口腔黏膜下纤维性变中u-PA和PAI-1的表达及意义

目的:了解尿激酶型纤溶酶原激活物(u-PA)及其抑制剂(PAI-1)在口腔黏膜下纤维性变(OSF)组织中的表达及其意义。方法:采用免疫组化染色法和图像分析法,对OSF早、中、晚期各10例及10例正常口腔黏膜组织中的u-PA及PAI-1的表达及分布进行分析。结果:OSF病变组织中u-PA和PAI-1有异常表达,其阳性物质主要分布于固有层血管内皮细胞、成纤维细胞及炎症细胞的胞质,PAI-1各期阳性表达强度均高于正常口腔黏膜组(P<0.05),u-PA早期阳性表达显著升高(P<0.05),中晚期则逐渐降低(与正常组比P>0.05)。结论:u-PA、PAI-1在OSF病变组织中的异常表达,可能是影响OSF发生发展的重要因素。     

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.     

外源性表皮生长因子对口腔鳞癌细胞系尿激酶型纤溶酶原激活剂及其抑制剂活性的影响

目的：探讨表皮生长因子（Epidermal growth factor,EGF）和口腔鳞癌侵袭转移的关系。方法：采用发色底物反应法测量不同浓度EGF作用于口腔鳞癌TSCCa细胞系及颈淋巴转移癌GNM细胞系后u—PA和PAI-1的活性,同时用Boyden Chamber观察EGF诱导口腔鳞癌TSCCa和GNM细胞转移作用。结果：不同浓度的EGF（0ng／ml,10ng／ml,20ng／ml,40ng／ml,80ng／ml）作用于两种细胞系后,随着外源性EGF浓度的增加,GNM和TSCCa细胞中u—PA和PAI-1的活性均有增加,与对照组相比差异有显著性（p〈0．05）;而以TSCCa细胞中u—PA和PAI-1的活性增加较为明显,u—PA和PAI-1活性与外源性EGF有剂量依赖关系,用不同浓度10ng／ml,20ng／ml,40ng／ml,80ng／ml的EGF培养TSCCa和GNM细胞2h,GNM和TSCCa细胞穿过滤膜数均有增加,在相同的浓度内,TSCCa细胞较GNM细胞增加较为明显,两者相比差异有显著性（p〈0．05）。结论：EGF可促进口腔鳞癌细胞侵袭转移。     

Increased lysyl oxidase activity in fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan

Growth characteristics and lysyl oxidase activity of fibroblasts derived from human normal mucosa (NM) and oral submucous fibrosis (OSF) associated with betel nut chewing were compared in cell cultures. The growth rates of cultured cells were identified by plating 5×105 cells/35 mm culture dish (Day 0) and every 24 hours cell proliferation was determined by quantifying the cell number (using a hemocytometer). The third to seventh passages were used. A medium without serum but supplemented with 5 mg/ml bovine serum albumin was substituted for the original medium at the subconfluent period and cultured for an additional 24 h. The medium was collected and used for assays of protein content and lysyl oxidase activity. Lysyl oxidase activity was assayed with [4,5-³H] -lysine labelled purified chick - embryo aorta elastin substrate. After incubation for 10 h at 37°C, the enzyme activity was measured from 3HHO (tritiated water) separated by ultrafiltration using Amicon C-10 micro-concentrators. The results showed the mean doubling time of OSF fibroblasts was 3.2 days and of NM fibroblasts was 3.6 days. NM fibroblasts became confluent at day 6 as determined by cell number, while OSF fibroblasts were confluent by Day 5. Furthermore, the immunoenzymatic assay for BrdUrd incorporation revealed that OSF fibroblasts proliferate significantly faster than NM fibroblasts under standard culture conditions. Both total protein content (10.84±1.15 mg/ml) and lysyl oxidase activity (3558.6±345.5 cpm/106 cell) in OSF fibroblasts were greater than in NM fibroblasts (6.35±0.96 mg/ml and 2436.0±352.6 cpm/10⁶ cell). The results of this study provide evidence that fibroblasts derived from oral submucous fibrosis (OSF) tissue and normal mucosa (NM), although similar in many respects, exhibit specific differences in proliferation rates and lysyl oxidase activity. Moreover, collagen deposition in OSF tissue may, at least in part, be ascribed to increased lysyl oxidase activity.     

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.     

