槟榔提取物抑制人类口腔黏膜成纤维细胞生长的实验研究

目的通过比较正常口腔黏膜和口腔黏膜下纤维性变(OSF)组织中成纤维细胞(FB)增殖差异、检测槟榔提取物(ANE)对成纤维细胞增殖的影响,来探讨OSF的发病机理.方法对人类口腔黏膜成纤维细胞进行分离培养,然后用四唑盐(MTT)比色试验法检测OSF患者和正常人口腔黏膜FB增殖状况,并且观察ANE对FB增殖的影响.结果表示增殖水平的OD值在OSF-FB为0.254±0.045,高于NM-FB的OD值0.236±0.012(P<0.05),ANE以浓度-效应依赖关系抑制FB增殖.结论 OSF-FB细胞增殖率较NM-FB高;ANE对口腔黏膜FB有细胞毒作用,提示槟榔及其有效成分不完全是通过直接刺激FB增殖而诱发OSF.     

槟榔提取物对口腔粘膜成纤维细胞表达细胞间粘附分子-1的影响

目的:探讨成纤维细胞(FB)与细胞间粘附分子-1(ICAM-1)在口腔粘膜下纤维化(OSF)发生中的可能作用。方法:分别从正常(NM)及OSF患者的口腔粘膜中培养出FB,向培养基中加入不同浓度的槟榔提取物(ANE)培养48 h后,用细胞酶联免疫方法检测FB表达的ICAM-1水平。结果:表示ICAM-1水平高低的OD值在OSF-FB为01386? 01099,高于NM-FB的OD值01324?01030(P<0105);ANE在50~150Lg/ml的范围内以浓度)效应依赖关系刺激 FB产生ICAM-1。结论:ANE能上调FB表达ICAM-1的水平,提示ICAM-1及其介导的细胞与细胞或细胞与基质间的相互作用在OSF的发生、发展中可能起重要作用。     

γ—干扰素对OSF成纤维细胞的生物学效应

目的 了解γ-干扰素（IFN-γ）对口腔黏膜下纤维化（OSF）患者颊黏膜成纤维细胞（OSF-FB）的生长增殖及胶原合成的影响,探讨IFN-γ用于治疗OSF的可能性。方法 取6例OSF患者及5例正常人颊黏膜,行成纤维细胞原代培养并传代,采用MTT比色法及羟脯氨酸试剂盒分别观察IFN-γ对NM-FB及OSF-FB的生长增殖及胶原合成的影响,并通过电镜观察IFN-γ对NM-FB及OSF-FB超微结构的影响。结果 IFN-γ明显抑制NM-FB及OSF-FB的生长增殖及胶原合成,电镜发现IFN-γ使NM-FB及OSF-FB的粗面内质网、游离核糖体减少、线粒体减少并有肿胀。结论 IFN-γ是OSF形成过程中的一种负性调节因子,有可能用于临床治疗OSF。     

TNF-α和IFN-γ对OSF成纤维细胞的增殖影响

目的 :研究TNF α、IFN γ对正常人颊黏膜成纤维细胞 (NM FB)和口腔黏膜下纤维化 (OSF)患者颊黏膜成纤维细胞 (OSF FB)增殖活性的影响。方法 :取 6例OSF患者及 5例正常人颊黏膜组织 ,采用组织块法进行成纤维细胞原代培养并传代 ,用MTT比色法观察不同浓度TNF α及IFN γ对NM FB和OSF FB的增殖效应。结果 :浓度为 10 0～ 10 0 0 0U/ml的TNF α能明显促进NM FB和OSF FB的增殖(P <0 .0 5) ;浓度为 40 0～ 40 0 0U/ml的IFN γ则明显抑制两者的生长 (P <0 .0 5) ,且 40 0U /ml的IFN γ其抑制率接近峰值 (与 40 0 0U/ml的IFN γ组比 ,P >0 .0 5)。结论 :TNF α促进NM FB和OSF FB的增殖 ;而IFN γ对两者的增殖活性具有抑制作用     

