Costea Daniela Elena, Johannessen Anne Christine, Vintermyr Olav Karsten
Department of Odontology-Oral Pathology and Forensic Odontology, Faculty of Dentistry, University of Bergen, Norway.
Differentiation. 2005 Apr;73(4):134-41. doi: 10.1111/j.1432-0436.2005.00017.x.
This study aimed to investigate the role of underlying fibroblasts on morphogenesis of in vitro epithelium reconstituted with normal and neoplastic human oral keratinocytes at various stages of malignant transformation. Primary normal human oral keratinocytes (NOKs), early neoplastic/dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ 15 cell line) were organotypically grown on top of a collagen type I matrix with or without primary normal human oral fibroblasts. Morphogenesis of the reconstituted epithelia was assessed by histomorphometry, immunohistochemistry (Ki-67, cyclin D1, cytokeratin 13 (CK13), collagen IV, E-cadherin, p53, CD40), and the terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end-labelling method. Reproducible in vitro models of multistage oral carcinogenesis were established. Presence of fibroblasts in the collagen matrix significantly increased cell proliferation in all three models (p<0.05), and induced an invasive pattern of growth in the neoplastic cell lines (p<0.05). In normal, but not in neoplastic oral keratinocytes fibroblasts induced the expression of CD40, and polarized the expression of E-cadherin and p53 to the basal cell layer. In both normal and early neoplastic keratinocytes (DOK cell line), fibroblasts induced the expression of CK13 and collagen IV. In the neoplastic oral keratinocytes (PE/CA-PJ 15 cell line), the presence of underlying fibroblasts did not change the expression of any of the protein markers assessed. This study showed that (1) major steps of oral carcinogenesis can be reproduced in vitro, and (2) the tight control exerted by fibroblasts on epithelial morphogenesis of in vitro reconstituted normal human oral mucosa is gradually lost during neoplastic progression.
本研究旨在探讨在恶性转化不同阶段，人正常和肿瘤性口腔角质形成细胞体外重构上皮形态发生过程中，成纤维细胞的潜在作用。原代人正常口腔角质形成细胞（NOKs）、早期肿瘤性/发育异常的人口腔角质形成细胞（DOK细胞系）和肿瘤性人口腔角质形成细胞（PE/CA-PJ 15细胞系）在含或不含原代人正常口腔成纤维细胞的I型胶原基质上进行器官样生长。通过组织形态计量学、免疫组织化学（Ki-67、细胞周期蛋白D1、细胞角蛋白13（CK13）、IV型胶原、E-钙黏蛋白、p53、CD40）以及末端脱氧核苷酸转移酶介导的dUTP原位缺口末端标记法评估重构上皮的形态发生。建立了可重复的体外口腔癌多阶段模型。胶原基质中存在成纤维细胞显著增加了所有三种模型中的细胞增殖（p<0.05），并诱导肿瘤细胞系出现侵袭性生长模式（p<0.05）。在正常而非肿瘤性口腔角质形成细胞中，成纤维细胞诱导CD40表达，并使E-钙黏蛋白和p53的表达极化至基底细胞层。在正常和早期肿瘤性角质形成细胞（DOK细胞系）中，成纤维细胞均诱导CK13和IV型胶原的表达。在肿瘤性口腔角质形成细胞（PE/CA-PJ 15细胞系）中，下层成纤维细胞的存在并未改变所评估的任何蛋白质标志物的表达。本研究表明：（1）口腔癌发生的主要步骤可在体外重现；（2）在肿瘤进展过程中，成纤维细胞对体外重构的人正常口腔黏膜上皮形态发生的严格控制逐渐丧失。
