Wnt1在口腔鳞癌中的表达及其与淋巴结转移关系的研究

目的 :研究Wnt信号传导通路中Wnt1蛋白在口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)中的表达水平,初步探讨Wnt1在OSCC淋巴结转移中的作用。方法:采用免疫组织化学方法,检测60例OSCC中Wnt1的表达情况;采用Western blot法检测5对OSCC临床标本和配对癌周组织中Wnt1的表达;通过实时定量PCR法和Western blot法分别检测高、低转移潜能的HN-12和HN-4口腔鳞癌细胞系中Wnt1基因和蛋白的表达水平;采用生长曲线、划痕实验和Transwell法,分别检测重组蛋白Wnt1对2种细胞的生长情况、迁移能力和侵袭能力的影响。结果:24例淋巴结转移的OSCC中高表达Wnt1,16例未有淋巴结转移的OSCC中低表达Wnt1,Wnt1的表达在两组之间差异有统计学意义(P<0.01);高转移细胞系HN-12较低转移细胞系HN-4在m RNA水平和蛋白水平上Wnt1表达显著增高;重组蛋白Wnt1,能够提高肿瘤细胞的生长、迁移和侵袭能力。结论:Wnt信号通路中的关键分子Wnt1在OSCC中有表达,且与淋巴结转移相关,为进一步探讨Wnt信号通路影响肿瘤转移的分子机制奠定基础。     

转录因子XBP1在口腔鳞状细胞癌中的表达

目的:检测口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)组织中X盒结合蛋白1 (X box binding protein 1,XBP1)的表达,并探讨其意义。方法:应用qRT-PCR法检测50例OSCC肿瘤和癌旁正常黏膜上皮中剪切型XBP1(XBP1 splicing, XBP1-s)的表达;应用组织芯片技术和免疫组化染色相结合的方法检测100例OSCC癌旁上皮、肿瘤中心、侵袭前沿及淋巴结转移灶中XBP1蛋白的表达。结果:qRT-PCR显示:XBP1-s mRNA表达显著上升(log2(倍数)≥1)的病例与颈淋巴结转移相关(P＜0.05)。免疫组化结果显示:XBP1蛋白在OSCC侵袭前沿和淋巴结转移灶中的表达均高于癌旁上皮和肿瘤中心(P＜0.05);且在淋巴结转移灶中的表达明显高于侵袭前沿(P＜0.05)。此外,XBP1蛋白表达还与OSCC组织病理学分级相关(P＜0.05)。结论:XBP1可能是促进口腔鳞状细胞癌侵袭和转移的一个重要因子。     

口腔鳞癌转移与特异性核基质结合区结合蛋白质1（SATB1）表达的关系

目的：检测特异性核基质结合区结合蛋白质1在口腔鳞状细胞癌中的表达及其与浸润转移的关系。方法：采用免疫组织化学染色法及逆转录-聚合酶链反应检测SATB1在各组织中的表达。结果：52例口腔鳞癌原发灶中,SATB1蛋白在病理分级Ⅱ~Ⅲ级组（中~低分化鳞癌）表达明显高于Ⅰ级组（高分化鳞癌）（P〈0.05）;在临床分期Ⅲ~Ⅳ期组高于Ⅰ~Ⅱ期组（P〈0.05）。SATB1蛋白和mRNA的表达在16例淋巴结转移组高于36例无淋巴结转移组（P〈0.05）。SATB1mRNA的表达量在6例淋巴结转移灶高于16例转移组原发灶（P〈0.05）。结论：SATB1蛋白及SATB1mRNA在口腔鳞癌中的表达上调与口腔鳞癌的恶性程度及转移相关;SATB1可能在口腔鳞癌的发展、转移过程中起到促进的作用。     

Reduced expression of the CD44 variant exons in oral squamous cell carcinoma and its relationship to metastasis