槟榔提取物对人类口腔黏膜上皮角朊细胞分泌内皮素的影响

目的 通过观察槟榔提取物(areca nut extract, ANE)对体外培养的人类口腔黏膜上皮角朊细胞(keratinocytes, KC)分泌内皮素(endothelin, ET)的影响， 初步探讨KC和ET在口腔黏膜下纤维性变(oral submucous fibrosis, OSF)发生中的作用。方法 分别从正常(NM)及OSF的口腔黏膜组织中培养出KC， 向培养基中加入不同浓度的ANE， 用放射免疫法测定培养细胞上清液中ET的水平。结果 OSF－KC 48 h的ET分泌量为96.983±17.802 pg/ml， 高于NM－KC分泌量14.641±15.796 pg/ml(P＜0.05)； ANE在5～150　μg/ml范围内以浓度—效应依赖关系刺激KC分泌ET。结论 人类口腔黏膜上皮KC能分泌一定量的ET， 且OSF－KC分泌的ET水平高于NM－KC， ANE能刺激KC分泌ET， 提示ANE可能是一种能引起KC分泌ET水平上调的外源性刺激物， ANE可能是通过改变KC分泌细胞因子的水平而诱发OSF。     

整合素α1在口腔黏膜下纤维性变发病机制中的作用

目的:观察口腔黏膜下纤维性变(OSF)组织中整合素α1的表达与分布,及槟榔碱对体外培养的人口腔黏膜成纤维细胞(FB)表达整合素α1mRNA的影响,探讨整合素α1在OSF发病中的作用与机制.方法:①采用免疫组织化学染色方法,观察整合素α1在10例正常口腔黏膜组织和OSF早、中、晚期各20例病变组织中的表达与分布,并采用半定量方法检测正常口腔黏膜组和OSF各组FB上整合素α1的表达强度.②采用RT-PCR法检测体外培养FB分别在0、10、20、40μg/ml浓度组别槟榔碱刺激下,整合素α1mRNA的表达水平.结果:①正常口腔黏膜组织中,整合素α1表达低,主要分布于上皮基底细胞和血管内皮细胞,呈胞膜或胞质阳性,FB极少见阳性表达;而OSF病变组织中,除表皮基底细胞及血管内皮细胞外,FB细胞膜及胞质内均见整合素α1的大量表达,炎症细胞上亦可见少量表达.OSF各期FB表达整合素α1均强于正常组(P<0.05),早中期组表达增高,晚期组表达降低,与中期组有统计学差异(P<0.05).②对照组整合素α1mRNA表达量低,在槟榔碱10、20 μg/ml浓度组表达逐渐升高与对照组存在显著性差异(P<0.05).40μg/ml组出现下降与10μg/ml组无显著性差异(P>0.05);结论:槟榔碱通过刺激口腔黏膜FB表达与分泌整合素α1,进而诱导胶原合成增多,可能是OSF发生机制之一.     

槟榔碱对口腔黏膜成纤维细胞分泌整合素α2的影响

目的：通过研究槟榔碱对体外培养的人口腔黏膜成纤维细胞（FB）表达整合素α2的影响,探讨整合素α2在口腔黏膜成纤维性变（OSF）发病机制中的可能作用。方法：体外培养人正常口腔黏膜成纤维细胞（FB）,培养基中加入不同浓度（0、10、20、40、80、160μg/ml）的槟榔碱孵育,MTT法12h后检测FB的增殖水平。RT-PCR法24h后检测0、10、20、40μg/ml浓度组整合素α2mRNA的表达水平。结果：槟榔碱浓度为20μg/ml时,FB增殖活性开始下降,与对照组相比无统计学差异（p〉0.05）,40μg/ml时下降明显与对照组相比有统计学差异（p〈0.05）。正常对照组整合素α2mRNA表达量低,随槟榔碱浓度增高其表达量呈增高趋势（p〈0.05）。结论：槟榔碱具有刺激FB表达整合素α2的作用,提示FB分泌整合素α2增多,进而诱导胶原合成增多,可能是OSF发生机制之一。     

血管内皮细胞上清液对口腔颊黏膜成纤维细胞增殖活性的影响

目的：观察血管内皮细胞上清液（ECS）对正常人口腔颊黏膜成纤维细胞（NM-FB）增殖活性的影响。方法：收集不同时间点、并配成不同比例的ECS干预NM-FB。结果：ECS12h能促进NM-FB的增殖,随着时间延长ECS促增殖能力逐渐增强,ECS48h促FB增殖能力达到最大（P〈0.05）;ECS/（ECS＋RPMI-1640）比值为1/4时可促NM-FB增殖,ECS稀释倍数越小,NM-FB的增殖率越高（P〈0.05）。ECS能促进FB的增殖,存在时效和量效依赖关系。结论：血管内皮细胞能通过分泌某些介质影响口腔颊黏膜成纤维细胞的生物学活性。     

