口腔黏膜上皮细胞和成纤维细胞的复合培养

目的 为癌前病变临床病理研究建立一种简便、迅速和有效的口腔黏膜上皮细胞和成纤维细胞复合培养的方法,实现体外模拟组织工程化口腔黏膜的发生发展。方法 用DispaseⅡ分离上皮和皮下组织,用KGM培养口腔黏膜上皮细胞。用细胞培养法和组织块培养法获取验用的口腔黏膜上皮细胞和成纤维细胞,并采用复合培养法对两种细胞进行共同培养。结果 用DispaseⅡ可成分地分离上皮和皮下组织。KGM可明显促进口腔黏膜上皮细胞的分裂繁殖。复合培养的HE染色切片显示薄层的结缔组织之上有方形的基底细胞、颗粒细胞和角化层。结论 KGM可明显促进口腔黏膜上皮细胞的分裂和成熟。采用组织培养法获取原代成纤维细胞是适合口腔黏膜取材等特点的有效方法。气液相培养的口腔黏膜下结缔组织可促进上皮细胞的分层和分化。     

Areca-treated fibroblasts enhance tumorigenesis of oral epithelial cells

Several hundred million Asians chew areca nut, which is strongly associated with oral carcinogenesis in people of this region. The impacts of areca nut extract on oral target cells are largely unclear. This study hypothesized an inductive role for areca-nut-exposed stromal cells in the progression of oral carcinomas in an at-risk population. Oral fibroblasts with chronic subtoxic areca nut extract treatment exhibited growth arrest and MMP-2 activation. The supernatant of arrested oral fibroblasts activated the AKT signaling pathway in oral carcinoma cells. The enhancement of proliferation, migration, and anchorage-independent growth of oral carcinoma cells elicited by such supernatant could be abrogated by blockers against MMP-2 or AKT. Subcutaneous co-injection of arrested oral fibroblasts into nude mice significantly enhanced the tumorigenicity of xenographic oral carcinoma cells. This study concludes that areca nut extract may impair oral fibroblasts and then modulate the progression of oral epithelial oncogenesis via their secreted molecules.     

Transforming growth factor-β response and expression in junctional and oral gingival epithelial cells

The junctional (JE) and oral gingival (OGE) epithelium show distinct morphological phenotypes and express different cell surface and keratin markers. Transforming growth factor-β (TGF-β) has been shown to stimulate extracellular matrix formation and inhibit proteolytic matrix degradation in periodontal wound healing. To elucidate potential roles of TGF-β in gingival epithelial regeneration and reattachement, the present study examined the effects of TGF-β on JE and OGE cell growth and determined the patterns of expression of mRNAs for the TGF-β isotypes β1, β2 and β3 and TGF-β receptor types I, II and III. Primary cell cultures were initiated from JE and OGE and the cell phenotypes confirmed using monoclonal antibodies to specific keratins. TGF-β induced a significant growth inhibition in OGE cells derived from 6 different patients with a mean inhibition of 46% and a range of 16–70% (p=0.031). Although responses varied between patients, in general maximum inhibition occurred at 10 ng/ml TGF-β. JE cells from 5 patients showed no significant growth inhibition by TGF-β (p=0.125). Greater expression of TGF-β2 and receptor type I mRNA was found in OGE than JE cells and thus appeared to be associated with differentiating epithelial cells. JE cells expressed more TGF-β type II receptor specific mRNA than did OGE cells, but TGF-β1 mRNA expression was similar in JE and OGE cells. JE or OGE cultures derived from 2 of 3 patients showed expression of mRNA for the TGF-β type III receptor. TGF-β3 mRNA was not detected in any of the JE or OGE samples examined. The greater sensitivity of OGE than JE to the growth inhibiting effects of TGF-β correlated with higher expression of receptor type I mRNA which, together with the type II receptor, is required for sensitivity to growth inhibition by TGF-β. The results suggest that, in addition to structural differences, the development of functional differences in the responses of JE and OGE to TGF-β may be associated with the formation of JE from OGE cells and the reformation of attachement after periodontal surgery.     

Shosaikoto increases calprotectin expression in human oral epithelial cells

Hiroshima Y, Bando M, Kataoka M, Shinohara Y, Herzberg MC, Ross KF, Inagaki Y, Nagata T, Kido J. Shosaikoto increases calprotectin expression in human oral epithelial cells. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01203.x. © 2009 John Wiley & Sons A/S Background and Objective: Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad‐spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up‐regulated by interleukin‐1α (IL‐1α). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL‐1α in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL‐1α in oral epithelial cells. Material and Methods: Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 μg/mL) in the presence or absence of anti‐IL‐1α or IL‐1 receptor antagonist. The expression of S100A8‐ and S100A9‐specific mRNAs was examined by northern blotting. Calprotectin expression and IL‐1α secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL‐1α was analyzed by RT‐PCR and by quantitative real‐time PCR. Results: Shosaikoto (25 μg/mL) significantly increased the expression of S100A8‐ and S100A9‐specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL‐1α‐specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL‐1α or IL‐1 receptor antagonist inhibited SST up‐regulated S100A8/S100A9 mRNA expression. Conclusion: These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up‐regulation of calprotectin may be partially induced via IL‐1α.     

