like protein, human rRNA gene upstreamsequence binding transcription factor, uracil DNA glycosylase,Annexin A2 and p300/CBP-associated factor werepreliminarily identified.CONCLUSION: These differentially expressed proteinsmight play an importance role during malignanttransformation of SHEEC from SHEE. The identification ofthese proteins may serve as a new way for studyingesophageal carcinoma associated genes.Xiong XD, Xu LY, Shen ZY, Cai WJ, Luo JM, Han YL, Li EM.Identification of differentially expressed proteins between humanesophageal immortalized and carcinomatous cell lines by twodimensionalelectrophoresis and MALDI-TOF-massspectrometry.

以人食管癌细胞系EC109作为驱赶方（driver），以被一氧化氮（nitric oxid, NO）诱导的ECl09作为实验方（tester），应用抑制消减杂交（suppression subractive hybridization, SSH）、反向mRNA斑点印迹和RNA印迹等技术手段研究了NO诱导的食管癌细胞中基因的过表达情况然后对过表达基因的表达序列标签（expressed seqnencetag，EST）实施序列测定，并与GenBank进行BLAST同源性比较和序列突变分析结果先后两次从69个SSH阳性克隆中共鉴定出6个线粒体DNA（milochondrial DNA，mtDNA）编码的基因，即ND 4L、ND 4、COX-2、Lys-tRNA、ATP -8和ATP-6表明NO可以诱导食管癌细胞mtDNA编码的基因过表达另外，在ND-4L/ND-4基因的片段（10736～11449）上发现了三处同型单核苷酸置换（10872→C，11001A →G，11346A→G），在COX-2/Lys-Trna/ATP-8/ATP-6基因片段（8011～8 589）上发现了一处单核苷酸缺失（8380A）氨基酸序列分析表明，在NO诱导的EC109中可能存在着一种结构异常的ATP-8肽链（一条在N端被截短的只有11个氨基酸残基的肽链，而正常的ATP-8肽链为68个氨基酸残基）这些研究结果为深入揭示NO对肿瘤细胞的作用机制提供了新的重要线索。

为研究NGAL （neutrophil gelatinase associated lipocalin）基因在永远生化食管上皮细胞恶性转化中的表达情况，以永生化食管上皮细胞系SHEE食管癌细胞系SHEEC互为对照，用cDNA微列阵进行筛选，用RNA印迹和RT-PCR进行鉴定，cDNA克隆测序后与GenBank进行BLAST分析比较，结果表明 NGAL基因在SHEEC中出现显著差异过表达，其cDNA序列与小鼠24p3、大鼠NRL（neu-related lipcalin）、人中性粒细胞NGAL和卵巢癌NGAL具有较高的相似性，这提示NGAL基因在永生化食管上皮细胞恶性转化中可能发挥着重要作用，可能是一种新癌基因或促癌基因。