Smad蛋白在口腔黏膜肌成纤维细胞转分化中的作用

目的:探索口腔黏膜成纤维细胞向肌成纤维细胞分化的机制。方法:利用免疫组织化学方法检测成纤维细胞表达α平滑肌肌动蛋白之前表达Smad蛋白的改变。结果:口腔黏膜成纤维细胞(FB)组核阳性率为(17.38±10.36)%;FB与口腔黏膜角质形成细胞(KC)共同培养24hSmad2/3蛋白核表达为(35.33±7.68)%,与FB组相比p=0.015结论:口腔黏膜FB向肌成纤维细胞(MFB)分化的机制与TGFβ-Smad信号途径有关。。     

TNF-α和槟榔碱对口腔黏膜成纤维细胞增殖和羟脯氨酸蛋白表达的影响及姜黄素对其影响的作用

目的:探讨TNF-α和槟榔碱对口腔黏膜成纤维细胞增殖和羟脯氨酸蛋白表达的影响及姜黄素对其影响的作用。方法:体外培养口腔黏膜成纤维细胞,分别用100～10 000 U/mL TNF-α和10～40μg/mL槟榔碱作用于细胞24～72 h, CCK-8实验检测细胞增殖,ELISA测定细胞中羟脯氨酸的表达。用5～40μmol/L的姜黄素作用于2种处理的细胞,检测细胞增殖和羟脯氨酸蛋白的表达。结果:TNF-α促进细胞增殖和羟脯氨酸蛋白表达(P<0.05),槟榔碱抑制细胞增殖和羟脯氨酸蛋白表达(P<0.05)。40μmol/L的姜黄素作用于TNF-α和槟榔碱刺激的口腔黏膜成纤维细胞,在24 h和48 h时抑制细胞增殖(P<0.05)。不同浓度的姜黄素作用于TNF-α和槟榔碱刺激的口腔黏膜成纤维细胞,均能抑制羟脯氨酸合成(P<0.05)。结论:TNF-α可促进口腔黏膜成纤维细胞增殖和羟脯氨酸蛋白表达,槟榔碱能抑制口腔黏膜成纤维细胞增殖和羟脯氨酸蛋白表达,较高浓度姜黄素能减弱TNF-α的刺激作用,增强槟榔碱的抑制作用。     

槟榔提取物刺激口腔黏膜角朊细胞培养上清对成纤维细胞增殖活性的影响

目的：探讨槟榔提取物刺激口腔黏膜角朊细胞在黏膜下纤维性变发病中的作用。方法：采用不同浓度槟榔提取液刺激体外培养的角朊细胞,取细胞培养上清,MTT法观察细胞培养上清对成纤维细胞增殖的影响。结果：一定浓度槟榔提取液刺激的角朊细胞培养上清能促进成纤维细胞增殖;细胞培养上清对成纤维细胞增殖的促进作用存在个体差异。结论：槟榔成分可能通过改变口腔黏膜角朊细胞的活性而导致口腔黏膜下纤维性变的发生。     

槟榔提取物抑制人类口腔粘膜角朊细胞生长的实验研究

目的：建立人类口腔粘膜上皮角朊细胞体外原代培养方法：探讨口腔粘膜下纤维性变患者上皮厚度改变的机理;方法;先对人类口腔粘膜上皮角朊细胞进行分离,培养和鉴定,然后用四唑盐比色试验检测ＯＳＦ患者和正常人口腔粘膜上皮ＫＣ增殖状况,并且观察槟榔提取物对ＫＣ增殖的影响。     

The fibroblast population in oral submucous fibrosis

The purpose of the investigation was to compare the morphology of fibroblasts cultured from healthy oral mucosa and mucosa of patients with oral submucous fibrosis (OSF) and to collate the occurrence of cell types of similar morphology. Cells cultured from biopsy specimens from the buccal mucosa of six subjects who did not chew the areca nut and six patients with OSF who chewed areca nut were grown according to standard techniques. Ninety cells per cell line were recorded daily for 8 days, classified into types F1, F2 and F3 according to their morphology, and the results statistically analyzed. We found that there was a relative increase of F3 cells in relation to Fl cells in OSF resulting in the ratio of F3 to F1 cells being significantly larger in OSF than the ratio in the controls. As it has been reported that F3 cells m rat connective tissues produce significantly more collagen types I and III than F1 cells, we concluded that a change of fibroblast population has occurred in OSF and that this relative increase of F3 cells in humans, which could be committed to the production of large quantities of collagen, can be an explanation for the excessive collagen formation in OSF.     

