组织蛋白酶B在口腔黏膜上皮癌变过程中的表达及意义

目的：探讨组织蛋白酶B（Cathepsin B,CB）在口腔鳞状细胞癌中的表达及临床意义。方法：以口腔黏膜上皮永生化细胞系（human immortalized oral epithelia cell line,HIOEC）及经过苯丙芘[benzo（a）pyrene,B（a）P]诱导产生鳞状细胞癌细胞（HB）而形成的口腔鳞癌体外癌变模型为研究对象,通过双向凝胶电泳（2-DE）和质谱分析（LC-MS/MS）筛选和鉴定出差异蛋白质CB。采用实时定量PCR、Western印迹和免疫组化方法,检测口腔鳞癌细胞和30例原发口腔鳞癌标本中的mRNA及蛋白质表达水平。采用SPSS10.0软件包对数据进行非参数检验。结果：与永生化HIOEC相比,HB和CAL27细胞中CB的mRNA水平显著升高,HB、Tca8113、TSCC、CAL27和OSC细胞中CB蛋白表达水平明显升高。30例口腔鳞癌患者癌组织中CB的mRNA和蛋白表达水平均较癌旁组织升高（P〈0.01）。CB的表达水平与肿瘤大小、淋巴结转移、临床分期、病理分化程度无显著相关性。结论：CB在口腔鳞癌中的表达明显升高,提示其与肿瘤的发生、发展具有一定关系。     

体外二维和三维立体培养的CAL-27差异表达蛋白的筛选

目的：比较体外二维和三维培养的CAL-27的蛋白表达,试图获得CAL-27立体培养和平面培养之间蛋白质表达的差异。方法：将CAL-27分别进行体外二维与三维培养,提取细胞总蛋白质,蛋白质样品通过双向电泳分离,扫描获得数字化的电泳凝胶图像并用PDquest图像分析软件比较分析,识别二维和三维培养CAL-27间的差异蛋白质点。实验在相同条件下重复3次。结果：二维和三维培养的CAL-27抽提的总蛋白质,经双向电泳、凝胶扫描、图像PDQuest软件分析、鉴定,发现蛋白质表达有差异,二维培养的口腔鳞状细胞癌细胞双向电泳凝胶图像上有1875个蛋白质点;三维培养的口腔鳞状细胞癌有1630个蛋白质点,重合的蛋白质点有867个,两者差异明显的蛋白质点38个。结论：三维立体培养与二维培养的CAL-27相比,存在明显的蛋白质表达差异,这些差异表达的蛋白质,值得深入研究。     

口腔鳞癌组织蛋白差异表达的蛋白组学测定

目的 应用蛋白质组学(proteomics)方法建立人口腔鳞癌组织与正常口腔黏膜组织中的差异蛋白质表达。方法 收集10例口腔鳞癌组织及正常黏膜组织,提取蛋白质,进行双向凝胶电泳,经图像分析软件分析识别差异表达,应用液相色谱-质谱分析鉴定差异蛋白质。结果 分析鉴定出S100A7、S100A8、S100A9在口腔鳞癌中高表达。结论 S100A7、S1000A8、S100A9在口腔鳞癌发生中发生了改变,为进一步筛选口腔鳞癌特异性的分子标志物打下了坚实的基础。     

应用双向电泳和质谱技术筛选口腔扁平苔藓差异蛋白

目的:筛选口腔扁平苔藓及正常黏膜组织的差异表达蛋白,为口腔扁平苔藓的发病机制及诊断治疗的研究提供实验室依据.方法:收集口腔扁平苔藓组织及正常口腔黏膜组织,提取组织蛋白,蛋白定量,进行双向电泳,图象分析寻找差异点,基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析鉴定表达差异量较大的蛋白质点,确定所分析的蛋白质类型和功能.结果:(1)人口腔扁平苔藓及正常黏膜组织的双向电泳图谱的平均蛋白质点数分别为1 576±67和1 608±73,同一组织凝胶在蛋白质点位置上重复性较好.(2)通过比较人口腔扁平苔藓与正常黏膜组织的双向凝胶电泳图谱,得到差异表达蛋白质点数为13个,其中7个点在口腔扁平苔藓中为高表达,6个点在口腔扁平苔藓中为低表达.选择1O个表达差异量较大的蛋白质点进行质谱和生物信息学分析,鉴定了其中的4个点,包括有锰超氧化物歧化酶(Mn-SOD),膜联蛋白I,波形蛋白,未知蛋白等.结论:锰超氧化物歧化酶,膜联蛋白I,波形蛋白,未知蛋白可能参与了口腔扁平苔藓的发生和发展,其相关机制尚待进一步阐明.双向电泳-质谱分析技术为口腔扁平苔藓发生发展过程中差异表达蛋白的研究提供了有效的技术手段.     

