关键词: 二氧化铈, 树枝状介孔二氧化硅, 颌骨骨髓间充质干细胞, 成骨分化, 无翅型MMTV整合位点家族/β-连环蛋白信号通路, 活性氧

Abstract:

Objective To discuss the effects of CeO₂-loaded dendritic mesoporous silica nanoparticles （DS@CeO₂ NPs） on the osteogenic differentiation of mouse jaw bone marrow mesenchymal stem cells （JBMMSCs）， and to clarify its possible mechanism. Methods DS@CeO₂ NPs were synthesized. Transmission electron microscopy （TEM） and dynamic light scattering （DLS） method were used to detect the morphology and particle size of DS@CeO₂ NPs. Enzyme digestion method was used to isolate mouse JBMMSCs. Flow cytometry method was used to identify the cell surface markers on JBMMSCs. The cells were divided into blank control group and different concentrations of DS@CeO2 NPs （25， 50， 100， and 200 mg·L^-1） groups. Cell counting kit-8 （CCK-8） method and Calcein/propidium iodide （PI） staining method were used to detect the proliferation and survival of the cells in various groups. The cells were further divided into control group and different concentrations of DS@CeO₂ NPs osteogenic induction groups （0， 25， 50， and 100 mg·L^-1）. After 7 d of osteogenic induction， alkaline phosphatase （ALP） staining method was used to detect the ALP activities in the cells in various groups. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of ALP， Runt-related transcription factor 2 （Runx2）， bone sialoprotein 2 （BSP2）， and osteopontin （OPN） mRNA in the cells in various groups. Western blotting method was used to detect the expression levels of ALP， Runx2， and OPN proteins in the cells in various groups. In addition， osteogenesis group and osteogenesis+DS@CeO₂ NPs group were established. RT-qPCR method and Western blotting method were used to detect the expression levels of β-catenin mRNA and protein in the cells in two groups. Under hydrogen peroxide （H₂O₂） stimulation， the cells were divided into control group and DS@CeO₂ NPs pretreatment group. 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence staining method and flow cytometry method were used to detect the levels of reactive oxygen species （ROS） in the cells in two groups. Results The TEM and DLS results showed that DS@CeO₂ NPs exhibited a nearly spherical morphology with an average hydrated particle size of 378.2 nm. The flow cytometry results showed that the expression rates of CD73， CD90， CD105， and CD44 in the cells were all higher than 95%， while CD34 and CD45 were almost not expressed， so the cells could be identified as JBMMSCs. The CCK-8 method results showed that compared with control group， there was no statistically significant difference in the survival rates of the cells in 25， 50， and 100 mg·L^-1 DS@CeO₂ NPs groups （P>0.05）， while the survival rate of the cells in 200 mg·L^-1 DS@CeO₂ NPs group was significantly decreased （P<0.05）. The ALP staining results showed that compared with control group and 0 mg·L^-1 DS@CeO₂ NPs group， the cells in 25 and 50 mg·L^-1 DS@CeO₂ NPs groups exhibited darker ALP staining and larger positive staining areas， and the ALP activities in the cells were increased. The RT-qPCR and Western blotting results showed that compared with 0 mg·L^-1 DS@CeO₂ NPs group， the expression levels of ALP， BSP2， Runx2， and OPN mRNA as well as the expression levels of ALP， Runx2， and OPN proteins in the cells in 25 mg·L^-1 DS@CeO₂ NPs group were significantly increased （P<0.05）. Compared with pure osteogenic group， the expression levels of β-catenin mRNA and protein in the cells in osteogenic+DS@CeO₂ NPs group were significantly increased （P<0.05）. The DCFH-DA staining and flow cytometry results showed that compared with control group， the ROS level in the cells in DS@CeO₂ NPs pretreatment group was decreased. Conclusion DS@CeO₂ NPs show good biocompatibility and can promote the osteogenic differentiation of JBMMSCs； the mechanism may be related to activation of Wnt/β-catenin signaling pathway and antioxidant effect.

Key words: Cerium oxide, Dendritic mesoporous silica, Jaw bone marrow mesenchymal stem cells, Osteogenic differentiation, Wingless-type MMTV integration site family/β-catenin signaling pathway, Reactive oxygen species