煅烧牙粉影响人牙周膜干细胞体外矿化的自噬机制研究

A study on the effect of the calcined tooth powder on autophagy mechanism of the in vitro mineralization of human periodontal ligament stem cells

目的探讨自噬在煅烧牙粉调节牙周膜干细胞体外矿化中的作用,为牙周膜干细胞的定向诱导分化和牙周病的治疗提供参考。方法选用成人完整的牙齿在300℃的条件下煅烧后,研磨成粉,与α-MEM混合制备成20μg/mL牙粉条件培养基,与人牙周膜干细胞共培养。采用流式细胞技术检测其对牙周膜干细胞凋亡的影响,通过Western blot检测、免疫荧光染色、茜素红染色和ALP染色观察检测其对牙周膜干细胞自噬活性及体外矿化的影响。结果流式细胞技术结果显示牙粉对牙周膜干细胞的凋亡无明显影响(P>0.05);Western blot结果显示煅烧牙粉显著上调人牙周膜干细胞的自噬相关蛋白Beclin1,ATG5的表达和LC3Ⅱ/LC3Ⅰ的比值,以及明显下调了P62的蛋白水平(P<0.05);免疫荧光染色显示牙粉促进人牙周膜干细胞中LC3在胞质内点状聚集;茜素红染色显示牙粉促进牙周膜干细胞的体外矿化;ALP染色显示自噬活性抑制剂氯喹降低牙粉对牙周膜干细胞体外矿化的促进作用。结论煅烧牙粉通过激活自噬调节牙周膜干细胞的体外矿化作用。

Objective To evaluate the effects of calcined tooth powder on mineralization of human periodontal ligament stem cells(hPDLSCs) in vitro and related mechanism,to provide reference for the induced directional differentiation of periodontal ligament stem cells and treatment of periodontal diseases.Methods The human intact teeth were calcined under 300 ℃ and milled into powder,then mixed with α-MEM to make 20 μg/mL tooth powder as culture medum.The hPDLSCs were isolated and cultured with or without calcined tooth powder.The apoptosis of hPDLSCs was detected by flow cytometry.Western blot,immunofluorescence staining,alizarin red staining and alkaline phosphatase staining were performed to verify the effect of calcined tooth powder on autophagy and mineralization of hPDLSCs in vitro.Results There were no significant differences apoptosis between two groups of hPDLSCs.The protein level of Beclin1,ATG5 and the ratio of LC3Ⅱ/LC3Ⅰ of hPDLSCs cultured in calcined tooth powder conditioned medium were significantly higher than the control group hPDLSCs,while the expression of P62 was significantly lower than the control group.The calcium mineralization was significantly higher after osteogenic induction in the group of hPDLSCs calcined tooth powder treated than control group.Alkaline phosphatase expression levels were inhibited by inhibition of autophagy in the process of mineralization in vitro.Conclusion The calcined tooth powder can up-regulate mineralization of hPDLSCs via inducing autophagy in vitro.