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目的:对人牙周膜干细胞进行筛选纯化,骨向诱导后观察其成骨能力,并对成骨相分化过程中相关基因的表达变化进行检测。方法:用组织块联合磁珠分选法分离纯化人牙周膜干细胞,初步探讨其成骨性能,碱性磷酸酶(ALP)和茜素红染色观察其成骨情况,实时定量PCR检测其相关成骨基因的表达变化。利用SPSS12.0软件包对数据进行统计学分析。结果:组织块联合磁珠分选法能成功培养出高纯度的人牙周膜干细胞。体外培养中,PDLSCs具备成脂和成骨的双相分化能力。在成骨诱导过程中,FOXO1和RUNX2的表达先升高再降低,ALP和OCN的基因表达水平持续增高。结论:成骨相关基因ALP、RUNX2、FOXO1和OCN均参与其向成骨样组织分化的过程,其表达变化具有时间规律性,与成骨过程类似。
OBJECTIVE: To screen and purify human periodontal ligament stem cells, observe their osteogenic ability after osteogenic induction, and detect the expression changes of related genes in the process of osteogenic differentiation. METHODS: Human periodontal ligament stem cells were isolated and purified by tissue block combined with magnetic bead sorting. The osteogenic properties, alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the osteogenic behavior. The correlations were detected by real-time quantitative PCR Osteogenic gene expression changes. SPSS12.0 software package for statistical analysis of the data. Results: Tissue block combined with magnetic bead sorting method could successfully produce high purity human periodontal ligament stem cells. In vitro culture, PDLSCs possess adipogenic and osteogenic bipolar differentiation. During osteogenic induction, the expression of FOXO1 and RUNX2 increased first and then decreased, and the gene expression levels of ALP and OCN continued to increase. CONCLUSION: Osteogenesis-related genes ALP, RUNX2, FOXO1 and OCN are all involved in their differentiation into osteoblasts. The expression of ALP, RUNX2, FOXO1 and OCN is time-dependent and similar to osteogenic process.