In the present study, we examined the expression of CD44 variant exons in human oral squamous cell carcinoma (OSCC). Of ten cell lines from OSCCs, two (KB and H357), as well as the HeLa cell line, failed to express CD44 variant exons. In surgical specimens, all normal mucosa expressed CD44v9 (both mRNA and protein). Of 40 primary OSCCs, 19 (47.5%) showed downregulation of CD44v9, which correlated with tumor cell differentiation, primary metastasis to lymph nodes and secondary metastasis to lymph nodes. The results suggest that the downregulation of CD44v9 may play a role in lymphatic metastasis of OSCC and changes in its expression may be a useful diagnostic tool to determine the metastatic potential of OSCC to lymph nodes. Moreover, three cell lines that failed to express CD44 variant exons might become a useful experimental model to study the role of variant exons in the progression of OSCC.     

基质金属蛋白酶-2与口腔鳞癌颈淋巴结转移关系的研究

目的 探讨基质金属蛋白酶－2（MMP－2）与口腔鳞癌颈淋巴结转移的关系。方法 通过免疫组化法和蛋白明胶酶谱法对40例口腔鳞癌（OSCC）癌组织、正常口腔黏膜组织及血浆中（MMP－2）的表达和含量进行研究，并与口腔鳞癌的临床病理特征进行统计学分析。结果 OSCC癌组织、正常口腔黏膜中均表达MMP－2，而OSCC癌组织中MMP－2表达与正常口腔黏膜组织相比呈过量表达，MMP－2表达升高和活性增加与口腔鳞癌病理分级临床分期无关，但与口腔鳞癌颈淋韦结转移密切相关。结论 MMP－2可作为判断口腔鳞癌颈淋巴结转移的指标。     

口腔癌前沿细胞基质金属蛋白酶-2表达与颈淋巴结转移的关系

目的　探讨基质金属蛋白酶-2(MMP-2)在口腔粘膜鳞状细胞癌中的表达特点及其与颈淋巴结转移的关系。方法　应用免疫组织化学LsAB法观察MMP-2在口腔癌生长前沿区与中央区细胞中的表达分布特点,并分别与颈淋巴结转移发生进行统计学分析。结果　71例口腔癌原发灶均有MMP-2表达,在口腔癌生长前沿区细胞的表达明显强于中央区(P<0·01),呈条带状分布;口腔癌前沿区细胞MMP-2表达强度与颈淋巴结转移的发生呈正相关(P<0·05),而中央区MMP-2表达强度与颈淋巴结转移无关(P>0·05)。结论　口腔癌生长前沿区细胞MMP-2 表达明显强于中央区,与颈淋巴结转移有关,可作为口腔癌颈淋巴结转移的预测指标。     

P120 ctn/E-cad 在口腔鳞癌细胞迁移和侵袭中的功能

目的：分析P120-连环蛋白（P120-catenin,P120ctn）和E-钙粘蛋白（E-cadherin,E-cad）在口腔鳞癌（oral squamous-cell cancer,OSCC）细胞迁移和侵袭中的功能。方法：采用实时荧光定量PCR、Western blot检测HN4（人舌癌原发灶细胞）和HN12（人舌癌转移淋巴结细胞）中P120ctn、E-cad的mRNA和蛋白表达,通过Transwell细胞侵袭及细胞迁移试验等方法检测HOK（人口腔黏膜角化上皮细胞）、HN4和HN12细胞的迁移和侵袭能力。结果：HN4细胞中P120ctn和E-cad的mRNA和蛋白表达水平明显高于HN12,低于HOK细胞,差异均有统计学意义（P＜0．05）。HN4细胞的侵袭和转移能力显著高于HOK细胞,低于HN12细胞,差异均有统计学意义（P＜0．05）。结论：在OSCC细胞中P120 ctn与E-cad的表达下调,可能抑制了OSCC细胞的侵袭和转移。     

The downregulation of ANGPTL4 inhibits the migration and proliferation of tongue squamous cell carcinoma

