不同浓度氧化石墨烯对人牙周膜干细胞增殖及成骨分化的影响

Effects of different concentrations of graphene oxide on proliferation and osteogenic differentiation of human periodontal ligament stem cells

目的研究不同浓度氧化石墨烯(GO)对人牙周膜干细胞(hPDLSCs)增殖及成骨分化的影响。方法将人健康牙周组织分离培养鉴定的hPDLSCs放置于完全培养基中培养,加入含0,0.1,0.5,2.5,12.5,62.5μg/ml GO的完全培养基,通过细胞计数试剂盒法(CCK-8)测定1,3,5,7 d的吸光度值(OD)来检测细胞的增殖能力。茜素红染色观察矿化结节形成情况,实时荧光定量PCR检测细胞成骨标志基因的表达水平。结果加入含不同浓度GO的完全培养基后第1,3,5,7天,0.1,0.5,2.5,12.5μg/ml GO对hPDLSCs增殖无影响(P>0.05),从第3天起,62.5μg/ml GO抑制hPDLSCs增殖(P<0.001)。不同浓度GO成骨诱导液诱导14 d后,茜素红染色显示,随着GO成骨诱导液浓度递增,矿化结节形成逐渐减少。同时,与0μg/ml相比,0.1,0.5,2.5μg/ml GO不同程度地抑制成骨标志基因ALP、Runx2及OCN的表达。结论不同浓度GO对hPDLSCs产生不同影响,低浓度GO对hPDLSCs的增殖无影响,高浓度GO抑制hPDLSCs的增殖。同时,GO能抑制hPDLSCs成骨分化。

Objective To study the effects of different concentrations of graphene oxide(GO)on the proliferation and the osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs).Methods The hPDLSCs isolated and cultured from human healthy periodontal tissues were cultured with complete medium containing 0,0.1,0.5,2.5,12.5,62.5μg/ml GO,respectively.The cell counting kit method(CCK-8)was used to determine the optical density(OD)of cells at days 1,3,5,and 7,respectively in order to evaluate the proliferation ability.The formation of mineralized nodules was observed by Alizarin red staining,and the expression levels of osteogenic marker genes in cells were detected by real-time fluorescent quantitative PCR.Results The proliferation of hPDLSCs showed no significant difference at day 1,3,5,7 after adding complete medium containing 0.1,0.5,2.5,12.5μg/ml GO(P>0.05),while the proliferation of hPDLSCs was significantly inhibited from the third day by 62.5μg/ml GO(P<0.001).After 14 d treatment with different concentrations of GO osteogenic induction liquid,Alizarin red staining showed that as the concentration of GO osteogenic induction liquid increased,the formation of mineralized nodules were gradually decreased.Compared with 0μg/ml GO,0.1,0.5,2.5μg/ml GO inhibited the expression of osteogenic marker genes ALP,Runx2 and OCN.Conclusion Different concentrations of GO have different effects on hPDLSCs.Low concentrations of GO have no effect on the proliferation of hPDLSCs,while high concentrations of GO can inhibit the proliferation of hPDLSCs.At the same time,GO inhibits the osteogenic differentiation of hPDLSCs.