双向电泳分析口腔扁平苔藓蛋白表达差异

目的：建立人口腔扁平苔藓（OLP）及正常黏膜组织的双向凝胶电泳图谱，分析其差异表达的蛋白质，筛选分子标记物。方法：采用固相pH梯度（immobilized p gradient，IPG）双向凝胶电泳（two-dimensional gel electrophoresis，2-DE）分离OLP及正常黏膜组织的总蛋白质，银染显色，Imagemaster图像分析软件分析差异蛋白质点。结果：①人口腔扁平苔藓及正常黏膜组织凝胶的平均蛋白质点数分别为1576±67和1608±73，凝胶图谱分辨率、重复性较好。②通过比较OLP与正常黏膜组织的双向凝胶电泳图谱，差异表达蛋白质点数为13个，其中7个点在口腔扁平苔藓中为高表达，6个点在OLP中为低表达。结论：本研究建立了分辨率高且重复性较好的OLP及正常黏膜组织的双向凝胶电泳图谱，发现存在一些差异表达的蛋白质，为进一步筛选OLP特异性的分子标志物奠定了基础。     

为研究口腔鳞癌发生发展的机制建立实验平台

《口腔黏膜上皮细胞体外癌变模型的建立》通过口腔常见的化学致癌剂B（a）P诱导永生化口腔上皮细胞HIOEC细胞，促使细胞恶性转化，逐步筛选得到具有恶性表型的HIOEC-B（a）P细胞。建立包含正常口腔黏膜上皮细胞、永生化上皮细胞的体外癌变模型。这个过程中包含了生物因素（HPV）、化学因素[B（a）P]和血清中的生长因子等作用因素；细胞经历了从正常到永生化，逐步恶变的过程。这样一个多因素、多步骤、多阶段的长期体外恶变过程，可以在一定程度上反映口腔黏膜上皮细胞的恶变，为进一步研究口腔鳞癌发生、发展的机制建立了实验平台，具有重要的研究价值。国内尚无同类报道。研究结果值得推广和发表，建议以“快速通道”的形式发表。     

双向电泳分析口腔鳞癌组织蛋白表达差异

目的:利用蛋白质组学方法建立分辨率高和重复性好的人口腔鳞癌组织及正常口腔黏膜组织的双向凝胶电泳图谱.分析其差异表达的蛋白质,为进一步寻找口腔鳞癌标志物奠定基础.方法:采用固相pH梯度双向凝胶电泳分离人口腔鳞癌组织及配对的正常口腔黏膜组织的总蛋白质,凝胶经银染显色后,ImagingMaster 2D图像分析软件进行比较分析,识别差异表达的蛋白质.结果:①癌组织和正常口腔黏膜组织凝胶的平均蛋白质点数分别为2325±390个和2487±281个.②通过比较分析10例口腔鳞癌组织及正常口腔黏膜的双向凝胶电泳图谱,得到差异表达蛋白点数为29个,这29个点在癌组织中均为低表达.结论:本研究建立了分辨率高且重复性较好的人口腔鳞癌组织及其正常口腔黏膜组织的双向凝胶电泳图谱.发现两者间存在一些差异表达的蛋白质,为进一步筛选口腔鳞癌特异性的分子标志物打下了坚实基础.     

口腔鳞癌患者与健康人唾液差异蛋白组学的初步研究

目的:研究口腔鳞癌患者与健康人唾液蛋白差异表达情况.方法:收集口腔鳞癌患者唾液17例,健康人唾液17例,通过以双向凝胶电泳技术以及质谱鉴定和生物信息学分析,寻找在口腔鳞癌患者唾液中差异表达的蛋白质.结果:选取口腔鳞癌患者和正常人唾液中具有明显差异表达的10个蛋白质点,进行质谱鉴定和生物信息学分析,共鉴定出3种差异表达的蛋白质,其分别为S100A8、S100A8/S100A9及表皮角蛋白2(EK2)在口腔鳞癌患者唾液中均呈高表达.结论:S100A8、S100A8/S100A9及EK2可能与口腔鳞癌患者发生有关.     

