上皮间质转化相关基因在口腔鳞癌中的相关性及其与临床病理特征的关系研究

目的：观察上皮标志物和转录因子之间的相关性,从而确定这些因子在肿瘤上皮-间质转化（EMT）过程中所起的作用,进一步研究其与口腔鳞癌临床病理之间的关系,揭示其在口腔鳞癌发生发展中的作用及临床意义。方法：免疫组化SP法检测57例口腔鳞癌组织和10例正常口腔黏膜组织中E-cad、Snail、Slug、MMP-2、MMP-9表达,分析其与口腔鳞癌临床病理的关系及E-cad和Snail、Slug、MMP-2、MMP-9表达的相关性。结果：①．口腔鳞癌中E-cad、Snail、Slug、MMP-2、MMP-9的阳性表达率分别为29．82％、75．43％、82．46％、71．93％、78．95％。②．口腔鳞癌中E-cad的表达与分化程度有关（P＜0．05）,Snail、Slug、MMP-2、MMP-9的表达与分化程度无关（P＞0．05）;③口腔鳞癌中E-cad、Slug、MMP-2、MMP-9的表达与浸润和淋巴结转移有关（P＜0．05）,Snail与淋巴结转移和浸润无关（P＞0．05）。④．口腔鳞癌中E-cad的表达与Snail、Slug、MMP-9表达均呈负相关（P＜0．05）,而与MMP-2的表达无关（P＞0．05）。结论：转录因子与上皮标志物之间相关性明显,它们与口腔鳞癌的侵袭、淋巴结转移和分化程度相关。这些因子在EMT过程中不同阶段起作用,它们之间的相互作用是口腔鳞癌发生发展的重要环节。     

Twist、Snail、Slug在口腔鳞状细胞癌中的表达及其与各临床病理因素的关系

目的:探讨Twist、Snail、Slug在口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)中的表达水平及其与各临床病理因素之间的关系.方法:采用免疫组织化学方法检测术前未接受放化疗的有颈部淋巴结转移或无颈部淋巴结转移的60例原发OSCC患者肿瘤切除石蜡标本中Twist、Snail、...     

EMT在口腔鳞癌和扁平苔藓病理机制中的作用

上皮-间充质转化（epithelial-mesenchymal transition,EMT）,是上皮细胞通过特定的程序转化为间质细胞的生物学过程,EMT在胚胎发育、创伤愈合、肿瘤的侵袭迁移等过程中起重要作用,EMT在恶性肿瘤的侵袭迁移病理过程中的分子机制成为研究热点。EMT的发生涉及多种机制,与低氧微环境、炎症因子、细胞外基质及microRNA等有关。文中综合目前研究进展,就EMT在口腔鳞癌和扁平苔藓侵袭转移中的作用及其分子机制作一综述。     

上皮间质转化及其调控基因Twist在肿瘤侵袭转移中的作用

上皮间质转化(EMT)是胚胎发生、慢性炎症和纤维化以及肿瘤进展中的一个重要过程,以上皮细胞极性的丧失及其间质特性的获得为主要特征。Twist蛋白是属于碱性螺旋-环-螺旋蛋白家族中的高度保守的转录因子,最近的研究表明,Twist作为EMT过程中的关键调控因子,对肿瘤的侵袭和转移有重要影响。本文就EMT及其调控基因Twist在肿瘤侵袭转移中的作用研究作一综述。     