Observations on the effect of areca nut extracts on oral fibroblast proliferation

The effects of aqueous extracts of raw, baked and boiled areca nuts were tested on cultured human buccal mucosa fibroblasts. Cells were exposed to extract concentrations of 0, 50, 100, 150, 300 and 500 μg/ml. The arecoline and arecaidine content was determined in the extracts with HPLC and raw nut contained 5.5% in m. baked nut 6.6% mm and boiled nut 7.1% m/m. Extract concentrations of 50 to 150 μg/ml inhibited cell growth in a concentration-dependent manner but did not lead to total cell death during a 7 day period. However, total cell death did occur with concentrations of 300 and 500 μg/ml. It is concluded that areca nut extract is toxic to cultured fibroblasts and inhibits their proliferation in a concentration-dependent manner.     

槟榔碱对口腔颊黏膜成纤维细胞和血管内皮细胞增殖活性的影响

目的：比较槟榔碱对NM—FB、OSF—FB、EC增殖活性的影响。方法：0μ／ml、30μm／ml、60μg／ml、90μg／ml、120μg／ml槟榔碱干预三组细胞48h,60μg／ml槟榔碱干预三组细胞24h、48h、72h,倒置显微镜下观察细胞形态的改变,四唑盐（MTT）比色法检测细胞增殖活性的变化。结果：槟榔碱以浓度、时间-依赖关系抑制细胞增殖,相同条件下存活率EC低于NM—FB、NM—FB低于OSF—FB。结论：槟榔碱有细胞毒作用,细胞损伤有先后时序关系;推测槟榔碱并不直接通过刺激FB增殖诱发OSF,可能通过改变FB性能和EC活性促进OSF的发生。     

Growth of oral and skin fibroblasts from patients with oral submucous fibrosis

The purpose of the investigation was to evaluate and compare the proliferation (growth) of mouth fibroblasts and skin fibroblasts from patients with oral submucous fibrosis (OSF). Material comprised fibroblasts from fibrous bands situated in the buccal mucosa and from the inner aspect of the forearm of 8 patients with classic features of OSF as well as fibroblasts from 6 buccal mucosa and 8 skin biopsy specimens from healthy non-areca nut chewing individuals. Cells were cultured for 8 days according to standard techniques. Their growth was monitored daily, under optimal conditions as well as exposure to concentrations of arecoline. The data were analyzed using regression analysis, analysis of variance and the Kruskal-Wallis test. We found no statistically significant differences between the proliferation patterns of oral and skin fibroblasts from patients or between those from patients and controls. The reaction of the cells exposed to concentrations of arecoline was similar; at low concentrations (0.1–10 μg/ml) normal growth was maintained, while 100 μg/ml inhibited growth. It is concluded that fibroblasts from mouths affected by OSF have proliferation patterns which fall within normal parameters, that the excessive collagen formation in established OSF is not due to increased fibroblast proliferation and that arecoline does not stimulate fibroblast proliferation.     

Regulation of interleukin-6 expression by arecoline in human buccal mucosal fibroblasts is related to intracellular glutathione levels

OBJECTIVES: Cytokines play an important role in regulating fibroblast function and is likely to play a key role in regulating the initiation and progression of scarring in any fibrotic disease. Interleukin-6 (IL-6) has been implicated in the development of a variety of fibrotic diseases. The aim of this study was to compare IL-6 expression in fibroblasts cultured from normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce IL-6 expression. METHODS: mRNA level of IL-6 in fibroblasts from OSF was compared with normal buccal mucosa. The effects of arecoline, the major areca nut alkaloid, on IL-6 expression in normal human buccal mucosa fibroblasts (BMFs) were measured in vitro. mRNA was quantified with AlphaImager 2000. To determine whether glutathione (GSH) levels were important in the induction of IL-6 by arecoline, we pretreated cells with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or with buthionine sulfoximine (BSO) to deplete GSH. RESULTS: Fibroblasts derived from OSF exhibited higher IL-6 gene expression than BMF in mRNA levels (P < 0.05). The exposure of quiescent BMF to arecoline resulted in the elevation of IL-6 mRNA expression in a dose-dependent manner (P < 0.05). IL-6 gene regulated by arecoline correlated with intracellular GSH levels in BMF. Arecoline at a concentration of 129 muM induced about 2.7-fold IL-6 mRNA levels over the 6-h incubation period. However, BSO enhanced the IL-6 mRNA levels by 3.9-fold (P < 0.05). In addition, OTZ was found to marginally reduce the arecoline-induced IL-6 expression by about 1.7-fold (P < 0.05). CONCLUSIONS: Taken together, these results suggest that IL-6 expression is significantly upregulated in OSF fibroblasts in areca quid chewers and arecoline may be responsible for the enhanced IL-6 expression. In addition, the regulation of IL-6 expression induced by arecoline is critically dependent on the intracellular GSH concentrations.