精氨酸-甘氨酸-天冬氨酸-丝氨酸序列在生物活性玻璃对人牙髓细胞生物作用中的影响

Influence of the Arg-Gly-Asp-Ser sequence on the biological effects of bioactive glass on human dental pulp cells

目的:生物活性玻璃(bioactive glass,BG)对人牙髓细胞(human dental pulp cells,hDPCs)的增殖及矿化有积极的作用,本研究目的是研究精氨酸-甘氨酸-天门冬氨酸-丝氨酸序列(Arg-Gly-Asp-Ser,RGDS)在BG对hDPCs的黏附、增殖及矿化作用中的影响。方法:用含不同质量浓度RGDS(12.5 mg/L、25.0 mg/L、50.0 mg/L、100.0 mg/L、200.0 mg/L)的BG浸提液培养hDPCs,对照组为不含RGDS的BG浸提液。采用细胞活性噻唑蓝(MTT)比色法于接种后4 h检测细胞黏附数量,于第1、3、5、7、9天测定细胞增殖,第14、28天对细胞进行茜素红染色检测矿化活性。结果:BG浸提液加RGDS组与单纯BG浸提液组相比,hDPCs黏附数目降低,呈RGDS浓度依赖性;BG浸提液加RGDS组的增殖活性早期低于单纯BG浸提液组,但后期两组之间差异无统计学意义;BG浸提液能够促进hDPCs的矿化,加入RGDS组的矿化活性与单纯BG浸提液组差异无统计学意义。结论:游离态RGDS不影响BG对hDPCs的增殖和矿化促进作用,但会与hDPCs结合进而影响hDPCs向培养皿底的黏附。

Objective：Positive effects of bioactive glass （BG） on proliferation,mineralization,and differentiation of human dental pulp cells （hDPCs） was already verified in various former studies.The Arg-Gly-Asp-Ser sequence （RGDS） was confirmed of affecting cell adhesion.Before further investigation,the objective of this study is to investigate whether RGDS can affect the effects of BG on the adhesion,proliferation and mineralization of hDPCs.Methods： hDPCs were harvested from third molars of 18-25-year-old individuals after informed consent.Enzyme digestion technique was used.The 4th to 6th ge-neration of hDPCs were used for all experiments.The cells of the experimental groups were cultured in Dulbecco minimum essential medium （DMEM） containing ionic dissolution products of BG and RGDS of seve-ral concentrations （12.5 mg/L,25.0 mg/L,50.0 mg/L,100.0 mg/L,200.0 mg/L）.DMEM containing ionic dissolution products of BG without RGDS was used for cell culture as control group.Cell adhesion was tested 4 h after cell seeding by MTT assay.Cell proliferation was examined at 1,3,5,7,and 9 d after cell seeding by MTT assay.Cell mineralization was investigated on days 14 and 28 by alizarin red staining.After being stained and dried,mineralized nodules were dissolved by cetylpyridinium chloride （CPC） for semi-quantitative test.Results were statistically analyzed by one way ANOVA,SPSS （version 19.0） and P〈0.05 was considered to be significant.Results： Cell adhesion in BG group showed no difference from that in DMEM group.Compared with BG group,hDPCs in BG＋RGDS groups suggested weaker cell adhesion.When the concentration of RGDS increased,the adhered cell number decreased.hDPCs cultured with BG and RGDS showed lower proliferation activity in the early stage,while no significant difference was observed after 3 d.BG group promoted the mineralization of hDPCs compared with positive control group,negative control group and RGDS group.No significant difference was observed between BG＋RGDS group and BG group or between RGDS group and positive control group.Conclusion： BG promotes proliferation and mineralization without affecting cell adhesion of hDPCs.Unbounded RGDS inhibits cell adhesion,but has no influence on the positive effects of BG on the proliferation and mineralization of hDPCs.