ObjectiveTongue squamous cell carcinoma (TSCC) is the most common malignant cancer in the oral cavity, with a high rate of metastasis to the neck lymphoid node. Angiopoietin-like protein 4 (ANGPTL4) and microvessel density (MVD) may be novel indicators for tumor metastasis. The aim of the present study was to investigate the expression and function of ANGPTL4 in TSCC and the relationship between ANGPTL4 and MVD.MethodsThe expression levels of ANGPTL4 and MVD (CD34) were analyzed in 65 TSCC specimens and the adjacent non-cancerous tissues using immunohistochemistry (IHC). siRNA was delivered into TSCCA cells to downregulate ANGPTL4 expression. Subsequently, validation with real-time RT-PCR and western blot analyses was performed to analyze ANGPTL4 expression levels. In addition, a proliferation assay, migration and invasion assays were carried out.ResultsANGPTL4 expression was associated with tumor stage, lymph node metastasis and MVD expression. Cox regression analysis showed that high levels of ANGPTL4 expression were closely associated with poor survival time. In vitro analyses using qRT-PCR and western blot confirmed that ANGPTL4 was successfully inhibited in TSCCA cells. Suppressing ANGPTL4 resulted in the inhibition of cell proliferation and migration, but neither invasion nor cisplatin resistance was significantly affected.ConclusionHigh expression levels of ANGPTL4 are associated with the T stage, lymphatic metastasis, angiogenesis and poor overall survival in TSCC patients. The downregulation of ANGPTL4 inhibits the migration and proliferation of cells in TSCC. Taken together, ANGPTL4 may serve as a novel biomarker and therapeutic target for TSCC.     

口腔癌对侧颈淋巴结转移的临床研究

目的:探讨原发灶未过中线口腔癌的对侧颈淋巴结转移的相关临床病理因素,为口腔癌手术方法的选择提供依据。方法:收集2010年6月~2012年12月口腔癌238例,对年龄、性别、病程、原发灶部位、CT分期、颈清术式、病理分级、淋巴结转移等情况进行统计学分析。结果:单因素Logistic回归分析显示口腔癌对侧颈淋巴结转移在T3/T4期、中低分化、同侧淋巴结转移的患者中明显增加;多因素Logistic回归分析表明病理分级为口腔癌对侧颈淋巴结转移的高危因素。结论:在肿瘤未过中线时,如果T3/T4期、同侧颈淋巴结明确有转移、病理分级为中低分化等因素中出现两者或以上,为改善预后及提高患者治愈率,则有必要考虑同期行对侧颈清。     

口腔鳞癌粘着斑激酶表达与颈淋巴结转移关系的研究

目的 :探讨粘着斑激酶 (FAK)在口腔粘膜鳞状细胞癌中的表达特点及其与颈淋巴结转移的关系。方法 :应用免疫组化LsAB法检测FAK在口腔癌原发灶及颈淋巴结转移灶中的表达分布特点 ,并与颈淋巴结转移的发生进行相关性分析。结果 :80例口腔癌原发灶均有FAK表达 ,口腔癌生长前沿区细胞的表达明显强于中央区 (P <0 .0 1) ,呈条带状分布 ;FAK在转移灶中的表达强度与原发灶前沿区细胞一致 ;口腔癌前沿区细胞FAK表达强度与颈淋巴结转移的发生呈正相关 (P <0 .0 5 ) ,而中央区FAK表达强度与颈淋巴结转移无关 (P >0 .0 5 )。结论 :FAK在口腔癌中的表达主要分布于生长前沿区细胞 ,参与癌细胞的侵袭性行为 ,与口腔癌颈淋巴结转移的发生有关     

口腔鳞癌原发灶及淋巴结转移灶中巨噬细胞的检测及意义

目的：通过检测巨噬细胞在口腔鳞癌原发灶、淋巴结转移灶中的浸润情况,探讨其在口腔鳞癌中的功能及意义。方法：采用免疫组化SABC法分别检测CD68和CD34在42例口腔鳞癌、15例淋巴结转移灶和10例口腔正常组织的表达情况。结果：CD68在口腔鳞癌原发灶及淋巴结转移灶中的表达显著高于正常组（P〈0.01）;而且在淋巴结转移灶中的表达显著高于原发灶（P〈0.01）。同时CD34在肿瘤原发灶中的表达显著高于淋巴结转移灶和正常组织（P〈0.01）,而淋巴结转移灶和正常组织之间无显著性差异。结论：巨噬细胞在口腔鳞癌原发灶和淋巴结转移灶中的浸润情况并不一致,在淋巴结转移灶中,巨噬细胞的功能可能与原发灶中有所不同。     

