增龄因素对人牙髓干细胞生物学行为影响的研究

目的 对不同年龄患者的牙髓干细胞(human dental pulp stem cells,hDPSCs)进行分离培养,并比较其生物学特性。方法 应用组织块联合胰酶消化法培养不同年龄患者的hDPSCs,记录细胞爬出时间,显微镜下观察比较细胞形态。流式细胞术检测细胞表面抗原,并对细胞行体外成骨、成脂诱导,观察矿化结节和脂滴的形成。CCK8细胞增殖实验比较细胞增殖能力,并通过qRT-PCR比较各组细胞成牙/成骨相关基因牙本质涎磷蛋白(DSPP)、骨钙素(OCN)、核心结合因子α1(RUNX2)、骨桥蛋白(OPN)、Ⅰ型胶原(COL-1)的表达水平。结果 不同年龄患者的hDPSCs基本呈梭形,但随着年龄增长,不规则形态的细胞数目增多,且细胞爬出时间存在统计学差异(P<0.05)。流式细胞术结果显示:各组细胞CD90、CD73高表达,CD146低表达,CD34不表达。在成骨及成脂诱导下,各组细胞均能够形成矿化结节和脂滴。CCK8检测及qRT-PCR结果显示:随着年龄的增长,hDPSCs增殖能力下降,成牙/成骨基因表达水平明显降低(P<0.05)。结论 来自不同年龄供体的牙髓组织中均可提取出具有多向分化能力的干细胞。细胞增殖与年龄相关,各年龄段细胞在一定程度上均可分化为成牙本质细胞/成骨细胞,但随着供体年龄增长,成牙/成骨能力明显降低。

Effects of age on the biological behavior of human dental pulp stem cells

Objective To isolate and cultivate human dental pulp stem cells (hDPSCs) from patients of different ages and compare their biological characteristics. Methods hDPSCs from patients of different ages were cultivated by the enzymatic digestion of pulp tissue method. The time when cells appeared was recorded, and their morphology was observed and compared under a microscope. Flow cytometry was used to detect surface markers of hDPSCs. Cells were induced to differentiate into odontoblasts and adipocytes in vitro. The formation of mineralized nodules and lipid droplets was observed. The CCK8 assay was used to compare the ability of cell proliferation. Moreover, mRNA levels of odontogenesis/osteogenesis related genes DSPP, OCN, RUNX2, OPN and COL-1 of different groups were detected and compared by qRT-PCR. Results hDPSCs from donors of different ages were mainly spindle-shaped. However, number of irregular cells increased with age, and there existed significant difference among the time in which cells crawled out (P<0.05). The result of flow cytometry showed that CD90 and CD73 were expressed positively, but CD146 was expressed negatively, with no expression of CD34. Mineralized nodules and lipid droplets were successfully formed in all groups under the induction of osteogenesis and lipogenesis. The CCK8 assay and qRT-PCR demonstrated that cell proliferation and the expression of odontogenic/osteogenic genes descended markedly with age (P<0.05). Conclusion hDPSCs that own multilineage differentiation potential can be obtained from pulp tissue of donors in different ages. Cell proliferation is related to age. The hDPSCs of different ages can be turned into odontoblasts/osteocytes in a certain degree. However, as the donor ages, odontogenesis/osteogenesis differentiative potential of hDPSCs decreases obviously.