共培养诱导人骨髓间充质干细胞的定向分化

Differentiation of Mesenchymal Stem Cells Induced by Co-culture Method

目的探讨间接共培养体外诱导人骨髓间充质干细胞(MSCs)向上皮样细胞定向分化的可行性。方法消化培养法获取人结膜上皮细胞,密度梯度离心法联合贴壁培养法培养MSCs,并对所培养的细胞进行免疫细胞化学鉴定。将人MSCs和结膜上皮细胞进行非接触共培养,免疫细胞化学、RT-PCR、Western blotting对分化细胞进行鉴定。结果体外培养的人MSCs阳性表达CD29,不表达CD34。结膜上皮细胞贴壁生长,呈不规则圆形,阳性表达细胞角蛋白4(CK4)。MSCs与结膜上皮细胞共培养10d后,大部分细胞呈不规则圆形外观,基因和蛋白水平均表达CK4。结论与人结膜上皮细胞体外非接触共培养可诱导人MSCs向上皮样细胞定向分化。

Objective To investigate the potential differentiation of human mesenchymal stem cells （MSCs） into epithelium-like cells by an in vitro co-culture method. Methods The human conjunctival epitheli- um was obtained by digestion with dispase l] , and the MSCs were isolated by density gradient centrifugaliza- tion. All cells were identified according to their morphologies and cell-surface antigen profiles by immunocyto- chemical analysis. The MSCs underwent co-culture with conjunctival epithelium in the manner without cell-to-cell contract. The morphological characterizes of cells were observed under contrast microscope, and the cytokeratin-4 expressions of the differentiated cells were identified by immunocytochemistry staining, reverse transcriptase poly- merase chain reaction （RT-PCR）, and Western blotting. Results Immunocytochemistry showed that positive expression of CD29 and negative expression of CD34 in the in vitro cultured MSCs. Cytokeratins4 （CK4） was positively expressed in the human conjunctival epithelium. After co-cultured with conjunctival epithelial for 10 days, CK4 was detected in differentiated cells by immunocytochemistry, RT-PCR, and Western blotting, Conclusion MSCs can differentiate into epithel ium~!ike cells after having been co-cultured with human eonjunetival epithelium.