转移抑制基因Kiss-1在舌鳞癌原发灶和淋巴结转移灶的表达

目的：探讨肿瘤转移抑制基因Kiss-1在舌鳞癌原发灶和淋巴结转移灶的表达。方法：采用免疫组织化学SP法检测59例舌鳞癌标本原发灶癌组织和其中21例发生淋巴结转移的转移灶中Kiss-1蛋白的表达,并分析其与临床病理参数的关系。结果：59例原发灶标本中Kiss-1蛋白的表达率：无淋巴结转移组为97．37％（37／38）,有淋巴结转移组为90．47％（19／21）,两组相比差异无统计学意义（p〉0．05）;但有淋巴结转移组的表达强度低于无淋巴结转移组,两组差异有统计学意义（p〈0．05）。在21例发生淋巴结转移的病例中,淋巴结转移灶表达率为28．57％（6／21）,明显低于原发灶的90．47％（19／21）,两组差异有统计学意义（p〈0．01）。Kiss-1蛋白表达与患者的年龄、性别、临床分期、组织学分级、嗜烟酒等因素无关（p〉0．05）。结论：舌鳞癌中Kiss-1蛋白表达减弱或丢失可能是导致舌鳞癌转移能力增强的因素之一。     

An immunohistochemical study of thrombomodulin in oral squamous cell carcinoma and its association with invasive and metastatic potential

Thrombomodulin (TM) is a glycoprotein that was originally identified on vascular endothelium and characterized as a natural endothelial anticoagulant. We reported previously that TM was also expressed at cell-cell boundaries of squamous epithelium, except in the basal layer cells and upper granular layer cells. The expression pattern of TM in the squamous cells implies that this molecule might be associated with keratinocyte differentiation, as well as being a potent anticoagulant. In the present study we examined TM expression immunohistochemically in biopsy specimens from 65 patients with primary oral squamous cell carcinoma (OSCC). and compared the results with the biological behavior of the carcinomas. The patients with intense TM expression in the carcinoma showed a significantly lower frequency of lymph node metastasis and significantly more favorable survival than those with negative TM expression. The TM expression was a better marker than the other prognostic factors, such as differentiation degree, tumor size and invasion mode. When TM expression was compared between the primary and metastatic lesions in the 36 patients who had lymph node metastasis, 14 (39%) showed decreased TM expression. 19 (53%) showed no change, and 3 (8%) showed an increase in the metastatic lesions. Wilcoxon's signed-rank test indicated that tumor cells positive for TM expression were significantly rarer in the metastatic lesions than in the primary tumors (P<0.05). These results indicate that reduced expression of TM is associated with metastasis of carcinoma cells. The reduction of TM expression seems to play an important role in the metastatic process of OSCC, and to be related with a poor outcome for the patients.     

Chemokine Receptor Expression in Oral Squamous Cell Carcinoma: Correlation with Growth Factor- and Cytokine-mediated Cell Migration in vitro