槟榔碱及机械刺激构建大鼠口腔黏膜下纤维化模型

目的 利用槟榔碱和机械刺激构建Sprague-Dawley（SD）大鼠口腔黏膜下纤维化（OSF）模型。方法 采用两因素析因实验设计将48只大鼠分为8个组,每组6只。分别用不同浓度（0、0.5、2、8 mg·mL ^-1）槟榔碱涂擦及机械刺激 (有或无毛刷涂擦)。处理16周后测量开口度;取局部颊黏膜行苏木精-伊红（HE）染色观察病理变化;并检测组织内Ⅲ型胶原、转化生长因子-β1（TGF-β1）和γ干扰素（IFN-γ）的表达情况。结果 2和8 mg·mL ^-1（中、高）浓度槟榔碱处理16周,颊黏膜出现了典型的OSF病理特征;开口度显著减小并且Ⅲ型胶原、TGF-β1表达显著增加（P<0.05）。机械刺激虽可导致黏膜Ⅲ型胶原、TGF-β1和IFN-γ表达增高（P<0.05）,但无病理学改变,开口度改变不显著。结论 中、高浓度槟榔碱有致OSF作用;机械刺激无法导致大鼠OSF的发生。     

颊脂垫瓣在口腔黏膜下纤维性变术后缺损修复中的应用

目的：研究颊脂垫瓣在口腔黏膜下纤维性变（OSF）切除后组织缺损修复中的应用。方法：10例伴重度张口受限的OSF患者,应用颊脂垫瓣修复切除后遗留的组织缺损,观察颊脂垫瓣的愈合过程及修复效果。结果：术后9例患者张口度均大于3cm,颊脂垫瓣愈合良好,术后1周颊脂垫瓣明显水肿,组织瓣表面有薄层伪膜覆盖,1周后伪膜逐渐消失,2周后伪膜完全消失,2～3周后水肿明显消退,颊脂垫表面逐渐上皮化,6～8周再生的黏膜变得光滑,呈粉红色,类似于正常口腔黏膜。结论：颊脂垫瓣为修复OSF术后缺损提供了一种良好的手术方式,有利于改善张口受限症状。     

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.     

槟榔碱及机械刺激构建大鼠口腔黏膜下纤维化模型

目的 利用槟榔碱和机械刺激构建Sprague-Dawley（SD）大鼠口腔黏膜下纤维化（OSF）模型。方法 采用两因素析因实验设计将48只大鼠分为8个组,每组6只。分别用不同浓度（0、0.5、2、8 mg·mL ^-1）槟榔碱涂擦及机械刺激 (有或无毛刷涂擦)。处理16周后测量开口度;取局部颊黏膜行苏木精-伊红（HE）染色观察病理变化;并检测组织内Ⅲ型胶原、转化生长因子-β1（TGF-β1）和γ干扰素（IFN-γ）的表达情况。结果 2和8 mg·mL ^-1（中、高）浓度槟榔碱处理16周,颊黏膜出现了典型的OSF病理特征;开口度显著减小并且Ⅲ型胶原、TGF-β1表达显著增加（P<0.05）。机械刺激虽可导致黏膜Ⅲ型胶原、TGF-β1和IFN-γ表达增高（P<0.05）,但无病理学改变,开口度改变不显著。结论 中、高浓度槟榔碱有致OSF作用;机械刺激无法导致大鼠OSF的发生。     

HLA-antigens in oral submucous fibrosis

HLA–typing was carried out on 122 areca nut chewers who attended hospitals for complaints unrelated to the habit. The subjects were South Africans of Indian extraction. The study did not include haplotypes. Palpable fibrous bands in the mouth indicated oral submucous fibrosis. The subjects were divided into 4 groups based on specific oral symptoms and signs. Groups A and B were without fibrous bands. Group A (47 subjects) included those with one or no symptoms while group B (28 subjects) suffered from 2 to 7 oral symptoms. Group C (17 subjects) had oral symptoms and represented early or mild oral submucous fibrosis and exhibited at least one discrete palpable fibrous hand. Group D (30 subjects) were classic oral submucous fibrosis cases with multiple bands. The high occurrence of oral submucous fibrosis in this study group (39%) is similar to the occurrence in comparable age groups reported earlier–n South Africa and is conceivably due to the higher age range of the subjects and their relatively long exposure to the area nut. We were unable to demonstrate a specific pattern of HLA–antigen frequencies in chewers with or without the disease. Furthermore, there were no differences between the study population and the controls. It is concluded that there is not necessarily a HLA–associated susceptibility in oral submucous fibrosis.     

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.     

