地塞米松对人牙髓细胞增殖分化影响的研究

Effect of Dexamethasone on Proliferation and Differentiation of Human Dental Pulp Cells In Vitro

目的观察地塞米松对体外培养的人牙髓细胞增殖和分化的影响。方法收集拔除的无龋坏、无牙周疾病的健康前磨牙及第三磨牙,获得牙髓,酶消化联合组织块法体外培养人牙髓细胞(Human dental pulp cells,hDPCs),扩增后取第4代生长状态良好的细胞用于实验。分为两组,实验组hDPCs在含2%胎牛血清(Fetal bovine serum,FBS)的αMEM培养基中加入10-8mol/L地塞米松(Dexamethasone,Dex)培养,对照组单纯使用含2%FBS的αMEM培养基培养。在细胞培养第1、3、5天检测细胞增殖情况;细胞培养的第1、5、10天分别进行ALP染色及ALP定量分析检测细胞分化情况;细胞培养第10天进行茜素红染色观察钙化结节形成情况。结果细胞增殖活性检测显示,实验组培养第3天及第5天可见hDPCs增殖活性得到显著抑制,与对照组相比有统计学差异;ALP染色及定量测定:培养10 d后,地塞米松实验组ALP活性明显高于对照组;茜素红染色结果:培养10 d后两组细胞茜素红染色均为阳性,表明实验组与对照组均有钙化结节形成,但是实验组钙化结节形成数量较多。结论酶消化联合组织块法可以成功体外培养hDPCs。10-8mol/L地塞米松可显著抑制hDPCs的增殖活性,提高hDPCs的ALP活性及矿化能力。

Objective To investigate the effect of dexamethasone on proliferation and differentiation of human dental pulp cells in vitro. Methods Human dental pulp cells （hDPCs） were isolated from extracted healthy premolars and third molars for clinical reasons hke orthodontics and impaction. The teeth were immediately cracked open, and the pulp tissue was dissected and digested by tissue-explant enzyme digestion. The fourth passage of hDPCs were used for experiment. The cells were randomly divided into two groups, experimental group and control group. The cells in the experimental group were treated with 10^-8 mol/L Dexamethasone （Dex） in 2% FBS aMEM media. The cells in the control group were cultured in 2% FBS aMEM media only. At 1, 3 and 5 days after culture, quantitative determination of hDPCs proliferation activity was observed. At 1, 5 and 10 days after culture, Alkaline phosphatase （ALP） staining and quantitative measurement were examined histologically and biochemically. At 10 days after culture, Alizarin red staining was used to observe the calcified nodules. Results Cell proliferation detection： 10~s mol/L Dex significantly inhibited cell proliferation of hDPCs at 3 and 5 days after culture. There were significant difference between experimental group and control group; ALP staining and quantitative analysis： quantitative analysis of ALP activity showed at 10 days after culture, Dex significantly enhanced ALP activity of HDPCs compared with control group; Alizarin Red staining results： At 10 days after culture, both groups showed positive staining, which indicated the calcified nodule formation in both groups. However, the number of calcified nodule accumulated in experimental group was higher than the control group. Conclusion Tissue-explant enzymatic digestion method could successfully cultivate hDPCs in vitro. Dexamethasone significantly inhibited human dental pulp cell proliferation. Conversely, Dexamethasone strongly stimulated alkaline phosphatase activity and promoted calcified nodule formation.