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。     

口腔粘膜下纤维性变患者角朊细胞分泌细胞因子水平变化

目的：探讨口腔粘膜上皮角朊细胞（ＫＣ）和细胞因子在口腔粘上纤维性变（ＯＳＦ）发生中的作用。方法：分别从正常及ＯＳＦ组织培养出ＫＣ（ＮＭ－ＫＣ）和ＯＳＦ－ＫＣ０,用放射免疫法和酶联免疫吸附法测定培养细胞上清液中内皮素（ＥＴ）和β１转化生长因子（ＴＧＦβ１）的水平。结果：ＯＳＦ－ＫＣ分泌ＥＴ和ＴＧＦ１水平均高于ＮＭ－ＫＣ,且两者呈正相关关系。     

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。     

Development of an in vivo mouse model to study oral submucous fibrosis

BACKGROUND: Epidemiological data have shown an association of areca nut chewing with oral submucous fibrosis (OSF). Experimental evidence to confirm this has been limited. Fibrosis-promoting activity of areca nut was tested in an animal model. METHOD: Buccal mucosa of a group of 20 female BALB/c strain mice, 10-12 weeks of age, was treated twice daily 6 days per week with topical application of aqueous areca nut extracts for 300-600 days. A control group (n = 20) was treated with 50 mM NaCl. The influence of areca nut on the oral epithelium and connective tissue was recorded semiquantitatively by light microscopy. RESULTS: The areca nut-treated oral epithelium showed progressive changes in epithelial thickness leading to atrophy, increased cellularity of fibroblasts, fibrosis of connective tissue, focal infiltration of inflammatory cells and muscle atrophy. On killing after 600 days of treatment, the scores on cellularity, inflammation and muscle atrophy were significantly different to the control group (P = 0.03). CONCLUSION: The study provides further evidence that areca nut contributes to the development of OSF in treated animals. The model has the potential to test synergism of areca nut with other carcinogens and any therapeutic interventions.     

槟榔提取液诱发大鼠口腔粘膜下纤维性变的实验研究 Ⅱ.肥大细胞对胶原代谢的影响

借助组织化学、生化分析和透射电镜方法，探讨肥大细胞对水溶性槟榔提取液诱发的大鼠口腔粘膜下纤维性变（ＯＳＦ）的胶原代谢的影响。结果发现：在ＯＳＦ发生过程中，肥大细胞与成纤维细胞关系密切，其数量明显增多，与同期对照组比较具有显著性差异，且随刺激时间延长而逐渐增加；模型组颊粘膜胶原纤维大量堆积，组织羟脯氨酸含量明显高于同期对照组，且随刺激时间延长而升高；各时期模型组肥大细胞计数与组织羟脯氨酸含量均呈高度正相关；血清总羟脯氨酸含量各期各组间无显著性差异。提示：槟榔某些水溶性成份可能通过激发肥大细胞增殖与活化，干扰组织胶原代谢而诱发ＯＳＦ。     

Experimental study on aqueous areca nut extracts inducing oral submucous fibrosis in rats. II. Effection of mast cells on collagen metabolism]

The effection of mast cells (MC) on collagen metabolism of oral submucous fibrosis (OSF) induced by aqueous areca nut extracts (AANE) in rats was studied by transmission electron microscope, histochemical and biochemical analyses. The results revealed that there was a close relationship between MC and fibroblats (FB) in the pathogensis of OSF; MC which appeared in the buccal mucosa in the model groups increased significantly and became more active in the function. A great deal of collagen compacted in the buccal mucosa in the model groups. The contents of tissue hydroxyproline (Hyp) in the model groups were obviously higher than those in the control groups. Moreover, the longer the time of irritation continued, the higher the contents were. The number of MC had a higher positive correlation with the contents of tissue Hyp at the same time. Total serum Hyp had no significant difference among all the groups and any stages. The results indicated that some aqueous components of areca nuts might disturb collagen metabolism by accumulation and activation of MC.     

Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for oral submucous fibrosis

Oral submucous fibrosis (OSF), a chronic oral mucosal condition commonly found in south Asians, is a disorder characterized by a quantitative as well as a qualitative alteration of collagen deposition within the subepithelial layer of the oral mucosa. Since degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues, we have investigated phagocytosis of collagen- and fibronectin-coated latex beads by fibroblast cultures with an in vitro model system. Coated fluorescent latex beads were incubated with human oral mucosa fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 16 h contained a mean of 75% collagen phagocytic cells and 70% fibronectin phagocytic cells; however, about 15% and 10% of phagocytic cells individually contained more than twice the mean number of beads per cell. In contrast, cells from OSF tissues exhibited a 40% reduction of the proportions of collagen phagocytic cells (mean=35%) and a 48% decrease of the proportions of fibronectin phagocytic cells (mean=22%), none of the cells having a high number of beads as compared to normal fibroblasts. OSF lesions appear to contain fibroblasts with marked deficiencies in collagen and fibronectin phagocytosis. To investigate if inhibition of phagocytosis could be demonstrated in vitro, normal fibroblast cultures were incubated with areca nut alkaloids (arecoline, arecaidine). The cultures had a dose-dependent reduction in the proportions of phagocytic cells. On the other hand, corticosteroid used in the treatment of OSF exhibited a dose-dependent enhancement in the proportion of phagocytic cells. Therefore, our hypothesis for OSF, although over-simplified, is that betel nut alkaloids (arecoline, arecaidine) inhibit fibroblast phagocytosis and this provides a mechanism for the development of OSF. The benefit of a local intralesional injection of corticosteroid is also possibly, at least in part, through an enhancement of fibroblast collagen phagocytosis.     

槟榔提取物对内皮细胞分泌一氧化氮的影响

目的 :探讨口腔黏膜下纤维性变 (OSF)可能的发病机理。方法 :用槟榔提取物干预体外培养的内皮细胞 ,收集培养上清 ,用ELISA方法检测内皮细胞分泌一氧化氮 (NO)的浓度。结果 :槟榔提取物能抑制体外培养的内皮细胞分泌NO。结论 :OSF的发生可能与槟榔引起的内皮细胞合成NO减少有关。     

Copper stimulates human oral fibroblasts in vitro: a role in the pathogenesis of oral submucous fibrosis

Copper is implicated in the pathogenesis of several fibrotic disorders. Areca nut has been shown to have a high copper content and areca chewing is associated with oral submucous fibrosis (OSF). The effects of copper on human oral fibroblasts were investigated in vitro. Human oral fibroblasts were incubated with copper chloride (CuCl2) at concentrations ranging from 0.01 microM to 500 microM for 24 h, and in vitro cell proliferation was assayed by incorporation of tritiated-thymidine; soluble and non-soluble collagen synthesis was assayed using tritiated-proline. Addition of copper chloride at concentrations ranging from 0.1 microM to 50 microM increased the collagen synthesis by the oral fibroblasts compared with growth without copper (P<0.05). The addition of copper chloride neither increased the synthesis of non-collagenous proteins by the fibroblasts nor influenced their proliferation rate. We conclude that copper upregulates collagen production in oral fibroblasts. This appears to be concentration dependent, with peak collagen synthesis at 50 microM CuCl2. These in vitro results taken together with the recent findings of copper in oral biopsies from OSF subjects support the hypothesis that copper in areca nut acts as a mediator of OSF.     

Oral submucous fibrosis: a case-control study in Chennai, South India

BACKGROUND: Oral submucous fibrosis (OSF) is a pre-cancerous condition caused by the use of areca nut in various forms. There are very few published reports on areca nut use and OSF from Chennai, South India. METHODS: A hospital-based case-control study on habits and OSF was performed in Chennai over a 3-year period. A total of 185 consecutive patients with OSF were matched with age- and sex-matched controls. History was recorded in a pre-determined format by qualified dental surgeons. RESULTS: The male to female ratio of OSF cases was 9.9 : 1. All areca nut products were associated with OSF, with the risk being greatest for pan masala. The duration of the habit was more significant than the frequency of the chewing habit. CONCLUSION: The present study confirms the strong association between areca nut use and OSF and the increasing use of pan masala.     

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口腔黏膜下纤维性变(oral submucous fibrosis,OSF)是一种慢性、隐匿性且具有癌变倾向的疾病,主要发生于印度、巴基斯坦等东南亚国家以及我国湖南、台湾两省。OSF病因不明,咀嚼槟榔是其最主要的致病因素,且咀嚼槟榔的频率越高、年限越长,越易患OSF。所有OSF患者都有咀嚼槟榔史,咀嚼槟榔还与口腔白斑、口腔癌发病高度相关。此外,OSF的发生还与遗传因素、免疫反应、胶原相关性基因,营养缺乏等有关。本文就OSF致病因素做一简单介绍。     

