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.