Aromatase expression in normal human oral keratinocytes and oral squamous cell carcinoma

Aromatase is the enzyme that catalyzes the conversion of androgen to oestrogen. Aromatase expression in extra-gonadal sites and local oestrogen synthesis play an important role in the physiological conditions and in the growth of certain neoplasms. OBJECTIVE: The purpose of this study was to investigate aromatase expression in oral keratinocytes and oral squamous cell carcinoma (SCC). DESIGN: Immunocytochemistry and RT-nested PCR were used to detect aromatase protein and mRNA expression in primary human oral epithelial cell culture and in an oral SCC cell line. Immunohistochemistry was used to detect aromatase protein expression in frozen and archival human tissue sections of normal oral epithelium and oral SCC. RESULTS: Cytoplasmic immunostaining was found in normal oral keratinocytes and SCC cells in culture. The common coding region of aromatase mRNA was detected in the oral keratinocytes derived from five different normal individuals and in the SCC cell line. However, there were variations in aromatase exon 1 expression among normal oral keratinocyte samples. Cytoplasmic staining was found in normal oral epithelium and well-differentiated oral SCC but not in poorly differentiated oral SCC by immunohistochemistry. CONCLUSION: Aromatase was expressed in normal oral keratinocytes and oral SCC both in cell culture and in tissues, indicating local oestrogen synthesis in normal and neoplastic conditions of oral epithelium.     

Khat induces G1-phase arrest and increased expression of stress-sensitive p53 and p16 proteins in normal human oral keratinocytes and fibroblasts

Khat is a psychostimulant plant used by over 10 million people daily, mainly in eastern Africa and the Middle East. Previous studies have suggested an association between khat use and oral lesions such as hyperkeratosis and oral cancer. This study investigated the effects of an extract of khat on primary normal human oral keratinocytes (NOK) and normal human oral fibroblasts (NOF). Low (sublethal) concentrations of khat inhibited the proliferation of both cell types in a dose-dependent and time-dependent manner. Both NOK and NOF treated with khat accumulated in the G1-phase of the cell cycle and showed increased expression of the stress-sensitive p53 protein after 24 h. Normal human oral keratinocytes showed a profound increase in p16^INK4A (p16) after 24 h and showed morphological changes suggesting cell differentiation. Normal human oral fibroblasts showed growth inhibition and increased expression of p21^WAF1/CIP1 (p21) within 24 h. The concentrations of khat tested in this study were within the range of those found in the oral cavity of khat chewers. The results show that stress induced by khat modulates the cell cycle in oral keratinocytes and fibroblasts. It is further speculated whether khat could have similar effects in vivo , especially in keratinocytes.     

Analysis of proliferation,apoptosis and keratin expression in cultured normal and immortalized human buccal keratinocytes

The current study was undertaken to analyse growth and differentiation-related functions of normal keratinocytes (NOK) and an SV40T-immortalized keratinocyte line (SVpgC2a) from buccal mucosa, viewing the latter cell line as a model of a dysplastic epithelium. Morphological and immunohistochemical assessments of organotypic epithelia generated from 10 or 17 d of culture showed three- to five-fold higher apoptotic and proliferative activity in SVpgC2a relative to NOK. Conditions with or without serum (up to 10%) did not significantly influence these parameters in NOK whereas serum supported proliferation of SVpgC2a. Both cell types showed basal expression of collagen IV and laminin 1, indicating basal lamina, as well as vimentin, indicating an activated, proliferative state. Reduced expression of keratin, including the non-keratinizing marker K13, was seen in SVpgC2a. Assessment of proliferative monolayer cultures by microarray showed that NOK transcribed tissue-specific keratins, but also the epidermal keratin K2a, several simple epithelial keratins and low levels of hair keratins. SVpgC2a transcribed keratins seen in epithelial dysplasia, and K2a and hair keratins, albeit at low level. Overall, the results implied aberrant apoptosis, proliferation and keratin expression in the immortalized state of SVpgC2a. Comparison of NOK and SVpgC2a under identical culture conditions may serve to model the progression from a normal to a pre-neoplastic state of buccal epithelium.     

