人牙髓干细胞向成牙本质细胞定向分化过程中miRNAs表达谱筛选及鉴定

摘要

目的:筛选人牙髓干细胞向成牙本质细胞定向分化过程中差异表达的microRNAs(miRNAs)并进行初步鉴定.方法:体外分离、培养和鉴定人牙髓干细胞,miRNAs芯片筛选牙髓干细胞向成牙本质细胞分化过程中差异表达的miRNAs,并通过TargetScan数据库预测靶基因,实时定量PCR法对结果进行初步鉴定.结果:人牙髓干细胞vimentin、nestin、GFAP和I型胶原4 种细胞表型均表达;成脂诱导3 周后细胞内有油红O染色阳性脂滴出现;成牙本质诱导可见钙结节形成;基因芯片结果显示,牙髓干细胞向成牙本质细胞分化过程中,发生2 倍以上表达变化的miRNAs有6 条,其中上调3 条,下调3 条.通过miRNA靶标预测工具预测靶基因,发现hsa-miR-633和hsa-miR-210有与牙髓干细胞分化相关的靶基因;real time-PCR验证hsa-miR-633和hsa-miR-210表达变化与基因芯片结果相符.结论:人牙髓干细胞经诱导向成牙本质细胞分化过程中miRNAs表达谱具有显著变化,为牙髓干细胞分化机制研究提供了新的提示.%Objective: To screen and identificate the differential expression microRNAs(miRNAs) of human dental pulp stem cells (DPSCs) during directional differentiation into odontoblasts. Methods: Human DPSCs were isolated, cultrued and identificated in vitro, the differential expression miRNAs of DPSCs were examined during directional differentiation into odontoblasts by miRNAs chip method. The target genes were forecasted by the TargetScan database and identificated by real time-PCR. Results: DPSCs were found to express many different proteins examined by immunohistochemistry, including vimentin, nestin, GFAP and collagen type Ⅰ . DPSCs could be induced into odontoblast-like cells and adipocyte-like cells. During directional differentiation into odontoblasts, there were 6 kinds of differently expressed miRNAs including 3 up-regulated and 3 down-regulated. Forecasted target genes of bsa-miR-633 and hsa-miR-210 were found by the TargetScan database,the target genes were related with the differentiation of DPSCs. The results of RTPCR of hsa-miR-633 and hsa-miR-210 were accord with those of gene chip. Conclusion: The expression profile of miRNAs in human DPSCs during directional differentiation into odontoblasts may provide a new method in study of the differention mechanism of DPSCs.