Bryja A, Dyszkiewicz-Konwińska M, Chachuła A, Ciesiółka S, Kranc W, Bukowska D, Antosik P, Bruska M, Nowicki M, Zabel M, Kempisty B
Department of Anatomy, Poznan University of Medical Sciences, Poznan, Poland.
Department of Biomaterials and Experimental Dentistry, Poznan University of Medical Sciences, Poznan, Poland.
J Biol Regul Homeost Agents. 2016 Oct-Dec;30(4):951-960.
In recent years, buccal pouch oral mucosa cells were used as a source of potential biological grafting material in advanced tissue engineering. However, there are several limitations in the process of graft fabrication: donor and recipient patient availability as well as an incomplete knowledge of in vitro procedures related to tissue surgical recovery, in vitro cell culture (IVC) and/or tissue processing in “human somatic cell therapy.” Therefore, the animal model for oral mucosa grafting is still recognized as a source for xenografts and a useful model for biomedical research. In this study, the porcine buccal pouch oral mucosa cells were used in analysis of the stromalization/epithelialization process during short-term, in vitro real-time cell proliferation. We evaluated cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression as epithelial and stromal cell markers, respectively. The porcine buccal pouch oral mucosa cells were cultured in vitro for 168 h, and the protein expression/ distribution was analyzed every 24 h during real-time cell proliferation. In our analysis of protein expression using fluorescence intensity (FI), followed by confocal microscopic observations, we found the highest expression of CK18 occurred after 24 h of IVC, panCK after 72 h, and Vim after 48 h of IVC, as compared to other cultivation periods. We also found a substantial increase in Vim expression (3-4 fold) as compared to CK18 and panCK, and all of the investigated proteins were distributed in the cellular cytoplasm. The lag phase of cell proliferation occurred during the first 24 h of IVC, whereas the log phase was observed between 24 h-120 h of IVC. Throughout 7 days of IVC, statistically significant differences were found in Cell Index (CI) of the analyzed cells. Increased Vim expression in buccal pouch oral mucosa cells, as compared to CK18 and panCK, suggested that the stromal cells substantially predominated during in vitro cell cultivation. This may be a result of significant specificity of porcine oral mucosa cells isolated from the buccal pouch.
近年来，颊囊口腔黏膜细胞被用作先进组织工程中潜在生物移植材料的来源。然而，在移植制备过程中存在一些局限性：供体和受体患者的可获得性，以及对“人类体细胞治疗”中与组织手术恢复、体外细胞培养（IVC）和/或组织处理相关的体外程序的了解不完整。因此，口腔黏膜移植的动物模型仍被认为是异种移植的来源和生物医学研究的有用模型。在本研究中，猪颊囊口腔黏膜细胞被用于分析短期体外实时细胞增殖过程中的基质化/上皮化过程。我们分别评估了细胞角蛋白18（CK18）、细胞角蛋白8 + 18 + 19（泛CK）和波形蛋白（Vim）作为上皮和基质细胞标志物的表达。将猪颊囊口腔黏膜细胞体外培养168小时，并在实时细胞增殖过程中每24小时分析一次蛋白质表达/分布。在我们使用荧光强度（FI）分析蛋白质表达并随后进行共聚焦显微镜观察时，我们发现与其他培养时期相比，CK18在IVC 24小时后表达最高，泛CK在72小时后表达最高，Vim在IVC 48小时后表达最高。我们还发现与CK18和泛CK相比，Vim表达大幅增加（3 - 4倍），并且所有研究的蛋白质都分布在细胞质中。细胞增殖的延迟期发生在IVC的前24小时，而对数期在IVC的24小时 - 120小时之间观察到。在整个7天的IVC过程中，分析的细胞的细胞指数（CI）存在统计学上的显著差异。与CK18和泛CK相比，颊囊口腔黏膜细胞中Vim表达增加表明在体外细胞培养过程中基质细胞占主导地位。这可能是从颊囊中分离的猪口腔黏膜细胞具有显著特异性的结果。