口腔鳞癌上皮-间质转化的研究进展

上皮-间质转化（EMT）是肿瘤侵袭和转移的首要步骤,对于EMT的研究将为靶向抗肿瘤治疗奠定基础。本文概述了EMT的发生过程和调控机制,对近年来口腔鳞癌中有关EMT研究的最新进展进行综述。     

miR-181a抑制舌鳞癌细胞上皮-间充质转化及化疗耐药机制的研究

目的:探讨miR-181a调控上皮-间充质转化(EMT)促进舌鳞状细胞癌细胞顺铂耐药的机制。方法:分别在CAL27/CDDP、CAL27中转染miR-181a mimics、miR-181a LNA,免疫印迹及免疫荧光检测E-cadherin、Vimentin的表达;免疫印迹检测Twist1、Slug的表达;Transwell及划痕实验检测细胞的侵袭、迁移能力;MTT法检测细胞对顺铂的半抑制率。利用生物信息学方法:预测Twist1可能为miR-181a的靶基因。构建包含Twist1结合序列片段的荧光素酶报告基因质粒,进行双荧光素酶报告基因实验。采用SPSS17.0软件包对结果进行单因素方差分析。结果:转染miR-181a mimics可逆转CAL27/CDDP的EMT表型,E-cadherin表达增强,Vimentin、Twist1及Slug表达降低,细胞的侵袭、迁移能力显著降低,IC₅₀下降47.57%±6.23%（P<0.05）。转染miR-181a LNA可诱导CAL27细胞发生EMT,E-cadherin表达降低,Vimentin、Twist1及Slug表达增强,细胞侵袭、迁移能力显著增强,IC₅₀升高55.61%±7.20%（P<0.05）。双荧光素酶报告基因实验证实,Twist1是miR-181a的靶基因。结论:miR-181a靶向调控Twist1,从而抑制舌鳞癌细胞上皮-间充质转化与顺铂耐药。     

上皮-间质转化与恶性肿瘤的侵袭转移

转移是肿瘤发展的一个重要阶段,且多数患者逝于肿瘤转移,所以肿瘤转移一直备受关注。上皮—间质转化（EMT）在恶性肿瘤的侵袭转移过程中发挥着关键的作用,而研究EMT的始发因素及其下游通路在肿瘤生长、侵袭、转移中的作用,阻断这一机制的发生发展,对恶性肿瘤的侵袭转移前的早期诊断、早期治疗有着非常重要的意义。下面就上皮—间质转化及其在组织胚胎发育过程中的作用、涉及的有关信号通路、与肿瘤侵袭转移的关系和在口腔恶性肿瘤侵袭转移中的作用等作一综述。     

蜗牛蛋白调控腭部发生发育的机制

蜗牛蛋白（Snail）参与调控腭部融合过程中腭中缝上皮（MES）细胞的上皮间质转化（EMT）、程序性细胞死亡（PCD）或存活以及细胞迁移等生物学行为,Snail基因失去了转化生长因子-β3的抑制而表达水平升高,此时Snail作为MEE/MES细胞的存活因子,导致腭中线上皮（MEE）/MES细胞持续存在并诱发腭裂。Snail基因编码具有锌指结构的Snail1、Snail2和Snail3等转录因子,其家族具有相似的结构,可以结合到E-钙黏蛋白的启动子,抑制其表达,可以在胚胎发生发育和肿瘤转移过程中诱导EMT。Snail基因可防止细胞因为存活因子的减少或PCD因子的积累而死亡,故Snail基因是MEE/MES细胞的存活因子和细胞运动的诱导因子。微小RNA（miRNA）不仅在人类癌细胞中发挥着重要的作用,而且在胚胎健康发育过程中必不可少。miRNA表达模式的变异和miRNA在mRNA的结合位点的结构变异,可能导致颅面发育变异。本文就Snail基因结构与功能、Snail基因在侧腭突发育过程中表达、Snail基因与MEE/MES细胞的EMT、Snail基因与MEE/MES程序性细胞死、Snail基因是MEE/MES细胞的存活因子、微小RNA调控Snail在MEE/MES消失过程中的作用等研究进展作一综述。     