LMP-1转染人胎盘源间充质干细胞前后蛋白质组学研究

目的:采用蛋白质组学技术检测LIM矿化蛋白-1(LIM mineralization protein-1,LMP-1)转染人胎盘源间充质干细胞(Human placenta-derived mesenchymal stem cells,hPDMSCs)前后蛋白质组学改变。方法:提取hPDMSCs LMP-1和hPDMSCsvector细胞总蛋白,通过二维凝胶电泳(two-dimensional gel electrophoresis,2-DE)制备两组细胞的二维电泳图谱,并用PDQuest软件进行图像分析,寻找差异表达的蛋白质点,并用基质辅助激光解吸粒子-飞行时间电离质谱(matrix-assisted laser desorption-ionization time-of-flight tandem mass spectrometry,MALDI-TOF-MS)分析技术对这些差异表达的蛋白质点进行鉴定,并选取6个与成骨相关的蛋白(PCNA, vimentin, annexin A1, annexin A2, eEF2和peroxiredoxin-1)作为验证对象,蛋白质印迹法检测各蛋白在两组细胞中的表达水平。结果:采用2-DE技术建立了hPDMSCsLMP-1和hPDMSCsvector两组细胞的二维电泳图谱;PDQuest软件分析图像发现两组细胞有22个差异蛋白质点,采用MALDI-TOF-MS质谱初步鉴定出15个蛋白质点,其中表达上调9个,下调6个。Western blot检测结果与蛋白质组学一致。结论:LMP-1基因转染hPDMSCs后共筛选获得15个差异表达的蛋白,它们是成骨分化潜在的重要参与者,为进一步阐明LMP-1对hPDMSCs细胞功能和成骨分化影响的分子机制奠定基础。     

口腔鳞癌组织与口腔正常黏膜组织蛋白质差异表达的初步研究

目的:筛选口腔鳞癌及正常口腔黏膜组织的差异表达蛋白质,为研究口腔鳞癌发生机制提供实验依据.方法: 收集10 例口腔鳞癌组织及正常口腔黏膜组织,进行二维电泳,选择在表达差异量较大的29 个点进行质谱和生物信息学分析,确定所分析的蛋白质类型. 结果: 口腔鳞癌及相应正常口腔黏膜组织凝胶的平均蛋白质点数分别为2 325±390和2 487±281.双向凝胶电泳图显示,口腔鳞癌及正常口腔黏膜组织的差异表达蛋白质点数为29 个,这29 个点在癌组织中均为低表达,对其进行了质谱(PMF)和生物信息学分析,鉴定了其中的3 个点,它们是:β纤维蛋白(fibrin beta)、磷酸丙糖异构酶(triosephosphate isomerase TIM)、unknown蛋白.结论: β纤维蛋白、磷酸丙糖异构酶、unknown蛋白在口腔鳞癌发生发展过程中发生了改变,其机制尚待进一步阐明.     

Overexpression of cytokeratin 17 protein in oral squamous cell carcinoma in vitro and in vivo

Objective:  To determine the cytokeratin 17 (CK17) expression in oral squamous cell carcinoma (OSCC) both in vitro and in vivo .
Methods:  Comparative proteomic analysis of an in vitro cellular carcinogenesis model of OSCC (including a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells and another kind of cells (HB56 cells) at the early stage of carcinogenesis was performed to identify differentially expressed proteins. CK17 was further validated in vitro (cellular carcinogenesis model and other three OSCC lines) and in vivo (tissues from six healthy persons and 30 primary OSCC patients) by Western blotting and immunohistochemistry respectively.
Results:  Increased CK17 expression was identified by two-dimensional gel electrophoresis and liquid chromatography–tandem mass chromatography in the HB56 and HB96 cells over HIOECs. Western blotting confirmed the increased CK17 expression in the HB56, HB96 cells and other three OSCC lines. Immunohistochemistry confirmed the increased CK17 expression in the cancerous tissues from OSCC patients compared with the paired adjacent non-malignant epithelia.
Conclusion:  Increased CK17 expression may play an important role in the carcinogenesis progression of OSCC; however, further studies on the molecular function of CK17 are encouraged to clear the precise mechanism of CK17 in OSCC.     

Increased expression of Cathepsin B in oral squamous cell carcinoma

Previously, an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelial cells (HIOECs), a line of cancerous HB96 cells and another type of cell (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells. Cathepsin B was one of the significantly up-regulated proteins accompanying cellular transformation. Cathepsin B was further validated for its expression in the three cell lines and in clinical samples of tumour tissues and their adjacent normal epithelia from 30 primary OSCC patients. Western blot analysis and real-time PCR detected increased Cathepsin B protein and mRNA levels in the cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry and real-time PCR showed elevated Cathepsin B protein and mRNA levels in the tumour tissues over the adjacent non-malignant epithelia from OSCC patients. The results presented here suggest that the expression of Cathepsin B increases along with the cancerisation in OSCC both in vitro and in vivo, and it may serve as a candidate biomarker of OSCC.     

