Dannewitz B, Tomakidi P, Syagailo Y, Kohl A, Staehle H J, Eickholz P, Komposch G, Steinberg T
Department of Operative Dentistry and Periodontology, Dental School, University of Heidelberg, Heidelberg, Germany.
J Periodontal Res. 2009 Feb;44(1):62-72. doi: 10.1111/j.1600-0765.2007.01066.x. Epub 2008 Oct 7.
Collagen type I elevation in cyclosporin A-induced gingival overgrowth supports evidence that gingival fibroblasts play a decisive role in the manifestation of the phenotype. To analyze the role of gingival fibroblasts under more in vivo-like conditions, we evaluated the effect of cyclosporin A on collagen type I gene and protein expression in gingival overgrowth-derived gingival fibroblasts established as cocultures with gingival keratinocytes as well as in matched gingival fibroblast monolayers.
Monolayers and cocultures of primary gingival fibroblasts were treated with cyclosporin A for 6 and 72 h. The expression of collagen type I mRNA was analyzed by quantitative real time polymerase chain reaction, while expression and secretion of collagen type I protein was analyzed by indirect immunofluorescence and western blotting.
Compared with controls, significant elevation of collagen type I mRNA was restricted to cocultures after 6 and 72 h of treatment with cyclosporin A. In keratinocytes, collagen type I remained undetectable. In monolayers and cocultures, indirect immunofluorescence showed a slightly higher level of collagen type I protein in gingival fibroblasts in response to stimulation with cyclosporin A. Semiquantitative detection of collagen type I by western blotting demonstrated a nonsignificant increase for cell extracts in monolayers and cocultures. For secreted collagen type I, western blot analysis of the supernatants revealed elevated protein levels in cultures stimulated with cyclosporin A. Compared with the corresponding monolayers, the stimulatory effect of cyclosporin A on protein secretion was significant only in coculture.
Our results indicate that collagen type I is a target of cyclosporin A and that gingival fibroblasts are decisive for the manifestation of the gingival overgrowth-phenotype. Furthermore, the results suggest that cocultures of gingival overgrowth-derived gingival fibroblasts and gingival keratinocytes permit analysis of cyclosporin A-induced effects under more in vivo-like conditions.
环孢素A诱导的牙龈过度增生中I型胶原蛋白水平升高，这支持了牙龈成纤维细胞在该表型表现中起决定性作用的证据。为了在更接近体内的条件下分析牙龈成纤维细胞的作用，我们评估了环孢素A对与牙龈角质形成细胞共培养建立的牙龈过度增生来源的牙龈成纤维细胞以及匹配的牙龈成纤维细胞单层中I型胶原蛋白基因和蛋白表达的影响。
原代牙龈成纤维细胞单层和共培养物用环孢素A处理6小时和72小时。通过定量实时聚合酶链反应分析I型胶原蛋白mRNA的表达，同时通过间接免疫荧光和蛋白质印迹分析I型胶原蛋白蛋白的表达和分泌。
与对照组相比，用环孢素A处理6小时和72小时后，I型胶原蛋白mRNA的显著升高仅限于共培养物。在角质形成细胞中，未检测到I型胶原蛋白。在单层和共培养物中，间接免疫荧光显示环孢素A刺激后牙龈成纤维细胞中I型胶原蛋白蛋白水平略高。通过蛋白质印迹对I型胶原蛋白进行半定量检测表明，单层和共培养物中细胞提取物的增加不显著。对于分泌的I型胶原蛋白，对上清液的蛋白质印迹分析显示环孢素A刺激的培养物中蛋白质水平升高。与相应的单层相比，环孢素A对蛋白质分泌的刺激作用仅在共培养中显著。
我们的结果表明I型胶原蛋白是环孢素A的靶标，并且牙龈成纤维细胞对牙龈过度增生表型的表现起决定性作用。此外，结果表明牙龈过度增生来源的牙龈成纤维细胞和牙龈角质形成细胞的共培养允许在更接近体内的条件下分析环孢素A诱导的效应。