口腔鳞状细胞癌中Slug及E-cadherin的表达

目的探讨口腔鳞状细胞癌(OSCC)中Slug及E-cadherin表达及差异性。方法收集口腔鳞癌标本30例,正常口腔黏膜组织20例,使用rt-PCR的方法检测Slug及E-cadherin mRNA的表达情况,分析其病理特征相关性。结果口腔鳞癌组织中Slug的表达高于正常口腔黏膜组织(P<0.05),而口腔鳞癌组织中E-cadherin的表达低于正常口腔黏膜组织(P<0.05),Slug和E-cadherin表达与TNM分期、淋巴结是否转移相关(P<0.05),而与性别、年龄、细胞分化程度、肿瘤浸润大小无关(P>0.05)。结论口腔鳞状细胞癌中Slug和E-cadherin的表达与肿瘤的发展、侵袭、转移密切相关。     

Smad7蛋白在口腔鳞癌组织中的表达及其对口腔鳞癌细胞增殖和迁移能力的影响

目的：检测Smad7蛋白在口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）中的表达及临床意义,探讨其在口腔鳞癌细胞增殖和迁移中的作用。方法：利用免疫组织化学染色,检测31例原发性口腔鳞癌患者肿瘤组织及癌旁标本中Smad7的表达,并分析其与淋巴结转移之间的关系;利用CCK-8及细胞划痕实验检测Smad7基因沉默对肿瘤细胞增殖及迁移能力的影响;通过Smad7 siRNA干扰HN4细胞,影响Smad7蛋白的表达,利用免疫印迹及qRT-PCR检测其对上皮-间充质转化（epithelial- mesenchymal transition,EMT）相关指标蛋白及RNA表达的影响。采用SPSS18.0软件包对数据进行统计学分析。结果：在口腔鳞癌肿瘤组织中,Smad7蛋白在细胞核和细胞质的表达均显著高于对应的癌旁组织。Smad7的表达与患者颈淋巴结转移显著相关。Smad7 siRNA干扰HN4细胞后,细胞的增殖能力减弱,迁移能力增强。Smad7 siRNA干扰使得HN4细胞神经钙黏附素（N-cadherin）蛋白的表达量升高,而上皮钙黏蛋白（E-cadherin）的表达量下降,N-cadherin蛋白及波形蛋白（Vimentin）RNA表达量显著升高,与对照组相比有显著差异。结论：Smad7的表达与口腔鳞癌患者颈淋巴结转移相关,且Smad7在口腔鳞癌肿瘤细胞的增殖及侵袭转移中发挥重要作用。     

Various immunostaining patterns of CD31, CD34 and endoglin and their relationship with lymph node metastasis in oral squamous cell carcinomas

BACKGROUND: Intratumoral blood vessels are known to play an important role in cancer growth and metastasis. The discrepancy in previous reports using various endothelial markers individually suggested us to investigate both normal and various tumor areas with a wide panel of vascular markers. METHODS: Here, we used a panel of three antibodies (CD31, CD34, and endoglin) as blood vessel markers to investigate the distribution and properties of blood vessels in normal oral tissues and squamous cell carcinomas. RESULTS: Many microvessels with strong remodeling activity as well as undifferentiated tumoral vascular endothelial cells and immature endothelial cells were present in the cancer cell nest and marginal area of cancer infiltration. Our results showed different vascular distribution patterns using various immunostaining markers in normal and tumoral tissues. CONCLUSION: Vascular distribution and properties of endothelial cells appear to be closely associated with metastasis.     

Stem cells in head and neck squamous cell carcinoma

The existence of a small subpopulation of tumourigenic cancer stem cells in the bulk of human head and neck squamous cancers (SCC) has been recognised in recent reports. This subpopulation has self-renewal properties and is responsible for the production of differentiated daughter cells that form the bulk of the tumour. Stem cells in head and neck SCC can be identified functionally using their self-renewal properties, or by their characteristic surface markers. As their resistance to contemporary cancer treatments may eventually lead to the failure of treatment there is an urgent need to better understand their biology with the ultimate goal of developing new diagnostic markers and curative cancer treatments.     