Increased expression of Annexin A2 in oral squamous cell carcinoma

Previously, in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells, and another kind of cells (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells, and Annexin A2 shown as one of the significantly up-regulated proteins accompanying cellular transformation. Annexin A2 was further validated for its expression in the three kinds of cells and in the clinical samples of tumour tissues and their adjacent normal epithelia from primary OSCC patients. Western blot analysis and real-time PCR detected increased Annexin A2 protein and mRNA levels in cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry showed elevated Annexin A2 protein expression in tumour tissues over the adjacent non-malignant epithelia from OSCC patients; however, the mRNA levels between tumour and normal tissues did not change significantly. Interestingly, levels of Annexin A2 protein expression negatively correlated with the tumour differentiation grades. The results presented here suggest that Annexin A2 protein may play important roles in carcinogenesis of OSCC, and it may also serve as a candidate biomarker for pathologic differentiation grade of OSCC.     

HPV口腔黏膜上皮体外癌变模型与免疫治疗

口腔黏膜上皮体外癌变模型，模拟了口腔黏膜上皮在体外的一个多因素、多步骤的癌变过程，是研究口腔鳞癌发生发展机制和治疗实验的必不可少的研究手段。免疫治疗被认为是可以清除残存癌细胞和感染病毒细胞的最有效方法这一。本文旨在通过探讨HPV口腔黏膜上皮体外癌变模型及免疫治疗的研究进展，为口腔鳞癌发病机制和治疗手段提供思路。     

苯丙芘诱导口腔上皮细胞癌变过程的差异基因表达谱分析

目的：检测并分析苯丙芘诱导的口腔上皮细胞癌变过程中基因表达的变化，寻找在口腔上皮细胞癌变过程中起重要作用的基因。方法：使用课题组前期建立的一株永生化细胞系，以及经过苯丙芘诱导而逐步产生的具有成瘤性的肿瘤细胞系【首代成瘤细胞（HB-56P）及典型鳞状细胞癌细胞（HB-94P）】，通过Affymetrix U133 plus 2．0基因芯片。检测在永生化细胞（HIOEC）向成瘤细胞转化过程中的基因表达变化。使用GenSpring7．0进行标准化及单因素方差分析，通过Venn图分析差异基因在癌变不同阶段的变化，并进一步通过GO术语进行注释。对部分基因使用实时定量PCR在细胞系中进行了验证。结果：在HIOEC向HB-94P转化过程中，共有883条差异基因，大部分集中于肿瘤发生的早期阶段。差异基因主要涉及大分子代谢、信号传导等生物学过程。具有过渡金属离子结合能力、腺核苷酸结合能力及激酶活性。其蛋白产物主要是膜结合蛋白，位于核内或细胞骨架。IGFBP3、S100A8、MAP2K、KRT6B、GDF15和MET的表达基本符合芯片结果。结论：本研究检测了苯丙芘相关口腔上皮细胞癌变过程中基因表达的变化，为苯丙芘相关的口腔癌分子发病机制研究提供了基础。     

Profiling of differentially expressed genes in porcine epithelial cells derived from periodontal ligament and gingiva by DNA microarray

OBJECTIVE: It is unknown which genes are differentially expressed in cultured epithelial cells derived from the epithelial rests of Malassez (ERM) in periodontal ligament and oral gingival epithelium (OGE). This study analysed the different gene expression of OGE and ERM cells using a DNA microarray technique. DESIGN: Epithelial cells from ERM and OGE were isolated from porcine periodontal ligament and oral gingival epithelium. Each RNA sample extracted from the cells was reverse transcribed into cDNA and labelled with either cytidine 5-dUTP (Cy5) or cytidine 3-dUTP (Cy3). These labelled cDNA probes were then mixed and simultaneously hybridised to the Pig 13K microarray plate bearing 13,295 different genes (Operon, AL). Cellular enzyme-linked immunosorbent assay (CELISA) was performed to confirm the expression at protein level. RESULTS: There were nine genes common to the triplicate microarrays in ERM cells and one in OGE cells. Four of the nine genes including tissue factor (TF), FAT cadherin (FAT) and two unknown genes were expressed at levels more than threefold higher in ERM cells than in OGE cells. The protein levels of both TF and FAT in ERM cells were significantly higher than those in OGE. CONCLUSION: TF and FAT may act as markers to distinguish ERM cells from OGE cells in vitro.     

