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目的探讨体外诱导人骨髓基质干细胞(hMSCs)向结膜上皮样细胞(hCjEs)定向分化的可能性。方法体外分离、培养hMSCs和人hCjEs,分别从细胞形态学和免疫细胞化学的方法对细胞进行鉴定;Transwell非接触共培养法诱导hMSCs向结膜上皮样细胞定向分化;倒置相差显微镜观察分化细胞的形态学改变;免疫细胞化学和反转录聚合酶链反应分别对分化细胞角蛋白13(CK13)的蛋白和mRNA表达进行检测;并用单独培养的hMSCs和hCjEs做对照。结果实验获取的hMSCs以长梭形为主,融合呈漩涡状;hCjEs呈不规则圆形或多边形,融合的hCjEs成铺路石样;hMSCs阳性表达CD44和CD29,hCjEs阳性表达CK13;非接触共培养使hMSCs的形状由长梭形逐渐变为不规则多边形;免疫细胞化学和RT-PCR分别在蛋白和基因水平证实分化细胞表达CK13,与单独培养的结膜上皮细胞表达相似。结论在一定条件下,可以诱导体外培养的hMSCs形态发生变化、向具有结膜上皮细胞表型的上皮样细胞定向分化。
Objective To explore the possibility of inducing directional differentiation of human bone marrow stromal cells (hMSCs) into conjunctival epithelial cells (hCjEs) in vitro. Methods hMSCs and human hCjEs were isolated and cultured in vitro. Cells were identified by morphological and immunocytochemical methods respectively. Transwell non-contact co-culture method was used to induce hMSCs to differentiate into conjunctival epithelial cells. The morphological changes of differentiated cells were observed under inverted phase contrast microscope The immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression of differentiated cytokeratin 13 (CK13). The hMSCs and hCjEs were used as controls. Results hMSCs were mainly fusiform in shape and swirling in shape. The hCjEs were irregular round or polygons, hCjEs fused into paving stones, hMSCs positive for CD44 and CD29, hCjEs positive for CK13, non-contact co-culture The shape of hMSCs gradually changed from long fusiform to irregular polygon. Immunocytochemistry and RT-PCR confirmed the expression of CK13 in differentiated cells at the protein and gene level respectively, which was similar to the cultured conjunctival epithelial cells. Conclusion Under certain conditions, the morphological changes of hMSCs cultured in vitro can be induced to differentiate into epithelial cells with the phenotype of conjunctival epithelial cells.