Cytotoxicity of polymerized commercial cyanoacrylate adhesive on cultured human oral fibroblasts

Cyanoacrylate (CA) has been used as both a commercial and tissue adhesive. Dentists may have had the experience of patients repairing their own acrylic-based dentures using a cyanoacrylate (CA) adhesive known as 'super glue'. This study evaluated the cytotoxicity of commercial CA adhesives when fully polymerized, as well as the toxicity of substances released from polymerized commercial CA adhesives after incubation of these materials for various periods of time. Toxicity was tested on cultured oral fibroblasts. Dead cells found around the various CA-coated filter papers constituted inhibitory zones which varied from 200-1000 microns and which persisted for two weeks. Control oral fibroblasts grew to approach the wax-coated filter paper. Cell viability testing using MTT and crystal violet staining methods supported the conclusion that polymerized CA-coated filter paper released substances that are toxic to cells, while wax-coated filter paper gave the same result as the control. The crystal violet staining method was also used to investigate the cytotoxicity of various CA materials after incubation for one, three, seven and 14 days and showed that CA continued to release cytotoxic substances at about the same level for at least two weeks. It can be concluded that, if CA adhesive is used for repair of broken dentures, it will release substances which are toxic to human oral fibroblast cells. This release of substances may persist for at least two weeks.     

NGF mRNA在人牙周膜成纤维细胞的表达

目的:研究牙周组织中是否存在神经生长因子(NGF)的表达。方法:采用RT-PCR方法研究培养的人牙周膜成纤维样细胞(hPDLF)是否表达NGF mRNA。结果:通过RT-PCR技术,在培养的hPDLF中扩增出NGF mRNA阳性产物。结论:通过离体研究初次显示培养的人牙周膜成纤维样细胞(hPDLF)表达NGF mRNA。     

人口腔癌相关成纤维细胞的分离培养及鉴定

目的：分离培养及鉴定口腔癌相关成纤维细胞（ CAFs）,为探究CAFs可能在肿瘤微环境促进口腔癌细胞的侵袭转移中发挥重要作用奠定基础。方法：用组织块培养法和酶消化法分离培养、纯化原代口腔癌相关成纤维细胞CAFs和口腔黏膜正常成纤维细胞（ NFs）;细胞免疫荧光技术及免疫蛋白印迹法对口腔CAFs和NFs进行鉴定。结果：口腔CAFs和NFs分离培养成功,口腔CAFs呈长梭形,达到一定密度时呈多层生长,排列紊乱;NFs呈多胞质突的扁平星状,无重叠生长现象,排列有一定方向性。上皮标志物细胞角蛋白18（ CK18）免疫荧光染色在口腔CAFs和NFs均呈阴性,间质标志物波形蛋白（ Vimentin）染色均呈阳性。α?平滑肌动蛋白（α?SMA）、成纤维细胞特异性蛋白?1（FSP?1）,成纤维细胞激活蛋白（FAP）、血小板衍生生长因子受体α（ PDGFR?α）在CAFs染色呈阳性,而在NFs中染色呈阴性。结蛋白（ Desmin）在CAFs和NFs中染色均呈阳性。蛋白表达水平上,相对于NFs,CAFs中α?SMA、PDGFR?α蛋白表达呈升高趋势,FSP?1呈下降趋势,而FAP、Desmin在 NFs和CAFs中表达无明显差异。结论：CAFs与NFs相比,在形态结构、生长方式、蛋白表达等方面均有明显差异。     

Effects of nicotine on proliferation, cell cycle, and differentiation in immortalized and malignant oral keratinocytes

