Dyszkiewicz-Konwińska M, Bryja A, Jopek K, Budna J, Khozmi R, Jeseta M, Bukowska D, Antosik P, Bruska M, Nowicki M, Zabel M, Kempisty B
Department of Biomaterials and Experimental Dentistry, Poznań University of Medical Sciences, Poznań, Poland.
Department of Anatomy, Poznań University of Medical Science, Poznań, Poland.
J Biol Regul Homeost Agents. 2017 Oct-Dec;31(4):855-864.
Recently, using experimental animal model, we demonstrated that porcine buccal pouch mucosal cells reflect increased proliferation capability during primary cultivation in vitro. Although the histological structure and morphogenesis in oral cavity is well recognized, the molecular mechanisms which regulate this process still need further investigation. This study was aimed to analyze the molecular marker expression profile involved in morphogenesis and differentiation capacity of porcine buccal pouch mucosal cells during their long-term primary cultivation in vitro. The experiment was performed on buccal pouch mucosal cells isolated from 80 pubertal crossbred Landrace gilts. After collection, the cells were treated enzymatically and transferred into a primary in vitro culture (IVC) system and cultured for 30 days. The cells were collected for RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using the RTCA system. We found an increased expression of FN1 and SOX9 genes when calculated against ACTB after 7, and 30 days of IVC, (P less than 0.01, P less than 0.001, respectively). The CXCL12 mRNA was down-regulated after 7, 15 and 30 days of IVC, but not statistically significant. Similar expression profile was observed when calculated against HPRT, however, DAB2 was found to be higher expressed at day 15 of IVC, (P less than 0.05). The cell index measured during real-time cell proliferation was substantially increased between 96 h and 147h of IVC and reached the log phase. Since FN1 and SOX9 revealed significant increase of expression after long-term culture in vitro, it is suggested that expression of these differentiation and stemness genes is accompanied by cell proliferation. Moreover, FN1 and SOX9 might be recognized as new markers of buccal pouch mucosal cell proliferation and differentiation in pigs in in vitro primary culture model.
最近，我们利用实验动物模型证明，猪颊囊黏膜细胞在体外原代培养过程中增殖能力增强。尽管口腔的组织结构和形态发生已得到充分认识，但调节这一过程的分子机制仍需进一步研究。本研究旨在分析猪颊囊黏膜细胞在体外长期原代培养过程中参与形态发生和分化能力的分子标志物表达谱。实验采用从80头青春期杂交长白母猪分离的颊囊黏膜细胞进行。细胞收集后，经酶处理并转移至体外原代培养（IVC）系统中培养30天。在IVC培养7天、15天和30天后收集细胞用于RNA提取，并使用RTCA系统检测其实时增殖状态。我们发现，与ACTB相比，IVC培养7天和30天后，FN1和SOX9基因的表达增加（分别为P<0.01，P<0.001）。IVC培养7天、15天和30天后，CXCL12 mRNA表达下调，但无统计学意义。以HPRT为对照计算时观察到类似的表达谱，然而，发现DAB2在IVC培养15天时表达较高（P<0.05）。在IVC的96小时至147小时之间，实时细胞增殖过程中测得的细胞指数大幅增加并进入对数期。由于FN1和SOX9在体外长期培养后表达显著增加，提示这些分化和干性基因的表达与细胞增殖相关。此外，在体外原代培养模型中，FN1和SOX9可能被视为猪颊囊黏膜细胞增殖和分化的新标志物。