Oral submucous fibrosis: a clinico-histopathological study in Chennai.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous condition associated with the use of areca nut in various forms. There are very few reports to correlate the clinical stage to histopathological grading in OSF. MATERIALS AND METHODS: A hospital-based study was conducted on 75 OSF cases who visited our hospital in Chennai from 2000-2003. A detailed history of each patient was recorded along with a clinical examination. Biopsy was performed for histopathological correlation. Clinical stage of the disease in terms of the ability to open one's mouth was correlated with histopathological grading. RESULTS: The male to female ratio of OSF cases was 6:1. All forms of areca nut products were associated with OSF. Chewing of paanmasala was associated with early presentation of OSF as compared to chewing of the betel nut. Out of 57 cases, which were in clinical stage II, 91.2% had histological grading of I and II in equal proportions and 8.8% had histological grade III. Out of 13 cases that showed a clinical stage of III, 52% showed a histological grade of II, 40% grade III and 8% grade I. CONCLUSION: In the present study, there was no direct correlation between clinical stages and histopathological grading. The possibility of difference in the severity and extent of fibrosis in different regions of the oral mucosa and involved muscles were considered as contributory factors for this variation.     

Epidemiological survey of oral submucous fibrosis in Xiangtan City, Hunan Province, China

Abstract Oral submucous fibrosis is a high risk precancerous condition and is suggested to be caused by areca nut chewing. Areca nut chewing is popular in Hunan Province of China, and is more concentrated in Xiangtan City. Two and nine cases of oral submucous fibrosis (OSF) were first noticed in 1984 and 1985 respectively, and an epidemiologic survey was subsequently performed in 1986. The epidemiologic method of cluster sampling was used. The Yuhu District, one of the five urban districts of the Xiangtan City with a population of 100 000 was selected as a whole body in the survey. 57 independent units of various professions were randomly selected as group samples and more than 70% of subjects in each unit were examined. Definite fibrous band on palpation was used as a main diagnostic criterion for OSF. A total of 11 046 individuals were examined; among them were 3 907 areca nut chewers (35.37%) and 7 139 non-chewers (64.63%). 335 cases of OSF were found, comprising a prevalence rate of 3.03%. The disease involved mainly the middle third of the oral cavity. All of the OSF cases were areca nut chewers. No case had been found in non-chewers. Four cases of oral carcinoma were found on the basis of OSF, the malignant transformation rate was 1.19%. The high prevalence of OSF may be due to areca nut chewing plus extensive and heavy use of hot pepper in Xiangtan people. The result supports the role of the areca nut as an etiologic factor in the development of OSF. The low malignant transformation rate of 1.19% compared with the 7.6% in an Indian report may be because Xiangtan people chew areca nut without tobacco.     

Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis

Background

In humans, areca nut induces oral submucous fibrosis (OSMF), a potentially malignant disorder, characterised by the deposition of collagen in the lamina propria. This study aimed to determine whether OSMF-like characteristics develop in a rabbit model following treatment with areca nut and lime.

Increased plasminogen activator inhibitor-1/tissue type plasminogen activator ratio in oral submucous fibrosis

OBJECTIVE: Plasminogen activators and their inhibitors are thought to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix (ECM) turnover. However, little is known about the expression of plasminogen/plasmin system at the site of oral submucous fibrosis (OSF). METHODS: We compared the activities of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) between fibroblasts derived from normal buccal mucosa and OSF by using an enzyme-linked immunosorbent assay. Furthermore, arecoline, a major areca nut alkaloid, was challenged with normal buccal mucosal fibroblasts (BMFs) to elucidate whether the activities of t-PA and PAI-1 could be affected by arecoline. RESULTS: Both t-PA and PAI-1 were found to be increased in OSF than in BMFs (P < 0.01). In addition, there was a statistically significant difference in PAI-1/t-PA ratio between OSF and BMF (P < 0.01). The addition of arecoline upregulated not only PAI-1, but also t-PA in BMFs (P < 0.05). In addition, the ratio between PAI-1 and t-PA was found to be significantly increased by a linear regression assay (P < 0.01). CONCLUSION: These results suggest that OSF caused by areca quid chewing may be the result of an imbalance in the plasminogen/plasmin system, the net result of which is increased deposition of ECM.     

Role of chewing and smoking habits in the etiology of oral submucous fibrosis (OSF): a case-control study

Oral submucous fibrosis (OSF), a premalignant and crippling condition of the oral mucous membrane, was studied to identify its relationship to various chewing and smoking habits. Two hundred and thirty-six consecutive cases of OSF were compared with 221 control subjects matched for age, sex and socio-economic conditions. It was found that chewing of areca nut/quid or pan masala (acommercial preparation of areca nuts, lime, catechu and undisclosed colouring, flavouring and sweetening agents) was directly related to OSF. Also, pan masala was chewed by a comparatively younger age group and was associated with OSF changes earlier than areca nut/quid chewing. However, chewing or smoking tobacco with various other chewing habits did not increase the risk of developing OSF. It was also found that frequency of chewing rather than the total duration of the habit was directly correlated to OSF.