BACKGROUND: Numerous epidemiological studies have reported that tobacco smoking is a major risk factor for oral cancer, but relatively little is known about the effect of nicotine, a major product of cigarette smoking, on immortalized oral keratinocytes and cancer cells. METHODS: We investigated the effects of nicotine on the growth and differentiation of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12), and human skin keratinocytes (HaCaT), in the monolayer and in the three-dimensional (3D) raft cultures using the MTT assay, Western blotting, and cell cycle analysis. RESULTS: Nicotine inhibited the proliferation of immortalized and malignant keratinocytes in dose- and time-dependent manners as determined by MTT assay. The 3D organotypic culture showed that nicotine at high concentration (300 microM) inhibits epithelial maturation, surface keratinization, and decreased epithelial thickness. Flow cytometry showed that nicotine inhibited cell cycle progression by inducing G(0)/G(1) arrest of HaCaT, IHOK, HN4, and HN12 cells without causing apoptosis. Nicotine treatment increased p21 expression in immortalized cells (HaCaT, IHOK) and oral cancer cells (HN4, HN12), but decreased pRb and p53 expression in oral cancer cells. Moreover, after high-dose nicotine treatment, the involucrin expression increased markedly in immortalized cells, but not in oral cancer cells. CONCLUSIONS: We demonstrated that nicotine inhibits growth through cell cycle arrest at G(0)/G(1) phase probably by increasing the expression of p21(WAF1/CIP1). Nicotine also affects epithelial differentiation in immortalized and malignant oral keratinocytes. Malignant oral keratinocytes appear to be more resistant to the effects of nicotine on epithelial growth and differentiation as compared to the immortalized cells.     

苯妥英钠对人牙周膜成纤维细胞生物学活性影响的实验研究

目的研究苯妥英钠（PHT）对体外培养的人牙周膜成纤维细胞（hPDLF）生物学功能的影响,并探讨其用于促进牙周组织再生的可能性。方法将不同质量浓度的PHT（1、5、20、100、500、2 500 mg/L）加入体外培养的第4代hPDLF,检测其对细胞增殖活性、蛋白合成、碱性磷酸酶（ALP）活性的影响;Von Kossa染色法检测PHT（20、100、500 mg/L）对第4代hPDLF矿化结节形成能力的影响;应用ELISA法测定PHT（20、100 mg/L）对第3代hPDLF中骨形态发生蛋白- 2（BMP- 2）表达的影响。结果在20、100 mg/L的质量浓度时,PHT可显著促进hPDLF的增殖及分化（P<0.01）;在质量浓度100 mg/L时,PHT可增强hPDLF的蛋白合成（P<0.05）;同时,PHT在质量浓度100 mg/L可显著促进hPDLF的矿化能力及BMP- 2的表达（P<0.01）。但高质量浓度（2 500 mg/L）的PHT则严重抑制细胞的生物学活性。结论适当质量浓度的PHT对hPDLF的增殖和分化有促进作用,但过高质量浓度的PHT具有细胞毒性,不利于牙周组织的修复和再生。     

Dual effect of nitric oxide in immortalized and malignant human oral keratinocytes: induction of apoptosis and differentiation

BACKGROUND: Nitric oxide (NO) is known to act cytostatically on several tumor cell when functioning as an effector molecule of activated macrophages, but the differential effects of NO on immortalized and malignant oral keratinocytes have not been examined. METHODS: We investigated the influence of NO on the proliferation, cell cycle, apoptosis, and differentiation of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, sulforhodamine B (SRB) assay, flow cytometry, nuclear DNA staining, and Western blotting. RESULTS: The MTT and SRB assays indicated inhibited growth of IHOK and HN4 cells that were treated with sodium nitroprusside (SNP) at concentrations higher than 1 mM but not at lower SNP concentrations. The higher concentrations of SNP up-regulated the apoptosis-related protein expression, which is consistent with the analyses of sub-G(1) phase arrest, annexin V-FITC (fluorescein isothiocynate) staining, nuclear staining, and DNA fragmentation. On the other hand, the lower concentrations of SNP enhanced the expression of keratinocyte differentiation markers in IHOK and HN4 cells. CONCLUSIONS: These data suggest that high concentrations of NO can inhibit the growth of IHOK and HN4 cells through the induction of apoptosis, while low concentrations of NO can induce cytodifferentiation. The dual effects of NO, namely, the induction of apoptosis or cytodifferentiation, have important implications for the possible anti-oral cancer treatment.     