HLA typing in Taiwanese patients with oral submucous fibrosis

BACKGROUND: A significant association of certain human leukocyte antigens (HLA) and haplotypic pairs with oral submucous fibrosis (OSF) has been reported. However, controversial result of no HLA association with OSF has also been reported. In this study, the phenotype and haplotype frequencies of HLA-A, -B, -C, -DRB1, and -DQB1 in 135 Taiwanese OSF patients were calculated and compared with those in 540 healthy control Taiwanese. METHODS: The analysis of HLA-A, -B, and -C antigens, and of HLA-DRB1 and -DQB1 alleles in OSF patients and healthy control subjects, was performed by serologic typing and DNA typing using polymerase chain reaction with sequence-specific primers (PCR-SSP), respectively. RESULTS: We found that the phenotype frequency of HLA-B76 (3.0%) in OSF patients was significantly greater than that (0%) in healthy control subjects (corrected P (Pc) = 0.000). In addition, the haplotype frequencies of HLA-B48/Cw7 (3.0%), -B51/Cw7 (6.7%), and -B62/Cw7 (8.2%) in OSF patients were significantly greater than the corresponding haplotype frequencies (0, 0.7, and 1.9%, respectively) in healthy control subjects (Pc = 0.000). The relative risk (RR) values of haplotypes B51/Cw7 (9.57) and B62/Cw7 (4.7) were greater than the RR values of phenotypes B51 (1.81), B62 (2.31), and Cw7 (1.91) in OSF patients. In addition, the etiologic fraction (EF) value of haplotype B51/Cw7 (0.63) was higher than the EF values of phenotypes B51 (0.2) and Cw7 (0.59). CONCLUSIONS: We conclude that some Taiwanese areca quid (AQ) chewers with particular HLA phenotypes and haplotypes are prone to have OSF. In addition, some particular HLA haplotypes may play more important roles than the individual HLA phenotypes for the genetic susceptibility to OSF. However, the significantly increased HLA phenotype B76 and three of the common HLA haplotypes detected are present in only about 20% of incident cases of OSF.     

High incidence of autoantibodies in Taiwanese patients with oral submucous fibrosis.

BACKGROUND: Previous study has shown a high incidence of autoantibodies including antinuclear (ANA), antismooth muscle (SMA), antigastric parietal cell (GPCA), antithyroid microsomal (TMA), and antireticulin antibodies in a small group of 26 patients with oral submucous fibrosis (OSF). The reasons why some of the OSF patients have high titers of autoantibodies in serum have not been completely explained and no further study on autoantibodies in OSF patients has been done in a large group of patients. METHODS: In this study, we determined the serum levels of ANA, SMA, GPCA, and TMA in a large group of 109 male Taiwanese patients with OSF by an indirect immunofluorescence technique (for ANA, SMA, and GPCA), and by a semiquantitative microtiter particle agglutination test (for TMA). The presence of serum autoantibodies in OSF patients was further correlated with patients' oral habits and the severity of OSF measured by maximum mouth opening (MMO) and sites of involvement. RESULTS: We found that the frequencies of presence of serum ANA (23.9%), SMA (23.9%), and GPCA (14.7%) in OSF patients were significantly higher than those (9.2, 7.3, and 5.5%, respectively) in healthy control subjects (P < 0.01, P < 0.005, and P < 0.05, respectively). Although the frequency of presence of TMA (5.5%) in OSF patients was also greater than that (2.8%) in healthy control subjects, the difference was not significant (P > 0.05). The presence of serum GPCA in OSF patients was significantly associated with daily areca quid (AQ) consumption (P < 0.05). The presence of serum ANA in OSF patients associated with daily AQ consumption was of borderline statistical significance (P = 0.066). However, no significant correlations were demonstrated between the presence of serum autoantibodies in OSF patients and other variables of oral habits, MMO, and sites of involvement. CONCLUSION: In this study, all the 109 OSF patients had AQ chewing habit and 73.4% of the OSF patients swallowed the 'juice' of AQ during the chewing process. The presence of serum GPCA and ANA in OSF patients was associated with daily consumption of AQs. AQ chewing caused mucosal microtrauma, and ulcerations facilitated the diffusion of genotoxic and cytotoxic AQ ingredients into the oral and gastric tissues. Altered autoantigens released from AQ ingredients-damaged cells may induce autoantibody production. Higher frequencies of specific HLA-DR antigens in OSF patients may also help autoantibody production. Therefore, we conclude that the high incidence of autoantibodies in OSF patients may be due to AQ chewing habit, toxic AQ ingredients, and genetic susceptibility of the OSF patients.