The possible role of oral epithelial cells in tissue-type plasminogen activator-related fibrinolysis in human saliva

We studied the fibrinolytic activities of the following subfractions of unstimulated human whole saliva on plasminogen-rich fibrin plates: (1) submandibular saliva, (2) parotid saliva, and (3) smears of buccal epithelial cells from ten healthy males. A cell-bound plasminogen activator could be demonstrated in the sediments of all three subfractions of whole saliva. The incorporation of antibodies (goat IgG) against human two-chain tissue-type plasminogen activator (t-PA) could quench the assessed fibrinolytic activities, whereas additional experiments suggested the absence of urokinase-like and F XII-dependent activators of fibrinolysis. The determinations in growth medium from buccal-epithelial cell culture of t-PA antigen by means of enzyme-linked immunosorbent assay showed the presence of t-PA. These clinical and experimental findings suggest that buccal-epithelial cells produce t-PA, while the activity of t-PA in parotid and submandibular saliva is very low.     

Effect of Hangeshashinto on calprotectin expression in human oral epithelial cells

Oral epithelial cells produce antimicrobial peptides (AMPs) to prevent microbial infection. Calprotectin (S100A8/S100A9) is one of these AMPs in oral epithelial cells, the expression of which is up-regulated by interleukin-1α (IL-1α). Hangeshashinto (HST) is a traditional Japanese herbal medicine that has anti-inflammatory effects. The purpose of this study was to investigate the effect of HST on the expression of calprotectin through the regulation of IL-1α in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with HST in the presence or absence of anti-IL-1α antibody or IL-1 receptor antagonist, or with six major components of HST (3,4-dihydroxybenzaldehyde, baicalin, ginsenoside Rb1, glycyrrhizin, oleanolic acid and berberine). The expression of S100A8, S100A9, other AMPs and cytokine mRNAs was examined by RT-PCR and quantitative real-time PCR. Calprotectin expression and IL-1α secretion were investigated by ELISA. HST (6 μg/ml) increased the expression of S100A8/S100A9 mRNAs and calprotectin protein, and also up-regulated β-defensin 2 (DEFB4) and S100A7 expression. The expression of IL-1α mRNA and its protein was slightly but significantly increased by HST. A neutralizing antibody against IL-1α and IL-1 receptor antagonist inhibited HST-up-regulated S100A8/S100A9 mRNA expression. Although 3,4-dihydroxybenzaldehyde, baicalin and ginsenoside Rb1 as HST components increased S100A8/S100A9 expression, oleanolic acid and berberine decreased their expression. These results suggest that HST increases the expression of calprotectin, DEFB4 and S100A7 in oral epithelial cells. In response to HST, up-regulation of calprotectin expression may be partially induced via IL-1α.     

Attachment of cell fragments of Fusobacterium nucleatum to oral epithelial cells,gingival fibroblasts and white blood cells

The characteristic haemagglutination (HA) caused by Fusobacterium nucleatum strains was used for measuring their attachment to oral epithelial, gingival fibroblast and white blood cells. Whole cells and sonicated preparations of oral F. nucleatum strains VPI No. 4355, 10197 and ATCC No. 10953 haemagglutinated human and sheep red blood cells. Sonicated preparations of the organisms were tested for HA activity before and after absorption with human cells. Buccal epithelial, gingival fibroblast and white blood cells bound the HA-active fragments, as observed by: (1) decrease in the HA activity of the sonicated preparation after absorption, (2) increase in HA activity of the cells used for absorption, (3) presence of microbial fragments on the cells after absorption detected by fluorescent antibody. The HA-active fragments were released from the cells by EDTA; furthermore, galactose but not glucose inhibited the attachment of F. nucleatum to human cells. The role of cell binding in colonization by the organisms and in immune-stimulated damage to host cells is important.     

Type I interferon and interferon‐stimulated gene expression in oral epithelial cells

Oral epithelial cells (OEC) represent the first site of host interaction with viruses that infect the body through the oral route; however, their innate antiviral defense mechanisms yet to be defined. Previous studies have determined that OEC express pathogen‐, damage‐, or danger‐associated molecular patterns (PAMPs or DAMPs), but their expression of key antiviral innate immune mediators, including type I interferons (type I IFN) and interferon‐stimulated genes (ISGs) has not been studied extensively. We used the oral keratinocyte cell line, OKF6/TERT1, in the presence and absence of the viral mimics poly(I:C) and unmethylated CpG DNA, to define the expression of type I IFN and ISGs. We identified the basal expression of novel type I IFN genes IFNE and IFNK, while IFNB1 was induced by viral mimics, through the nuclear translocation of IRF3. Numerous ISGs were expressed at basal levels in OEC, with an apparent correlation between high expression and antiviral activity at the earlier stages of viral infection. Stimulation of OECs with poly(I:C) led to selective induction of ISGs, including MX1, BST2, PML, RSAD2, ISG15, and ZC3HAV1. Together, our results demonstrate that OECs exhibit a robust innate antiviral immune defense profile, which is primed to address a wide variety of pathogenic viruses that are transmitted orally.