口腔癌相关成纤维细胞的生长增殖特性研究

目的 :对口腔癌相关成纤维细胞 (CAFs)的生长增殖特性进行研究。方法 :复苏前期实验中冻存的第3代纯化舌CAFs及同部位的正常成纤维细胞 (NFs) ,在保证培养条件、接种细胞量、观察时间基本一致的情况下 ,比较二者的生长曲线、群体倍增时间、有丝分裂指数曲线 ,并用MTT法测定细胞的增殖活力。结果 :从总体上比较 ,CAFs的生长速度、分裂增殖能力及存活能力均较NFs明显增强 ( p <0 .0 5 )。CAFs和NFs的细胞群体倍增时间分别为 60 .19h和 76.61h。结论 :口腔CAFs的生长增殖特性发生了变化 ,推测可能是对肿瘤 --宿主界面微生态环境进行调控的机制之一     

雌激素对牙周膜细胞生物学性质的影响

目的：明确雌激素对体外牙周膜成纤维细胞迁移、增殖和碱性磷酸酶的影响，以探讨雌激素对牙周组织改建的影响。方法：SD大鼠来源的牙周膜成纤维细胞经传代培养至第四代，建立体外创伤模型，分别在普通培养基和含雌激素的培养基中培养，观察测量细胞迁移情况，运用MTT、酶联检测仪测定细胞的增殖速度，对细胞的ALP进行提取和测定。结果：MTT结果硅示加入雌激素对该细胞的增殖无明显影响；在含雌激素的培养基中成纤维细胞的迁移速度加快；同时牙周膜成纤维细胞的ALP表达娃著提高。结论：雌激素能明显促进牙周膜成纤维细胞的迁移，促进其分化。     

内毒素对牙龈成纤维细胞增殖活性及分泌基质金属蛋白酶的影响

目的观察内毒素对牙龈成纤维细胞增殖活性及分泌基质金属蛋白酶-1,3量的影响,探讨基质金属蛋白酶在牙周炎致病机制中的可能作用。方法通过改良酶消化组织块法进行牙龈成纤维细胞的原代培养,用内毒素对其进行刺激,运用MTT法观察内毒素对牙龈成纤维细胞增殖活性的影响,通过免疫组化法检测内毒素对牙龈成纤维细胞分泌基质金属蛋白酶-1,3的影响。结果MTT结果显示,低浓度内毒素在短期内可以促进牙龈成纤维细胞的增殖,但随着时间的延长,也表现为对细胞的毒性作用,抑制其增殖;高浓度内毒素抑制牙龈成纤维细胞的增殖;免疫组化研究结果表明,未受LPS刺激的牙龈成纤维细胞MMP-1,3表达弱阳性,受刺激后阳性表达增强,在12h时达到顶峰,且MMP-3的整体表达较MMP-1弱。结论LPS可以影响牙龈成纤维细胞的增殖活性;LPS可以促进牙龈成纤维细胞对基质金属蛋白酶的分泌,加快牙周细胞外基质的降解,加速牙周炎的进程。     

肿瘤相关成纤维细胞对口腔鳞癌细胞生长和迁移的影响

目的：研究肿瘤相关成纤维细胞（ cancer associated fibroblasts, CAFs）对口腔鳞癌细胞生物学行为的影响,初步探讨CAFs在口腔鳞癌发生发展中的作用。方法采用原代培养的方法,培养口腔鳞癌相关成纤维细胞,光学显微镜观察其形态,免疫组化和免疫印迹实验进行鉴定,CCK-8法检测细胞的增殖能力。 transwell迁移实验检测CAFs诱导口腔鳞癌细胞迁移的能力;萤火虫荧光素酶报告系统检测CAFs对肿瘤细胞增殖能力的影响;平板克隆形成实验评价 CAFs对肿瘤细胞克隆形成能力的影响。结果成功分离培养出口腔鳞癌来源的 CAFs。CAFs能促进口腔鳞细胞的迁移、增殖和克隆形成等生物学行为。结论 CAFs能显著影响口腔鳞癌的增殖和迁移,表明其在口腔鳞癌的发生发展中发挥着一定的作用。     

The effect of cyclosporin on cell division and apoptosis in human oral keratinocytes