头颈部肿瘤干细胞的表面标志物及其分选

越来越多的研究显示,头颈部肿瘤干细胞很可能是头颈部肿瘤形成、复发、转移和耐药的根源。本文就头颈部肿瘤干细胞表面特异分子标志物,头颈部肿瘤干细胞的分选等研究进展作一综述。     

Cancer stem cells - new and potentially important targets for the therapy of oral squamous cell carcinoma

There is increasing evidence that the growth and spread of cancers is driven by a small subpopulation of cancer stem cells (CSCs) - the only cells that are capable of long-term self-renewal and generation of the phenotypically diverse tumour cell population. Current failure of cancer therapies may be due to their lesser effect on potentially quiescent CSCs which remain vital and retain their full capacity to repopulate the tumour. Treatment strategies for the elimination of cancer therefore need to consider the consequences of the presence of CSCs. However, the development of new CSC-targeted strategies is currently hindered by the lack of reliable markers for the identification of CSCs and the poor understanding of their behaviour and fate determinants. Recent studies of cell lines derived from oral squamous cell carcinoma (OSCC) indicate the presence of subpopulations of cells with phenotypic and behavioural characteristics corresponding to both normal epithelial stem cells and to cells capable of initiating tumours in vivo. The present review discusses the relevance to OSCC of current CSC concepts, the state of various methods for CSC identification, characterization and isolation (clonal functional assay, cell sorting based on surface markers or uptake of Hoechst dye), and possible new approaches to therapy.     

Expression of p63 and CD44 in oral squamous cell carcinoma and correlation with clinicopathological parameters

Squamous cell carcinoma (SCC) is the sixth most frequent malignant tumor of the head and neck region. Despite advances in therapeutic options over the last decades, the rate of mortality and morbidity has not been improved markedly. A small subset of cells, cancer stem cells (CSCs), with self-renewal properties have become a major focus of current cancer research. CD44 and p63 are identified as candidate stem cell markers in normal squamous epithelium and SCC. The role of these markers in oral squamous cell carcinoma (OSCC) is still debatable. The aim of this study was to evaluate immunohistochemical expression of these markers in OSCC samples and also correlates the expression of these markers with some clinicopathological parameters of prognostic significance including histological grading, TNM staging, overall survival (OS) rate as well as patients’ age, gender, and tumor location. CD44 and p63 were expressed in all studied lesions with different degrees. Statistically significant difference was observed between CD44 and p63 expression with tumor grade and stage with higher expression in high grade and advanced OSCCs. No significant relationship was detected between markers immunoreactivity and patients age, gender, tumor location as well as OS. These markers can possibly advance our understanding of the initiating mechanisms and pathogenesis of OSCC and also result in novel therapeutic target in cancer treatment.     

唾液腺黏液表皮样癌干细胞的分离、鉴定及生物学特征分析

目的: 筛选出唾液腺黏液表皮样癌细胞的特异基因,进一步了解肿瘤干细胞的成瘤机制。方法: 通过有限稀释法对肿瘤细胞行单细胞培养,观察肿瘤干细胞单克隆生长的情况。应用免疫磁珠分离技术,确定唾液腺黏液表皮样癌干细胞的相对特异性表面标志。采用SPSS 11.5软件包对数据进行统计学分析。结果: CD133在唾液腺黏液表皮样癌细胞株M3SP2单克隆细胞株中表达较高,具有显著差异（P<0.05）。结论: CD133可能是唾液腺黏液表皮样癌细胞株M3SP2单克隆细胞株的表面特异性抗原标记物或组合之一。     

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line

ObjectiveCancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia.DesignWe used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers.ResultsHN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40 μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44⁺ cells, CD105⁺ cells, and STRO-1⁺ cells was significantly abolished by apigenin.ConclusionApigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia.     

