体外诱导人骨髓基质干细胞向角膜上皮样细胞的分化

发布时间：2018-06-05 18:10

本文选题：骨髓基质干细胞 + 分化　；参考：《解剖学报》2016年01期

【摘要】：目的探讨体外诱导人骨髓基质干细胞(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(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(CK12)在分化细胞的原位表达;反转录聚合酶链反应(RT-PCR)检测分化细胞CK12 m RNA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 the possibility of inducing the differentiation of human bone marrow stromal cells (HMSCs) into corneal epithelioid cells in vitro. Methods Human corneal stromal cells (HCFs) were cultured with tissue mass culture method, HMSCs were isolated by density gradient centrifugation, morphological characteristics of cells were observed by inverted phase contrast microscope, and cells were identified by immunocytochemistry. The HMSCs in the co-culture system was induced by the microenvironment of corneal epithelial cell growth, and the expression of keratin 12 (CK12), a specific molecular marker of corneal epithelial cells, was detected by immunocytochemistry. Reverse transcriptase polymerase chain reaction (RT PCR) was used to detect the expression of CK12 m RNA in differentiated cells. HMSCs cultured alone was used as control. Results the adherent cells of HMSCs and HCFs were mainly fusiform, and the cells close to confluence were arranged in a certain direction, and the positive expression of CD29HCFs and fibrin was observed in the form of whirlpool. With the extension of co-culture time, the shape of HMSCs gradually changed from long spindle to irregular polygon, similar to the morphology of epithelial cells. Immunocytochemistry and RT-PCR confirmed the expression of CK12 in differentiated cells at the protein and gene levels, respectively. Conclusion under certain induction conditions, cultured HMSCs can differentiate into epithelioid cells with phenotype of corneal epithelial cells.
【作者单位】：山东大学第二医院眼科;山东省肿瘤医院基础实验室;山东大学医学院人体解剖学研究所;
【基金】：山东省科技发展计划(2014GSF118006) 山东大学基本科研业务费资助自然科学专项(自由探索项目2015TS012)
【分类号】：R772

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