BACKGROUND AND OBJECTIVE: Gingival overgrowth (GO) is a side-effect of cyclosporin A (CsA) therapy and is characterised by enlargement of the gingiva with epithelial thickening and overproduction of extracellular matrix components. The pathogenesis of the epithelial thickening in GO remains obscure. The objective of the present study was to investigate the effects of CsA on the growth of oral epithelial cells in vitro and to test the hypothesis that CsA influences apoptosis in these cells. MATERIAL AND METHODS: Cyclosporin was cocultured with an immortalized normal human oral keratinocyte cell line (HOK-16B), an epitheloid cervical carcinoma cell line (HeLa) and primary oral keratinocytes. Cell division was quantified using a CyQUANT kit. Apoptosis was induced using tumour necrosis factor-alpha (TNF-alpha) and assayed by analysis of caspase-3 activity. Expression of the anti-apoptotic protein, Bcl-2, was measured by western blotting. RESULTS: CsA exhibited a dose- and time-dependent inhibition of cell division in all three keratinocyte cell cultures. Significantly, HOK-16B cells treated with high doses of CsA (10 alphag/ml) did not recover their proliferative capacity 3 d after withdrawal of CsA, indicating that CsA-induced inhibition of growth is not temporary. Concentrations of CsA that inhibited cell division (1 microg/ml) did not have any effect on constitutive or TNF-alpha -induced apoptosis or Bcl-2 expression in HOK-16B cells. CONCLUSION: CsA inhibits oral epithelial cell division and this effect is not associated with changes in apoptosis in these cells. The action of CsA on oral epithelial cells may be associated with a long-lasting stress signal, which might account for some of the pathological effects of this drug.     

Laser therapy accelerates initial attachment and subsequent behaviour of human oral fibroblasts cultured on titanium implant material. A scanning electron microscope and histomorphometric analysis

The aim of the study was to investigate the effect of low-level laser therapy (LLLT) on attachment and proliferation of human gingival fibroblasts (HGF) cultured on titanium implant material. HGF were exposed to gallium-aluminum-arsenide diode laser at dosages of 1.5 or 3 J/cm(2) and then cultured on commercially pure titanium discs. Cell profile areas were measured after 1, 3 and 24 h, using scanning electron microscopy and an automatic image analyzer. The results were expressed as percentage of attachment. In order to investigate the effect of LLLT on cellular growth after 8 and 10 days, HGF were cultured on titanium discs for 24 h and then exposed to laser irradiation on 3 consecutive days. Colony-forming efficiency (CFE) and clonal growth rates (CGR) were measured. Cell viability was determined by Hoechst and prodidium iodide staining. Non-lased cultures served as controls. Morphologically, the cells spread well on all titanium surfaces, indicating good attachment by both irradiated and non-irradiated cells. Fibroblasts exposed to laser irradiation had significantly higher percentages of cell attachment than the non-exposed cells (P<0.05). CFE and CGR were also enhanced for the irradiated cells (P<0.05). Cell viability was high (>90%) in the irradiated and control groups, without significant differences. It is concluded that in vitro LLLT enhances the attachment and proliferation of HGF on titanium implant material.     