舌癌单细胞培养建系与癌干细胞相关标志的检测

目的 以舌癌Tca8113M1细胞系单细胞培养建系为基础,观察Tca8113M1细胞系中舌癌干细胞存在的现象及其相关标志的变化规律。方法 选取Tca8113M1细胞系,以有限稀释法进行体外单细胞培养并建立细胞亚系,在证实其成瘤性的基础上,进一步采用流式细胞术检测癌干细胞相关标志CD44、CD184、细胞外可溶性抗原（ESA）的表达情况,着重观察单个细胞培养形成细胞克隆的形态与时间。结果 以有限稀释法获取192个Tca8113M1舌癌单个细胞,在96孔板中进行体外培养,获取12个细胞亚系（获取比例为6.25%）,均有高成瘤性。癌干细胞相关标志CD44 与ESA均为高水平表达,而CD184表达则在12个细胞系之间有差异。在单个细胞培养中,形成完全克隆、部分克隆与旁克隆3种形态,12个细胞亚系均源于单个细胞形成的完全克隆,均可进行连续传代与扩增,而部分克隆与旁克隆则在后续培养中逐渐衰老与消亡。结论 Tca8113M1细胞系中可能存在癌干细胞,而单细胞培养可形成完全克隆并建立细胞亚系,是进行舌癌干细胞后续研究重要的细胞培养模式。     

Markedly increased Oct4 and Nanog expression correlates with cisplatin resistance in oral squamous cell carcinoma

J Oral Pathol Med (2011) 40 : 621–628 Background: Oral squamous cell carcinoma (OSCC) is the sixth most prevalent cancer worldwide. Cancer stem cells (CSC) model theoretically contribute to tumor growth, metastasis, and chemo‐radioresistance. Cisplatin is a widely used chemotherapeutic agent for OSCC treatment. The aim of this study was to compare stemness genes expression in chemo‐sensitive and chemo‐resistant specimens and further explore the potential markers that may lead to induce chemo‐resistance in OSCC. Methods: The study method is the treatment of OC2 cells with cisplatin select cisplatin‐resistant OC2 cells. Self‐renewal ability was evaluated by cultivating parental and cisplatin‐resistant OC2 cells within sphere‐forming assay after serial passages. Differential expression profile of stemness markers between parental and cisplatin‐resistant OC2 cells was elucidated. The parental and cisplatin‐resistant OC2 cells were assessed for migration/invasion/clonogenicity tumorigenic properties in vitro. Expression of stemness markers in chemo‐sensitive and chemo‐resistant patients with OSCC was performed by immunohistochemistry staining in vivo. Results: Sphere‐forming/self‐renewal capability was increased in cisplatin‐resistant OC2 cells. Cisplatin‐resistant OC2 cells highly expressed the stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2). Furthermore, cisplatin‐resistant OC2 cells increased migration/invasion/clonogenicity ability. Notably, up‐regulation of Oct4 and Nanog expression was significantly observed in cisplatin‐resistant patients with OSCC (**P < 0.01). Conclusions: These data indicate that cancer stem‐like properties were expanded during the acquisition of cisplatin resistance in OSCC. Clinically, oral cancer stemness markers (Oct4 and Nanog) overexpression may promote the OSCC’s recurrence to resist cisplatin.     

p53、C-erbB-2癌基因蛋白在涎腺粘液表皮样癌中的表达及其临床病理学意义

目的探讨癌基因蛋白p53、C-erbB-2（p185）过度表达与粘液表皮样癌生物学行为的关系。方法利用微波免疫组织化学方法对32例人粘液表皮样癌的癌基因蛋白p53、C-erbB-2进行检测。结果正常诞腺组织p53、C-erbB-2均为阴性反应。癌旁导管上皮细胞两者阳性率分别为10.0％及15.0％,但腺泡细胞阴性。粘波表皮样癌组织中p53、C-erbB-2阳性率分别为40.6％及46.9％。p53、C-erbB-2在粘液细胞、表皮样细胞及中间细胞内均有表达。p53、C-erbB-2的表达与粘液表皮样癌肿瘤组织学类型、分化程度及肿瘤复发等肿瘤生物学行为密切相关（P＜0.01）。结论p53、C-erbB-2可作为粘液表皮样癌的分化性标志物,用于监测病情、判断预后等。