Metastasis is the chief cause of mortality in cancer patients. Recently, chemokines and chemokine receptors were shown to play an important role in the metastasis of various cancers. We examined the role of chemokine receptor-mediated signaling in the invasion potential of human oral squamous cell carcinoma (OSCC) cell lines that were derived from 5 primary tumors and 6 cervical lymph node metastases. Comprehensive analysis of the mRNAs for human chemokine receptors showed that the OSCC cell lines had uniform expression patterns of chemokine receptors. Overall, there were no consistent differences in the expression of chemokine receptors between primary site- and lymph node metastasis-derived cell lines. However, a highly invasive OSCC cell line (SAS-H1) expressed up-regulation of CCR5, CCR6, CCR7, CXCR1, CXCR6 and CX3CR1 compared to a poorly invasive OSCC cell line (SAS-L1). Then we examined whether factors in the tumor microenvironment regulated chemokine receptor expression in SAS-H1 cells. Specifically, transforming growth factor (TGF) -β1 enhanced the expression of CCR5, CCR6, CCR7 and CX3CR1. Pretreatment of SAS-H1 cells with transforming growth factor (TGF) -β1 increased the expression of CCR7 and CX3CR1, and then enhanced CCL21- and CX3CL1-induced directional migration (1.5-fold enhancement as compared with untreated control). In addition, CX3CL1 increased the adhesion of SAS-H1 cells on uncoated tissue culture plates. Neither chemokine stimulated cell proliferation. Treatment of SAS-H1 cells with CX3CL1 activated the phosphotidylinositol-3-kinase (PI3K) and MEK signal transduction pathways. Our results suggest that chemokine receptor-mediated signaling is involved in the local invasion and metastasis of human OSCC.     

口腔鳞癌中趋化因子受体CXCR4和CCR7表达及其与临床病理特征的关系

目的检测CXC类趋化因子受体4(chemokine CXC motif receptor 4,CXCR4)和CC类趋化因子受体7(chemokine CCmotif receptor 7,CCR7)在口腔鳞癌(oral squamous cell carcinoma,OSCC)中表达,探讨其与OSCC临床病理特点及颈淋巴结转移的关系。方法采用免疫组化和反转录聚合酶链反应(RT-PCR)检测64例OSCC组织原发灶、39例转移淋巴结组织和10例正常口腔黏膜组织中CXCR4及CCR7的表达,并分析其与临床病理参数的关系。结果 OSCC组织细胞中CXCR4、CCR7蛋白的阳性表达率分别为62.5%、65.6%,明显高于正常口腔黏膜组织细胞(P<0.01),其中有淋巴结转移组表达率分别为74.36%、84.62%,无淋巴结转移组表达率分别为44%、36%,差异均有统计学意义(P<0.05),而CXCR4、CCR7在淋巴结转移灶中的阳性表达率差异无统计学意义(P>0.05)。RT-PCR检测结果也证实,CXCR4及CCR7在OSCC细胞中均有阳性表达。此外,CXCR4及CCR7的表达与肿瘤的分化程度、侵袭程度和TNM分期密切相关(P<0.05),而与年龄、性别和肿瘤大小无关(P>0.05)。结论趋化因子受体CXCR4、CCR7的表达与OSCC侵袭发展和淋巴结转移密切相关。CXCR4、CCR7有可能成为OSCC治疗的新靶点。     

口腔鳞状细胞癌组织中USP22的表达及其意义

目的：检测USP22在口腔鳞状细胞癌（oral squamous cell carcinoma,0SCC）组织中的表达,并探讨其与0SCC的发生、发展及预后的相关性。方法：应用免疫组化染色法检测USP22在正常口腔黏膜、OSCC组织及转移淋巴结组织中的表达,并采用SPSSl7．0软件对结果进行统计学分析。结果：USP22在0SCC组织中的阳性率和染色强度随组织分化程度的降低而呈增高趋势（P〈O．05）,USP22在转移的淋巴结组织中的表达较OSCC组织更为明显（P〈O．05）,OSCC病人术后5年生存率随着USP22表达的升高而降低,差异有统计学意义（P〈O．05）。结论：USP22在0SCC的发生、发展及转移起到一定作用。USP22蛋白的高表达提示病人有一个差的预后。     

TEAD4与口腔鳞癌预后和细胞迁移侵袭的研究

目的:研究TEA转录因子4(TEAD4)在口腔鳞癌(OSCC)组织中的表达与临床病理参数、预后的关系。方法:免疫组化检测79例原发性OSCC标本和21例口腔正常上皮黏膜中TEAD4的表达水平,分析其与临床病理参数之间的关系。使用小干扰RNA(siRNA)转染沉默Cal27细胞内源性TEAD4,通过Western blot、qRT-PCR、划痕试验、Transwell侵袭等实验探究TEAD4沉默对OSCC细胞系Cal27迁移、侵袭的影响。结果:免疫组化结果显示TEAD4在OSCC组织中表达明显高于正常口腔黏膜(P<0.05)。TEAD4高表达与患者颈淋巴结转移、临床分期和患者总体生存率相关(P<0.05)。TEAD4沉默后Cal27细胞侵袭、迁移能力明显减弱。结论:TEAD4高表达与OSCC恶性表型及预后有关,参与OSCC细胞迁移、侵袭表型的调控。     