环孢素A和肿瘤坏死因子α对人牙龈成纤维细胞增殖的影响

目的 研究环孢素A(cyclosporinA)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)对体外培养人牙龈成纤维细胞增殖的影响,探讨牙龈炎症与药物性牙龈增生的关系及环孢素A所致牙龈增生的相关机制.方法用原代培养的方法获取5名健康人的牙龈成纤维细胞,体外培养、传代后取其中1个生长良好的组织块4～8代细胞用于实验.按以下条件进行实验分组:A组:空白对照组;B1组:10 μg/L环孢素A,B2组:50 μg/L环孢素A,B3组:250 μg/L环孢素A,B4组:1250 μg/L环孢素A;C组:5μg/L TNF-α; D1组:10 μg/L环孢素A+5μg/L TNF-α,D2组:50 μg/L 环孢素 A+5μg/L TNF-α,D3组:250 μg/L环孢素A+5μg/L TNF-α,D4组:1250 μg/L环孢素A+ 5 μg/L TNF-α.将条件培养液分别作用于牙龈成纤维细胞,培养3、5、7d后用甲基噻唑基四唑法测定细胞的增殖情况.结果不同质量浓度的环孢素A作用于成纤维细胞后,细胞的增殖受到抑制,A值下降,其中B1、B2、B3组与A组相比差异无统计学意义,B4组与A组相比差异具有统计学意义(P =0.001);5μg/L的TNF-α作用于成纤维细胞可以刺激细胞的增殖,A值(0.542)与A组(0.441)相比显著升高(P＜0.01).环孢素A和TNF-α共同作用于成纤维细胞后,D1、D2、D3组A值均较A组升高,但均较C组显著降低(P＜0.05).D4组细胞增殖显著增加,与C组相比差异具有统计学意义(P＜0.01).结论环孢素A对成纤维细胞的增殖无促进作用,高浓度时可抑制细胞的增殖;TNF-α可以促进成纤维细胞的增殖;高浓度环孢素A与TNF-α共同作用于成纤维细胞可促进成纤维细胞增殖,提示在一定浓度下环孢素A可能放大了TNF-α刺激成纤维细胞增殖的效应.     

口腔癌相关成纤维细胞对人淋巴管内皮细胞增殖、迁移、侵袭、成管的影响

目的 研究口腔癌相关成纤维细胞（CAFs）在口腔鳞状细胞癌淋巴管生成过程中的作用。方法 通过组织块法分离、培养口腔鳞状细胞癌患者的CAFs和正常成纤维细胞（NFs）,免疫组织化学染色鉴定CAFs和NFs。利用24孔的transwell细胞培养室分别将CAFs（实验A组）、NFs（实验B组）与人淋巴管内皮细胞（HLEC）共培养,以DMEM高糖培养基作为空白对照组,在倒置相差显微镜下观察各组HLEC的增殖、迁移、侵袭、成管能力。结果 培养96、144、192 h,实验A组HLEC的数目均多于实验B组和空白对照组,实验B组多于空白对照组（P<0.01）。培养96 h后,实验A组HLEC的迁移和侵袭数目均多于实验B组和空白对照组,实验B组多于空白对照组（P<0.01）。培养24 h后,实验A组HLEC的成管数目多于实验B组和空白对照组,实验B组多于空白对照组（P<0.01）。结论 口腔CAFs促进了HLEC的增殖、迁移、侵袭和成管作用,并且CAFs的促进作用大于NFs。     

Effects of HEMA on Nrf2-related gene expression using a newly developed 3D co-culture model of the oral mucosa

Objective2-Hydroxyethyl methacrylate (HEMA) is a component of many resin-modified materials and elutes from dental restorations into the oral cavity. Objective of our investigation was to determine the impact of HEMA on oral keratinocytes (OKF6/TERT2) and gingival fibroblasts (HGFs) in a newly established 3D co-culture model (3D-CCM) and to analyze the permeability of OKF6/TERT2 cells for HEMA.MethodsWell-characterized 3D-CCMs, consisting of confluent OKF6/TERT2 cells on cell culture inserts above HGF-containing collagen gels, were treated supra-epithelial with HEMA. Mass spectrometry was used to measure the supra- and sub-epithelial distribution of HEMA after 24 h. The impact of HEMA on nuclear factor erythroid 2-related factor 2 (Nrf2) target genes was measured by qRT-PCR and western blot analysis.ResultsMass spectrometry showed that HEMA was evenly distributed above and below the keratinocyte layer after 24 h. Analyzed target genes of Nrf2 were induced in both cell types on the mRNA-level but less pronounced in HGFs. On the protein-level, both cell types showed similar effects: At 5 mM HEMA, heme oxygenase-1 was induced 5.1-fold in OKF6/TERT2 cells and 4.1-fold in HGFs. NAD(P)H quinone dehydrogenase-1 was approximately induced 1.85-fold in both cell types.SignificanceOur 3D-CCM is suitable to analyze the biocompatibility of dental materials due to an improved simulation of the oral mucosa compared to monolayer cultures. Our results indicate that HEMA is able to penetrate a dense layer of keratinocytes and to activate the cellular oxidative defense response. This may be due to the activation of the Nrf2-pathway in both cell types.