Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022 China.
Cell Mol Biol Lett. 2019 Apr 4;24:3. doi: 10.1186/s11658-018-0133-x. eCollection 2019.
Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization.
Rat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs).
COMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63α ( = 0.9177), ABCG2 ( = 0.526), Ki67 ( = 0.0987), and CK3 ( = 0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) ( = 0.0038 for RT-qPCR, = 0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) ( = 0.0172 for RT-qPCR, = 0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) ( < 0.0001 for RT-qPCR, = 0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability ( = 0.0002) and tube formation ( = 0.0002) of HUVECs.
LNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.
自体培养的口腔黏膜上皮移植（COMET）是治疗角膜缘干细胞缺乏症的重要方法。但是术后周边角膜新生血管化会阻碍其应用。本研究旨在使用异体角膜缘巢细胞（LNCs）培养体系代替 3T3 细胞作为饲养层，以减轻术后新生血管化。
将大鼠口腔黏膜上皮细胞（OMECs）与大鼠 LNCs 或 3T3 细胞共培养。通过苏木精和伊红染色和免疫细胞化学鉴定不同培养体系培养的口腔黏膜上皮细胞（COMECs）。通过 RT-qPCR 和 Western blot/ELISA 分析与血管生成相关的因子的表达水平。通过人脐静脉内皮细胞（HUVECs）的细胞活力和管形成实验再次确认血管生成潜力。
成功从两种培养体系中获得了 COMECs。免疫细胞化学显示，不同组的 COMECs 中 p63α（=0.9177）、ABCG2（=0.526）、Ki67（=0.0987）和 CK3（=0.4000）的阳性染色细胞百分比大致相同。RT-qPCR 和 Western blot/ELISA 显示，LNC 组的 COMECs 表达的碱性成纤维细胞生长因子（bFGF）（=0.0038 为 RT-qPCR，=0.0026 为 Western blot）显著降低，但色素上皮衍生因子（PEDF）（=0.0172 为 RT-qPCR，=0.0253 为 Western blot）和可溶性 fms 样酪氨酸激酶-1（sFlt-1）（=0.0001 为 RT-qPCR，=0.0064 为 ELISA）表达增加。与 3T3 组的 COMECs 相比，LNC 组的 COMECs 可降低 HUVECs 的活力（=0.0002）和管形成（=0.0002）。
LNCs 可替代 3T3 细胞在体外扩增 OMECs，且该体系获得的 COMECs 不易引起术后新生血管化，为体外培养系统提供了另一种选择，并促进了 COMET 的应用。