Metalloproteinase expression in normal and malignant oral keratinocytes: stimulation of MMP-2 and -9 by scatter factor

Matrix metalloproteinases (MMPs) are Zn2+ dependent proteases produced by a variety of cell types. They have a fundamental role in tissue remodelling, tumour invasion and metastasis. Scatter factor (SF), secreted by fibroblasts, has a paracrine action on epithelial cells and binds the trans-membrane c-met receptor inducing loss of adhesion, cell motility and invasiveness in vitro. The purpose of this study was to test if SF can regulate the production of MMPs by epithelial cells. Supernatants from oral squamous cell carcinoma-derived cells (H375 and H376), a human keratinocyte line (UP), and primary cultures of oral mucosal keratinocytes, grown in the presence or absence of SF, were analysed using 0.1% gelatin zymography. MMPs were characterised by comparison with human recombinant enzymes and by the use of specific inhibitors. Oral mucosal keratinocytes, UP, and H357 cells expressed MMP-2 and MMP-9, whilst H376 cells only expressed MMP-2. SF increased the expression of MMP-9 in UP and MMP-2 in H376 supernatants. Both MMP-2 and MMP-9 activity were increased in H357 and normal keratinocyte supernatants. This could be blocked using a human recombinant anti-SF antibody. In all epithelial lines tested, c-Met, the cell surface receptor for SF, could be detected. The results indicate that SF stimulates MMP expression in UP, H376, H357, and normal oral mucosal cells and points to a role for SF in the regulation of oral keratinocyte behaviour in wound healing and neoplasia.     

两种基因芯片数据分析软件在口腔黏膜下纤维化差异表达基因分析中的应用

目的 运用基因芯片数据分析软件对口腔黏膜下纤维化(oral submucosa fibrosis,OSF)差异表达基因进行分析,研究其隐含的生物学意义.方法 利用DAVID和Onto-express两种基因芯片数据分析软件对前期筛选的865个OSF差异表达基因进行染色体定位、GO分析(gene ontology)和遗传关联疾病分析.结果 差异表达基因主要定位于1、2、5、6、7、11和12号染色体上(P＜0.01);GO分析显示差异表达基因主要参与免疫反应、防御反应等生物过程,并主要与细胞外基质、细胞骨架、细胞膜的组成和蛋白结合,细胞外基质结构组成和信号转导激活等分子功能相关;而与这些基因有遗传关联的疾病主要有感染,免疫和心血管疾病等.结论 运用基因芯片数据分析软件可快速分析大量的基因芯片数据,实现对差异基因初步的功能归类,为OSF的发病机制和流行病学研究提供新的思路.     

小分子锌指蛋白1在4-硝基喹啉-N-氧化物诱导SD大鼠颊黏膜癌变中的表达研究

目的 研究口腔黏膜癌变中小分子锌指蛋白1（LMO1）在基因转录和蛋白水平的表达变化。方法 取本课题组前期4-硝基喹啉-N-氧化物（4NQO）饮水法构建鳞癌动物模型的49例样本进行苏木精-伊红（HE）染色,依据上皮异常增生程度确定实验组病理分级并确定实验分组;免疫组织化学染色定性确定LMO1的表达部位;实时荧光定量聚合酶链式反应（RT-qPCR）和Western blot检测5组样本中LMO1 mRNA和蛋白的表达。结果 HE染色确定对照组7例,轻度组6例,中度组11例,重度组9例,OSCC组16例。免疫组织化学染色检测可见,LMO1主要表达于细胞质,对照组、轻度组、中度组、重度组和OSCC组的阳性表达率分别为14.3%、33.3%、81.8%、88.9%、93.8%。RT-qPCR检测可见,对照组LMO1 mRNA的表达量最低,OSCC组最高,对照组与轻度组间mRNA表达差异无统计学意义（P>0.05）,其余各组组间两两比较,mRNA的表达差异均有统计学意义（P<0.05）。Western blot检测可见,随着上皮异常增生程度的加重,LMO1的蛋白表达量逐渐上升,在OSCC组中表达最高。结论 口腔黏膜癌变进程中,LMO1 mRNA和蛋白异常表达,且mRNA和蛋白表达量随上皮异常增生程度的加重而增加。