趋化因子受体CXCR4在口腔鳞癌中的表达和临床意义

目的研究口腔鳞癌中趋化因子受体CXCR4蛋白及mRNA的表达情况，探讨其与口腔鳞癌临床病理特点及颈淋巴结转移的关系。方法采用免疫组化SABC法和原位杂交法检测64例口腔鳞癌中CXCR4蛋白的表达及在CXCR4阳性表达的口腔鳞癌中CXCR4 mRNA的表达情况，并分析其与临床病理参数的关系。结果CXCR4 mRNA和CXCR4蛋白在口腔鳞癌中呈阳性表达，CXCR4蛋白的阳性表达率为62.5%。同时，CXCR4在伴有淋巴结转移病例中的表达显著高于无淋巴结转移者（P=0.014 4）。此外，CXCR4的表达与肿瘤的分化程度（P=0.004 2）、TNM分期（P=0.001 2）、侵袭程度（P=0.002 3）密切相关，而与年龄、性别和肿瘤大小无关。结论CXCR4在口腔鳞癌中有阳性表达，其表达水平与口腔鳞癌侵袭发展和淋巴结转移有关。CXCR4有可能成为口腔鳞癌治疗的新靶点。     

Endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma

J Oral Pathol Med (2012) 41 : 124–130 Background: Loco‐regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl‐2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl‐2 expression in lymphatic endothelial cells. Methods: Endothelial cells were selectively retrieved from paraffin‐embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection. RT‐PCR was used to evaluate Bcl‐2 expression in tumor‐associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)‐C on the expression of Bcl‐2 in primary human lymphatic endothelial cells. Results:  We observed that Bcl‐2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage‐matched tumors without metastasis. VEGF‐C induced Bcl‐2 expression in lymphatic endothelial cells via VEGFR‐3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF‐C and induce Bcl‐2 in lymphatic endothelial cells. Conclusions: Collectively, this work unveiled a mechanism for the induction of Bcl‐2 in lymphatic endothelial cells and suggested that endothelial cell Bcl‐2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.     

Fos‐related activator‐1 is overexpressed in oral squamous cell carcinoma and associated with tumor lymph node metastasis

J Oral Pathol Med (2010) 39 : 470–476 Background: Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB cells. Then, expression microarray analysis showed that the gene encoding fos‐related activator‐1 (Fra‐1) was significantly upregulated in the cancerous HB cells compared with HIOECs. Methods: To confirm the expression of Fra‐1 at mRNA and protein levels by real‐time PCR and western blotting analysis in the carcinogenesis model of OSCC and CAL27 cells. To investigate Fra‐1 expression in clinical samples from 30 primary OSCC patients by immunohistochemistry. Results: Fra‐1 expression was increased both at mRNA and protein levels in this carcinogenesis model of OSCC and CAL27 cells. Nuclear and cytoplasmic Fra‐1 protein expressions both increased in the cancerous tissues compared with those in the paired adjacent non‐malignant epithelia (nuclear: P < 0.001, cytoplasmic: P = 0.003). A higher level of nuclear Fra‐1 expression was seen in the tumor samples of patients with lymph node metastasis than those without lymph node metastasis (5.07 ± 1.33 vs 3.81 ± 1.33, P = 0.023). Higher level of Fra‐1 expression was also found in the tumor invasive margin than tumor center. Conclusions: Fra‐1 is a positive gene of OSCC development and progression, Fra‐1 can be used as a potential therapeutic target gene and an additional marker for evaluation of lymph node metastasis.