目的 探讨体外诱导人骨髓基质干细胞（human bone marrow mesenchymal stem cells, HMSCs）向角膜上皮样细胞分化的可能性。 方法 组织块培养法原代培养人眼角膜基质细胞(Human corneal fibroblasts, HCFs)并进行传代培养；密度梯度离心法分离、培养HMSCs；倒置相差显微镜观察细胞的形态学特征；免疫细胞化学对细胞进行鉴定。利用角膜上皮细胞生长的微环境对处于共培养体系的HMSCs进行诱导培养；免疫细胞化学检测角膜上皮细胞的特异性分子标志角蛋白12（cytokeratins12, CK12）在分化细胞的原位表达；逆转录聚合酶链反应（reverse transcriptase polymerase chain reaction, RT-PCR）检测分化细胞CK12 mRNA的表达，用单独培养的HMSCs做对照。 结果 HMSCs和HCFs贴壁生长，以梭形为主，接近汇合的细胞排列有一定方向性，呈漩涡状；HMSCs阳性表达CD29、HCFs阳性表达波形纤维蛋白；随着共培养时间的延长，HMSCs形状逐渐由长梭形变为不规则多边形，与上皮细胞的形态相似；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CK12。结论 在一定诱导条件下，体外培养的HMSCs可向具有角膜上皮细胞表型的上皮样细胞分化。
Abstract
Objective To investigate whether human bone marrow mesenchymal stem cells（HMSCs） are able to differentiate, in vitro, into corneal epithelial-like cells. Methods The primary human corneal keratocytes (HCFs) were obtained from the corneal stromal pieces by tissue culture, and the HMSCs were isolated by density gradient centrifugalization. Both the HMSCs and HCFs were identified according to their morphological characterizes, and the protein expression of CD29 and vimentin were analyzed by immunocytochemistry. To induce cells differentiation, HMSCs were cultured by establishing epithelial culture microenvironment in a co-culture model. Cells were characterized by phase contrast microscopy observation. Using immunocytochemistry，we confirmed the protein presence of cornea-specific cytokeratin 12 (CK12) in differentiated cells. What is more, the CK12 mRNA in differentiated cells was analyzed by reverse transcriptasepolymerase chain reaction（RT-PCR）.Results Most of the adherent HCFs and HMSCs were in spindle-shape, and perfectly align in a certain direction of the whirlpool-like shape while cells were grown to near confluence. HMSCs positively stained for CD29 and HCFs expressed Vimentin. HMSCs changing shape toward irregular polygon morphology after co-culture with HCFs over time, and the differentiating cells formed cell clusters some of which resembled epithelial-like cells. The expression of CK12 was positive for immunocytochemistry staining in differentiated cells, and further confirmed by RT-PCR. These results indicated that the differentiated cells acquired characteristics similar to those of corneal epithelial. Conclusion HMSCs isolated from human bone marrow were capable of differentiating into epithe1ial-like cells and possess the phenotypical characteristics of corneal epithe1ial.
关键词
骨髓基质干细胞 /
细胞培养 /
分化 /
角膜
Key words
Mesenchymal stem cells /
Cell culture /
Differentiation /
Cornea
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